Materials and Methods for Assaying for Glyoxylate

• US 20100143897A1
• Filed: 09/15/2006
• Published: 06/10/2010
• Est. Priority Date: 09/16/2005
• Status: Abandoned Application

• Current Assignee: University of South Florida (State University System of Florida)
• Original Assignee: University of South Florida (State University System of Florida)
• Inventors: Carpenter, Sarah E., Merkler, David J.
• Application Number:
  US12/066,922
• Publication Number:
  US 20100143897A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: Y10T 436/200833 Carbonyl, ether, aldehyde or ketone containing

Abstract

The subject invention concerns enzyme-based methods for detecting and assaying for glyoxylate. In particular, the invention is directed to methods for assaying for glyoxylate produced by the reaction of peptidylglycine α-amidating monooxygenase (PAM). The subject invention also concerns methods for assaying for the enzyme peptidylglycine α-amidating monooxygenase. The detection of glyoxylate using the present invention is indicative of the presence of PAM. The subject invention also concerns methods for screening for peptide hormones and any N-acyl-glycine or N-aryl-glycine conjugated molecule.

Claims

1. A method for detecting glyoxylate in a sample, said method comprising contacting said sample with one or more reagents, wherein the reaction of glyoxylate in said sample with said reagents results in the production of a detectable reaction product;

and detecting said detectable reaction product.

2. The method according to claim 1, wherein said detectable reaction product is hydrogen peroxide.

3. The method according to claim 1, wherein said detectable reaction product is detected visually or using a fluorescent assay, a luminescent assay, or a spectrophotometric assay.

43. The method according to claim 1, wherein said method comprises contacting said sample with ascorbate.

44. The method according to claim 43, wherein said method further comprises contacting said sample with ascorbate oxidase after contact with ascorbate.

4. A method for detecting glyoxylate in a sample, said method comprising:

a) contacting said sample with acetyl-CoA, malate synthase, and malate dehydrogenase, wherein NADH is produced if glyoxylate is present in said sample;

b) detecting said NADH produced, wherein said NADH corresponds to the presence of glyoxylate in said sample.

5. The method according to claim 4, wherein said NADH is detected using phenazine methosulfate (PMS) and a tetrazolium compound.

6. The method according to claim 5, wherein the reaction product produced using phenazine methosulfate and a tetrazolium compound is a reduced formazan product that is detected spectrophotometrically, optionally at about 490 nm.

7. The method according to claim 5, wherein said tetrazolium compound is 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxylphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS).

8. The method according to claim 4, wherein step a) further comprises contacting with NAD⁺.

57. The method according to claim 4, wherein said method comprises contacting said sample with ascorbate.

58. The method according to claim 57, wherein said method further comprises contacting said sample with ascorbate oxidase after contact with ascorbate.

9. A method for detecting glyoxylate in a sample, said method comprising:

a) contacting said sample with lactate dehydrogenase and glycolate oxidase, whereby glycolate and hydrogen peroxide are produced if glyoxylate is present in said sample; and

b) detecting said hydrogen peroxide produced in the presence of glyoxylate.

10. The method according to claim 9, wherein said hydrogen peroxide is detected using MBTH and DMAB and a peroxidase.

11. The method according to claim 10, wherein the reaction product produced using MBTH and DMAB and a peroxidase is an indamine dye that is detected spectrophotometrically, optionally at about 590 nm.

12. The method according to claim 9, wherein said hydrogen peroxide is detected using a peroxidase and a substrate that reacts in the presence of said peroxidase to produce a fluorescent molecule, wherein said substrate is optionally Amplex Red, whereby Amplex Red is oxidized to Resorufin in the presence of hydrogen peroxide.

13. The method according to claim 10, wherein said peroxidase is horseradish peroxidase.

59. The method according to claim 9, wherein said method comprises contacting said sample with ascorbate.

60. The method according to claim 59, wherein said method further comprises contacting said sample with ascorbate oxidase after contact with ascorbate.

14. A method for detecting glyoxylate in a sample, said method comprising:

a) contacting said sample with glycolate oxidase or glyoxal oxidase, whereby oxalate and hydrogen peroxide are produced if glyoxylate is present in said sample;

b) detecting said hydrogen peroxide produced from step a).

15. The method according to claim 14, wherein said hydrogen peroxide is detected using MBTH and DMAB and a peroxidase.

16. The method according to claim 15, wherein said reaction product produced using MBTH and DMAB and a peroxidase is detected spectrophotometrically, optionally at about 590 nm.

