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Carpenter, Sarah Elizabeth.
Enzyme linked spectroscopic assays for Glyoxylate :
b the use of Peptidylglycine alpha-Amidating Monoxygenase for the discovery of Novel alpha-Amidated hormones
h [electronic resource] /
by Sarah Elizabeth Carpenter.
[Tampa, Fla] :
University of South Florida,
ABSTRACT: Peptide hormones are responsible for cellular functions critical to the survival of an organism. Approximately 50% of all known peptide hormones are post-translationally modified at the C-terminus. Enzymatic oxidative conversion of C-terminal glycine extended peptide precursors results in an a-amidated peptide and glyoxylate. Peptidylglycine a-amidating monooxygenase (PAM) is the single known enzyme responsible for catalyzing this reaction. PAM is an O2, Cu(II), and Zn(II) dependent bifunctional enzyme. Initially, PAM hydroxylates the glycyl a-carbon followed by dealkylation of the hydroxylated intermediate to an a-amidated product and glyoxylate. PAM is also responsible for the conversion of glycine extended fatty acids to fatty acid amides and glyoxylate. PAM catalyzes the activation of all glycine-extended prohormones including biomolecules ranging from neuro to physio-homeostatic hormones. Identification of a-amidated hormones from a biological source has been s everely hindered by the lack of a specific assay for this distinctive class of biological hormones, indicating that numerous a-amidated hormones remain undiscovered. Based on the selective in situ chemistry of PAM, a novel and specific assay was developed for the discovery of a-amidated hormones. The identification of novel a-amidated hormones will lead to an increased understanding of post-translational modifications and will pioneer a new understanding of a-amidated hormone biosynthesis, regulation, and bioactivity. Discovery of novel a-amidated biomolecules could also lead to their use as pharmaceuticals as there are several currently marketed a-amidated peptide based pharmaceuticals.Inhibition of PAM in cell culture leads to the accumulation of glycine-extended hormones in the conditioned medium. The medium was fractionated by chromatographic techniques and each specific fraction was then assayed by the newly developed platform technology for the presence of a-amidated hormones For every a-amidated hormone synthesized by PAM, glyoxylate is also formed. Based on this 1:1 molar ratio, several novel spectrophotometric, fluorescent, and chemi-luminescent enzyme linked assays for glyoxylate were developed, which when utilized on cell culture fractions proved positive for the identification of a-amidated hormones. Each novel spectroscopic assay was independently verified by a variety of known methodologies. Moreover the assay was utilized to identify two known a-amidated hormones accumulated from cell culture, which were further verified by Mass Spectral analysis.
Dissertation (Ph.D.)--University of South Florida, 2006.
Includes bibliographical references.
Text (Electronic dissertation) in PDF format.
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Adviser: David J. Merkler, Ph.D.
Mouse joining peptide.
Calcitonin gene related peptide.
High performance liquid chromotography.
t USF Electronic Theses and Dissertations.
Enzyme Linked Spectroscopic Assays for Glyoxylate; The Use of Peptidylglycine alpha-Amidating Monoxygenase for the Discovery of Novel alpha-Amidated Hormones by Sarah Elizabeth Carpenter A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: David J. Merkler, Ph.D. Robert Potter, Ph.D. Randy Larsen, Ph.D. Nozer Mehta, Ph.D. James Garey, Ph.D. Date of Approval: January 23, 2006 Keywords; glyoxylate, platform technololgy, mouse joining peptide, glycolate oxidase, chemi-luminescence, calcitonin gene re lated peptide, high performance liquid chromotography Copywright 2006, Sarah Elizabeth Carpenter
Dedication I would like to dedicate this disserta tion to Mrs. Charlotte R. Carpenter.
Acknowledgements I would like to acknowledge the help and support of my coll eagues Neil R. McIntyre and Edward W. Lowe Jr., and my parents Melissa A. Leffler, Herbert L. Carpenter and Allan T. Leffler, M.D. I would also like to ac knowledge the support of my major professor David Merkler Ph.D., and my committee members for the guidance and direction provided to me as a graduate student. In conclusion, I w ould also like to acknowledge the following people: Terrance C. Owen Ph.D ., Angelo P. Consalvo, Ted Gauthier Ph.D., Patricia Mueisner Ph.D., Ellen Verdel Ph.D., Julie Harmon Ph.D., Brian Space Ph.D., James Garey Ph.D., Steven Grossman Ph.D., the USF Chemistry Department Faculty and Staff, and Unigene Laboratories, Inc.
i Table of Contents List of Tables vi List of Figures vii Abstract x Chapter One: Peptidylglycine -Amidating Monooxygenase (PAM) 1 The Catalytic Role of Peptidylglycine -Amidating Monooxygenase 1 -Amidated Peptide Hormones; Bios ynthesis, Identification, and Applications 3 -Amidated Peptide Hormones and Their Biosynthesis 3 The Rate Limiting Role of PAM 4 Current Methodologies Used for the Identification of -Amidated Hormones 6 Method of Tatemoto and Mutt 6 Method of Hill & Flannery 9 Immunological Detection of -Amidated 11 Hormones Computer Based Analysis for -Amidated peptides 11 Therapeutic Uses of the Known -Amidated Peptides 12 Impetus for the Design of a Novel, Broadly Applicable Assay for the Discovery of -Amidated Peptide Hormones 15 Utilizing PAM as a General Tool for -Amidated Hormone Discovery 15 Platform Technology Process and Design 17 Development of a Series of Glyoxylate Assays; Glyoxylate as a Signal Molecule for the Detection of -Amidated Hormones 19 Prior Art for the Detection of Glyoxylate; A Literature Review 20 The Rimini-Schryver Chemical Spectrophotometric Assay for Aldehydes 20 Determination of Glyoxylate by Capillary Electrophoresis 23 The Fluorescent / Spectrophotometric Adduct of Glyoxylate and Resorcinol 25 Authentication of the Proposed Platform Technology 26 Authentication of the Newly Designed Glyoxylate Assays 27 Authentication for the PAM Dependent Discovery of Novel -Amidated Peptides 26
ii Chapter Two: Glyoxylate as a Signal Molecule for the Detection of -Amidated Peptides 28 PAM Activity as a Key to the Discovery of -Amidated Peptides 28 Identification of PAM Produced Glyoxylate 29 Introduction of Spectrophotometric En zyme Dependent Assays for Glyoxylate 32 Introduction to the Malate Synt hase / Malate Dehydrogenase Glyoxylate Consuming Enzymes 33 The Glyoxylate Dependent Formation of a Formazan Dye 34 Introduction to the Glyoxylate Consuming Enzyme Glycolate Oxidase 38 The Glyoxylate Dependent Production of H2O2 39 Introduction to the Glyoxylat e Consuming Enzyme Glyoxylate Reductase 42 Independent Verification of Glyoxylat e Concentration as a Reference Standard for the Newly Developed Assays 43 Material and Methods 45 Materials 45 Methods 46 Malate Synthase / Malate De hydrogenase (MS / MD Assay) Spectrophotometric Assay for Glyoxylate 46 Standardization of the MS / MD Dependent Formazan Assay with NADH 46 Standardization of the MS / MD Assay with Glyoxylate 46 Measurement of the Base-Catal yzed Production of Benzamide and Glyoxylate from -Hydroxyhippurate Utilizing the MS / MD Assay 47 Measurement of PAM Produced Benzamide and Glyoxylate from Hippurate Utilizing the MS / MD Assay 48 Glycolate Oxidase / Horse Radish Peroxidase (GO / HRP) Linked DMAB / MBTH Spectrophotometric Assay for Glyoxylate 49 Standardization of the Glycol ate Oxidase/Horse Radish Peroxidase Assay with H2O2 as Substrate 49 Standardization of the Glycolate Oxidase / Horse Radish Peroxidase Assay with Glyoxylate as Substrate 50 Standardization of the Glycolate Oxidase / Horse Radish Peroxidase Assay for PAM Pr oduced Glyoxylate as Substrate 51 Glyoxylate Reductase Spectrophoto metric Assay for Glyoxylate 52 Purification of Glyoxylate Reductase from Spinacia oleracea 52 Standardization of the Glyoxylate Reductase Assay for Glyoxylate 53 Standardization of the Glyoxylate Reductase Assay for PAM Produced Glyoxylate 54
iii Independent Analysis; Reverse Phase C18 HPLC Separations for the Quantification of Benzamide / Glyoxylate 55 Reverse-Phase HPLC Separation of N-dansyl-Tyr-Val-Gly and N-dansyl-Tyr-Val-NH2 55 Reverse-Phase HPLC Separation of Hippurate, -Hydroxyhippurate, and Benzamide 56 Results and Discussion 57 MS / MD Spectrophotometric Assay for Glyoxylate 57 MS / MD Assay for Chemically and Enzymatically Produced Glyoxylate 57 Glycolate Oxidase/Horse Radish Pe roxidase Linked DMAB / MBTH Assay for Glyoxylate 61 Glycolate Oxidase / Horse Radish Peroxidase Linked DMAB / MBTH Detection of H2O2 61 Glycolate Oxidase Dependent DMAB / MBTH / HRP Detection of Standard Glyoxylate 63 Glycolate Oxidase Dependent DMAB / MBTH / HRP Detection of PAM-Produced Glyoxylate 64 Glyoxylate Reductase Depende nt Assay for Glyoxylate 66 Glyoxylate Reductase Detection of PAM-Produced Glyoxylate 66 Chapter Three: Fluorescent and Chem i-Luminescent Assays for Glyoxylate 68 Fluorescent Assays for the Det ection of Hydrogen Peroxide 69 Enhanced Sensitivity of Amplex Red Fluorometric Detection 70 Fluorescent Assays for Glyoxylate 72 The Flavin Dependent Glyoxylate Consuming Enzyme Glycolate Oxidase 72 Utilization of Glycolate Oxidase as a Fluorescent Assay for Glyoxylate 74 The Lignolytic Degrading En zyme Glyoxal Oxidase 76 Utilization of Glyoxal Oxidase for the Quantification of Glyoxylate 77 Chemi-luminescent Assays for Hydrogen Peroxide 79 Materials and Methods 82 Materials 82 Methods 83 Standardization of the Fluorophore Resorufin 83 Standardization of H2O2 Produced Fluorescence 83 Glyoxal Oxidase Fluorescent Assay for Glyoxylate 84 Standardization of the Fluores cent GLOX Assay with Standard Methyl Glyoxal and Glyoxylate 84 Standardization of the Fl uorescent GLOX Assay with PAMProduced Glyoxylate 85 Glycolate Oxidase Fluorescent Assay for Glyoxylate 86
iv Standardization of the Fluorescent Glycolate Oxidase Assay with Glycolate and Glyoxylate 86 Standardization of the Fluores cent Glycolate Oxidase Assay with PAM-Produced Glyoxylate 87 Chemi-luminescent Assays for Glyoxylate 88 Hydrogen Peroxide Chemi-luminescent Standard Curve 88 Chemi-luminescent Glycolate Oxidase Assay with Glyoxylate 83 Chemi-luminescent Glycolate Oxidase Assay for PAM Produced Glyoxylate 90 Preparative RP-HPLC for the Collection of the PAM Substrate N-dansyl-T yr-Val-Gly; An Empirical Trial For the Use of the Platform Technology to Detect a Glycine-Extended Peptide 90 Results and Discussion 93 Resorufin Fluorescent Standard Curve 93 Standardization of H2O2 Produced Fluorescence 94 Glyoxal Oxidase Fluorescent Assay for -Amidated Peptides 96 Glycolate Oxidase Fluorescent Assay for -Amidated Peptides 99 Chemi-luminescent Glycolate Oxidase Dependent Assay for Glyoxylate 102 Chapter Four: Platform Technology Process 107 Application of the Glyoxylate Assays to the Discovery of -Amidated Hormones 107 Utilization of the Mouse P ituitary Cell Line in the Platform Technology 108 Utilization of the Rat Neuro-intermediate Cell Line in the Platform Technology 110 Material and Methods 111 Materials 111 Methods 112 At-T20 Cell Growth Conditions 112 At-T20 Cell Growth for the mJP-Gly Accumulation Methodology 113 Extraction of mJP-Gly from At-T20 cells 114 Purification of Cellula r Extract on a Sep-Pak Plus Cartridge 115 Preparation of the Cellular Extr act for HPLC Purification 116 RP-HPLC Separation of the Accumulated mJP-Gly 117 Definition of Collected Sample Content for mJP-Gly Characterization 119 Treatment of RP-HPLC Fractions with PAM; Production of mJP-NH2 / Glyoxylate 121 Analysis of mJP-Gly Depende nt Glyoxylate Production by Luminescence 121 Matrix Assisted Laser Desorpti on Ionization Â– Time of Flight
v Mass Spectrometry Analysis for mJP-Gly Accumulation 122 Demonstration of the Platform Technology by a Blind Experiment; Analysis of Peptide-Gly Samples Se nt by Unigene Laboratories, Inc. 123 Unigene Cell Growth Conditions 123 Unigene Extraction of CGRP-Gly From Rat CA-77 Cells 124 Unigene Purification of Rat GGRP-Gly by RP-HPLC 125 Analysis of Unigene Fractions for CGRP-Gly by the Developed Platform Technology 126 Matrix Assisted Laser Desorption Ionization Â– Time of Flight Mass Spectrometry Analysis for CGRP-Gly Accumulation 127 Results and Discussion 128 Demonstration of the Platform Technology to Identify mJP-Gly Accumulation by the Inhi bition of PAM in Cell Culture 128 RP-HPLC of the mJP-Gly Standard 128 RP-HPLC Separation of the Accumulated and Spiked mJP-Gly from At-T20 Cell Culture 129 Analysis of mJP-Gly Dependent Glyoxylate Production by Luminescence 130 MALDI-TOF Analysis of Standard mJP-Gly and mJP-NH2 134 MALDI-TOF Analysis of Accumu lated and Spiked mJP-Gly 136 Demonstration of the Platform Technology to Identify CGRP-Gly Accumulation by the Inhibition of PAM in Cell Culture; 140 Analysis of Unigene Fractions for CGRP-Gly by Chemi-luminescence 140 Analysis of Unigene CGRP-Gly Standard by the MALDI-TOF 141 Analysis of Unigene Fractions for CGRP-Gly by MALDI-TOF 142 Conclusion 143 Future Work for Novel Peptide Id entification and Characterization 145 Literature Cited 148 Bibliography 158 Appendices 168 Appendix A: Abbreviations 169 About the Author End Page
vi List of Tables Table 1 An Abbreviated List of -amidated Mammalian Peptide Hormones 5 Table 2 Examples of Currently Marketed Peptide Hormones 12 Table 3 Comparison of PAM Produ ced Glyoxylate to Benzamide 59 Table 4 Kinetic Constants for Se veral Aldehyde Substrates of Glyoxal Oxidase 78 Table 5 Definition of Platform Technology Sample Notation 120 Table 6 Spiked vs. Non-spiked Cellu lar Extract Analysis for mJP-Gly 133
vii List of Figures Figure 1 Peptide Amidation Reaction Catalyzed by PAM 2 Figure 2 Tatemoto & Mutt Methodology 6 Figure 3 Hill & Flannery Method for the Discovery of -Amidated Peptides 9 Figure 4 Central Dogma for the PAM Dependent Discovery of -Amidated Peptides 17 Figure 5 Rimini-Schryver Chemical Assay for Glyoxylate 21 Figure 6 Allantoin Dependent Production of Glyoxylate 21 Figure 7 Assay for Glyoxylate as Described by Zarembeski 25 Figure 8 Reaction Stoichiometry of -Amidated Peptide Production as Catalyzed by PAM 29 Figure 9 Spectrophotometric Assays for Glyoxylate 32 Figure 10 Catalytic Role of PMS 35 Figure 11 Malate Synthase / Mala te Dehydrogenase Glyoxylate Assay 36 Figure 12 Enzymatic Reaction of Glycolate Oxidase 38 Figure 13 H2O2 Dependent Indamine Dye Production 40 Figure 14 Glycolate Oxidase Based H2O2 Assay 41 Figure 15 Enzymatic Reaction of Glyoxylate Reductase 42 Figure 16 Reference Reactions for the Independent Analysis of the Developed Glyoxylate Assays 44 Figure 17 Glyoxylate-Depende nt Oxidation of MTS 58 Figure 18 Linear Regression of the DMAB / MBTH / HRP Detection of H2O2 62
viii Figure 19 Linear Regression of the DMAB/MBTH/HRP Detection of Glyoxylate 63 Figure 20 Linear Regression of the DMAB/MBTH/HRP Detection of PAM-Produced Glyoxylate 64 Figure 21 Glyoxylate Reductase Determination of PAM Produced Glyoxylate 66 Figure 22 Reaction Catalyzed by Glyoxal Oxidase 68 Figure 23 Fluorescent Analysis for H2O2 Quantification 69 Figure 24 Spectra of the Fluorophore Resorufin 70 Figure 25 Flavins 73 Figure 26 Amplex Red Fluorescent Detection of H2O2 75 Figure 27 Glyoxal Oxidase pH Profile 77 Figure 28 A Jablonski Diagram 79 Figure 29 Mechanism of Luminol Chemi-luminescence 81 Figure 30 Fluorescent Response of Resorufin 93 Figure 31 Resorufin Standard Curve for Detection of H2O2 at pH 6.0 94 Figure 32 Resorufin Standard Curve for Detection of H2O2 at pH 7.8 95 Figure 33 Glyoxal Oxidase Activity Standard Curve 97 Figure 34 Standard Curve of the Glyc olate Oxidase Dependent Fluorescent Assay for Glycolate vs. Glyoxylate 99 Figure 35 FAD / Resorufin Fluorescent Spectra 100 Figure 36 Standard Curve for th e Glycolate Oxidase Dependent Fluorescent Enzyme Assay for PAM Produced Glyoxylate 101 Figure 37 Chemi-luminescent Standard Curve for Hydrogen Peroxide 104
ix Figure 38 Glycolate Oxidase Chemi-luminescent Standard Curve for PAM produced glyoxylate 104 Figure 39 Linear Detection Ra nge of Chemi-luminescence 105 Figure 40 Proteolytic Processing of the Mouse pro-ACTH / Endorphin Homologue 108 Figure 41 mJP Sequence 109 Figure 42 CGRP Peptide Sequence 110 Figure 43 Standard Curve for the Ab sorbance at 278nm of mJP-Gly as Analyzed by RP-HPLC 128 Figure 44 Chemi-luminescent Anal ysis of Accumulated mJP-Gly from At-T20 Cells 130 Figure 45 Chemi-luminescent Analysis of Spiked mJP-Gly from At-T20 Cells 131 Figure 46 Chemi-luminescent Analysis of Accumulated mJP-Gly 132 Figure 47 Chemi-luminescent Analysis of Spiked mJP-Gly 132 Figure 48 Mouse Joining Peptide Precursor 134 Figure 49 Mouse Joining Peptide 135 Figure 50 MALDI-TOF of Accumulated mJP-Gly 137 Figure 51 MALDI-TOF of Spiked mJP-Gly 138 Figure 52 MALDI-TOF of Accumulated Vs. Spiked mJP-Gly 139 Figure 53 Chemi-luminescent Analysis of Unigene Fraction set # 1 for CGRP-Gly 140 Figure 54 MALDI-TOF of Standard CGRP-Gly 141 Figure 55 Fraction 29 142
x Enzyme Linked Spectroscopic Assays For Glyoxylate; The Use of Peptidylglycine alpha-Amidating Monooxygenase for the Discovery of Novel alpha-Amidated Hormones Sarah Elizabeth Carpenter ABSTRACT Peptide hormones are responsible for cellular f unctions critical to the survival of an organism. Approximately 50% of all known peptide hormones are po st-translationally modified at the C-terminus. Enzymatic oxidative conversion of C-terminal glycine extended peptide precur sors results in an -amidated peptide and glyoxylate. Peptidylglycine -amidating monooxygenase (PAM) is the single known enzyme responsible for catalyzing th is reaction. PAM is an O2, Cu(II), and Zn(II) dependent bifunctional enzyme. Initiall y, PAM hydroxylates the glycyl -carbon followed by dealkylation of the hydroxylated intermediate to an -amidated product and glyoxylate. PAM is also responsible for the conversion of glycine extended fatty acids to fatty acid amides and glyoxylate. PAM catalyzes the activation of all glycine-extended prohormones including biomolecules ranging from neuro to physio-homeostatic hormones. Identification of -amidated hormones from a biological source has been severely hindered by the lack of a specific as say for this distinctiv e class of biological
xi hormones, indicating that numerous -amidated hormones remain undiscovered. Based on the selective in situ chemistry of PAM, a novel and specific assay was developed for the discovery of -amidated hormones. The identification of novel -amidated hormones will lead to an increased understand ing of post-translational modifications and will pioneer a new understanding of -amidated hormone biosynt hesis, regulation, and bioactivity. Discovery of novel -amidated biomolecules could al so lead to their use as pharmaceuticals as there are several currently marketed -amidated peptide based pharmaceuticals. Inhibition of PAM in cell culture leads to the accumulation of glycine-extended hormones in the conditioned medium. The medium was fractionated by chromatographic techniques and each specific fraction was then assayed by the newly developed platform technology for the presence of -amidated hormones. For every -amidated hormone synthesized by PAM, glyoxylate is also forme d. Based on this 1:1 molar ratio, several novel spectrophotometric, fluorescent, and chem i-luminescent enzyme linked assays for glyoxylate were developed, which when utili zed on cell culture fractions proved positive for the identification of -amidated hormones. Each novel spectroscopic assay was independently verified by a variety of known methodologies. Moreover the assay was utilized to identify two known -amidated hormones accumulated from cell culture, which were further verified by Mass Spectral analysis.
