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Detection of Glyoxylate by Glyoxylate Reductase

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Detection of Glyoxylate by Glyoxylate Reductase US 20070254328A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0254328A1 Carpenter et al. (43) Pub. Date: Nov. 1, 2007 (54) GLYOXYLATE ASSAYS AND THEIR USE OF Publication Classification INDENTIFYING NATURAL AMDATED COMPOUNDS (51) Int. Cl. CI2O I/26 (2006.01) (75) Inventors: Sarah E. Carpenter, Caldwell, NJ (52) U.S. Cl. ................................................................ 435/25 (US); Dave J. Merkler, Valrico, FL (US); Duncan A. Miller, Morris Township, NJ (US); Nozer M. Mehta, (57) ABSTRACT Randolph, NJ (US); Angelo P. Consalvo, Monroe, NY (US) Methods for detecting and assaying for glyoxylate, include Correspondence Address: enzyme-based assays and/or assays for hydrogen peroxide OSTROLENK FABER GERB & SOFFEN following liberation of hydrogen peroxide from glyoxylate, 118O AVENUE OF THE AMERICAS are disclosed. In some embodiments, the invention is NEW YORK, NY 100368403 directed to methods for assaying for glyoxylate produced by the reaction of peptidylglycine alpha-amidating monooxy (73) Assignee: Unigene Laboratories Inc. genase (PAM). The subject invention also concerns methods for assaying for the enzyme peptidylglycine alpha-amidat (21) Appl. No.: 11/654,211 ing monooxygenase and/or its Substrates. The detection of (22) Filed: Jan. 17, 2007 glyoxylate is indicative of the presence of PAM and/or its Substrates. The Subject invention also concerns methods for Related U.S. Application Data screening for peptide hormones, amidated fatty acids, any N-acyl-glycine or N-aryl-glycine conjugated molecule, and (60) Provisional application No. 60/761,681, filed on Jan. generally compounds having a glycine reside in free acid 23, 2006. form and attached to a carbonyl group. Detection of Glyoxylate by Glyoxylate Reductase - O08 OO7 O06 OO5 s <g OO4 2 - y=00063 +0.0002 RO9912 4 6 B O 2 Glyoxylate=dns-YW-Gly M Patent Application Publication Nov. 1, 2007 Sheet 1 of 15 US 2007/0254328A1 Glyoxylate Acetyl-CoA 1 CoA-SH Malate NAD" PMS Tetrazolium OxaloacetateX.X. NADH X X FORMAZAN FIG. 1 O 1.0 E E s O.80 0.80 wre s ss s 0.40 or 0.40 9 S 0.20 3 0.20 ae a. 0.0 O.O O O 20 30 40 SO O 10 20 30 40 Glyoxylate), (LM) Benzamide = Glyoxylate, (M) FIG. 2A FIG. 2B Patent Application Publication Nov. 1, 2007 Sheet 2 of 15 US 2007/0254328A1 O Lactate Dehydrogenase O E.C. 11.1.27 + NADPH + H NADP' + HO ON OH pH = 7.8 OH glyoxylic acid glycolic acid Glycolate Oxidase O E.C.1.1.3.15 O -- H2O2 + O. ON pH = 7.8 HO OH OH lvoxvlic acid glycolic acid glyoxy MBTH 3-methyl-2-benzothiazolinone hydrazone DMAB 3-(dimethylamino) benzoic acid 1U/ml HRP HO dependent indamine dye a F590nm e = 53,000Mcm FIG. 3 Patent Application Publication Nov. 1, 2007 Sheet 3 of 15 US 2007/0254328A1 LD-GO, GO Assay for Glyoxylate/o-Amidated Peptide Hormones O7 O6 0.5 o4 lf is oa <g O2 O1 O O \ FIG. 4 O O Glyoxylate Reductase + NADPH +H" - E.C.I.1.179 - Ho NADP n - pH 8.0 O O glycolate glyoxylate FIG. 5 Patent Application Publication Nov. 1, 2007 Sheet 4 of 15 US 2007/0254328A1 Detection of Glyoxylate by Glyoxylate Reductase 4 6 8 O Glyoxylate=dns-YW-GlypM FIG. 6 Patent Application Publication Nov. 1, 2007 Sheet 5 of 15 US 2007/0254328A1 Amplex Red Resorufin Horseradish HO O OH Peroxidase HO O C pH 8.0 E.C.1. 