17. The method according to claim 14, wherein said hydrogen peroxide is detected using a peroxidase and a substrate that reacts in the presence of said peroxidase to produce a fluorescent molecule, wherein said substrate is optionally Amplex Red, whereby Amplex Red is oxidized to Resorufin in the presence of hydrogen peroxide.

18. The method according to claim 14, wherein said hydrogen peroxide is detected visually or using a fluorescent assay, a luminescent assay, or a spectrophotometric assay.

19. The method according to claim 14, wherein said hydrogen peroxide is detected by contacting said sample with luminol and detecting luminescence.

20. The method according to claim 19, wherein said contacting step is performed at a basic pH and in the presence of a catalyst.

21. The method according to claim 20, wherein said catalyst is horseradish peroxidase.

22. The method according to claim 15, wherein said peroxidase is horseradish peroxidase.

23. The method according to claim 17, wherein said method further comprises contacting said sample with flavin adenine dinucleotide (FAD).

24. The method according to claim 14, wherein said method is performed at a basic pH.

25. The method according to claim 24, wherein said pH is about 8.0.

61. The method according to claim 14, wherein said method comprises contacting said sample with ascorbate.

62. The method according to claim 61, wherein said method further comprises contacting said sample with ascorbate oxidase after contact with ascorbate.

26. A method for detecting glyoxylate in a sample, said method comprising:

a) contacting said sample with glyoxylate reductase and NADPH, whereby glycolate and NADP⁺ is produced if glyoxylate is present in said sample; and

b) detecting said NADP⁺.

27. The method according to claim 26, wherein said NADP⁺ is detected spectrophotometrically by measuring absorbance of said sample, optionally at about 340 nm.

63. The method according to claim 26, wherein said method comprises contacting said sample with ascorbate.

64. The method according to claim 63, wherein said method further comprises contacting said sample with ascorbate oxidase after contact with ascorbate.

28. A method for assaying for a glycine extended molecule in a sample, wherein said method comprises:

a) contacting said sample with PAM, wherein glyoxylate is produced if a glycine extended molecule is present in said sample; and

b) assaying for the presence of glyoxylate, whereby the presence of glyoxylate is indicative of the presence of a glycine extended molecule.

29. The method according to claim 28, wherein the presence of glyoxylate is assayed using a method selected from:

i) contacting said sample with one or more reagents, wherein the reaction of glyoxylate in said sample with said reagents results in the production of a detectable reaction product; and

detecting said detectable reaction product;

or,

ii) a) contacting said sample with acetyl-CoA, malate synthase, and malate dehydrogenase, wherein NADH is produced if glyoxylate is present in said sample; and

b) detecting said NADH produced, wherein said NADH corresponds to the presence of glyoxylate in said sample;

or,

iii) a) contacting said sample with lactate dehydrogenase and glycolate oxidase, whereby glycolate and hydrogen peroxide are produced if glyoxylate is present in said sample; and

b) detecting said hydrogen peroxide produced in the presence of glyoxylate;

or,

iv) a) contacting said sample with glycolate oxidase or glyoxal oxidase, whereby oxalate and hydrogen peroxide are produced if glyoxylate is present in said sample; and

b) detecting said hydrogen peroxide produced;

or,

v) a) contacting said sample with glyoxylate reductase and NADPH, whereby glycolate and NADP⁺ is produced if glyoxylate is present in said sample; and

b) detecting said NADP⁺ produced.

30. The method according to claim 28, wherein said glycine extended molecule is a glycine extended peptide.

31. The method according to claim 28, wherein said glycine extended molecule is a glycine extended fatty acid.

32. The method according to claim 28, wherein said glycine extended molecule is a glycine extended prohormone.

33. The method according to claim 28, wherein said glycine extended molecule is an N-acyl-glycine or an N-aryl-glycine conjugated molecule.

34. The method according to claim 28, wherein step (a) further comprises contacting said sample with ascorbate and/or hydrogen peroxide.

35. The method according to claim 28, wherein step (a) further comprises contacting said sample with catechol and/or hydrogen peroxide.