1 Chapter One Peptidylglycine -Amidating Monooxygenase (PAM) Introduction The Catalytic Role of Peptidylglycine -Amidating Monooxygenase Peptidylglycine -amidating monooxygenase (PAM; E. C. 184.108.40.206) is the sole known enzyme responsible for the bioconversion of inactive glycine-ex tended prohormones to their bioactive -amidated product. PAM exhibits a br oad range of subs trate specificity; the enzyme can catalyze the post translational -amidation of both glycine-extended fatty acids, and glycine-extended pept ides. Ultimately, it is the C-terminal glycyl residue which is necessary for PAM catalysis. PAM is a bifunctional enzyme comprised of two catalytically independent domains, which synergistically convert a gl ycine extended substrate to an -amidated product and glyoxylate (Fig.1). Peptidylglycine -hydroxylating monooxygenase (PHM) is an O2, Cu(II), and ascorbate (reductant) dependent domain, which removes the pro-S hydrogen for the hydroxylation of the glycyl -carbon. The second catalytic domain, peptidylamidoglycolate lyase (PAL), is a Zn (II) dependent enzyme which dealkylates the
2 hydroxyglycine interm ediate to the -amidated product and glyoxylate. PAM is the only known enzyme that catalyzes this unique form of post-translational modification; glycine extended prohormones generally remain inactive prior to -amidation. Several alternately spliced isoforms of PAM exist within a single organism. Often, in more primitive organisms PAM is found as two ca talytically independent domains PHM and PAL, and in some cases only PHM is found . PAM is both the rate determining, and last step in the catalytic cas cade of events for the synthesis of bioactive -amidated hormones [2, 3] Publis hed data has demonstrated that the inhibition of PAM in rats, and in cultu red mammalian cells leads [4, 5]to a decrease in -amidated peptide formation, resulting in the accumulation of the glycine-extended precursors. Peptide -Amidation Reaction as Catalyzed by PAM HN O OH O2H2O2 Ascorbate2 Semidehydroascorbate PeptideHN O OH PeptideOH Zn(II)2 Cu(II) O O Oglyoxylate Peptide O NH2 peptide amide Fi g ure 1. Peptide amidation reaction catal y zed b y PAM The bifunctional enzyme is comprised of two separate catalytic domains: peptidylglycine hydroxylating monooxygenase (PHM) and peptidylamidoglycolate lyase (PAL).
3 -Amidated Peptide Hormones; Biosynth esis, Detection, and Applications -Amidated Peptide Hormones and their Biosynthesis Conversion of a glycine-extended hormone to the -amidated product is in most cases necessary for full potency or activation of a peptide hormone . Peptide hormones containing a C-terminal -amide functionality are widely important, found in mammals [7, 8, 9], insects [10, 11], cnidarians [ 12], and plants . Although, reports of amidated peptides in plants exist, (T RH-like tripeptide, pyroGlu-Tyr-Pro amide, ) unpublished work from the Merkler lab indicat es that a PAM-like enzyme does not exist in plants (Carpenter and Merkler, unpublis hed). The absence of a PAM-like enzyme (sequence, and catalytic sim ilarity) suggests that plant -amidated peptides are produced via a PAM-independent pathway. A catalytic cascade of sequen ce specific proteolytic events takes place to produce the glycine-extended prohormone from a larger pol ypeptide. A C-terminal glycine extended prohormone is generally excised from a larger peptide precursor. Peptides whose fate is to become glycine-extended have a pair of ba sic amino acid flanking th e internal glycine. The collective endoproteolytic activity of subs tilin-like proprotein convertase (SPC), and carboxypeptidase E (CPE) liberate the once internal glycine by removing the juxtaposed basic residues, thereby producing th e glycine-extended peptide.
4 The Rate Limiting Role of PAM Approximately 50% of all mammalian peptide hormones have an -amide functionality at their C-terminus , in several cases conversion of the gl ycine-extended peptide (PAM) to the -amide is required for bioactivation. To furthe r this study Merkler and Kreil defined this by a numerical value the potency ratio [6, 9] as the bio-activity contribution of the mature -amidated peptide as compared to the non-amidated prohormone (see Table 1a). Evidence to support this incl udes the identification of several glycine-extended peptide precursors from cell homogenates, in contrast to small quantities of the -amidated peptides [6, 7]. Moreove r, glycine-extended adrenomedulin from human plasma was found to be 5.4-fold hi gher in concentration as compared to the mature -amidated adrenomedulin . Accumu lation of the PAM substrates defines PAM as the rate limiting step in the biosynthetic cascade of -amidated peptide formation. Refer to Table 1, for several exam ples of alternate peptide hormones, their potency ratios, and length. Further studies on the role of PAM in -amidated peptide physiology, utilized a cell line known to express PAM. This cell line when treated with PAM anti-sense mRNA resulted in the under-expression of PAM. PAM under-expression resulted in a peptideNH2 / Peptide-Gly ratio of ~0.3 as compared to the wild type ratio of wild ~ 1.0. These
5 Table 1. An abbreviated list of mammalian -amidated peptide hormones, their length, Cterminus, and potency ratio [6, 9]. The potency ratio is a numerical value defined as the contribution of the C-terminal amide to the activity of the peptide hormone. aThe potency ratio is defined as the activity of -amidated peptide divided by the corresponding C-terminal amino acid analog. For example, the activ ity of TRH-pro-NH2 divided by the activity of TRH-pro is 4,400. An Abbreviated list of -Amidated Mammalian Peptide Hormones results provide further evidence that PAM is involved in catalyzing the rate limiting step in -amidated peptide biosynthesis . Peptide Hormone Length C-terminus Potency Ratio a Thyrotropin-Releasing Hormone (TRH) 3 amino acids Pro-NH2 4,400 Calcitonin 32 amino acids Pro-NH2 1,700 Corticotropin-Releasing Factor (CRF) 41 amino acids Ala-NH2 1,000 Lutenizing Hormone-Releasing Factor (LHRH) 10 amino acids Gly-NH2 1,000 Adrenomedulin 52 amino acids Tyr-NH2 > 330 Substance P 11 amino acids Met-NH2 100-1,000 Nueropeptide Y 36 amino acids Tyr-NH2 >225 Secretin 27 amino acids Val-NH2 10
6 Current Methodologies Used for the Identification of -Amidated Hormones Method of Tatemoto and Mutt Tatemoto and Mutt Methodology H2N H N N H H N NH2 R 1 O R2 O R 3 O R4 O H2N COOH H2N COOH H2N COOH H2N CO R1 R2 R3 R4 NH2 + + + Novel -amidated peptide Amino acid/Amino acid amide Mixture Dye Extraction of Amino Acid amide Amine Labeling Dye HN CO R4 NH2 Identification of Amino Acid Amide (Chromoatographic separation and comparison t o s t an d a r d s / M a s s S p e c) Figure 2. Tatemoto & Mutt Methodology. Procedure for the identification of novel -amidated peptides developed by Tatemoto & Mutt.
7The majority of -amidated peptides have been isolat ed, to date, utilizing bioassays for identification. The use of bi oassays for the discovery of -amidated peptides is not uniquely specific to -amidated peptides, and is both laborious and expensive. For example, the -amidated peptide neuromedulin C was isolated using a uterine contractile assay and monitoring platelet cAMP concentrat ions on a set of column fractions. The use of bioassays is not limited to -amidated peptides only and due to these limitations other chemical based assays have been developed. The most successful approach for the identification of novel -amidated hormones was developed by Tatemoto & Mutt . Tatemo to & Mutt used their procedure to isolate and identify a number of -amidated peptides such as ga lanin , pancreastatin , PHI  and PYY . The key to the Ta temoto-Mutt procedure is the proteolytic fragmentation of a target pep tide (Fig. 2). Proteolytic frag mentation of a target peptide resulted in a degradation mixture of peptid es, amino acids, and a single C-terminal amino acid amide which could be chemically identified. This degradation mixture was subjected to dansylation, and the result ant hydrophobic dansylated-amino acids were extracted into an organic solvent. Two-di mensional TLC was then employed to isolate the peptides and dansylated amino acids, with a detection limit of approximately 1 nanomole. Since the development of the Tatemoto & Mutt procedure in the 1970Â’s, improvements in the separation and detection of derivitized amino acid amides have been made, [21, 22, 23, 24] with an increased de tection limit in the pi comole range .
8Despite improvements the Tatemoto & Mutt procedure is extremely laborious and inefficient for a variety of reasons. Incomplete proteolytic fragmentation coupled to inefficiencies in the extraction and labeli ng procedures proved deleterious to assay sensitivity as compared to model studies. This methodology has not been widely used, and is especially non-amenable to high-thr oughput analysis. Consequently, Tatemoto & Mutt have been the primary users of th is technology for the discovery of novel amidated peptides, while most others have ad apted the procedure to test for and identify the -amidated C-terminus in an othe rwise purified, bio active peptide.
9 Method of Hill and Flannery. NH2 NH2 O NH NH2 NH NH2 NH NH O O R O O O R O R R O Dye I ( OC ( O)C F 3)2 + H20I OH O + CO2+ 2CF3COOH Dye Hill and Flannery  developed a more ch emical approach for the identification of amidated peptides from a mixture of pe ptides purified from a biological source. Acetylation of peptide free amines followe d by conversion of the N-acetylamides to amines by a Hoffman rearrangement allows for detection of the resultant amine by ninhydrin (Fig. 3). Amide derived amines separated into column fractions can be correlated to the presence of a C-terminal glycine by conversion of glycine to 2thiohydantoin. This assay is based on the pr esumption that the C-terminal glycine and the amide will co-elute, and that their co-eluti on is strongly indicative for the presence of an -amidated peptide. Overall, this assay is only sensitive to the millimole range, and is completely reliant on the co-elution of two species which may or may not co-elute in most cases. Furthermore, this assay will always be fraught with false positives for any Hill & Flannery Procedure for -Amidated Peptide Discovery Figure 3 Hill method for the discovery of -amidated peptides. Outline of the technique designed by Hill et al for the identification of -amidated peptides utilizing chemical dyes.
10Asn and Gln containing peptides. Adapting th e assay for improved sensitivity by the use of other amine dyes  may improve sensitivity; however it is unlikely that this procedure could ever find a widespread use. The Hill & Flannery procedure has never been successfully used to identify an -amidated peptide, and a Web of Science search indica tes that this paper has only been cited once since Feng & Johnson .
11 Immunological Detection of -Amidated Peptides A library of antibodies could theoretically be used to exploit the physiolo gical difference between a glycine-extended peptide and an -amidated peptide. This approach would necessitate the use of collection of 20 anti bodies, each one specific for a particular amino-acid amide. The Grimmelikhuijzen group has used a sim ilar approach by generating an antibody against the dipeptide amide, Arg-Phe-NH2 to discover -amidated peptides in cnidarians . This approach would re quire a collection of 400 antibodies in order to test all of the possible di-peptid e permutations of the 20 common amino acids. Computer Based Analysis for -Amidated Peptides With the advent of the genomic database and specific peptide sequence information, it has been suggested that the search for -amidated peptides become a computer-based dry technique, which could be followed by wet ch emistry. Unfortunatel y, without a defined model peptide sequence to search for, da tabase searching would produce ambiguous results. Recall, that the only defining fact or for a peptide whose fate is to become amidated, is the basic residues flanking the initially internal glycine. The outcome of a computer based search on different perm utations of the basic amino acid residues would yield results entirely t oo non-specific and numerous.
12 Therapeutic Uses of the Known -Amidated Peptides Table 2 Examples of Currently Ma rketed Peptide Hormones Drug Name (Company) Compound Delivery Dose Indications Lupron (TAP) Nonapeptide analog of LHRH Subcutaneous, daily 1.0 mg daily Advanced prostate central, precocious puberty DDAVP (Sanofi-Aventis) analog of 8-Arg vasopressin Oral tablets 0.1 to 0.8 mg daily Central diabetes insipidus, primary nocturnal enuresis Cortrosyn (Organon) -(1-24)-corticotropin i.m. or i.v. injections or i.v. infusion 250 g Diagnostic agent for adrenocortical deficiency Sandostatin (Novartis) Cyclic octapeptide analog of somatostatin s.c or i.v. injections 50-1500 g daily Acromegaly, carcinoid tumors, VIPomas Thyrel TRH (Ferring) Synthetic tripeptide i.v. injection 500 g Diagnostic assessment of thyroid function Miacalcin (Novartis) Salmon calcitonin s.c. injection 100 I.U. daily Postmenopausal osteoporosis, PagetÂ’s disease, hypercalcemia Miacalcin Nasal (Novartis) Salmon calcitonin Nasal spray 200 I.U. daily Postmenopausal osteoporosis Geref (Serono) GHRF(1-29)-NH2 s.c. injection 30 g/kg daily Pediatric, idiopathic growth hormone deficiency Acthrel (Ferring) Ovine CRF(1-41)-NH2 i.v. injection 1.0 g/kg single injection Differentiates pituitary and ectopic production of ACTH in CushingÂ’s syndrome Secretin-Ferring (Ferring) Porcine secretin(1-27)NH2 Slow i.v. injection 1-2 CU/kg Testing for pancreatic function and gastrinoma Byetta (Amylin) Exenatide(1-39)NH2 (Exendin-4) s.c injection 5-10 g b.i.d. Adjunctive therapy in Type 2 diabetes Fuseon (Roche) T-20 peptide s.c. injection 90 mg daily Treatment of HIV-1 infection in combination with antiretroviral agents Symlin (Amylin) Analog of Amylin(137)NH2 s.c. injection 15-60 g for Type 1 and 60-120 g preprandial for Type 2 diabetes Adjunct treatment for Type 1 and Type 2 diabetes Fortical (UpsherSmith) Salmon calcitonin Nasal Spray 200 I.U. daily Postmenopausal osteoporosis Forteo (Lilly) Parathyroid hormone(134) s.c. injection 20 g daily Postmenopausal osteoporosis with high risk of fracture Table 2. Examples of currentl y marketed peptide hormones. A summary of the currently available therapeutic amidated peptide hormones, their manuf acturers and pharmacological usage.
13Several -amidated peptide hormones have already been discovered, and a few have proven to be useful pharmaceutical therapeu tics (Table. 2). Considering the large marketability of the known -amidated peptides, the likelihood that undiscovered amidated peptide hormones could lead to the development of novel diagnostics and pharmaceuticals is of extremely high probability. Based on the literature it is evident that some tissues know n to express high levels of PAM , do not produce si milar quantities of known -amidated hormones (exocrine), and vice versa (autocrine). Autocrine cells both synthesize and utilize the machinery necessary for their cell growth. Conversely, exocrine cells ge nerally synthesize materials for exportation to other cells, and acquire ma terials via a receptor mediated pathway. PAM is localized in the secret ory vesicles, and has been linked to both autocrine growth factor loops [28, 29], such that PAM is bot h highly expressed and functional within the same tissue, and exocrine growth loops [30, 31]. Gl ycine-extended hormones may therefore be expressed in cells which do not also express PAM, and conversely cells known to express large quantities of PAM may not co-express any glycine-extended hormones. Thus, it is likely that several undiscovered -amidated hormones exist both in tissues known to express large quantities of PAM, and in tissues which do not express large quantities of PAM. Cultured cell lines known to express larg e quantities of PAM, yet not known to express many -amidated peptides could serve as an initial source material for the discovery of novel -amidated hormones. It is possible that many cell lines which do not express PAM, may expre ss glycine-extended hormones. There are many tissues to search for glycine-extended peptides, without a specific assay in place for
14their discovery, the probability that several -amidated peptides remain to be discovered is highly likely. A review on the literature of PAM strongly suggests that th e main physiological function of PAM is to hydroxylate (PHM) extended prohormones (glycine-extended peptides, fatty acyl-glycines), and to further dealkylate the -hydroxyglycyl intermediate (PAL). Currently, no data exists to suggest an alte rnate physiological function of PAM, or to provide reason for high levels of PAM in tissues not known to pr oduce and/or utilize amidated peptides. This fact combined with the large amounts of PAM in certain tissues and lack of a glycine-extended hormone assa y provides impetus for the assertion that several amidated hormones exist which have yet to be discovered.
15 Impetus for the Design of a Novel, Broadly Applicable Assay for the Discovery of amidated Peptide Hormones Utilizing PAM as a General Tool for -Amidated Hormone Discovery From a chemical perspective there is little to differentiate an -amidated hormone from a non-amidated hormone. Any assay developed on the basis of an amide specific reagent would be fraught with high backgrounds and fa lse-positives due to the cross reactivity with any Asn or Gln containing peptides. It is evident in the literature on -amidated peptides and their discovery methods, that no single methodology has proven effective as a general and specific assay for the presence of an -amidated peptide/hormone. The lack of a general assa y for the discovery of -amidated peptides has left a void in the knowledge of -amidated peptide hormones as a w hole, their biosynthesis, tissue distribution, and physiological role. Capitali zing on the very unique role of PAM and the stoichiometric production of glyoxylate: -amidated peptide, identification of PAM produced glyoxylate could lead to the id entification of yet to be discovered -amidated hormones. Utilization of enzymatic assay sy stems for the detecti on of glyoxylate allows for a signal specific assay, and decreases the loss of sensitivity due to high backgrounds created by non specific interactions. As previously discussed, other -amidated peptide discovery methods to date have been fraught with non-specific / insufficient interactions for -amidated peptide detection. Quantitative analysis of PAM produced glyoxylate is both stoichiometric fo r the presence of -amidated peptides and their glycine-extended
16prohormone precursors. Ultimately this assay has one very positive feature in that it is non-specific for a particular class of PAM s ubstrates, thereby incr easing the application of this assay to not only the discovery of several -amidated peptides, but also novel amidated fatty acid amides.
17 Platform Technology Process and Design N H O HO NH2O O Oglyoxylate Hormone Hormone = = The utilization of PAM is para mount to the development and us e of a robust and specific assay for biologically generated -amidated hormones. Capitalizing on the unique chemistry of PAM allows for the generation of an unequivocal rout e to both purify and discover novel -amidated hormones from a biological source. The key to designing an assay for -amidated peptides utilizing the uni que physiological role of PAM is glyoxylate For every -amidated peptide produced via PAM, a molecule of glyoxylate results (Fig. 4). Employing glyoxylate as a signal molecule for the discovery of novel peptides leads to the design of an assay system enriched with a specific yet general method for detection of -amidated peptides. As mentioned, several tissues known to express PAM at high levels are not known to produce co rrespondingly high levels of amidated peptide hormones [30, 32]. Figure 4. Central dogma for the PAM dependent discovery of -amidated peptides. Based on the stoichiometry for every mole hormone-Gly consumed one mole of hormone-amide and glyoxylate is produced. Sensitive detection of glyoxylate was used to define the presence of an -amidated hormone. Central Dogma for the PAM Dependent Discovery of -Amidated Peptides
18Cell lines generated from tissues known to express high levels of PAM will serve as an initial source material for the discovery of novel -amidated hormones. These cells cultured in the presence of a PAM inhibi tor accumulate the glyc ine-extended hormone precursors. Mains and Eipper [4, 33] demons trated that the glycine-extended peptide mouse joining peptide (mJP-Gly) did in fact accumulate when grown in the presence of a PAM inhibitor. Glycine-extended precursor s accumulated from cell culture when HPLC purified [4, 34], result in a series of semi-purified frac tions containing the glycineextended hormones. Treatment of the semi-purified fractions with PAM results in glyoxylate production only in the presence of a PAM substrate. Namely, fractions positive for PAM produced glyoxylate, are also posi tive for the glycine-extended precursor. This unique method allows for the identification of any glycine-extended molecule permitting the identification of a broad range of novel PAM substrates. A sensitive high-throughput assay for glyoxylate provi des a signal that is independent of the peptide (or acyl group) upstream of the term inal glycine. All fractions positive for glyoxylate can be further analyzed for conten t, and the structure of novel substrates determined by mass spectrometry. Comparis on of the cells treated with the PAM inhibitor to non-treated cells for glyoxylate content removes the interference of any intrinsic glyoxylate re sultant of other meta bolic processes.
19 Development of a Series of Glyoxylate Assays ; Glyoxylate as a Signal Molecule for the Detection of -Amidated Hormones A series of enzyme linked glyoxylate assa ys have been designed and coupled to spectrophotometric, fluorescent, and chemiluminescent detection. Each assay was developed with the interest of ease of use, sensitivity, specificity, and high-through put analysis. Based on the absolute necessity of a specific and sensitive technique, the establishment of robust glyoxylate assays is the foundation of this novel platform technology. Three spectrophotometric, two fluor escent, and two chemi-luminescent for a total of seven novel enzymatic assays were created each able to detect PAM produced glyoxylate. Each of these assa ys will be discussed in detail in the following chapters. Enzymes provide a degree of selectivity far grea ter than that of typical chemical assays, based on their inherent substrate specificity. The glyoxylate assays constructed for this platform technology were designed on the premis e of specificity and as a result all newly developed assays are based on the enzymatic de tection of glyoxylate. Utilizing natureÂ’s tools provides an unrivaled degree of sensitivity as compared to organic / chemical methods for glyoxylate detection.