11.7 2 orsN N H2O2 O2 O O O O Glycolate Oxidase H2O + susN O pH=8.0 O E.C.1.1.3.15 glyoxylate oxalate pH 8.0 Fig. 7A Amplex Red Resorufin Horseradish HO O OH Peroxidase HO O C pH 8.0 E.C.1.11.1.7 2 orsN N H2O2 O2 O O O O Glyoxal Oxidase HaO + oulsN O pH=8.0 O glyoxylate oxalate Fig. 7B Patent Application Publication Nov. 1, 2007 Sheet 6 of 15 US 2007/0254328A1 y=12.1 x + 12.3 R’=0.975 T -: &. ... s . d-TYGuM FIG. 8A Patent Application Publication Nov. 1, 2007 Sheet 7 of 15 US 2007/0254328A1 g y=15.417x - 1.5886 5. R’=0.9722 9 . 3 asO , ?t d-TYG) uM FIG. 8B Patent Application Publication Nov. 1, 2007 Sheet 8 of 15 US 2007/0254328A1 3. OH OH PAM To O Oh : OH e pH 6 s VV Gly is + - CuSO - N - VVV NH . 2. Ot O OH -- aschate One hour at 37 C, 0.015 U/ml Ascorbate Oxidase H63 EC 1033 One hour at 37 C, 2 U/m Or ro-,aul GlycipidaseA |- towoww. SéninN pHHRP 10.5 --h |; O -EC P 1.1.3.15 - H2O, : V t , ::::::::::::::::::::::::: SEE. 3::::::::::::::. Patent Application Publication Nov. 1, 2007 Sheet 9 of 15 US 2007/0254328A1 Detection of Glyoxylate? Glycine-Extended/o-Amidated Peptides by Luminescence O O 2O 3O 40 SO 60 — H2O/d-TYG nM FIG 11 Extract Cell Spent Media in 0.1%TFA, Grind cells in ground glass homogenizer and combine. Elute peptide extract from Sep-Pak with 0.1%TFA/80% CH3CN, and lyophilize. Resuspend sample in 200 0.1%TFA/0.001%Triton-X. Aliquot extract into 4,50ul samples for HPLC injection. Two samples of accumulated mP-Gly. Two samples of accumulated mP-Gly with 2.5 nmoles mP-Gly Collect fractions 26-35 (mP-Gly R - 30 t / Treat Cells with PAM and Determine Presence of Glyoxylate by Luminescence. Assay Fractions on MALDI-TOF for Presence of MP-GLY FIG. 12 Patent Application Publication Nov. 1, 2007 Sheet 10 of 15 US 2007/0254328A1 Luminescent Analysis of Spiked mP-Gly in At-T20 Cells 900000 8OOOOO 700000 - 6OOOOO SOOOOO 2 400000 30OOOO 200000 1OOOOO 30 blank 31 blank 32 blank FIG. 13A Luminescent analysis of RP-HPLC fractions 29-32; spiked mP-Gly. Fractions were analyzed by the described method for glyoxylate dependent peroxide production. A blank solution containing all necessary enzymes and cofactors was analyzed between fractions to establish the luminescent baseline signal. Patent Application Publication Nov. 1, 2007 Sheet 11 of 15 US 2007/0254328A1 Luminescent Analysis of Accumulated mJP-Gly in At-T20 Cells 200000 - 180000 - 160000 - 40000 - 20000 - D 100000 80000 - 60000 - 40000 - 2OOOO - 30 blank 31 sample FIG. 13B Luminescent analysis of RP-HPLC fractions 29-32; accumulated mP-Gly. Fractions were analyzed by the described method for glyoxylate dependent peroxide production. A blank Solution containing all necessary enzymes and cofactors was analyzed between fractions to establish the luminescent baseline signal. Patent Application Publication Nov. 1, 2007 Sheet 12 of 15 US 2007/0254328A1 Fraction 31 of -- - - xtracts of Accumulated in P-Gly 5150. FIG. 14A boo FIG. 14B bso d : 200 : . Patent