36. The method according to claim 28, wherein after step (a) said sample is contacted with ascorbate oxidase.

37. A method for detecting peptidylglycine α

-amidating monooxygenase (PAM) in a sample, said method comprising detecting the presence of glyoxylate using a method selected from;

i) contacting said sample with one or more reagents, wherein the reaction of glyoxylate in said sample with said reagents results in the production of a detectable reaction product and detecting said detectable reaction product;

or,

ii) a) contacting said sample with acetyl-CoA, malate synthase, and malate dehydrogenase, wherein NADH is produced if glyoxylate is present in said sample; and

b) detecting said NADH produced, wherein said NADH corresponds to the presence of glyoxylate in said sample;

or,

iii) a) contacting said sample with lactate dehydrogenase and glycolate oxidase, whereby glycolate and hydrogen peroxide are produced if glyoxylate is present in said sample; and

b) detecting said hydrogen peroxide produced in the presence of glyoxylate;

or,

iv) a) contacting said sample with glycolate oxidase or glyoxal oxidase, whereby oxalate and hydrogen peroxiderroduced if glyoxylate is present in said sample; and

b) detecting said hydrogen peroxide produced;

or,

v) a) contacting said sample with glyoxylate reductase and NADPH, whereby glycolate and NADP⁺ is produced if glyoxylate is present in said sample; and

b) detecting said NADP⁺ produced,

wherein the presence of glyoxylate is indicative of the presence of PAM.

38. A method for assaying for the presence of a glycine extended substrate, said method comprising detecting the presence of peptidylglycine α

-amidating monooxygenase (PAM) according to the method of claim 37, wherein the presence of PAM activity is indicative of the presence of a glycine extended substrate, and assaying for the presence of a glycine extended substrate in the sample that is determined to contain PAM.

39. A method for screening for the presence of an α

-amidated peptide or fatty acid in a sample, said method comprising;

a) growing cells in a medium in which said cells will grow;

b) preparing cell extract and/or obtaining spent media from the cells grown in step a);

c) chromatographically fractionating said cell extract and/or spent media from step b);

d) contacting the fractionated samples obtained from step c) with peptidylglycine α

-amidating monooxygenase;

e) assaying said fractionated sample for the presence of glyoxylate using a method selected from;

i) contacting said sample with one or more reagents, wherein the reaction of glyoxylate in said sample with said reagents results in the production of a detectable reaction product; and

detecting said detectable reaction product;

or,

ii) a) contacting said sample with acetyl-CoA, malate synthase, and malate dehydrogenase, wherein NADH is produced if glyoxylate is present in said sample; and

b) detecting said NADH produced, Wherein said NADH corresponds to the presence of glyoxylate in said sample;

or,

iii) a) contacting said sample with lactate dehydrogenase and glycolate oxidase, whereby glycolate and hydrogen peroxide are produced if glyoxylate is present in said sample; and

b) detecting said hydrogen peroxide produced in the presence of glyoxylate;

or

iv) a) contacting said sample with glycolate oxidase or glyoxal oxidase, whereby oxalate and hydrogen peroxide are produced if glyoxylate is present in said sample; and

b) detecting said hydrogen peroxide produced;

or,

v) a) contacting said sample with glyoxylate reductase and NADPH whereby glycolate and NADP is produced if glyoxylate is present in said sample; and

b) detecting said NADP produced,

wherein fractions that test positive for glyoxylate are indicative of the presence of an α

-amidated peptide or fatty acid.

40. The method according to claim 39, wherein said cell medium comprises an inhibitor of PAM.

41. The method according to claim 39, wherein said method further comprises characterizing said detected peptide by mass spectrometry.

42. The method according to claim 39, wherein said method further comprises determining the amino acid sequence of said detected peptide.

45-56. -56. (canceled)

65. A kit comprising in one or more containers:

acetyl-CoA;

malate synthase; and

malate dehydrogenase;

or

lactate dehydrogenase; and

glycolate oxidase;

or

glycolate oxidase or glyoxal oxidase;

a peroxidase; and

a substrate that reacts in the presence of said peroxidase to produce a fluorescent molecule, wherein said substrate is optionally Amplex Red;

or

glyoxylate reductase.

66. The kit according to claim 65, further comprising:

phenazine methosulfate; and

a tetrazolium compound.

67. The kit according to claim 66, wherein said tetrazolium compound is 3-(4,5-dimethylthiazol-2-yl)-5-(3 -carboxymethoxylphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS).

68. The kit according to claim 65, further comprising:

NAD⁻

.

69. The kit according to claim 65, further comprising:

3-methyl-2-benzothiazolinone hydrazone (MBTH);

3-(dimethylamino)benzoic acid (DMAB); and

a peroxidase.

70. The kit according to claim 65, further comprising:

flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN).

71. The kit according to claim 65, further comprising ascorbate and/or ascorbate oxidase and/or catechol and/or PAM.