20 Prior Art for the Detection of Glyoxylate; A Literature Review The Rimini-Schryver Chemical Spectrophotometric Assay for Aldehydes Detection of PAM produced glyoxylate is the ne xus of a definitive and sensitive assay for -amidated peptides. Moreover, the ability to detect glyoxylate is the foundation on which this platform technology resides. A great deal of effort has been made towards the production of glyoxylate assays which are amen able to high throughput analysis, feasible within the working conditions of many laboratories, and foremost sensitive detection without ambiguity. Methodologies utilizing chemical detectio n rely on the reactivity of a specific functionality constituen t of the analyte of interest. Often a chemical detection assay design, when applied to biochemical analyses results in large background signals as a result of the increased complexity of bioche mical solutions. Detection of analytes from biochemical samples requires analyte specific detection (enzymatic detection), as biochemical samples are often grossly im pure. Specifically, the Rimini-Schryver colorimetric reaction was developed as a met hod for the detection of formaldehyde . Originally developed by Rimini the assay was developed to detect aldehydes in the presence of phenylhydrazine and sodium hydroxide it was later adapted by Schryver  to increase sensitivity by the addition of ferriccyanide (Fe(III)) in the presence of
21hydrochloric acid (Fig. 5). The assay has in mo st part been utilized to detect allantoin content in various biological fluids [37, 38], allantoin is indicative of purine metabolism as it is the end-produc t of purine metabolism (Fig. 6) . O O OglyoxylateHN H2N phenylhydrazine + HN NH H O O C glyoxylate phenylhydrazon e H+K3Fe(CN)6 O HN N H N H NH2O O allantoin NH2 N H COON H NH2 O O H2O H+Allantoinase E.C. 220.127.116.11 H2ONH 2 O H2N urea Allantoicase E.C.18.104.22.168OH O HO H N O NH2 ureidoglycolateO O Oglyoxylate + CO2+ 2NH3Ureidoglycolate hydrolase E.C. 22.214.171.124 Figure 6. An excerpt of the AMP catabo lic pathway as it applie s to allantoin metabolism. A portion of the AMP catabolism cascade displays the enzymatic cascade for the production of glyoxylate from allantoin. Determination of allantoin content in blood samples was of interest in early metabolic investigations of purine metabolism. Figure 5 Rimini-Schryver chemical assay for glyoxylate The condensation reaction developed by Rimini and adapted by Schryver, for the detec tion of the colorimetric condensation adduct of glyoxylate and phenylhydrazine, glyoxylic acid phenylhydrazone respectively . Rimini-Schryver Chemical Assay for Glyoxylate An excerpt of the AMP Cata bolic Pathway as it Applies to Allantoin Metabolism
22The following is a list of molecules whic h in the presence of phenylhydrazine also produce a hydrazone colorimetric adduct detect able at the same wavelength as the glyoxylate: phenylhydrazone a dduct: xanthine, hypoxanthine glycerol, pyruvic acid, malic acid, tartaric acid, alloxan, aloxantin, glyc ine, chloral, lactic and uric acids, and aldehydes in general . Alternately, 2,4-di nitrophenylhydrazine can also be used to produce the condensation product glyoxylic aci d 2,4-dinitrophenylhydrazone (glyoxylate 2,4-DNPH) . Production of keto-acid 2,4-DN PH adducts has long since been used as a method to characterize both aldehydes, a nd oximes by melting point and color. In addition to the non-specificity, of the Ri mini-Schryver method, analyses are laborious, not very sensitive (~ 10 M), time consuming, and not completely amenable to highthroughput analysis as the co lorimetric product fades quic kly. Although the RiminiSchryver method has been used to detect PAM activity, its use is only amenable to the detection of glyoxylate formed w ith highly purified enzyme. In addition, this assay is not useful in the detection of PAM activity in non-pure extracts, resul ting in a very limited use of this technique.
23 Determination of Glyoxylate by Capillary Electrophoresis Calcium oxalate is the major constituent of kidney stones  and ~50-60% of urinary oxalate ( OOC-COO ) is derived from the enzyma tic oxidation of glyoxylate (HCOCOO )  As a consequence of the metabol ic importance and ro le of glyoxylate in kidney stone formation, a number of assays have been developed for glyoxylate. Existing assays for the determination of gl yoxylate include colorimetric methods [36, 4246], fluorometric methods [47, 48], the iodome tric or potentiometic titration of the bisulfite adducts  and th e use of capillary electrophore sis with direct UV detection [50, 51]. Generally, these are insensitive, nonspecific, or both. Capillary electrophoresis has been the detection method of choice for the separation and quan tification the organic acids contained in urine. Capillary electrophor esis (CE) separation is based on the movement of an analyte velocity within an el ectric field. The inherent velocity of an analyte is a function of its electrophoretic mobility in relation to an applied voltage coupled to spectrophotometric detection. Nishijima et al. [ 50] developed a CE separation on a polyamide fused silica column at a cons tant voltage of -30k V. Urine derived organic acids including glyoxylat e elute over an eight minute period, with a detection limit for glyoxylate of 8 9 M . Although separation and quantification can be achieved by the Nishijima  CE method, the practicality of this technique for the quantification of glyoxylate from alternate sources is of limited value . In addition to
24the limited utility a detection method ba sed on the inherent spectrophotometric absorption of glyoxylate itself results in a limited degree of sensitivity.
25 The Fluorescent / Spectrophotometric A dduct of Glyoxylate and Resorcinol An alternate spectrophotometric assay for gl yoxylate was develope d by Zarembski and Hodgkinson in 1965 , based on the colo red product produced by the reaction of glyoxylate and resorcinol in the pr esence of an acid extract of Psuedomonas oxalaticus The glyoxylate : rescorcinol adduct is spectrophotometrically visible with a max of 490nm, and detection limit of thirteen micro-molar. In addition the colored product is also visible by fluorescence within a pH rang e of 7 to 9. This procedure while not explicitly stated in the refe rence is dependent upon the en zymatic activity of glyoxylate dehydrogenase which is a metabo lic product of th e growth of Psuedomonas only under high oxalate conditions. The Zarembski me thod is reliant on th e oxalate supported Psuedomonas extract, an extract which is extremel y time and preparation intensive to produce in any appreciable qu antities. The Zarembeski method has not found general use, and has been cited only once since its 1965 publication. O O OglyoxylateOH HO resorcinol O H H O OH O O + Assay for Glyoxylate as Described by Zarembeski Figure 7. Assay for glyoxylate as described by Zarembeski Proposed reaction catalyzed in the presence of Psuedomonas oxalaticus for the formation of a glyoxylate dependent spectrophotometricly active adduct. The proposed glyoxylate-resorcinol adduct was verified by Vieles and Badre .
26 Authentication the Proposed Platform Technology Authentication of the Newly Designed Glyoxylate Assays A variety of techniques were necessary to pr ove the usefulness and feasibility of all the newly developed assays intended for the platform technology. Exigent to the use of a glyoxylate assay as a paragon for the identification of novel -amidated hormones was the external standardization of each deve loped assay via an independent confirmed methodology. This served as a reference comparison for each assay in order that all newly developed techniques coul d be referenced against a know n control. Furthermore, all PAM produced glyoxylate is a consequence of an enzymatic reaction with a 1:1 ratio of glyoxylate : -amidated peptide; measurement of produced -amidated peptide as compared to glyoxylate should result in a ra tio of 1. All PAM produced glyoxylate determinations were indexed against -amidated peptide concentr ations assayed via wellestablished methodologies. Once the assay wa s standardized against known procedures, it was then utilized for determination of unknown gl yoxylate, and PAM produced glyoxylate. Several techniques were utilized as standardization procedures, each to ascertain the ratio of glyoxylate: amidated pept ide. This analysis has proven invaluable in the determination of PAM produced glyox ylate concentrations, in context to the affirmation of newly developed techniques. Details of all authentication procedures will be fully outlined in the following chapter.
27 Authentication for the PAM Dependent Discovery of Novel -Amidated Peptides Prior to the utilization of the assay to di scover novel hormones from a biological source, the assay was initially used to demonstrat e its ability to identify an already known amidated hormone (mouse joining peptide, mJPGly). It was imperative to the utility of the proposed assay to demonstrate this ne w methodology could be used to identify a known -amidated hormone from cell culture prior to employing the assay for the discovery of novel -amidated hormones. A mouse corticotropic cell line known to produce mouse joining peptide (mJP-Gly), and accumulate mJP-Gly upon the inhibition of PAM as outlined by Mains et. al.  provided the necessary data for the verification of the newly developed platform technology. Th e demonstration of the platform technology with a known glycine-extended pe ptide served both as an internal control and also as a template for the empirical design of future assays for the discovery of novel -amidated hormones. Delineating the assay feasibility from tissue cult ure to the utility of glyoxylate as a signal for a known -amidated peptide by mass spectrometry is discussed in detail in this work. A comprehensive discussion of all authen tication methodologies, enzymatic spectral assays for glyoxylate, and the use of these assays towards the platform technology are outlined in the following chapters, preceded by a thorough discussion of the newly designed platform technology.
28 Chapter Two Glyoxylate as a Signal Molecule for the Identification of -Amidated Peptides Introduction PAM Activity as a Key to the Discovery of Amidated Peptides The use of glyoxylate as indicative for the presence of -amidated hormones is truly innovative. Development of a glyoxylate based route for -amidated hormone detection exploits the inherent biochemistry of their production. This was the method of choice for a variety of fundamental reasons. Often th e discovery of novel enzyme substrates is hindered by the lack of a general non-substrate dependent assay. Glyoxylate is always a product of PAM catalysis. This feature impa rts an Â“analyticalÂ” quality to PAM which had not been exploited to its full potential, prior to the development of this platform technology. Capitalizin g on this Â“tool-likeÂ” quality bri ngs a novel functi onality to PAM beyond typical mechanistic biochemistry. Mo reover, PAM directs both the qualitative and quantitative discovery of -amidated peptides via the use of glyoxylate as a signal. Simple chemical techniques lack this unique culmination of qualities, resulting in their definite inferiority.
29HN O OH glycine O Peptide O PeptideNH2 +O O Oglyoxylate Identification of PAM Produced Glyoxylate Manipulating the biochemical role of PAM for the discovery of -amidated peptides mandates the facile detection of glyoxylate as a PAM product. This feature requires that glyoxylate assays be amenable to PAM reacti on conditions. As a result, prior to the analysis of glyoxylate, a series of sample pre-treatment procedures was necessary pending the glyoxylate detection system. In th is chapter, the development of several spectrophotometric assays is detailed, fo llowed by the development of more sensitive fluorescent and chemi-luminescent assays in chapter three. Reaction Stoichiometry of -Amidated Peptide Pro duction as Catalyzed by PAM Figure 8. Reaction Stoichiometry of amidated peptide production as catalyzed by PAM The stoichiometric production of glyoxylate : peptide-NH2 is the central dogma of the developed platform technology. A series of novel spectrophotometric (Chapter 2), fluorescent (Chapter 3), and chemiluminescent (Chapter 3) assays have been developed for the detection of standard glyoxylate, PAM produced glyoxylate, and -amidated peptides. The differences between standard glyoxylate detection a nd PAM produced glyoxylate detection is a ramification of the necessary additional reagen ts used for PAM catalysis. Detection of PAM produced glyoxylate necessita tes the consideration of seve ral factors relating to the
30tandem activity of PAM, and glyoxylate consuming enzymes. Although glyoxylate consuming enzymes do not need to be cataly tically active in tandem with PAM, they must be able catalytically active in the presen ce of the necessary PAM reagents. This is a direct consequence of this novel platform technology, such that glycine-extended peptides are discovered through the use of PAM and glyoxylate consuming enzymes as analytical tools. PAM is a redox active, copper-dependent monooxygenase that relies on the presence of a reductant for reduction of Cu(II) for catalytic turnover. Additionally, PAM requires the presence of a radical/peroxid e scavenger to protect the enzyme from OH radicals, and H2O2, produced as a result of the Fenton chemistry between Cu(II) and ascorbate (reductant). Each glyoxylate assay must be designed with the presence of these molecules in mind. Circumvention of the tandem catalysis of PAM and glyoxylate consuming enzymes was readily achieved. Utilizi ng PAM first for the production of all glyoxylate from the glycine-extended substrates allows for inactiv ation of PAM after catalysis, followed by optimization of reaction conditions for glyoxyl ate consuming enzymes. Inactivation of PAM is necessary such that reaction conditions may be alte red to optimize for glyoxylate detection. It is necessary to remove PAM co-substrates, adjust pH conditions, and / or add cofactors for the subsequent detecti on of glyoxylate. Ascorbate inhibits many glyoxylate consuming enzymes and interferes wi th methodologies utilized to detect their activity. Therefore it is imperative that asco rbate be removed after PAM catalysis. This is accomplished by the oxidation of ascorbate by the enzyme ascorbate oxidase. Likewise, in some cases addition of different buffering reagents is required to facilitate
31enzymatic detection of glyoxylate. Mani pulation of the PAM produced glyoxylate conditions was particular to the enzyme(s) utilized for glyoxylate detection and will be discussed as they apply to each particular glyoxylate assay.
32 Introduction of Spectrophotometric Enzy me Dependent Assays for Glyoxylate Enzyme linked spectrophotometric assays we re developed around two chemistries: the glyoxylate-dependent oxidation / reduction of NADPH / NAD+ and subsequent formazan dye production, and the glyoxylate dependent formation of hydrogen peroxide (H2O2). Spectrophotometric Assays for Glyoxylate HO O Oglyoxylate "gem diol" + Acetyl-CoA malate synthase E.C.126.96.36.199 pH = 7.4OH O -O O Omalate + CoA-S H + glyoxylate reductase NADP+ + H2O +HO O O+ H+NADPH glycolate E.C. 188.8.131.52 pH 8.0O O-O-O oxalate + OH-O2+ H2O + glycolate oxidase E.C. 184.108.40.206 pH 8.0OH HO O Oglyoxylate "gem diol"OH HO O Og l y o x y l a t e" g e m di o l"OH A B C+ H2O2malate dehydrogenase E.C. 220.127.116.11 NAD O O-O O ONADH + Figure 9. Spectrophotometric assays for glyoxylate Overview of the spectrophotometric assays developed for the analysis of glyoxylate. (A) The malate synthase / malate dehydrogenase assay, detection is based upon the concomitant reduction of NADH and oxidation PMS to yield a tetrazolium dye. (B) The glycolate oxidase assay for the stoichiometric production hydrogen peroxide based on glyoxylate consumption. (C) The glyoxylate reductase dependant glyoxylate reduction upon NADPH oxidation visible at 340nm.
33 Introduction to the Malate Synthase / Malate Dehydrogenase Glyoxylate Consuming Enzymes Malate synthase catalyzes the condensati on of glyoxylate and acety l-Co-A to produce malate, and is an enzyme of the glyoxylate cy cle of eubacteria, plants, and fungi . The glyoxylate cycle is involve d in the regeneration of th ree carbon molecules which become depleted during the TCA cycle [ 56]. Malate synthase activity can be spectrophotometrically measured by the re lease of free CoA upon condensation. Free CoA reacts with 5Â’,5Â’-dithio-bis-(2-nitrobenz oic acid) (DTNB / EllmanÂ’s reagent) to form a TNB anion (thio-bis-2-nitrobenzoat e) and a CoA-TNB adduct with a max of 412nm and a literature extinction value of = 13.6 mM-1cm1. Malate dehydrogenase is a member of the citric acid cycle; it catalyzes the last reaction of the TCA cycle via production of oxaloac etate and NADH from malate and NAD+. Malate dehydrogenase activity is thermodynamically regulated and plays a major role in the regulation of NADH production. In the forward direction the malate synthase oxidation of malate to oxaloacetate coupled to the reduction of NAD+ to NADH, is a highly unfavorable reaction, with a GoÂ’ = +29.7 kJmol-1
34 The Glyoxylate Dependent Formation of a Formazan Dye The concomitant oxidation of malate and reduction of NADH necessary for glyoxylate detection is a non-spontaneous re action of malate dehydrogenase in vitro The large GoÂ’ value for the reduction of NAD+ by malate dehydrogenase n ecessitates the use of an electron shuttling reagent for the catalyt ic activity in the forward direction in vitro An electron shuttling reagent is a catalytic redox ac tive cycling reagent, responsible for affecting the equilibrium of a reaction by consumption of the product. An electron shuttling reagent is the terminal electron acceptor, which upon reduction can auto-catalytically become re-oxidized. This catalytic mechanism allows a single shuttling molecule to shuttle numerous times thereby imparting a significant effect on the turnover of the terminal oxidant (NADH / NAD+) (Fig. 10). Utilization of an electron shuttling reagent for NAD+ reduction by malate dehydrogenase is a manifestation of Le Chatliers principle. For malate dehydrogenase activity, the use of an electron shuttling reagen t is essential to shift the eq uilibrium in favor of product formation (NADH). This affect is a ramification of the instantaneous removal (via oxidation) of any NADH production. In the abse nce of an electron sh uttling reagent the malate dehydrogenase forward reaction is catalytically inco mpetent as a consequence of thermodynamic regulation. Several mol ecules including, cysteine, quinones and phenazines play the role of el ectron shuttling reagents in biochemical systems. A major
35prerequisite for a functional electron shuttling reagent is that it must have the correct redox potential to be catalytical ly competent in a given system. Definitively, the electron shuttling reagent redox potential (EoÂ’) must be a value between the EoÂ’ of the reductant and the oxidant . Catalytic Role of PMS OH O-O O OmalateO O -O O O oxaloacetate malate dehydrogenase NAD+ + H+NADH N N N N O O N N N N N S O OH O SO3 N N N H N N S O OH O SO3 MTS Formazan PMSoxPMSred Figure 10. Catalytic role of PMS. The electron shuttling pathway for the NADH dependent stoichiometric reduction of MTS, the reduced MTS, formazan, is an intensely colored product visible at max 490nm.
36 Malate Synthase / Malate De hydrogenase Glyoxylate Assay O O Oglyoxylate + Acetyl-CoA Malate Synthase E.C.18.104.22.168 pH = 7.4OH O -O O Omalate + CoA-S H Malate Dehydrogenase E.C.22.214.171.124 + NAD+ O O-O O Ooxaloacetate + NADH + H+ PMSred NAD+ ox PMSoxMTS FORMAZAN Figure 11 Malate synthase / malate dehydrogenase glyoxylate assay. The malate synthase / malate dehydrogenase coupled assay for glyoxylate dependent upon the stoichiometric reduction of a tetrazolium dye.
37The electron shuttling reagent chosen for the stoichiometric NADH formation and subsequent tetrazolium dye (MTS, 3 -(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxylphenyl)-2-(4-sulfophenyl)-2H-tetr azolium, inner salt) reduction was phenazine methosulfonate (PMS) . PM S is also known as pyocyanin an electron shuttling pigment of the facultative anaerobe Psuedomonas aeruginosa . PMS cycles electrons from NADH to a tetrazolium dye via 2, 1eprocesses (Fig. 10). The reduced tetrazolium dye formazan product has a max of 490nm and a molar extinction coefficient of = 25mM-1cm-1 . This formazan dependant spectrophotometric assay is 4 fold more sensitive than the analysis of NAD H production based on extinction coefficient alone. Moreover, malate dehydrogenase is catalytically inactive in the absence of phenazine methosulfonate, which drives th e reaction towards concomitant glyoxylate oxidation and NAD+ reduction (Figs.10, 11).
38 Introduction to the Glyoxylate Consuming Enzyme Glycolate Oxidase Glycolate oxidase, (E.C.126.96.36.199) is a flavin de pendent, oxidoreductase enzyme found in plants , protists  and mammals [62, 63, 64]. Enzymatic Reaction of Glycolate Oxidase HO O Oglycolate + O2 O O Oglyoxylate + H2O 2 g l y c o l a t e oxidase Figure 12. Enzymatic reaction of glycolate oxidase The oxidation of glycolate and concomitant reduction of bimolecular oxygen as catal yzed by the enzyme glycolate oxidase. Glycolate oxidase is a peroxisomal enzyme, whic h in plants is part of the photosynthetic oxidative C2 cycle. The main function of the plant C2 cycle is for the complete oxidation of C2 units to carbon dioxide and water. The mammalian enzyme is involved in the production of oxalate from carbohydrates and seri ne . Glycolate oxidase catalyzes the oxidation of both glyoxylate and glycolate, utilizing O2 as the electron acceptor  and has a pH range between 8.0 and 8.5 . At pH values above 3.5 glyoxylate is found in the hydrated gem diol form, HC(OH)2 Â– COOH, in aqueous so lutions [67, 68]. The glyoxylate gem diol is oxidized by glycolate oxidase to oxalat e as dioxygen is reduced to hydrogen peroxide (Fig. 12).