Application Publication Nov. 1, 2007 Sheet 13 of 15 US 2007/0254328A1 FIG. 14C FIG. 14D Patent Application Publication Nov. 1, 2007 Sheet 14 of 15 US 2007/0254328A1 20000 - 10000 - 100000 - 90000 - 80000 70000 - 60000 - blank 25 blank 26 blanksample .27 blank 28 blank 29 blank FIG. 15 Figure 15. PAM/glycolate oxidase enzyme linked luminescent assay of RP-HPLC fractions 25 through 29. Blanks or buffer washes are performed between fractions. Relative luminescence indicated the presence of rat CGRP-Gly in fraction 29. Patent Application Publication Nov. 1, 2007 Sheet 15 of 15 US 2007/0254328A1 a E - a c i? to Eil 29 good as Fox - esco - soo 400 3. acco - FIG 16 Figure 16. MALDI-TOF Mass Spectrometry analysis of fraction 29 from conditioned medium purified by chromatography on a Hypersil BDS C18 column. US 2007/0254328A1 Nov. 1, 2007 GLYOXYLATE ASSAYS AND THEIR USE OF Biochem. 1:240-248, and the use of capillary electrophoresis INDENTIFYING NATURAL AMDATED with direct UV detection (Nishijima, S. et al. (2001) “Gly COMPOUNDS oxylate Determination in Rat Urine by Capillary Electro phoresis'Int. J. Urol. 8:S63-S67; Garcia, A. et al. (2001) 0001. This invention was made with government support “Measurement of Nephrolithiasis Urinary Markers by Cap under the National Institute of Health SBIR grant number illary Electrophoresis'.J. Chromatogr: B 755:287-295. 1-R43-DK063812-01. The government has certain rights in the invention. 0005 Generally, these assays are insensitive and/or non specific. Increased sensitivity has been provided by the BACKGROUND OF THE INVENTION separation and quantification of the colored or fluorescent glyoxylate derivative by HPLC (Bongers, J. et al. (1992) 0002 C-Terminal amidation of glycine extended prohor “Semisynthesis of Human Growth Hormone-Releasing Fac mones is a post-translational modification necessary for the tors by C.-Amidating Enzyme Catalyzed Oxidation of Gly activity of many peptide hormones. Amidated peptide hor cine-Extended Precursors' Peptide Res. 5:183-189: Funai, T. mones are an important class of hormones found in mam et al. (1986) “High-Performance Liquid Chromatographic mals, insects, and cnidarians. The discovery of novel ami Determination of Glyoxylate in Rat Liver'J. Biochem dated hormones has been severely hindered by the lack of a 99:579-589; Mentasi, E. et al. (1987) “High-Performance good assay specific to this class of bioactive hormones. The Liquid Chromatographic Determination of Glyoxylic Acid formation of all amidated peptide hormones is dependant and other Carboxyl Compounds in Urine'J. Chromatogr: B. upon the activity of Peptidylglycine alpha-Amidating 417:253-260, 1987; Petrarulo, M. et al. (1988) “High Monooxygenase (PAM). Glycine extended precursors Performance Liquid Chromatographic Determination of reacted with PAM result in the formation of an alpha Glyoxylic Acid in Urine'J. Chromatogr. 432:37-46; Lange, amidated peptide and glyoxylate. The general reaction is M. et al. (1994) “Fast Method for the Simultaneous Deter shown below in Scheme 1: mination of 2-Oxo Acids in Biological Fluids by High Performance Liquid Chromatography'.J.
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