39 The Glyoxylate Dependent Produ ction of Hydrogen Peroxide Several techniques exist for the spectrophotome tric detection of hydrogen peroxide [69, 70, 71, 72]. The glycolate oxidase mediat ed conversion of gl yoxylate to hydrogen peroxide was chosen as the basis for a novel spectrophotometric glyoxylate assay. The horse radish peroxidase (HRP), cataly zed oxidation of substrates 3-methyl-2benzothiazolinone hydrazone (MBTH), and 3-(d imethylamino)benzoic acid (DMAB), in the presence of hydrogen peroxide pr oduces an indamine dye adduct, 47mM-1cm-1 53mM-1cm-1 [73, 74] (Fig. 13). The MBTH / DMAB / HRP assay for hydrogen pe roxide detection was chosen over other methods as a few key characteristics provide d this assay with higher sensitivity, and ease of use. Several alternate HRP substrates, (homovanillic acid, resorufin, etc) undergo auto-oxidation at rates apprec iably higher than that of th e indamine dye production. Auto-oxidation of HRP substrates leads to the generation of background values approaching levels sufficiently large to affect the dynamic linear range of analyte detection. Spectrophotometric detection is fundamentally li mited at high concentrations on account of light scattering due to precip itation (solubility issu es at high analyte concentration), and by incomplete percent transmission of highly colored solutions (Abs = 2 log % T). Auto-oxidation becomes increasingly limiting for end-point enzymatic assays as long incubations at 37oC are required for reaction completion. Particularly, in the case of glycolate ox idase which has an incubation period of 40
40minutes for glyoxylate detection (Fig. 14). Coll ectively, these issues significantly affect the both the limit of detection and dynamic linear range of analyte detection as compared to the DMAB / MBTH / HRP assay where auto-oxidation is comparably much less. Moreover, the DMAB / MBTH / HRP assa y has a comparably large extinction coefficient. A glyoxylate depe ndent stoichiometric assay ut ilizing glycolate oxidase and the DMAB/MBTH peroxidase detection syst em has been developed based on this chemistry. H2O2 Dependent Indamine Dye Production O HO N dimethylaminobenzoic acidS N N NH2 3-methyl-2-benzothiazolinone hydrazone + S N N N N O HO H2O2HRP + H 2 O + 1/ 2 O 2 Figure 13. H2O2 dependent indamine dye production The mechanism for hydrog en peroxide detection, based upon the HRP dependent oxidative coupling of DMAB and MBTH to produce the blue colored indamine dye adduct. The glycolate oxidase assay for glyoxylate based on stoichiometric hydrogen peroxide detection is an enzyme coupled assay reliant on catalytically competent HRP. In order for the assay to be stoichiometric (linear over a defined range) for gl yoxylate, the rate of HRP dye production must be on the order of at least 10-fold higher than glycolate oxidase activity. Such that color fo rmation is directly proportiona l to the glyoxylate dependent
41hydrogen peroxide production, and not affected by the rate of indamine dye formation. Such an assay requires the vmax of HRP to far exceed that of the glycolate oxidase, for an almost instantaneous color forma tion upon hydrogen peroxide production. Glycolate Oxidase Based H2O2 Assay Glycolate Oxidase E.C.188.8.131.52HO O Oglyoxylate hydrated O2+ pH = 8.0MBTH3-methyl-2-benzothiazolinone hydrazoneDMAB3-(dimethylamino) benzoic acid HRP H2O2 dependant MBTH/DMAB Oxidative Coupling max = 590nm 53,000M-cm +H2O2+ + + O O O-O oxalateOH Figure 14. Glycolate oxidase based H2O2 assay The glycolate oxidase assay developed for glyoxylate detection coupled to the peroxidase indamine dye detection system.
42 Introduction to the Glyoxylate Consuming Enzyme Glyoxylate Reductase Glyoxylate Reductase (GR) is a NADPH dependant reductase and catalyzes the reduction of glyoxylate to glycolate with the concomitant oxidation of NADPH (or NADH) (Fig. 15). Glyoxylate reductase was first isolated from the leaves of Spinachea olericea by Zelitch and Gotto in 1962 . Glyoxylate reductase is also found in bacteria , green algae , protists , yeast  and humans . Liver metabolic studies on glyoxylate reductase have shown that alterati on in glyoxylate reductase activity in vivo has been linked to hype roxaluria , a ramificat ion of altered glyoxylate and oxalate metabolism. In plants, glyox ylate reductase is an enzyme of the photo-respiratory cycle, Kleczkowski and Blev ins defined the kinetic constants of the spinach isozyme, as having a higher affinity for glyoxylate (KM=0.085 mM) in the presence of NADPH, as compared to NADH (KM=1.10mM) . Enzymatic Reaction of Glyoxylate Reductase Figure 15. Enzymatic reactio n of glyoxylate reductase Oxidation of NADPH drives the reduction of glyoxylate to glycolate. O O Oglyoxylate + Glyoxylate Reductase E.C.184.108.40.206 pH 8.0 NADP++HO O Oglycolate + H+NADPH
43 Independent Verification of Glyoxylate Concentration as a Reference Standard to Newly Developed Assays The development of novel assays requires an independent verification of analyte concentration. The PAM dependent cleavage of the substrate glycyl C bond results in a 1:1 ratio of glyoxylate and amidated product [83, 84]. Each novel glyoxylate assay developed herein, was compared to th e production of the corresponding amide. Independent analysis functions to both va lidate each glyoxylate, as well as demonstrate the utility of each assay for the discovery of novel -amidated peptides. Three reference assays were used to validate glyoxylate production; (a) the PAM catalyzed oxidation of N-dansyl-Ty r-Val-Gly to N-dansyl-Tyr-Val-NH2 and glyoxylate, (b) the PAM catalyzed oxidation of hippurat e (N-benzoylglycine) to benzamide and glyoxylate, and (c) the base-c atalyzed dealkylation of -hydroxyglycine to benzamide and glyoxylate (Fig. 16). For each reference reaction, a RP-HPLC sepa ration of substrate and amide product was developed and used to determine the concentr ation of amide formed. The concentration of the amide product was then compared to the glyoxylate concentrat ion, as determined using the newly developed assays.
44Reference Reactions for the Indepe ndent Analysis of the Developed Glyoxylate Assays S N O O O NH2O NH O N H O OH Cu(II) ascorbate dehydroascorabteS N O O O NH2O NH O N H2 O O O glyoxylate PAM+ N -dansyl-Tyr-Val-Gly N-dansyl-Tyr-Val-NH2 H N O O Oascorbate semidehydroascorabte Cu(II) O2O H2N O O O Glyoxylate+ H i p p u r a t e Benzamide H N O O OO H2N O O O Glyoxylate+alpha-Hydroxyhippurate Benzamide Base Figure 16 Reference reactions for the independent anal ysis of the developed glyoxylate assays Three reference reactions for the independent analysis of gly oxylate utilized as a standard for all newly developed glyoxylate assays. (A) The PAM cataly zed oxidation of the dansylated tr i-peptide substrate, amide product quantified by RP-HPLC coupled to fluorescent detection. (B) The PAM catalyzed oxidation of hippurate to benzamide and glyoxylate, ben zamide product quantified by RP-HPL C coupled to spectrophotometric detection. (C) The base catalyzed dealkylation of -hydroxyhippurate, product quantified by RP-HPLC separation and UV analysis. A B C
45 Materials and Methods Materials B. stearothermophilus malate synthase, porcine heart malate dehydrogenase, Cucurbita ascorbate oxidase, Spinachea olericea glycolate oxidase, a cetyl-CoA, MBTH, DMAB, HRP, lactate dehydrogenase, nicotimamide adenine dinculeotide, 5Â’,5Â’-dithio-bis-(2nitrobenzoic acid) (DTNB), NADH, NADP H, FAD, FMN, ammonium sulfate, glyoxylate, and PMS were purchased from Sigma, -hydroxyhippuric acid was from Aldrich, Mimetic blue was from Prometic, MTS was a gift from Dr. T. C. Owen, and recombinant rat peptidylglycine -amidating monooxygenase (PAM) was a gift from Unigene Laboratories, Inc. A ll other reagents were of the highest quality commercially available.
46 Methods Malate Synthase / Malate Dehydrogenase (MS / MD Assay) Spectrophotometric Assay for Glyoxylate Standardization of the MS / MD Depe ndent Formazan Assay with NADH. A working stock solution of 1500 M MTS / 82.5 MPMS was prepared in water, stored at 4 C, and small aliquots removed as needed to reduce light and air exposure . NADH production is frequently linked to tetrazolium reduction because the detection limit for the resulting formazan is lower than that possible for the spectrophotometric determination of NADH . To verify th at NADH will drive the conversion of MTS to a formazan, MTS reduction was initiated by the addition of NADH to a solution of 100mM triethanolamine-HCl pH 7.8, 150 M / 8.25 M MTS/PMS, and 0-50 M NADH to a final volume of 1mL. After a 5 min. incubation at 37 C in the dark, the absorbance at 490nm was measured using a Jasc o Model V-530 UV-VIS spectrophotometer equipped with the Spectra Analysis softwa re package. The small amount of MTS reduced for the zero NADH contro l was subtracted from that obtained in the presence of NADH. Reaction conditions (pH, temperatur e, and choice of buffer) were chosen to minimize the pH dependent spontaneous reducti on of MTS in the absence of NADH [59, 86] and the optimal pH values for malate s ynthase and malate dehydrogenase [87, 88].
47 Standardization of the MS / MD Assay with Glyoxylate The enzyme-coupled assay for glyoxylate was initiated by the addition of malate synthase and malate dehydrogenase. The assay containe d a standard solution of 100 mM TEA-HCl pH 7.8, 150 M / 8.25 M MTS/PMS, 10mM MgCl2, 400 M acetyl-CoA, 500 M NAD+, 0-50 M glyoxylate, 6U/mL malate synthase, and 6U/mL malate dehydrogenase in a final volume of 1mL. The absorban ce at 490 nm was measured after 1 hr incubation at 37 C in the dark. The small amount of MTS reduced for the zero glyoxylate control was subtracted from that obtained in th e presence of glyoxylate. Measurement of the Base-Catalyzed Produc tion of Benzamide and Glyoxylate from Hydroxyhippurate Utilizing the MS / MD Assay Glyoxylate is a product of the base-catalyzed N-dealkylati on of carbinolamides . Incubation of 2.5mM -hydroxyhippurate (C6H5-CO-NH-CH(OH)-COO ) in 1.0M NaOH for 12 hr at 37 C resulted in the conversion of -hydroxyhippurate to benzamide as determined by reverse-phase HPLC. The resultant glyoxylate concentration was determined via the enzyme-coupled assa y after appropriate dilution with H2O to a final glyoxylate concentration of 40 M.
48 Measurement of PAM Produced Benzamide and Glyoxylate from Hippurate Utilizing the MS / MD Assay Hippurate (C6H5-CO-NH-CH2-COO ) is a PAM substrate whic h is amidated to produce benzamide and glyoxylate [83, 84]. Hippurate amidation at 37 C was initiated by the addition of PAM (0.6 mg) to 0.5mL of 100mM MES pH 6.0, 2.0 M Cu(NO3)2, 1.0mM ascorbate, and 3.5mM hippurate. At 10 min intervals, a 45 L aliquot was removed and added to 10 L of 6% (v/v) TFA to terminate th e reaction. Percen t conversion of hippurate to benzamide was determin ed at each time interval by C18 RP-HPLC. Approximately 20 nanomoles of glyoxylate wa s added to a 0.9mL solution containing the necessary components for the glyoxylate assay, excluding the malate synthase, malate dehydrogenase, and MTS/PMS. Ascorbate was eliminated from all samples, prior to glyoxylate determination, with 1 hour incubati on in the presence of 2U/mL of ascorbate oxidase at 37 C. Ascorbate, a co-substrate for the PAM reaction, r eadily reduces MTS and must be removed prior to addition of the MTS/PMS reagent. After ascorbate elimination, addition of a 100 L solution of 10X concentrated malate synthase, malate dehydrogenase, and PMS/MTS resulted in a fi nal 1.0mL reaction at standard conditions. The glyoxylate concentration was determined by measuring the absorbance increase at 490nm after incubation at 37 C for 1 hr ( 490nm = 25.6mM-1cm-1). The small amount of MTS reduced for a control lacking hippur ate was subtracted from each time point.
49 Glycolate Oxidase / HRP (GO / HRP) Sp ectrophotometric Assay for Glyoxylate Standardization of the Glycolate Oxidase / Horseradish Peroxidase Assay with H2O2 A standard working solution of 0.6mM MBTH, 30mM DMAB, and 50U/mL HRP was prepared and stored under N2 in the absence of light. The working solution must be prepared fresh daily to avoid blank values of high absorbance. The quantification of a standardized hydrogen peroxide solution was carried out in 80mM phosphate buffer pH 7.8. The reaction was initiated by the addition of 72 L of the working solution in a final volume of 250 L. The formation of the blue colored indamine dye was followed at 595nm. Linear regression analysis of the obt ained data was analyzed against literature regression curves for extinction coefficient analysis.
50 Standardization of the Glycolate Oxidas e / Horseradish Peroxidase Assay with Glyoxylate Following standardization of the MBTH / DMAB / HRP dependent system for the quantification of hydrogen pe roxide, the detection syst em was applied to the quantification of glyoxylate production. A stan dard glyoxylate solution was utilized to develop a standard curve for the detecti on of glycolate oxidase produced hydrogen peroxide. All reactions we re performed in 80mM phospha te buffer pH 7.8, 0.1mM FAD, 0.48U/mL glycolate oxidase, and 0 Â– 4 M glyoxylate at a final volume of 250 L. Reactions were allowed to proceed for forty minutes at 37oC prior to spectrophotometric analysis at 595nm.
51 Standardization of Glycolate Oxidase / Horseradish Peroxidase Assay for PAM Produced Glyoxylate PAM reactions were carried out in 100mM MOPS pH 7.1 containing 10U/mL HRP, 1mM sodium ascorbate, 1 M Cu(NO3)2, 20 M dansyl-Tyr-Val-Gly with 3 g/mL PAM, the reaction proceeded for one hour at 37 C. The resultant concen tration of the amidated PAM product was measured by RP-HPLC to quantify the exact concentrations of [dansyl-Tyr-Val-NH2] and [glyoxylate] (Chapter 3, dansyl assay procedure). Aliquots of the PAM reaction pertaining to variable concentrations (1, 3, 5, 7, and 9 M) of glyoxylate were taken for analysis by the MBTH / DMAB / HRP assay. Prior to analysis of glyoxylate by the afore described assay, the PAM reaction underwent a sample pretreatment procedure to remove no reacted (non-oxidized) ascorbate. An aliquot of 2U/mL ascorbate oxidase was added to the PAM reaction, incubated for a one hour time period at 37oC prior to glyoxylate analysis. Anal ysis of PAM produced glyoxylate was carried out under the describe d conditions for 40 minutes.
52 Glyoxylate Reductase Spectrophot ometric Assay for Glyoxylate Purification of Glyoxylate Reductase from Spinach Glyoxylate reductase was purified from spin ach leaves by the methods of Kleczkowski and Blevins . The enzyme was partia lly purified by a 49 / 60 ammonium sulfate precipitation. The enzyme was homogenized in an extraction buffer containing 40 mM Tricine pH 7.8, 2mM MgCl2, 1mM EDTA, 1mM benzamidine, 5 M leupeptin, and 14mM -mercaptoethanol. The specific activ ity and protein concentration of the resuspended ammonium sulfat e pellet was analyzed by the method of Kleczkowski .
53 Standardization of Glyoxylate Reductase Assay for Glyoxylate A standard and PAM produced glyoxylate solu tion was utilized to develop a standard curve for the glyoxylate reductase detection of glyoxylate. All reactions were performed in 100mM MOPS buffer pH 7.1, 0.2mM NADPH. 0.4 mg of enzyme at a final volume of 1mL. Glyoxylate dependent loss of NADPH was monitored at =340nm on a Jasco Model V530 spectrophotometer equipped with a Spectral Analysis software package. Reactions were initiated by the addi tion of substrate (1, 3, 5, 7, and 9 M glyoxylate) and the 340nm was obtained after 10 minutes at 25oC. All A340nm measurements were obtained by the NADPH dependent loss of A340nm in the presence of glyoxylate, subtracted from the loss A340nm in the presence of NADPH without substrate.
54 Standardization of Glyoxylate Reductas e Assay for PAM Produced Glyoxylate A standard solution of PAM produced glyoxylat e was produced as described (chapter 2). The solution was utilized for standard curve analysis of the glyoxylate reductase dependent analysis of PAM produced glyoxylate.
55 Independent Analysis; C18 Reverse-Phase HPLC Separations for the Quantification of Benzamide / Glyoxylate C18 Reverse-Phase HPLC Separation of dan syl-Tyr-Val-Gly and dansyl-Tyr-Val-NH2 HPLC assays were performed with a He wlett-Packard 1100 liquid chromatograph equipped with a quaternary solvent delivery system, a heated column compartment, an auto-sampler, and an auto injector. Analytes were detected at excitation = 300nm, and emission = 380nm using an in-lin e Gilson model 121 fluorom eter equipped with appropriate filters. Separations were ach ieved using a Keystone ODS Hypersil C18 column (100 4.6 mm, 5 particle size). Dansyl-T yr-Val-Gly (rete ntion time = 1.2 min), dansyl-Tyr-Val-Gly (retention time = 2.1 min) were resolved at 50 C using an isocratic mobile phase of 100mM sodium acetat e pH 6.0 / acetonitrile (52/48) at flow rate of 1.0mL/min . The percent conversion of dansyl-Tyr-Val-Gly dansyl-Tyr-ValNH2 was calculated based on peak height values obtained using a Hewlett Packard HP3392A integrator. The ratio of nmoles/peak area for dansyl-Tyr-Val-Gly, and dansylTyr-Val-NH2 was obtained from linear standard cu rves generated from 0-50 nanomoles of each. Data resulted in the ultimate dete rmination of the quantity of PAM produced amidated product and glyoxylate (Fig. 17).
56 C18 Reverse-Phase HPLC Separation of Hippurate, -Hydroxyhippurate, and Benzamide. HPLC assays were performed with a He wlett-Packard 1100 liquid chromatograph equipped with a quaternary solvent delivery sy stem, a heated column compartment, an auto sampler, and an auto injector. Analyt es were detected at 254nm using an in-line variable wavelength UV / VIS spectrophotometer. Separati ons were achieved using a Keystone ODS Hypersil C18 column (100 4.6 mm, 5 m particle size). Hippurate (retention time = 3.1min), -hydroxyhippurate (retention time = 2.9 min), and benzamide (retention time = 5.9 min) were resolved at 50 C using an isocratic mobile phase of 100mM sodium acetate pH 6.0 / acetonitrile ( 82/18) at flow rate of 1.1mL/min. The percent conversion of hippurate benzamide or -hydroxyhippurate benzamide was calculated based on peak area values obt ained using a Hewlett Packard HP3392A integrator. The ratio of nmol es/peak area for hippurate, -hydroxyhippurate, and benzamide was obtained from linear standard curves generated from 0-50nmoles of each (Fig. 17, & Table 3).
57 Results and Discussion Malate Synthase / Malate Dehydrogenase Spectrophotometric Assay for Glyoxylate MS / MD Assay for Chemically and Enzymatically Produced Glyoxylate The increase in absorbance at 490nm from the NADH-dependent reduction of MTS (at limiting NADH) was linear with the NADH concentration and yielded an 490nm = 0.0282M-1cm-1 (data not shown). The value was within 10% of that reported by Debnam & Shearer , 0.0256M-1cm-1. The sequential actions of malate synthase and malate dehydrogenase convert glyoxylate and acetyl-CoA to oxaloacetate and NADH (Fig. 9 A). PMS-dependent NADH oxidation drives the reduction of MTS to a purplecolored formazan that produces an increase in absorbance at 490nm proportional to [glyoxylate] (Fig. 10). The extinction coefficient derived from these data, 490nm = 0.0239M-1cm-1, was within 7% of the published value (Fig. 17).
58 0.0 0.20 0.40 0.60 0.80 1.0 01020304050Absorbance at 490 nm[Glyoxylate], (M)A 0.0 0.20 0.40 0.60 0.80 1.0 010203040Absorbance at 490 nm[Benzamide] = [Glyoxylate], (M)B Figure 17. Glyoxylate-Dependent Oxidation of MTS. The increase in absorbance obtained using glyoxylate (A) and that obtained by the base-catalyzed dealkylation of -hydroxyhippurate to benzamide and glyoxylate (B). The data points are the average of 3-10 determinations and the error bars represent the standard deviation of the measurements. Carbinolamides undergo base-catalyzed N-dealkylation to produce an amide and glyoxylate  R-CO-NH-CH(OH)-COO + OH R-CO-NH2 + HCO-COO + H2O (Fig. 16 C). The N-dealkylation of -hydroxyhippurate provided a test for the newly developed glyoxylate assay. Per cent conversion analysis of -hydroxyhippurate to benzamide by HPLC served as an independent test for the [glyoxylate] values. The Ndealkylation of 2.5mM -hydroxyhippurate with NaOH (as described in the Materials and Methods) resulted in a solution of 0.6mM unreacted -hydroxyhippurate, 1.9mM benzamide, and 1.9mM glyoxylate (76% conve rsion) (Fig.17 B). Analysis of this solution using the enzyme-coupled glyoxylate assay yielded an 490nm = 0.0277M-1cm-1 (Fig. 17 A).
59 Table 3. Ratio of [Glyoxylate] Produced to [Benzamide] Produced by the PAM Treatment of Hippurate Time (min) Glyoxylate Produced (mM) Benzamide Produced (mM) [Glyoxylate]/[Benzamide] 40 0.69 0.58 1.2 50 0.67 0.70 0.96 60 0.71 0.80 0.89 70 0.75 0.90 0.83 80 0.77 0.98 0.79 90 1.3 1.1 1.2 100 1.3 1.2 1.1 110 1.3 1.3 1.0 Average standard deviation = 1.0 0.16 Note. Reactions were initiated by the addition of PAM to 2.5 mM hippurate as described in the Materials and Methods. At the indicated time, an aliquot was removed and assayed for benzamide by HPLC and glyoxylate using the malate synthase/malate dehydrogenase/MTS/PMS system. Table 3 Comparison of PAM Produced Glyoxylate to Benzmide Production. The ratio of PAM produced glyoxylate as quantified by the newly developed MS/MD assay as compared to PAM produced benzamide analyzed by RP-HPLC. PAM-catalyzed cleavage of the glycyl C-N bond of glycine to form an amide and glyoxylate requires O2 and ascorbate as co-substrate s [83, 84]. Ascorbate, a common contaminant of biological samples, reduces MTS  and must be elim inated in order to use the enzyme-coupled glyoxylate assay to measure PAM activity. Ascorbate was effectively removed by treatment of PAM produced glyoxylate-containing samples with 2U/mL ascorbate oxidase. The PAM-catal yzed amidation of hippurate produced glyoxylate and benzamide at a ratio of 1.0 0.16 [glyoxylate] / benzamide] (Table 3), within experimental error of the expe cted stoichiometry of 1:1.
60Thus, the developed malate synthase / mala te dehydrogenase assay effectively quantifies PAM produced glyoxylate. For the MTS-derived formazan, this translates into a detection limit of ~2 M glyoxylate ( 490nm = 0.05-0.06). The enzyme-coupled glyoxylate assay can be adapted to a microtiter plate format with a final assay volume of 0.1mL providing a detection limit of ~0.2nmoles of glyoxylate. In conclusion, an enzyme-based method that links glyoxylate oxidati on to tetrazolium reduction has been described. Tetrazolium reduction produced a colored formazan that enabled the spectrophotometric determinatio n of glyoxylate. Methods to eliminate ascorbate enabled the successful determina tion of glyoxylate in samples initially contaminated with excess ascorbate. The enzyme-coupled glyoxylate assay is amenable for use in 96-well microplate format that wi ll increase the sensitivity of detection and may facilitate a high throughput analysis for glyoxylate.
61 Glycolate Oxidase / Horse Radish Peroxidase Linked DMAB / MBTH Assay for Glyoxylate Glycolate Oxidase/Horse Radish Peroxidas e Linked DMAB / MBTH / HRP Detection of H2O2 An increase in absorbance at 595nm from the H2O2 driven HRP dependent oxidative coupling of DMAB / MBTH was linear with the H2O2 concentration and yielded an 595nm = 0.0456M-1cm-1 (Fig. 18). This value was with in 4.20% of that reported by Ngo & Lenhoff  595nm = 0.0476M-1cm-1 at pH 6.5, and within 16.2 % of the value reported by  595nm = 0.0530M-1cm-1 at pH 4.5. The HRP dependent oxidative coupling of DMAB/MBTH was essentially instantaneous.
62Linear Regression of the DMAB / MBTH / HRP Detection of H2O2 y = 0.0456x 0.0008 R2 = 0.9892 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0246810 [H2O2] MA 595nm Figure 18. Linear regression of the DMAB / MBTH / HRP detection of H2O2 Peroxide dependant formation of an indamine dye as analyzed at an absorbance of 595nm by a Bio-Rad Model 550 micro-plate reading spectrophotometer.
63 Glycolate Oxidase Dependent DMAB / MBTH / HRP Detection of Standard Glyoxylate Glycolate oxidase in the presence of the glyoxylate gem diol form and O2 produces a stoichiometric quantity of H2O2 and oxalate (Fig. 9 B). Ox idation of the glyoxylate gem diol drives the H2O2 HRP dependent oxidative coupling of MBTH and DMAB to produce an increase in absorbance at 595nm pr oportional to [glyoxylate] (Fig. 13). The extinction coefficient derived from these data, 595nm = 0.0450M-1cm-1, was within 3.2 % of the Ngo & Lenhoff  published value (Fig. 19). Linear Regression of the DMAB / MBTH / HRP Detection of Glyoxylate y = 0.045x + 0.0245 R2 = 0.98360 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 00.511.522.533.54[glyoxylate] MA 595nm Figure 19. Linear regression of the DMAB / MBTH / HRP detection of glyoxylate. Calibration of glycolate oxidase activity with glyoxyl ate as a substrate, demonstration fo r the use of glycolate oxidase as an enzyme to detect stoichiometric quantities of glyoxylate.
64 Glycolate Oxidase Dependent DMAB / MBTH / HRP Detection of PAM-Produced Glyoxylate The PAM-catalyzed amidation of dansyl-Tyr-V al-Gly proceeded to approximately 100% as analyzed by RP-HPLC product formation. Thus, PAM produced a stoichiometric quantity of [glyoxylate]f = [dansyl-Tyr-Val-Gly]i for the glycolate oxi dase analysis. The glycolate oxidase assay resulted in a ratio of 1.0 0.17 ( 595nmglyoxylate = 0.0450M-1cm-1 / glyoxylatePAM produced = 0.0375M-1cm-1), for the compared values of standard glyoxylate vs. PAM produced glyoxylate, a value within expe rimental error of 1 : 1 stoichiometry (Fig. 20). Linear Regression of the DMAB / MBTH / HRP Detection of PAM-Produced Glyoxylate y = 0.0375x + 0.0072 R2 = 0.99530 0.1 0.2 0.3 0.4 0.5 0.6 02468101214[d-TYG] = [d-TVNH2] = [glyoxylate] M A 595nm Figure 20. Linear regression of the DMAB/MBTH/HRP detection of PAM-produced glyoxylate. Stoichiometric detection of PAM produced glyoxylate generated from a glycine-extended peptide substrate. [d-YVG] = [d-YVNH2] = [glyoxylate] M
65The developed glycolate oxida se assay effectively quantif ies PAM produced glyoxylate. For the H2O2 produced indamine, this translates into a detection limit of ~0.5 M glyoxylate ( 595nm = 0.05-0.06). The enzyme-coupled gl yoxylate assay was adapted to a micro-titer plate format with a final assa y volume of 0.25mL providing a detection limit of ~125 picomoles of glyoxylate.
66 Glyoxylate Reductase Dependent Assay for Glyoxylate Glyoxylate Reductase Detectio n of PAM-Produced Glyoxylate The standardized solution of PAM produced glyoxylate as an alyzed by the oxidation of NADPH resulted in a A 340nm=0.0063 M-1cm-1. This value is within 1% of the reported 340nm value for NADPH of 340nm=0.0062 M-1cm-1(Fig. 21). Glyoxylate Reductase Determinatio n of PAM Produced Glyoxylate Figure 21. Glyoxylate Reductase Determination of PAM Produced Glyoxylate The glyoxylate reductase dependent assay for glyoxylate determination. y = 0.0063x + 0.0002 R2 = 0.99120 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 024681012[Glyoxylate] = [d-YVG] MLoss A 340nm
67In conclusion, two enzyme based systems ha ve successfully been developed for the stoichiometric detection of PAM produced glyoxylate. Both assays provide novel methodologies for the sensitive detection of glyoxylate, glycine-extended peptide, and amidated peptides. It is important to note that the glycolate oxidase assay is also amenable to the sensitive spectrophotometric detection of glycolate as well. Both assays have proven to be valid techniques for the de tection of PAM activity and have provided a framework for the design of other more sens itive novel techniques for the stoichiometric detection of -amidated peptides.
68 Chapter Three Fluorescent and Chemi-Luminescent Assays for Glyoxylate Introduction The spectrophotometric assays described in Ch apter 2 are robust, stoichiometric assays for glyoxylate and subsequently PAM activity. However, more sensitive assays for glyoxylate are required for the development of a platform technology to discover amidated peptides. Several methods exist for the sensitive detection of enzyme catalysis. In particular, fluorescent and chemi-luminescent assays for hydrogen peroxide have detection limits in the femtomole range. To take advantage of these assays, the PAMdependent production of glyoxylat e must be linked to hydr ogen peroxide production. This can be accomplished utilizing two enzymes, glycolate oxidase (chapter 2, Fig. 9 B) and glyoxal oxidase (Fig. 22). As described in this chapter, both were used for the successful detection of femtomole levels of PAM produced glyoxylate. Reaction Catalyzed by Glyoxal Oxidase HO O OHO + O2 glyoxal oxidaseHO O OO + H2O 2 g l y o x y l a t e g e m d i o l o x a l a t e Figure 22. Reaction catalyzed by glyoxal oxidase The glyoxal oxidase dependent production of hydrogen peroxide from glyoxylate.
69 Fluorescent Assays for the Det ection of Hydrogen Peroxide Several fluorophores exist for the fluores cent determination of hydrogen peroxide generated in solution. Fluorescence dyes for hydrogen peroxide determination have been designed as substrates for the enzyme horsera dish peroxidase. For example, horseradish peroxidase (E.C. 220.127.116.11.) utilizes Ampl ex Red as an electron donor in a disproportionation reaction of hydrogen peroxi de to water and molecular oxygen (Fig. 23). Amplex red becomes oxidized to the in tensely fluorescent co mpound resorufin, in a stoichiometric ratio to hydrogen peroxide consumption. Amplex red can be utilized in a continuous assay format for enzymatic activ ity assays which produce stoichiometric quantities of hydrogen peroxide. Amplex Red has a quantum yield ( ) at pH = 9 ( = photons absorbed / photons emitted) of 0.75, rendering Amplex Red a highly fluorescent fluorophore . Fluorescent Analysis for H2O2 Quantification Figure 23. Fluorescent analysis for H2O2 quantification The production of the highly fluorescent dye resorufin, is dependent upon the horseradish peroxidase oxidative catalyzed oxidation of amplex red. Resorufin production is stoichiometric to glyoxylate consumption. N O N O OH HO O O -O Na+ H2O2H2O HRP
70 Enhanced Sensitivity of Amplex Red Fluorometric Detection Spectra of the Fluorophore Resorufin Figure 24. Spectra of the fluorophore resorufin The Stokes shift excitation and emission spectra for the oxidized Amplex Red (resorufin) fluorophore . Resorufin fluorescence is typically measured with an excitation of 560nm and emission of 589nm. The sensitivity of Amplex Re d, like all fluorophores, can be compromised in a biochemical assay format as a result of a high signal / noise ratio. Fluorescence is a process allowing for greater sensitivity as result of the StokesÂ’ shift (Fig. 24). The StokesÂ’ shift describes the distance of th e red shift exhibited by a fluorophore from a shorter excitation wavelength to the longer em issive wavelength. Quantification of the F l u o r e s c e n c e
71emission, measures an observable event from no light emission (excitation) to emission resulting in a smaller signal / noise ratio, thereby allowing for a greater degree of sensitivity. Biochemical based fluoresce nt assay fluorescent detection can be compromised by auto-oxidation of the fluor ophore caused by enzymes and / or enzyme cofactors and substrates. This phenomenon will be discussed as it applies to the two enzymatic based assay systems for glyoxylate de scribed in the results section of this chapter.
72 Fluorescent Assays for Glyoxylate The Flavin Dependent Glyoxylate Co nsuming Enzyme Glycolate Oxidase Glycolate oxidase (hydroxyacid oxidase, E.C. 18.104.22.168) is an oxidoreductase enzyme that typically catalyzes the oxidation of a primary alcohol in the presence of O2 to a ketone and hydrogen peroxide (chapter 2, Fig. 9 B) Glycolate oxidase is a flavoenzyme, an enzyme that requires a flavin prosthetic group for electron transfer from donor to acceptor (Fig. 25). Classically, glycolate ox idase has been known as a FMN (flavin mononucleotide) dependent flavoprotein, howev er published data provides evidence for the ability of this enzyme to be catalytically competent in the presence of alternate flavin prosthetic groups, namely, FAD . The flav in-binding domain of glycolate oxidase is deeply seated within the interior of the en zyme, and tightly bound to the interior flavin binding domain with dissociation constants of 10-8 to 10-10M . Riboflavin is the simplest of all the flavins in that it contains the basic isoalloxazine Â“flavinÂ” domain and a D-ribitol group. FMN is riboflavi n with a free phosphate attach ed to the D-ri bitol, and, lastly, FAD is a conjugate of FMN cont aining an AMP moiety via a phospho-diester bond (Fig. 25).
73 Flavins N H2N N N P O O ON NH O N OH OH OH NO O P O O O O HO OH P O O ON NH O N OH OH OH NO O N NH O N OH OH OH OH N O riboflavin FMN FAD Figure 25. Flavins. The structure of flavin cof actors FMN, FAD, and riboflavin, glycolate oxidase catalysis is dependent upon the presence of either FMN or FAD
74 Utilization of Glycolate Oxidase as a Fluorescent Assay for Glyoxylate. Fluorescence assays are limited in most pa rt by signal/noise, due to the spontaneous oxidation (or reduction) of fluor ophores resulting in significant background fluorescence. In addition, fluorescence detection can be furt her compromised in biological samples by the intrinsic fluorescence of biological molecu les (peptides, proteins etc), thus further affecting signal/noise. In the presence of FMN, Amplex Red non-enzymatically oxidizes to resorufin to the extent that glycolate oxi dase activity cannot be measured via an HRPdependent Amplex Red assay. However, non-enzymatic oxidation of Amplex Red is significantly reduced in the pres ence of FAD. FAD is a cof actor that comp letely supports glycolate oxidase activity . In order to link glycolate oxida se activity to an HRP/Amplex Red based assay, the cofactor must be FAD (Fig.26).
75 Amplex Red Fluorescent Detection of H2O2 O O OOO N O HO OH Ho r se r a d i s h Peroxidase pH 8.0 E.C.22.214.171.124 H2O2O2 Glycolate Oxidase / Glyoxal Oxidase pH = 8.0 A m p l ex R e d oxalateHO O OH2O + OH glyoxylate gem diol O N -O O Resorufin Na+ Figure 26. Amplex Red fluorescent detection of H2O2. The fluorescent Amplex Red dependent assay system for glyoxylate, as catalyzed by glycolate oxidase, and glyoxal oxidase.
76 The Lignolytic Degrading Enzyme Glyoxal Oxidase Glyoxal oxidase (GLOX) (Fig. 20) is a basi diomycete fungal enzyme, one of the three enzymes used in the degradation of lignin. The fungal lignin degradation pathway plays a major role in the decompositi on of detritus, an integral part of the global carbon cycle . Lignin is the second most abundant substance on the pl anet second only to cellulose, and forms the Â“woodyÂ” tissue of plan ts. Collectively, lignin is comprised of several monolignols, namely p -courmaryl, sinapyl, and coniferyl alcohols which compromise the basic set of monomers for th e lignin polymer . Degradation of lignin is imperative to the regeneration of car bon, eventually producing atmospheric CO2. The catalytic role of glyoxal oxidase is the oxi dation of aldehydes to carboxylic acids coupled to the concomitant reduction of dioxygen to hydrogen peroxide . GLOX is a copper metalloenzyme, containing a free radical-coupled copper active site. The radical-copper catalytic motif comprises the two-electron re dox active site. More importantly, GLOX is isolated in the reduced form. Activation of the reduced enzyme requires oxidation via treatment with a strong oxida nt such as Ir(IV) or Mo(V), or the presence of lignin peroxidase (LiP) or horser adish peroxidase (HRP) [97, 98, 99] for cata lytic activity.
77 Utilization of Glyoxal Oxidase for the Quantification of Glyoxylate Glyoxal oxidase has broad substrate specific ity among simple aldehydes (table 4). Methylglyoxal the prefe rred substrate has a Km = 0.64mM as compared to glyoxylic acid Km = 2.5mM, and a kcat/KM ratio of 12.4 % the activity for glyoxylate compared to methylglyoxal  (Table. 4). Moreover, GLOX has an acidic pH optimum (Fig. 27) which when applied to the Amplex Red detection system (Fig. 26), compromises detection limits because the reduced resorufin at pH 6.0 from 0.75 to 0.11, an overall 85% resorufin reduction at pH 6.0. Glyoxal Oxidase pH Profile 0 20 40 60 80 100 120 44.555.566.577.58 pH% Activity Figure 27. Glyoxal oxidase pH profile pH profile of glyoxal oxidase shown in this figure was adapted from Kersten and Kirk . GLOX functions in the detection system of glyoxylate like glycolate oxidase: the oxidation of the glyoxylate gem diol to oxala te with the concomitant production of H2O2. H2O2 production is coupled to Am plex Red oxidation to reso rufin as catalyzed by HRP (Fig. 26).
78 Table 4. Kinetic Constants for Alternate Aldehyde Substrates for Glyoxal Oxidase Substrate KM, mM kcat, s-1 kcat / KM, M1 s-1 % relative activity Methylglyoxal 0.64 198 309,600 100 Glyoxylic acid 2.5 96 38,200 12 Glycoaldehyde 8.3 208 25,000 8.1 Acetaldehyde 8.3 148 17,800 5.7 Formaldehyde 23 254 11,000 3.6 Glyoxal 12 118 10,400 3.3 Dihydroxyacetone 38 188 5,000 1.6 DL-Glyceraldehyde 42 148 3,600 1.1 Table 4. Kinetic constants for several aldehy de substrates of glyoxal oxidase Figure adapted from Kersten (1990) PNAS. Vol. 87 pp. 2936-2940 . GLOX is isolated in the catalytically inactive, reduced form requiring the presence of an oxidant for catalysis. However, the use of strong oxidants must be avoided as this results in the spontaneous oxidation of Amplex Red. Thus, HRP has a dual role in this detection system as it both activates the reduced gl yoxal oxidase, and converts Amplex Red to resorufin via a stoichio metric reaction with H2O2 (Fig. 25).
79 Chemi-luminescent Assays for Hydrogen Peroxide Chemi-luminescence is a similar process to th at of fluorescence however excitation to the excited state is dependent upon a chemical reaction rather than incident light ( ex i.e. fluorescence) (Fig. 28). A Jablonski Diagram Figure 28 A Jablonski diagram. Display of the different types of emission from an electronically excited state to the ground state. Blue describes absorption, green fluorescence, and red phosphorescence. Fluorescence and chemi-luminescence, undergo excitation, internal conversion, followed by an observable emission. Internal convers ion is a non-photo-emissive transition of electrons between two states of the same spin number (Fig. 28), and relates to the quantum yield of a fluorophore ( ). Phosphorescence also undergoes a non-emissive transition, however initially it is between tw o different spin states. This type of nonemissive transfer is described as intersystem crossing and is followed by internal
80conversion. Chemi-luminescence has been u tilized for biochemical applications much like fluorescence due to its enhanced sensitivity. Luminol in the presence of an oxidative (1 or 2 eoxidant) metal catalys t such as Mn (II) , Cu(phen)3 2+ , HRP; (Fe(III)) [104,105,106,107,108,109], K4Fe(CN)6Â•3H2O; (Fe(III)) , and Co(II)  and hydrogen peroxide, becomes chemically excited to a triplet state (Fig. 29). Excitation is followed by internal conversion to a singlet state, and lastly photo-emission. The amount of lumi nol photo-emission is proportional to the concentration of hydrogen peroxide present. Luminol prior to becoming excited must be in the doubly de-ionized form requiring that luminol chemi-luminescence be carried out in a basic environment. Of all possible metal oxidants it was found that use of HRP resulted in the greatest li ght emission by luminol [109, 106].
81 Mechanism of Luminol Chemi-luminescence NH NH NH2 O O -OH -OH N-NO O N N OO O O O O Singlet excited stat e OOO +hv+ N2 keto-enol tautomerizism + H2O2+ H2OOFigure 29. Mechanism of luminol chemi-luminescence Initially, luminol is oxidized by a metal catalyst, the oxidized luminol then reacts with hydrogen peroxide to produce the excited aminopthalate anion. The amino-phthalate undergoes relaxation emitting blue light with a wavelength maximum at 425 nm. The amount of light given off by the excited amino-phthalate is proportional to the concentration of hydrogen peroxide consumed.
82 Materials and Methods Materials Amplex red, resorufin, and luminol were purchased from Molecular Probes, (Eugene, OR); glycolate oxidase, HRP, FMN, FAD, MES, sodium glycolat e, N-dansyl-Tyr-ValGly, were purchased from Sigma-Aldrich; recombinant rat PAM was a gift from Unigene Labs, Inc. (Fairfield, NJ); and glyoxal oxidase (source: Phanerochaete chryosporium ) was a gift from Dr. James Whittaker (OGI School of Science and Engineering, Oregon Health and Science University, Beaverton, OR). Black flat-bottom, and U-shaped well plates were purchased from Corning. All ot her reagents were of the highest quality commercially available.
83 Methods Standardization of the Fluorophore Resorufin A standard solution of resorufin was initially used to standardize the micro-plate fluorometer for resorufin fluorescence. C oncentrations ranging from 30nM to 9 M of were analyzed for fluorescent response at ex = 530nm and em = 584nm to generate a resorufin standard curve. Samples were anal yzed in black U-shaped microplates, in a Fluoroskan II microplate readi ng fluorometer equipped with the MTX software analysis package. Standardization of H2O2 Produced Fluorescence A standard hydrogen peroxide solution was utilized for th e preparation of a hydrogen peroxide standard curve for resorufin producti on. Concentrations ranging from 30nM to 9 M peroxide were analyzed in a solution containing, HRP (1U/mL) and 50 M Amplex Red in 50mM phosphate buffer pH 6.0, and pH 8.0.
84 Glyoxal Oxidase (GLOX ) Fluorescent Assay for Glyoxylate Standardization of the Fluorescent GLOX Assay with Standard Methyl Glyoxal and Glyoxylate. The GLOX assay consisted of a standard solution of 50 mM sodium phosphate pH 6.0, 50 M Amplex red, HRP (1U/mL), and either 0.03 Â– 2.3 M methyl glyoxal or 0.3 Â– 1.7 M glyoxylate. The reaction was initiated by the addition of GLOX (final concentration = 0.4mg/mL) and fluorescence was determined after 1 hour at 37Co in the dark for the glyoxylate substrate and after a 30 min 37Co incubation for methyl glyoxal ( ex = 530nm and em = 584nm). The fluorescence produced from Amplex red oxidation in the absence of glyoxylate or methyl glyoxal was subtracted from that obtained in the presence of glyoxylate/methyl glyoxal.
85 Standardization of the Fluorescent Gl yoxal Oxidase Assay for PAM Produced Glyoxylate Glyoxylate production was initiated by the a ddition of PAM (15U/L) to a solution containing 40mM MES / NaOH pH 6.0, 10U/mL HRP, 1.0mM catechol, 0.5 M Cu(SO4), 20 M dansyl-Tyr-Val-Gly, the reaction proceeded for one hour at37Co. It is necessary to note that catechol was used as the reductant to support PAM catalysis, as the Fenton chemistry produced by ascorbate in th e presence of copper (generation of OH, and H2O2) resulted in the complete auto-oxida tion of Amplex Red. The complete PAM dependent conversion of 20 M N-dansyl-Tyr-Val-Gly to N-dansyl-Tyr-Val-NH2 and glyoxylate was verified by RP-H PLC to quantify the exact c oncentrations of [N-dansylTyr-Val-NH2] and [glyoxylate] (100% conversion, 20 M N-dansyl-Tyr-Val-NH2 and glyoxylate). Aliquots of the PAM produ ced glyoxylate pertaining to variable concentrations (0.3, 0.7, 1.6, and 2.3 M) were utilized for analysis by the GLOX assay. Aliquots of glyoxylate were added to a so lution of 50mM sodium phosphate pH 6.0, 50 M Amplex red, at HRP (1U/mL) and a fina l concentration of 0.4mg/mL GLOX. All samples were incubated at 37Co for one hour (as described chapter 3).
86 Glycolate Oxidase (GO ) Fluorescent Assay for Glyoxylate Standardization of the Fluorescent Glycolate Oxidase (GO) Assay with Glycolate and Glyoxylate. The glycolate oxidase assay c onsisted of a standard soluti on of 70mM sodium phosphate pH 7.8, 50 M Amplex red, 0.1mM FAD, 1U/mL HRP, and either 0 Â– 10 M glycolate, or 0 Â– 10 M glyoxylate in a fina l volume of 300 L. Commercially available glycolate oxidase as purchased from Sigma contains 2mM FMN. Excess FMN was removed from the enzyme solution by dialysis against 3L of 10mM Tris-HCl pH 7.4. 0.75mM FAD was then added to the apo-enzyme, and pre-inc ubated for at least 1 hour in order to be fully catalytically competent in the glyoxylate assay. It is necessary to note that the enzyme must be pre-incubated with the FAD cofactor prior to its use. The reaction was initiated by the addition of glycolate oxidase (final concentration = 0.2mg/mL) and fluorescence determined after 1 hour at 37Co for the glyoxylate substrate, and after a 20 min. incubation for the glycolate substrate. Fluorescence produced from the Amplex Red oxidation in the absence of glyoxylate or gl ycolate was subtracted from that obtained in the presence of glyoxylate / glycolate.
87 Standardization of the Fluorescent Glycola te Oxidase (GO) Assay for PAM Produced Glyoxylate Glyoxylate production was initiated by the ad dition of PAM (0.015U/mL) to a solution containing 40mM MES/NaOH pH 6 .0, 10U/mL HRP, 1.0mM catechol, 0.5 M Cu(SO4), 20 M dansyl-Tyr-Val-Gly, the reaction proceeded for one hour at 37oC. The PAM dependent conversion of 20 M N-dansyl-Tyr-Val-Gly to N-dansyl-Tyr-Val-NH2 and glyoxylate was verified to be 100% by a RP-H PLC assay (chapter 2), to ensure a 20 M PAM produced glyoxylate solution. Aliquots of the PAM produced gl yoxylate pertaining to variable concentrat ions (1.0, 3.0, 5.0, 7.0 and 9.0 M) were taken for analysis by the glycolate oxidase assay. The aliquots of glyoxylate were added to a solution of 70mM sodium phosphate pH 7.8, 50 M Amplex red, and HRP (1U/mL) in a final volume of 300 L. All fluorescent reactions were initia ted by the addition of 0.2mg/mL GO, 0.1mM FAD (final concentration). All samp les were incubated for 1 hour at 37Co prior to fluorescent analysis of resorufin production.
88 Chemi-Luminescent Assays for Glyoxylate; A Hydrogen Peroxide Chemi-Luminescent Standard Curve A standard working solution of 0.1mM Luminol in 0.1M NaHCO3 pH 10.5 was prepared and purged in an atmosphere of N2, (20 min. N2/100mL solution) and stored at 4Co in the dark, the stock was prepared fresh daily. Black flat-bo ttom 96 well microplates were prepared by the addition of 8 L HRP (11mg/mL) and 67 L of sodium bicarbonate (0.4M), microplates were incubated for at le ast 45 min. in the dark prior to luminetric analysis. A Berthold / Tropix TR-717 binary injector micr oplate luminometer, with a 500 L dead volume was utilized for all microplate luminescent assays. The luminometer dead volume is defined as the volume of sample retained in-line after injection prior to reaching the detector for measurement. The primary injector was programmed to inject 200 L of the sample (hydrogen peroxide) and the offset injector was programmed to inject 25 L of the luminol stock. Thus, each well contained a final concentration of 0.3 g/ L HRP, 8.0 M Luminol, 0.1M NaHCO3 pH 10.5, in a final volume of 300 L. Standard hydrogen peroxide concentr ations (5nM, 7nM, 10nM, 20nM, 50nM) were prepared within a 600 L final sample volume for luminetr ic analysis. Microplates were prepared such that the injection of 200 L peroxide, 25 L luminol, in addition to the HRP / NaHCO3 prepared plates resulted in a final well volume of 300 L. Chemi-luminetric
89measurements (RLU = Relative Luminescence) were obtained in the flash kinetic mode, with a 1.6sec. time delay between sample and luminol injection. All measurements were obt ained immediately following lu minol injection for a time duration of 10 seconds. Three sequential 200 L injections of the standard were necessary to analyze the total 600 L hydrogen peroxide sample. A water blank control was injected prior to and in between each standard H2O2 solution in order to define a baseline background RLU chemi-luminescent signal, for signal / noise analysis. A standard curve was generated from the to tal RLU generated per standard peroxide solution.
90 Chemi-Luminescent Glycolate Oxidase Assay with Glyoxylate Following standardization of the chemiluminescent dependent system for the quantification of hydrogen pe roxide, the detection syst em was applied to the quantification of gl yoxylate dependent H2O2 production. A standa rd glyoxylate solution (7nM, 20nM, 50nM, 80nM, 200nM) was utilized to develop a standard curve for the detection of glycolate oxidase produced hydrogen peroxide. All glycolate oxidase reactions were performed in 100mM phosphate buffer pH 7.8, with a final concentration of 0.48U/mL glycolate oxidase, 0.2mM FMN in a final volume of 600 L. It was not necessary to dialyze the FMN containing enzyme as FMN does not interfere with the chemi-luminescent detection system. Microplates were prepared with 8 L HRP (11mg/mL) and 67 L of sodium bicarbonate (0.4M), in cubated for one hour, prior to the analysis of glyoxylate dependent H2O2 production. Each 600 L reaction was injected over three wells as 200 L aliquots per well. In total, the addition of sample (200 L), luminol (25 L), HRP (8 Ll) and NaHCO3 (67 L), resulted in a final concentration per well of 0.3 g/ L HRP, 8.0 M luminol, 0.1M NaHCO3 pH 10.5, in a final volume of 300 L. The background RLU was determined by the addition of all reagents excluding the glyoxylate, and the blank was injected pr ior to and between each standard glyoxylate concentration. A standard curve was genera ted by the addition of RLU response per well for each standard glyoxylate sample. The detection limit was defined as the signal over background which fit a linear re gression of RLU vs. [glyoxylate].
91 Chemi-luminescent Glycolate Oxidase Assay for PAM produced Glyoxylate Preparative RP-HPLC for the Collection of the PAM substrate N-dansyl-Tyr-Val-Gly; An Empirical Trial for the Use of the Platform Technology to Detect a GlycineExtended Peptide A 20 M sample of the PAM substrate N-dans yl-Tyr-Val-Gly was injected onto a Keystone ODS Hypersil C18 column (100 4.6 mm, 5 particle size) RP-HPLC column, equipped with a Bio-Rad Model 1200 in-line fr action collector at 1.0 min intervals. The analyte was separated and co llected by an isocratic mob ile phase of 100 mM sodium acetate pH 6.0 / acetonitrile (52/48) at flow ra te of 1.0mL/min . Fractions were collected over the 4 minute separation, lyophilized to dryness, and reconstituted in PAM assay conditions consisting of 40mM ME S-NaOH pH 6.3, 1mM sodium ascorbate, 0.5 M CuSO4, 10U/mL HRP, 0.015U/mL PAM in a final volume of 300 L. The PAM reaction proceeded for 1 hour at 37Co, 10 L aliquots per fraction were removed for percent conversion analysis of N-dansyl -Tyr-Val-Gly to Ndansyl-Tyr-Val-NH2 by the described RP-HPLC assay . Conversion of the N-dansyl-Tyr-Val-Gly substrate to the products N-dansyl-Tyr-Val-NH2 and glyoxylate, was analyzed by a RP-HPLC PAM activity assay (Chapter 2), to ensure 100 % conversion of the s ubstrate in order to verify the reaction products at [N-dansyl-Tyr-Val-NH2] = [glyoxylate] = 20 M. Following the PAM reaction, and product analysis, an aliquot of 2U/mL of ascorbate oxidase was added
92to all PAM reactions at 40mM MES pH 6.3 to oxidize the remaining ascorbate. It was necessary to oxidize all rema ining ascorbate in the PAM reaction, as the reductive properties of ascorbate suppress the oxidation of luminol, necessary for light production. The resultant solution was utilized as a st andard PAM produced glyoxylate stock, for use in the glycolate oxidase assay for PAM produced glyoxylate. Aliquots of the PAM reaction corresponding to variable conc entrations of glyoxylate (7nM, 20nM, 80nM, 200nM) were taken for analys is by the chemi-luminescent glycolate oxidase assay. Aliquots were added to a solution containing 100mM sodium phosphate buffer pH 7.8, reactions were initiated by the addition of 0.48U/mL glycolate oxidase, 0.2mM FMN and reacted for one hour at 37Co at a final volume of 600 L. The RLU response per [glyoxylate] sample was an alyzed as described (chapter 3).
93 Results and Discussion Resorufin Fluorescent Standard Curve A solution of resorufin standard was prepar ed from a purified solid sample. A stock concentration of 15 M was utilized to obt ain the slope of 33.5RFU M-1 (RFU = relative fluorescent units) was obtained (F ig. 30). This value acts as an empirical standard for the concentration dependent fl uorescence of Resorufin at ex = 560nm and em = 584nm. Fluorescent Response of Resorufin; ex = 560nm, em = 584nmy = 33.46x + 5.1914 R2 = 0.9665 0 10 20 30 40 50 60 70 80 90 00.511.522.5[Resorufin] M Fluorescence Figure 30. Fluorescent response of resorufin An initial standard curve for the detection limit of Resorufin was generated as a reference standard.
94 Standardization of H2O2 Produced Fluorescence An increase in fluorescence from the H2O2 driven HRP dependent oxidation of Amplex Red was linear with H2O2 concentration and yi elded a slope of 40.5RFU M-1 at pH 6.0, and a slope of 42.5RFU M-1 at pH 7.8 (Figs. 31 & 32). Hydrogen peroxide detection provided an value for which all enzymatic ba sed developed assays could be compared, and was within 17 % error of the resorufin standard. Resorufin Standard Curve for Detection of Hydrogen Peroxidey = 40.53x 2.8473 R2 = 0.9964 0 20 40 60 80 100 120 00.511.522.5 [H2O2] M Fluorescence Figure 31 Resorufin standard curv e for detection of H2O2 at pH 6.0. An initial standard curve for the detection limit of hydrogen peroxide produced resorufin was generated as a reference standard at pH 6.0. Resorufin Standard Curve for Detection of H2O2 at pH 6.0
95 Resorufin Standard Curve for Detection of Hydrogen Peroxidey = 42.365x 1.6788 R2 = 0.9971 0 20 40 60 80 100 12000.511.522.5[H2O2] MFluorescence Figure 32 Resorufin Standard Curve for Detection of H2O2 at pH 7.8. Initial standard curve developed to define the detection limit of h ydrogen peroxide produced resorufin at pH 7.8. Resorufin Standard Curve for Detection of H2O2 at pH 7.8
96 Glyoxal Oxidase (GLOX) Fluorescent Assay for -Amidated Peptides Glyoxal oxidase oxidizes the glyoxylate gem diol in the presence of O2 to produce a stoichiometric quantity of H2O2 and oxalate (Fig. 22). Glyoxylate oxidation drives the H2O2, HRP dependent oxidation of Amplex Red to produce an increase in fluorescence proportional to [glyoxylate] (Fig. 31 & 32). The extinction coeffi cients derived from these data are, = 25.8RFU M-1 (methyl glyoxal) and = 12.8RFU M-1 (glyoxylate). The methylglyoxal slope is within 36% error of the value for H2O2, and within 23 % error of the value obtained for standard resorufin. The value of 12.8 obtained for glyoxylate is 3 fold reduced from the standard values. The cause for this market decrease in sensitivity is a ramification of the significantly reduced kcat/KM value for methyl glyoxal as compared to glyoxylate. The e fficiency of glyoxal oxidase is reduced approximately 4-fold under gl yoxylate oxidation c onditions (Fig. 33). This decrease in efficiency results in the reduced sensitivity on account of two main factors. First, the incubation time for glyoxylate oxidation is 50 % longer as co mpared to methyl glyoxal, resulting in the formation of a higher b ackground fluorescence fo r the zero glyoxylate control. Secondly, as mentione d in the introduction to chapter three, low signal to noise is the foremost cause of sensitivity loss in fluorescent assays. Th erefore, on account of the reduced enzyme efficiency the detection limit of this assay suffers. The ultimate detection limit for glyoxylate with this assay was 0.3 nanomoles.
97 Glyoxal Oxidase Activity Standard Curve y = 25.799x + 1.4488 R2 = 0.9973 y = 12.823x 0.7666 R2 = 0.98270 10 20 30 40 50 60 70 00.511.522.5 [glyoxylate] [methyl glyoxal] MFluorescence methyl glyoxal glyoxylate Figure 33. Glyoxal Oxidase Activity Standard Curve Glyoxal oxidase activity for methyl glyoxal as compared to glyoxylate was analyzed to determine the stoichiometric response for glyoxal oxidase glyoxylate oxidation. The error bars represent the standard deviation of triplicate samples. Glyoxal oxidase activity is severe ly inhibited in the presence of a reductants. Kurek and Kersten  define catechol as a severe inhibitor of glyoxal oxida se activity, therefore catechol could not replace ascorbate in the PAM reaction for the glyoxal oxidase dependent glyoxylate fluorescen t assay. Furthermore, a treatment of the catechol supported PAM reaction with catechol oxidase wa s tried as an avenue for the removal of catechol. Unfortunately, the oxidized dione pr oduct is intensely dark which inhibits all resorufin fluorescence. Several other know n PAM reductants were analyzed, however their presence also markedly hindered the abil ity of the glyoxal oxidase assay to detect PAM produced glyoxylate. Recall from chapter three that glyoxal oxidase must be in the oxidized form to support catalysis, on account of its copper radical mechanism, therefore presence of such reductants inactivates gl yoxal oxidase. For th ese reasons, it was 0 0.5 1.0 1.5 2.0 2.5 [methyl glyoxal] or [glyoxylate] M
98decided that glyoxal oxidase was not the en zyme of choice for th e detection of PAM produced glyoxylate.
99 Glycolate Oxidase Fluorescent Assay for -Amidated Peptides Glycolate oxidase oxidizes glycolate in the presence of O2 to glyoxylate and H2O2 (chapter 2, Fig 12). The gem diol of glyoxyl ate an alternate substrate for glycolate oxidase is oxidatively (stoichiometric O2 required) converted to H2O2 and oxalate (Chapter 2, Fig. 14). Glyoxylate / gl ycolate oxidation drives the H2O2, HRP dependent oxidation of Amplex Red to produce an increase in fluorescence proportional to [glyoxylate] / [glycolate] consumed (Fig. 34). The extinction coefficients derived from standard curve analysis are = 5.83RFU M-1, (glycolate) and = 3.78RFU M-1 (glyoxylate). Figure 34. Standard curve of the glycolate oxidase dependent fluorescent assay for glycolate vs. glyoxylate Glycolate oxidase activity for glycolate as compar ed to glyoxylate was analyzed to determine the stoichiometric response for glyoxal oxidase glyoxylate oxidation. Standard Curve of the Glycolate Oxidase Dependent Fluorescent Assay for Glycolate vs. Glyoxylate y = 5.8333x 0.1 R2 = 0.9954 0 10 20 30 40 50 60 0246810[glycolate] M Fluorescence y = 3.6667x + 2.7333 R2 = 0.9848 0 5 10 15 20 25 30 35 40 45 0246810[glyoxylate] M Fluorescence
100 The glycolate and glyoxylate slopes are within 36% error of the each other and approximately 10-fold reduced for the value obtained by H2O2 Amplex Red oxidation. Glycolate oxidase is a flavin dependent en zyme, therefore requires a minimal amount (0.05mM) flavin prosthetic for activity. The FAD supported catalysis for the data presented in chapter three shows a ten fold d ecrease in sensitivity as a result of the presence of FAD. The fluor escence spectra of FAD has a ex = 450nm and a em = 530nm, which overlays with the fluorescen t spectra of FAD (Fig. 35). The result of fluorescence overlap is decreased sensitivity since the observed fluorescent emission in this case is not a Â‘darkÂ’ process. Th e excitation/emission overl ap greatly hinders the sensitivity of this particular fluorescent as say. The two values ob tained for glycolate ( = 5.83RFU M-1-) and glyoxylate ( = 3.67RFU M-1) are within 36% error, the difference owing to decreased enzyme efficiency for glyoxylate. 0 50 100 150 200 250 300 350 400 450 550560570580590600610620630640650660670680670680 wavelength nmFluorescence FAD resorufin Figure 35. FAD / Resorufin fluorescent spectra. An equi-molar overlay of the fluorescent spectra of FAD and resorufin at a ex = 530nm. FAD / Resorufin Fluorescent Spectra
101 Data obtained for the detecti on of PAM produced glyoxylate wa s linear with respect to glyoxylate, and well within error of the reported value for glyoxylate (Fig.36). Comparatively, the PAM produced glyoxylate standardization was within 14%, of that obtained from a standard glyoxyl ate solution. This value rela tes to the effectiveness of the proposed assay to stoichiometricly detect -amidated/glycine-extended peptides. In conclusion, the fluorescent glycolate oxidase assay was able to detect -amidated peptide production at a sensitivity of 0.3 nanomoles (0.5 M in 300 L), whereas the glyoxal oxidase assay had considerable difficulties with detection of PAM-produced glyoxylate. Figure 36. Standard Curve for the glycolate oxid ase dependent fluorescent enzyme assay for pAM produced glyoxylate Aliquots of independently quantified gly oxylate were subjected to analysis by the glycolate oxidase assay for glyoxylate analysis.. Standard Curve for the Glycolate Oxidase Dependent Fluorescent Enzyme Assay fo r PAM Produced Glyoxylate y = 4.2x + 2.2 R2 = 0.9443 0 5 10 15 20 25 30 35 40 45 50 0246810 [d-YVG] M Fluorescence
102 Chemi-Luminescent Glycolate Oxidase Dependent Assay for Glyoxylate The H2O2 dependent HRP catalyzed luminol chemi-luminescent RLU response (RLU = relative chemi-luminescence) wa s linear over a range from 10 to 80nM with respect to [H2O2] concentration and yiel ded a slope of 618.2RLUnM-1 (Figs. 37 & 38). This was within 7.0 % of the value obtained by the glycolate oxidase dependent H2O2 production from [glyoxylate] / [d-YVG] of 574.7RLUnM-1 (Fig. 35). Chemi-lu minescent standard curve analysis of PAM produced glyoxylate was provided results comparable to exogenous H2O2. The RLU value deviates from linearity approaching 100nM (30 picomoles). The assay is sensitive within error to 5nM hydrogen peroxide and 15nM [glyoxylate]. The chemi-luminescent assay has sensitivity well with in the nM range (~515nM). The microplate formatted assay has a detection limit of approximately 1.5 x 10-12moles peroxide, 4.5 X 10-12moles glyoxylate at a final volume of 300 L. The luminescent assay displays a marked increase in sensitivit y as a result of the large difference in light emission of luminol as compared to resorufin. Recall, that one major contributing factor to the detection of peroxide in the fl uorescence assay was the overlap of FAD and resorufin. The red emission at 584nm of Amplex red in the fluorescent assay is affected by the orange emission of FAD at 525nm. Lu minol light emission is within the range of em 440-480nm. The drastic blue shift in em ission markedly decreases the background
103emission of flavin allowing for greater se nsitivity. Additionally, intrinsic protein (enzyme) fluorescence is not an additive f actor for a low signal / noise ratio chemiluminescent assays, as proteins do no t intrinsically chemi-luminescence.
104 Chemi-luminescent Standard Curve for Hydrogen Peroxidey = 618.23x 1280.9 R2 = 0.9973 0.00E+00 5.00E+03 1.00E+04 1.50E+04 2.00E+04 2.50E+04 3.00E+04 3.50E+04 0102030405060 [H2O2] n M RLU dbl Int Figure 37. Chemi-luminescent standard curve for hydrogen peroxide Data was obtained by taking the double integration with limits of RLU signal per time and RLU signal per well. Chemi-Luminescent Curve for PAM Produced Glyoxylatey = 574.69x + 8550.8 R2 = 0.9854 0.00E+00 2.00E+04 4.00E+04 6.00E+04 8.00E+04 1.00E+05 1.20E+05 1.40E+05 050100150200250 [d-YVG] nMRL U Figure 38. Glycolate oxidase chemi-luminescent standard curve for PA M produced glyoxylate The tri-peptide dansyl-Tyr-Val-Gly was collected from a RP-HPLC separatio n, lyophilized, and treated with PAM. Glycolate Oxidase Chemi-luminescent Standard Curve for PAM Produced Glyoxylate Chemi-luminescent Standard Curve for Hydrogen Peroxide
105 2 2.5 3 3.5 4 4.5 5 5.5 0100200300400500600 [H2O2] n M log10 dbl Int Figure 39. Linear detection range of chemi-luminescence. A log10 plot was created in order to determine the extent to which the observed chemi-luminescence was linear. The glyoxal oxidase assay (data not shown) pr ovided the least sensitive determination of glyoxylate dependent production of peroxide. In addition to the decreased sensitivity the enzyme is not readily available and woul d require the growth of wood rot fungus ( Phanaerochaete chrysosporium ), as a source for enzy me purification. Therefore, the glycolate oxidase assay was chosen as the premiere assay for peptide discovery. In conclusion, the chemi-luminescent assa y proved most sensitive of all developed assays. Fluorescent assays are often very sens itive into the nM range (Fig. 30) of analyte. However the flavin interfer ence, in addition to long in cubation times increasing background fluorescence, presented a definite loss in fluorescent sensitivity. The luminescent assay was chosen as the premiere assay for -amidated peptide discovery. Luminol light emission is gene rally within the range of 440nm to 480nm, thus there is no flavin interference to adversely affect the si gnal. Albeit, the deve lopment of three novel L o g R L U Linear Detection of Chemi-Luminescence
106assays for glyoxylate, not all demonstrated usefulness towards the outlined platform technology.
107 Chapter Four Platform Technology Process Introduction Application of the Glyoxylate Assays to the Discovery of Amidated Hormones In order to establish the feas ibility of the proposed platfo rm technology to identify novel -amidated peptides from cell culture, it must first be demonstrated as working prototype for the discovery of a known -amidated peptide accumulated from cell culture. Amidated peptide secreting cells when gr own in the presence of a PAM inhibitor, accumulate the glycine-extended precursors (PAM substrates). Recall, from chapters 2 and 3 that each developed glyoxylate assay was applied to the detection of PAM produced glyoxylate (from a peptide s ubstrate) as an initial f easibility study. To further this work, glyoxylate detection was applied to the discovery of an -amidated/glycineextended peptide accumulated in cell culture. The detection of a known glycine-extended peptide accumulated in cell culture, served as an empirical trial to establish the performance of the glyoxylate assay(s) as a general tool fo r the detection of -amidated hormones.
108 Utilization of the Mouse Pituitary Cell Line in the Platform Technology A cell line known to express PAM, and an -amidated hormone in appreciable quantities was chosen for the cell culture empirical trial. For this purpose a mouse neurointermediate pituitary, corticotropic tumo r cell line (AtT-20) known to express high levels of PAM and mouse joining peptide (mJP-Gly; an -amidated peptide) was chosen as a pragmatic trial. mJP-Gly, in addition to -MSH-Gly are the proteolytic cleavage products of the parent peptide pro-adrenoco rticotropin (ACTH/endo rphin) (Fig. 37). Following dibasic proteolytic clea vage (Lys-Arg, mJP; Lys-Lys, -MSH) the corresponding glycine-extended prohormones are produced . Both mJP-Gly and MSH-Gly are substrates for PAM. Mouse joining peptide (mJP) was chosen as the peptide of interest for this study based prev ious data which established mJP-Gly as the peptide in majority (Fig. 40 & 41). Signal Peptide16 K Fragment ACTH LPH 3MSHmJP MSHCLIP MSHMetT r pArg-Lys Arg-Arg Lys-Arg Arg-Arg Lys-Arg Lys-Arg Lys-Lys Lys-LysFigure 40 Proteolytic processing of the mouse pro-ACTH / endorphin homologue Processing and the respective nomenclature of each resulting peptide . Proteolytic Processing of the Mouse pro-ACTH / Endorphin Homologue
109Mains et. al  report an eighty perc ent inhibition of mJP-Gly amidation in At-T20 cells when grown in the presence of 0.5 M Â– 2.0 M disulfiram (1,1'-dithiobis(N,Ndiethylthioformamide), a copper chelating PAM inhibitor. Di sulfiram was chosen based on previous work which demonstrated its use as a PAM inhibitor in AtT-20 cells, and the resultant accumulation of mJP-Gly. Moreover, disulfiram is not extremely lethal to cell growth, and is a reversible inhibitor of PAM, as the disulfiram is metabolized over time, PAM activity is restored. Ala-Glu-Glu-Glu-Ala-Val-Trp-Gly-Asp-Gly-Ser-Pro-Glu-Pro-Ser-Pro-Arg-Glu-Gly Ala-Glu-Glu-Glu-Ala-Val-Trp-Gly-Asp-Gly-Ser-Pro-Glu-Pro-Ser-Pro-Arg-Glu-NH2 Figure 41. mJP Sequence. Sequences of both the glycine-extended form of mJP-Gly (19-mer) and the PAM amidated product (18-NH2) .
110 Utilization of the Rat Neuro-intermediate Cell Line in the Platform Technology Rat CA-77 cells which produce glycine-extende d calcitonin gene-related peptide (CGRPGly, Fig. 42) were cultured by Un igene Laboratories, Inc. As a blind analysis Unigene Labs sent samples to USF for the verifica tion of the presence of CGRP-Gly from a cellular extract. Figure 42. CGRP Peptide Sequence. Sequence of the glycine-extended and amidated forms of the rat thyroid CGRP peptide . Ser-Cys-Asn-Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-SerArg-Ser-Gly-Gly-Val-Val-Lys-Asp-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-SerGlu-Ala-Phe-Gly Ser-Cys-Asn-Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-SerArg-Ser-Gly-Gly-Val-Val-Lys-Asp-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-SerGlu-Ala-Phe-NH2
111 Materials and Methods Materials Mouse pituitary AtT-20 cells, HamÂ’s F-12K Medium, horse serum, and bovine serum albumin were purchased from the American Type Cell Culture Collection (www.atcc.org). Benzamide, MES buffer, ascorbate oxidase, and all HPLC grade solvents were purchased from Sigma-Aldr ich. All cell culture flasks (T-125, 100mm Petri dishes) were purchased from Gibco-BRL. Disulfiram was purchased from Fluka. Sep-Pak Plus C18 cartridges, and an Atlantis dC18, 4.6 x 250 mm, 5 m column was purchased from Waters Corp. The mouse joining peptide-Gly (mJP-Gly) and mouse joining peptide-NH2 (mJP-NH2) were synthesized in-house at the USF core peptide synthesis and Mass Spec facility run by Dr Ted Gauthier. CGRP -Gly was purchased from American Peptide, Inc. PAM was a generous gift from Unigene Labs, Inc. All other reagents and solvents were of the highest quality available.
112 Methods At-T20 Cell Growth Conditions Mouse pituitary cells were grown in Hams F-12K culture medium supplemented with 15% (v/v) horse serum, 2.5% (v/v) fetal bovine serum, containing 1% (v/v) of the antimicrobial Pen-Strep (10,000 units Penicillin (Base)/mL and 10,000 g Streptomycin (Base)/mL in 0.85% NaCl (liquid). Cells were rapidly thawed from liquid N2, added to fresh media and incubated in the presence of 5% CO2 at 37oC. The AtT-20 cells were non-adherent, therefore cells we re passed at a ratio of 1:3 into fresh media after 40% confluency was reached in each culture flask. A sterile laminar flow hood in combination with sterile technique was utilized for all cell culture work. Cells were grown in a 37oC incubator equipped with a constant flow of 5% CO2 to maintain cell growth.
113 At-T20 Cell Growth for the mJPGly Accumulation Methodology A 2.5mM disufiram stock solution was prepared in 70% EtOH for addition to cell culture suspensions. The AtT-20 cells were gr own in T-75 cell culture flasks ( 20mL of media+cells/flask), prior to cell harvesting for accumulation studies. A total of 5, T-75 flask cell suspensions (Vt of cells & media 100mL) were collected for disulfiram dependent PAM inhibition. The cell suspensi on was collected in 15m L centrifuge tubes, centrifuged (2,000 x g) for 3 min., and th e supernatant spent media removed by aspiration. The centrifugati on and aspiration step was re peated until all the entire suspension was collected from the 8, T-75 cultu re flasks. A total of 7 centrifuge tubes were utilized to collect all AtT-20 cells. Seven, 100mm cell culture flasks were prepared for disulfiram inhibition by the addition of 7mL of media containing 5 M disulfiram (16 L of 2.5mM stock). The cells were collected from the 7 centrifuge tubes by resu spension in 1mL media, and added to the 100mm culture flasks. The final volume of each flask was 8mL media containing 5 M disulfiram, flasks were incubated at 37oC, in a 5% CO2 atmosphere, for 20 hours. The entire procedure was repeated for the non-disu lfiram treated negativ e control cells, with substitution of simply the 70% EtOH carrier for disulfiram.
114 Extraction of mJP-Gly from At-T20 Cells Cells were collected by centrifugation, the supernatant spent media was decanted and acidified to 0.1% (v/v) TFA w ith a 6% TFA stock (1.6 mL 6% TFA added to 100 mL). Whole cells were homogenized at 4 C in a ground glass homogeni zer with an acid extract containing 0.1M HCl, 5% (v/v) formic acid, 1% (w/v) NaCl, 1% (v/v) TFA . The homogenate was centrifuged, the supernatant co llected and added to the acidified media. The remaining cell pellet was again homoge nized, centrifuged, and th e supernatant added to the acidified media. The solution was approximately 125mL of 0.1% TFA spent media including the homogenized extract. Th e same procedure was followed for the nontreated cells.
115 Purification of Cellular Extract on a Sep-Pak Plus Cartridge The acid extract was initially purified by solid phase extraction on a Waters Sep-Pak Plus cartridge prior to HPLC separation. This initial step served to both de-salt and concentrate the spent media/cellular extract containing the accumulated glycine-extended peptides. A 5mL syringe was used to load the Sep-PakPlus cartridge with 5mL of a column pre-wetting solution composed of 0.1%(v /v) TFA/80% Acetonitrile at a flow rate 2.0mL/min. The cartridge was then rinsed with an aqueous so lution of 0.1 % (v/v) TFA at a flow rate of 5.0mL/min. The entire spent media/cell extract (Vt = 125mL) was loaded onto the cartridge at a rate of 3.0mL/min, followed by a wash of 20mL of 0.1% TFA (v/v). Elution of the desired peptide mixture was accomplishe d by the addition of 3mL of 0.1% (v/v) TFA/80% at a flow rate 0.5mL/min. The 3mL eluent was collected and lyophilized on a Savant SpeedVac concentrator equipped with a Savant VLP 120 Vaccuum pump, then resuspended in 250 L of 0.1% TFA (v/v)/0.001 % Triton-X for injection on the HPLC.
116 Preparation of the Cellular Extract for HPLC Purification The entire 250 L portion of the resuspended cellula r extract was divi ded into four separate samples for further an alysis. Two of the four samp les were purely experimental for the analysis of mJP-Gly accumulation by PAM inhibition in cell culture, by both MALDI-TOF and glyoxylate analysis (samples A and B). The remaining two were prepared as spiked standards and 2.5 nmoles of the standard mJP-Gly was added to the 60ul aliquot prior to HPLC separation (sampl es C and D). For experimental purposes samples were denoted as A-D which correspond ed to the sample in jected onto the HPLC and 1-70 to indicate fraction number. To exclude any cross contamination or analyte carry-over, the non-spiked cell extract samples A and B were injected and separated prior to the injection of spiked samples C and D.
117 RP HPLC Separation of the Accumulated mJP-Gly A quaternary solvent delivery HP-1100 LC unit equipped with a autosampler / autoinjector, a heated column compartment, and a ChemStation software package was utilized to perform a binary linear grad ient separation of 100% 0.1 % TFA to 52 % acetonitrile over the duration of sixty-five mi nutes. A flow rate of 1.0mL/min at a detection of 278nm at 37oC was utilized to define the standard retention time and signal of pure mJP-Gly. An initial RP-HPLC sta ndard curve was performed on an mJP-Gly standard as a control for the confirmati on of analyte retention time and signal. Additionally, RP-HPLC of an equimo lar mixture of mJP-Gly and mJP-NH2, verified that both the amidated and glycine extended forms co-elute as stated by Mains et. al. . A 60 L sample injection volume (Vt = 65 L for internal standard samples C and D) of the crude AtT-20 peptide extracts was loaded onto a Atlantis dC18, 4.6 x 250mm, 5 m, reverse phase analytical HP LC column. A Bio-Rad 1200 fr action collector was set inline to collect HPLC fractions at the time interval of 1.0mL / min and was operator controlled. To avoid any HPLC sample ca rry over spiked fr actions containing 2.5nanomoles mJP-Gly internal standard were analyzed after accu mulated samples. Following HPLC fraction collection all fractions were lyophilized to complete dryness on a Savant Speed Vac concentrator equipped with a Savant VLP 120 vacuum pump and stored at -80oC. It must be noted that no true qualitative and/or quantitative analysis of
118mJP-Gly accumulation can be made based solely on the results of the RP-HPLC separation data, and theref ore RP-HPLC was solely a preparative method.
119 Definition of Collected Sample Co ntent for mJP-Gly Characterization For the initial characterization of mJP-Gl y accumulation in cell culture samples, a duplicate set of samples were prepared and analyzed by two independent methods. As previously described samples were designated A through D to inform of the contents, for example non-spiked (A, B) and spiked (C, D) For notation purposes a subscript number following the letter informs of the trial number, and a regular font sized number records the fraction number. For example, A2-35 defines the sample as non-spiked, trial 2, fraction number thirty-five. Fraction sample sets were split, each se t to be designated as glyoxylate analysis (odd numbers A1,3,5 through D1,3,5) or MALDI-TOF analysis (even numbers A2,4,6 through D2,4,6) (see Table 5.). A total of samples A1-6 through D1-6 were analyzed by both methodologies, for a total of three sets of glyoxylate vs. mJP-Gly analysis. Initially, samples A1-D1 were characterized by the methods of the newly developed platform technology for the verifica tion of mJP-Gly via gl yoxylate detection. Thus, the duplicate sample sets A2 D2 demonstrated inhibition of PAM in cell culture via MALDI-TOF analysis for the presence of the mJP-Gly. After de tection of glyoxylate in samples A1 D1, and mJP-Gly in samples A2 D2, duplicate sets were then analyzed.
120 Definition of Platform Technology Sample Notation Table 5. Definition of platform technology sample notation. Samples were defined by analyte, detection method, and fraction number. A designated notation was necessary to keep the large sample numbers organized. Sample Name Analyte Detected Method of Detection A1; 1 -70 Accumulated mJP-Gly/ glyoxylate Chemi-luminescence B1; 1 70 Accumulated mJP-Gly/ glyoxylate Chemi-luminescence C1; 1 70 Spiked mJP-Gly/ glyoxylate Chemi-luminescence D1; 1 70 Spiked mJP-Gly/ glyoxylate Chemi-luminescence A2; 1 70 Accumulated mJP-Gly MALDI-TOF Mass Spec B2; 1 70 Accumulated mJP-Gly MALDI-TOF Mass Spec C2; 1 70 Spiked mJP-Gly MALDI-TOF Mass Spec D2; 1 70 Spiked mJP-Gly MALDI-TOF Mass Spec
121 Treatment of RP-HPLC Fractions with PAM; Production of mJP-NH2/Glyoxylate Fractions 26 through 35 from samples A1;26-35 through D1; 26-35, were treated with PAM. Lyophilized samples were resuspended in 100 L, of a PAM reaction mixture containing 40 mM MES buffer pH 6.3, 1.0mM sodium ascorbate, 0.5 M CuSO4, 10U/mL HRP, and 0.1 mg PAM, and reacted in a 37oC water bath overnight. An aliquot of 2U/mL ascorbate oxidase was added, and the reaction incubated for 1 hour at 37oC. Analysis of mJP-Gly Dependent Glyoxyla te Production by Chemi-luminescence Fractions 26 Â– 35 of sample set A1 through D1 were brought up to 600 L in glycolate oxidase reaction conditions. The pH was verifi ed as pH 7.8, and samples were tested for glyoxylate utilizing the newly developed chem i-luminescent glyoxylate assay (chapter 3).
122 Matrix Assisted Laser Desorption Ionizatio n Â– Time of Flight Mass Spectrometry Analysis for mJP-Gly Accumulation Fractions 26 Â– 35 of sample set A2 through D2 were removed from storage at -80oC and resuspended in 20 L of 0.01% (v/v) TFA for Mass Spec analysis. A matrix of -cyano4-hydroxy-cinnamic acid was spotted in a 1:1 ratio of matrix : analyte on a 384 carbon plate and evaporated to dryness to allow for complete sample : matrix co-crystallization. A MALDI-TOF equipped with an N2 laser of 337nm was opera ted in the positive ion analysis mode, utilizing an acceleration velocity of 19.00kV at the initial ion source, a 130nanosecond time delay, and a second ion sour ce at the extraction plate of 16.35kV. All MALDI-TOF data was collected from the reflector detector a nd calibrations were performed externally with a st andard peptide mixture.
123 Demonstration of the Platform Technology by a Blind Experiment; Analysis of Peptide-Gly Samples Sent by Unigene Laboratories, Inc. Unigene Cell Growth Conditions Rat CA-77 cells which produce CGRP-Gly we re grown in T75 flasks to 60 Â– 90% confluency in DMEM:F10 media (supplemente d with insulin, transferrin and selenium) and 10% fetal bovine serum (FBS). Cells were collected and washed twice with PBS to remove residual serum/media. The cells were then grown in serum free media (DMEM:F10) supplemented with transferrin and selenium for either 24 or 48 hours under accumulation conditions. 0.1 M of the dexamethasone secretagogue and, 100 M of diethyldithiocarbamic acid a PAM inhibitor were added to the media for the accumulation of CGRP-Gly. Insulin, present at high concentration in the medium, was omitted from the accumulation growth medium to avoid potential interference during purification. Conditioned me dium was harvested and cellular debris was removed by centrifugation.
124 Unigene Extraction of CGRP-Gly From Rat CA-77 Cells Approximately 270mL the conditioned medium was loaded onto a Bio-Cad Sprint Perfusion LC System equipped with an Am berchrom CG300M column (1.1cm x 14.5cm) equilibrated with 0.1% TFA (v/v) / 2% acetonit rile. After sample loading the column was washed with 1% (v/v) TFA / 10% acetonitrile to remove cell culture by-products. The absorbance was monitored at 220nm, with a flow rate of 27Ml / min. Peptides were eluted with a mobile phase containing 0.1% TFA (v/v) / 50% acetonitrile and the resultant peak, which had previously been show n to contain the majority of the peptides in the conditioned medium, was collected. The peptide fraction was concentrated approximately 10-fold by lyophilization, an d acetonitrile was added to a final concentration to 5%. All particulate matter follo wing the acetonitrile addition was removed by centrifugation.
125 Unigene Purification of Rat CGRP-Gly by RP-HPLC The concentrated peptide fraction was loaded onto a Rainin HPLX, HPLC equipped with a Hypersil BDS C18 column (4.6mm x 250mm) equilibr ated with 0.1% (v/v) TFA / 20% acetonitrile, and set in-line to an LKB Brom ma Model 2112 Redifrac fraction collector. The column was initially washed with 0.1% TFA (v/v) / 20% acetonitrile for 18 minutes. Peptides were eluted with a linear gradie nt from 0.1% TFA (v/v ) / 20% acetonitrile to 0.1% TFA (v/v) / 52% MeCN over a duration of 60minutes. The column was operated at 1.2mL / min, and the effluent monitored at an absorbance of 220nm by an Applied Biosystems model 683 programmable spectr ophotometric detector. Approximately 1.5mL fractions were collected over 60 minut es. Fractions 15 through 41 (fraction set # 1) were collected and concentrated to dryness by lyophilization, and sent to the University of South Florida for CGRP-Gly analysis. The retent ion time of CGRP-Gly was determined by the injection of a pure CGRP -Gly standard. Duplicate fraction sets # 13 were prepared by the described methodology.
126 Analysis of Unigene Fractions for CGRP-G ly by the Developed Platform Technology Unigene fractions 20-29 from fraction set # 1 were selected to undergo the described platform technology. The fractions were resuspended in 100 l of PAM reaction conditions, 40mM MES pH 6.3, 1.0mM sodium as corbate, 0.5mM CuSO4, 10U/mL HRP, and 0.015U/mL PAM. The PAM react ion conditions for CGRP-Gly amidation were specified by Unigene Laboratories, Inc. The reaction proceeded for 1 hour at 37oC, and was followed by a 1 hour incubation with 2U/mL of ascorbate oxidase. The samples were brought to 600 L in 100mM Phosphate Buffer pH 7.8, and analyzed by the developed chemi-luminescent assay for gl yoxylate. The plat form technology was repeated on fractions 18 Â– 34 from fraction set #1, and fractions 27 Â– 32 of fraction set # 3.
127 Matrix Assisted Laser Desorption Ionizatio n Â– Time of Flight Mass Spectrometry Analysis for CGRP-Gly Accumulation Fractions 20 Â– 31 of fraction set # 2 were se nt from Unigene Laboratories, Inc. for Mass Spectral Analysis of CGRP-Gly. A standard CGRP-Gly (American Peptide, Inc.) was initially analyzed to establish the CGRP-Gly parent ion ( m/z ), by the described methods (chapter 4). Fractions 27 -32 were resuspended in 10 L of 0.1% (v/v) TFA, and spotted in a 1 : 1 ratio (matrix : an alyte) on a 384 carbon plate and evaporated to dryness. The MALDI-TOF procedure for fraction analysis wa s performed as described (chapter 4).
128 Results and Discussion Demonstration of the Platform Technology to Identify mJP-Gly Accumulation by the Inhibition of PAM in Cell Culture RP-HPLC of the mJP-Gly Standard Injection of standard mJP-Gly resulted in an average Rt of 31.42 0.0564. The average Rt and standard deviation was calculated over a total of 21 in jections (Fig. 43). Standard Curve for the Absorbance at 278nm of mJP-Gly as Analyzed by RPHPLC; mJP-Gly Rt = 31.417 +/0.0564y = 171.36x + 173.8 R2 = 0.9903 0 500 1000 1500 2000 2500 024681012 mJPGly nanomolesPeak Area mAU 278nm Figure 43. Standard curve for the absorbance at 278nm of mJP-Gly as analyzed by RP-HPLC. Standard curve anlaysis of mJP-Gly resulted in the determination of the average Rt and RP-HPLC sensitivity of the standard peptide.
129 RP-HPLC Separation of the Accumulated and Spiked mJP-Gly from At-T20 Cell Culture The mJP-Gly and mJP-NH2 standards, confirmed that both the mJP-Gly and mJP-NH2 co-elute in the described separa tion. Thus, both the mJP-Gly MW = 1999g/mol, and mJPNH2 = 1941g/mol, appear in the same HPLC fr action by Mass Spectral an alysis (Fig. 50). Moreover, RP-HPLC coupled to spectrophotometr ic detection is not a technique sensitive enough to detect and quantify the accumula ted mJP-Gly peptide within the linear detection range of mJP-Gly peptide (Fig. 43).
130 Analysis of mJP-Gly Dependent Glyoxyla te Production by Chemi-luminescence Fractions 29-32 of A1,3,5 and B1,3,5 show a positive luminescent signal for fractions 30 and 31. Although results were not stoichiometric ly quantifiable, the signal of mJP-Gly accumulation is relative to (Figs. 44, 46 &. 45, 47). The chemi-luminescent signals define fractions 30 and 31 positive for glyoxyl ate: mJP-Gly in both accumulated and spiked samples. These fraction numbers ar e within standard deviation of the RP-HPLC Rt of mJP-Gly as determined by standard analysis, the non-disu lfiram treated cell extract displayed no positive luminescent signal for fractions 29 -32. Chemi-luminescent Analysis of Accumulated mJP-Gly from At-T20 Cells 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000 blank29blank30blank31blank32blank sampleRLU Figure 44. Chemi-luminescent analysis of accumulated mJP-Gly from At-T20 cells Fractions 29 Â– 32 were analyzed by the described method for glyoxylate dependent peroxide production. A blank solution containing all necessary enzymes and cofactors was analyzed betw een fractions to establish the luminescent baseline signal.
131 Chemi-luminescent Analysis of Spiked mJP-Gly from At-T20 Cells 0 100000 200000 300000 400000 500000 600000 700000 800000 900000 blank29blank30blank31blank32blank sampleRL U Figure 45 Chemi-luminescent Analysis of Spiked mJP-Gly from At-T20 Cells Fractions 29 Â– 32 were analyzed by the described method for glyoxylate dependent peroxide production. A blank solution containing all necessary enzymes and cofactors was analyzed betw een fractions to establish the luminescent baseline signal.
132 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000 29303132 FractionRLU 0 100000 200000 300000 400000 500000 600000 700000 800000 900000 29303132FractionRLU Chemi-luminescent Analysis of Accumulated mJP-Gly from At-T20 Cells Figure 46. Chemi-luminescent Analysis of Accumulated mJP-Gly from At-T20 Cells Fractions were analyzed by the described method for glyoxylate dependent peroxide production. Values represent RLU minus the average blank value obtained between samples. Chemi-luminescent Analysis of Spiked mJP-Gly from At-T20 Cells Figure 47. Chemi-luminescent analysis of spiked mJP-Gly from At-T20 cells. Fractions were analyzed by the described method for glyoxylate dependent peroxide production. Values represent RLU minus the average blank value obtained between samples.
133 Summary of Spiked vs. Non-Spiked Cellular Extract for mJP-Gly Fraction # Spiked Non-spiked 29 0 20811 30 767759 177711 31 68905 72778 32 74788 0 Table 6. Summary of spiked vs. nonspiked cellular extract for mJP-Gly. Values are obtained from the total RLU gained minus the average blank va lue from each fraction anal yzed for glyoxylate:H2O2 content. The luminescent data demonstrates an approximate 4-fold increase in mJP-Gly in the spiked extract. Thus, the approximate amount of mJP-Gly accumulated is 0.625 nanomoles as compared to the 2.5nanomole spike.
134 MALDI-TOF Analysis of St andard mJP-Gly and mJP-NH2 Mass Spectral analysis of the purif ied standards defined the absolute m/z values for mJPGly and mJP-NH2. Amidation of mJP-Gly result s in a total mass loss of 59 [C2 H3O2] from the glycine extended peptide. The amidated product gain s one proton on the terminal -amine during catalysis, resulting in a tr ue difference of 58 mass units between the glycine-extended and amidated forms of mJP-Gly. Respectively, the m/z value for mJP-Gly is 1999 and 1941 for -amidated mJP-Gly (Figs. 48, 49). Figure 48. Mouse joining peptide precursor MALDI-TOF of a standard mJP-Gly sample, depending on calibration standards the parent ion is found 1998 m/z 2001. 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 m/z 50 100 150 200 250 300 350 400 a.i
135 1300 1500 1700 1900 2100 2300 2500 m/z 500 1000 1500 2000 2500 3000 3500 a.i Figure 49. Mouse joining peptide. MALDI-TOF of a standard mJP-NH2 sample, depending on calibration standards the parent ion is found 1940 m/z 1942.
136 MALDI-TOF Analysis of Accumulated and Spiked mJP-Gly Based on the HPLC co-elu tion of mJP-Gly/mJP-NH2 and incomplete PAM inhibition both parent ions (mJP-Gly, mJP-NH2) are present by MALDI-T OF within the same fraction (Figs. 50-52). MALDI-TOF is not a quantitative methodology based on several variables which affect the si gnal of a given analyte. Th ese variables include, matrix composition, matrix : analyte co -crystallization, and sample i onization. Other parent ions evident within the analyzed fractions are result of incomplete purity of cellular fractions and most likely are remnants of the cell cultu re media / serum. Although the identity of these other molecules is not known, their existen ce is not harmful as it is evident that they do not interfere with the platform technology. Likewise, the existence of other peaks is expected, and will vary among cell lines, and culture mediums used for cell growth. MALDI-TOF analysis demonstrates the disulf iram dependent inhibition of PAM in cell culture and thereby further validate s the developed platform technology.
137 mJP-NH2 MALDI-TOF of Accumulated mJP-Gly 1940.204 1960.208 1972.181 1999.195 0 100 200 300 400 500 600Intens. [a.u.] 1930 1940 1950 1960 1970 1980 1990 2000 m/ z Figure 50 MALDI-TOF of accumulated mJP-Gly MJP-Gly accumulated in sample B230, accumulated mJP-Gly was found in samples B2 30 Â– 31. mJP-Gly
138 1999.374 1940.422 0 100 200 300 400 500 600Intens. [a.u.] 1900 1920 1940 1960 1980 2000 2020 m/ z MALDI-TOF of Spiked mJP-Gly Figure 51. MALDI-TOF of spiked mJP-Gly MJP-Gly from spiked sample D2-30, samples D2 30 32 were positive for mJP-Gly. mJP-Gly mJP-NH2
139 1998.253 1940.275 0 20 40 60 80 100 Intens. [a.u.] 00 1850 1900 1950 2000 2050 2100 m/ z 1998.916 1997.919 1999.917 2000.900 0 100 200 300 Intens. [a.u.] 1985.0 1987.5 1990.0 1992.5 1995.0 1997.5 2000.0 2002.5 2005.0 2007.5 m/ z A B MALDI-TOF of mJP-Gly Accu mulated Vs. Spiked mJP-Gly Figure 52. MALDI-TOF of accumulated vs. spiked mJP-Gly (A) An example of MALDI-TOF data from accumulated sample set A4 Â– 31, and (B) spiked sample D4 31 displaying the isotopic resolution of mJP-Gly within the spiked sample.
140 Demonstration of the Platform Technology to Identify CGRP-Gly Accumulation by the Inhibition of PAM in Cell Culture Analysis of Unigene Fractions fo r CGRP-Gly by Chemi-luminescence Fractions 20-29 of Unigene sample set #1 were analyzed by the described chemiluminescent assay for glyoxylate and displaye d a positive signal for rat CGRP-Gly in fraction 29 (Fig. 53). Repeat analysis on sample set #1 of fractions 18-34, also defined fraction 29 as positive for gl yoxylate (data not shown). Chemi-luminescent Analysis of Unigene Fraction Set # 1 for CGRP-Gly 20000 30000 40000 50000 60000 70000 80000 90000 100000 110000 120000 blank25blank26blank27blank28blank29blank sampleRL U Figure 53. Chemi-luminescent analysis of Unigene fraction set # 1 for CGRP-Gly Luminescent analysis defines sample 29 as positive for glyoxylate.
141 Analysis of Unigene CGRP-Gly Standard by the MALDI-TOF A rat CGRP-Gly standard was supplied by Unigene for analysis of the CGRP-Gly parent ion. The parent ion at m/z of 3860 corresponds to the standard molecular weight of rat CGRP(Fig. 54). MALDI-TOF of Standard CGRP-Gly Figure 54. MALDI-TOF of standard CGRP-Gly. The CGRP-Gly standard supplied by Unigene had a m/z of 3860, this value corresponds to the calculated molecular weight. 3600 3700 3800 3900 m/z 0 200 400 600 800 1000 1200 1400 1600 a.i
142 Analysis of Unigene Fractions for CGRP-Gly by the MALDI-TOF Fractions 21-32 of Unigene fraction set #2 analyzed by MALDI-TOF displayed the parent ion of 3860 m/z (Fig. 55). 3600 3700 3800 3900 4000 m/z 0 100 200 300 400 500 600 700 800 900 a.i Figure 55. Fraction 29. Fraction 29 from the Unigene fraction set #2. Analysis of fractions 21 -32 displays the presence of CGRP-Gly ( m/z 3860) accumulated from cell culture.
143 Conclusion The cellular accumulation of the glycine-extend ed peptides is the premise on which this newly designed platform resides, without the accumulation of the glycine-extended peptides detection of PAM produced glyoxyl ate is futile. The production of glyoxylate upon treatment of cellular fracti ons with PAM is compelling for the accumulation of the glycine-extended peptides but not definitiv e. Mass spectral analysis of glyoxylate positive fractions for the presence of the glycine-extended peptides provides not only verification of the glyoxylate signal, but also it defines the framework for platform technology. The utilization of PAM presents a novel method for the discovery of amidated peptides. This method relies on the ability to detect glyoxylate, a stoichiometric product of the PAM dependent amidation of glycine-extended peptides. This platform technology has been thor oughly optimized towards the design of both specific and sensitive assays for glyoxylate. This body of work has described the devel opment of seven novel spectroscopic enzyme based assays for glyoxylate. Detection of gl yoxylate is not only usef ul as a PAM assay, but is also useful in a clin ical setting. The presence of glyoxylate in urine is used a marker for monitoring kidney stone formati on. To date, many of the prior glyoxylate assays have been developed for this use, or for detection of ureidoglycolate activity (chapter 1).
144 Each novel glyoxylate assay can also be us ed for the detection of PAM-produced glyoxylate, moreover the chemi-luminescent assa y has proven itself most useful for the identification of glycine-extended peptides. The development of this new platform technology has proven useful as a Â‘proof of conceptÂ”, as this technology has been successful at identification of model glycin e-extended peptides accumulated from cell culture. The platform technology is now poised to be efficacious for the identification of novel glycine-extended peptides accumulated from cell cultur e. Although work remains to be done as different cell lines may require alternate PAM inhibito rs, and identification of a novel peptides will undoubtedly be diffi cult, an efficient and robust platform technology for glycine-extended peptid e discovery has been developed.
145 Future Work for Novel Peptide Id entification and Characterization The platform technology is now poised for novel peptide discovery from a variety of sources. Future work relies on the choice of source materi al, namely cell line choice. Classically, hormones are chemical messengers which carry messages from one cell to another. By definition, this doe s not imply that both PAM and an -amidated hormone are both synthesized in the same tissue, (cel l source) which ultimately means that the cell lines used in the platform technology do not have to express PAM. However, as mentioned in chapter two, PAM has been link ed to both exocrine and autocrine growth loops. This feature imparts a unique decisi on making option to cell line choice for the platform technology; cells which do expre ss PAM and cells which do not express PAM may both be logical choices. One definite aspect of future work lies in the process for discovery of a novel peptide. The putative peptide must ini tially be purified to near homogeneity by a variety of chromatographic techniques including reve rse-phase and ion-exchange chromatography, coupled to analytical HPLC for purity assess ment. It is possible that the platform technology may find a glycine-ex tended protein fragment, alt hough this is not likely as there are no known endoproteases which cleave at the C-termin al side of glycine. A variety of techniques when employed in tandem will easily dissolve any issues concerning the identity of al l novel peptides discovered by this platform technology.
146Initially, it is most important to determine th e peptide sequence, a goa l easily achieved by a variety of techniques, including N-terminal sequence analysis (Edman degradation) or LC-MS-MS. Alternatively, if Edman degradat ion fails sequences da ta can be obtained by enzymatic C-terminal sequencing in conjunc tion with amino acid analysis. Sequence data will provide the initial in formation to discover if in fa ct peptides are novel, screening peptide databases with the put ative novel peptide sequence will discover if this peptide is simply a fragment of a larger known peptide and/or protein, and if this peptide is an already documented -amidated peptide. Often, peptides are classed into families because they exist together in one readi ng frame (e.g. mJP-Gly of the mouse pro-ACTH homologue fig. 40). It is likely that a putat ive peptide may be contained within an open reading frame known to contain other peptid e hormones. One key feature of all the amidated peptides is the proteolytic pathway to their formation. R ecall, from chapter 1 that all peptides which ultimately become glycine-extended undergo a series of sequence specific proteolytic events. The signature se quence for all peptides to become glycineextended is a pair of dibasic am ino acids flanking the internal glycine. This hallmark of -amidation will most definitely provide useful information as to if the discovered peptide hormones; are indeed truly novel -amidated hormones. In conclusion, there are three main factors which must be defined fo r identification of newly discovered peptides; if the sequence has the correct proteolytic processing site s, is entirely contained within one open reading frame, and cont ains a C-terminal glycine. Defining the identity newly discovered amidated hormones ma y prove trivial as compared to in vitro characterization of the newly discover ed peptide. It will most likely
147require a great deal of metabolic and genomic studies to define physio logical the role of any newly discovered peptide. The peptide will need to be produced in large enough quantities for bio-characterization, a goal accomplished by recombinant overexpression of the peptide/peptides in E. coli Parameters such as tissue distribution, disease relevance, and bio-characterization will re quire time, and will most likely differ among newly discovered peptides. Nort hern blot analysis can provide valuable information as to the population of peptide transcripts within a specific tissue, a nd aid in gathering information as to the population in various disease states. Gene knockout and or siRNA can also provide information as to the phenot ypical and/or metabolic role of the novel peptide in cell culture or animal studies. Several tools for the in vitro characterization of the newly discovered peptide exist, and ma ny can be develop as information of the peptides role unfolds during initial studies. The use of this assay in time may lead to the discovery of many novel peptid es essential to the study of -amidated hormones and their physiological role. Moreover, peptides are the chemical messengers that keep living systems in a homeostatis and abnormalities arising from their over/under production leads to the onset of hormone induced diseases (T able 2). A platform technology for the discovery of novel -amidated peptides is currently in pl ace, and the ramifications of this technique will provide the scientific community with the basic the knowledge to pioneer the biochemistry of novel -amidated hormones.
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169 Appendix A: Abbreviations CGRP-Gly Â– calcitonin gene related peptide Dansyl 5-dimethylamino-na phthalene-1-sulfonyl chloride DMAB 3-(dimethylamino)benzoic acid GLOX Â– glyoxal oxidase GO Â– glycolate oxidase GR Â– glyoxylate reductase HRP Â– horse radish peroxidase Luminol 3-aminophthalhydrazide MALDI-TOF MS Â– matrix assist ed laser deionization Â– time of flight mass spectrometry MBTH 3-methyl-2-ben zothiazolinone hydrazone mJP-Gly Â– mouse joining peptide MOPS 3-(N-morpholino)-propanesulfonic acid MS malate synthase MD malate dehydrogenase MS / MD Assay Â– malate syntha se / malate dehydrogenase assay MTS 3-(4,5-dimethylthiazol-2-yl)-5 -(3-carboxymethoxylphe nyl)-2-(4-sulfophenyl)2H-tetraolium, inner salt NAD+/NADH nicotinomide adenine dinucleot ide/nicotinamide adenine dinucleotide reduced form PAM Â– peptiylglycine -amidating monooxygenase
170PMS phenazine methosulfate RFU Â– relatice fluorescent units RLU Â– relative chemi-luminescent units RP-HPLC Â– reverse phase high pe rformance liquid chromatography TEA triethanolamine TFA trifluoroacetic acid
171 About the Author Sarah Elizabeth Carpenter rece ived a Bachelor of Arts De gree in Biology from Franklin Pierce College (1997). During her college car eer, Sarah held a summer position at the Baltimore Medical Examiners, where she assisted pathologists in autopsies to determine cause of death. Sarah also received a Bachel or of Science in Biochemistry and Molecular Biology from the University of Massachusetts Amherst (2000). As an undergraduate at UMASS she performed independent research in the laboratory of Dr. Jennifer Normally, where she was awarded two Howard Hughe s Medical Scholarships, and a National Science Foundation Summer Support Grant to continue her research over the summer semester. She also performed research in the laboratory of Dr. John P. Burand in the Department of Microbiology and Entomology at UMASS. Prior to beginning her Ph.D. work at The University of South Florida, she also worked as a substitute high school teacher in Agawam, Mass. As an undergraduate she was also hired by Spectrum Analytical, Inc., as an analyst in the Orga nic Extraction Department. Sarah was also hired as an adjunct Natural Sciences facu lty member at Manatee Community College while finishing her Ph.D. Sarah has also participated and performe d research on many other projects in Dr. MerklerÂ’s laboratory in the Department of Ch emistry. Sarah has studi ed classical ballet, pointe, jazz, and modern dance at the Fireth orn School of Dance, and was a member of the competitive dance team. Her performan ce repertory at the Tampa Bay Performing
172Arts Center includes; Paquita, Don Quiote, and Giselle variati ons. She was also selected to perform in the Moscow BalletÂ’s Great Russian Nutcracker, in the Arabian variation.