Stabilization of oxidase

• US 4,543,326 A
• Filed: 11/12/1982
• Issued: 09/24/1985
• Est. Priority Date: 11/12/1981
• Status: Expired due to Fees

• Current Assignee: WAKO Pure Chemical Industries Limited (Fujifilm Holdings Corporation)
• Original Assignee: WAKO Pure Chemical Industries Limited (Fujifilm Holdings Corporation)
• Inventors: Satomura, Shinji, Miyashita, Yoshinobu
• Primary Examiner(s): Kepplinger, Esther M.
• Application Number:
  US06/441,156
• Time in Patent Office: 1,047 Days
• Field of Search: 435/14, 435/188, 435/190, 435/20, 435/28, 435/25, 435/15, 435/18, 435/19, 435/810, 435/805, 435/11, 424/94
• US Class Current: 435/15
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

An oxidase selected from the group consisting of glycerol-3-phosphate oxidase, choline oxidase and glucose oxidase can be stabilized by adding thereto an acidic amino acid or a salt thereof. The resulting stabilized composition can be used for quantitatively determining the content of glycerol-3-phosphate, choline, triglyceride, glucose, etc., in a biological fluid.

Claims

1. A process for stabilizing an oxidase which comprises adding an acidic amino acid or a salt thereof in an amount of 1 to 5% by weight to an aqueous solution of an oxidase selected from the group consisting of glycerol-3-phosphate oxidase, and choline oxidase.

2. A process according to claim 1, wherein the oxidase is glycerol-3-phosphate oxidase.

3. A process according to claim 1, wherein the oxidase is choline oxidase.

4. A process according to claim 1, wherein the acidic amino acid or a salt thereof is a buffer solution-soluble salt of acidic amino acid.

5. A process according to claim 1, wherein the acidic amino acid or a salt thereof is represented by the formula:

##STR6## wherein A is a lower alkylene group having 1 to 5 carbon atoms; and

Y and Y'"'"' are independently hydrogen, a NH₄ group or an alkali metal.

6. A process according to claim 1, wherein the acidic amino acid or a salt thereof is sodium glutamate.

7. A process according to claim 1, wherein the acidic amino acid or a salt thereof is sodium aspartate.

20. A process according to any of claims 1, 8, 11, 14, or 17 in which the acidic amino acid is selected from the group consisting of glutamic acid, aspartic acid, alpha-aminoadipic acid and salts of these acidic amino acids.

8. A process for quantitatively determining the glycerol-3-phosphate content in a biological fluid which comprises adding to the biological fluid an aqueous solution containing

glycerol-3-phosphate oxidase in an amount of 1 to 20 units/ml,

a buffer solution in an amount sufficient to maintain the pH near neutral value,

an acidic amino acid or a salt thereof in an amount of 1 to 5% by weight based on the weight of the aqueous solution containing the oxidase,

peroxidase, 4-aminoantipyrine, and an oxidizable color producing indicator in amounts sufficient to produce a color,

and measuring the color produced colorimetrically.

9. A process according to claim 8, wherein the acidic amino acid or a salt thereof is an alkali metal salt of acidic amino acid.

10. A process according to claim 9, wherein the alkali metal salt of acidic amino acid is sodium aspartate or sodium glutamate.

11. A process for quantitatively determining the triglyceride content in a biological fluid which comprises adding to the biological fluid an aqueous solution containing

glycerol-3-phosphate oxidase in an amount of 1 to 20 units/ml,

a buffer solution in an amount sufficient to maintain the pH near neutral value,

an acidic amino acid or a salt thereof in an amount of 1 to 5% by weight of the aqueous solution,

lipase, glycerol kinase, peroxidase, 4-aminoantipyrine and an oxidizable color producing indicator in amounts sufficient to produce a color,

and measuring the color produced colorimetrically.

12. A process according to claim 11, wherein the acidic amino acid or a salt thereof is an alkali metal salt of acidic amino acid.

13. A process according to claim 12, wherein the alkali metal salt of acidic amino acid is sodium aspartate or sodium glutamate.

14. A process for quantitatively determining the choline content in a biological fluid which comprises adding to the biological fluid an aqueous solution containing

choline oxidase in an amount of 1 to 20 units/ml,

a buffer solution in an amount sufficient to maintain the pH near neutral value,

an acidic amino acid or a salt thereof in an amount of 1 to 5% by weight, based upon the weight of the aqueous solution,

peroxidase, 4-aminoantipyrine and an oxidizable color producing indicator in sufficient amounts to produce a color, and

measuring the color produced colormetrically.

15. A process according to claim 14, wherein the acidic amino acid or a salt thereof is an alkali metal salt of acidic amino acid.

16. A process according to claim 15, wherein the alkali metal salt of acidic amino acid is sodium aspartate or sodium glutamate.

17. A process for measuring the activity of choline esterase in a biological fluid which comprises adding to the biological fluid an aqueous solution containing

choline oxidase in an amount of 1 to 20 units/ml,

a buffer solution in an amount of 1 to 5% by weight based on the weight of the solution,

peroxidase, choline benzoyl chloride, 4-aminoantipyrine and an oxidizable color producing indicator in amounts sufficient to produce a color,

adding to the resulting mixture a reaction stopper solution, and

measuring the color produced colorimetrically.

18. A process according to claim 17, wherein the acidic amino acid or a salt thereof is an alkali metal salt of acidic amino acid.

19. A process according to claim 18, wherein the alkali metal salt of acidic amino acid is sodium aspartate or sodium glutamate.

21. A stabilized composition comprising

an oxidase selected from the group consisting of glycerol-3-phosphate oxidase, and choline oxidase,

an acidic amino acid or a salt thereof in an amount equal to 1 to 5% by weight of the composition, and

a buffer solution in an amount sufficient to maintain the pH near neutral value.

22. A composition according to claim 21, wherein the oxidase is glycerol-3-phosphate oxidase.

23. A composition according to claim 21, wherein the oxidase is choline oxidase.

24. A stabilized composition according to claim 21, wherein the acidic amino acid or a salt thereof is represented by the formula:

##STR7## wherein A is a lower alkylene group having 1 to 5 carbon atoms; and

Y and Y'"'"' are independently hydrogen, a NH₄ group or an alkali metal.

25. A composition according to claim 21, which further comprises a color producing reagent.

26. A composition according to claim 21, wherein the acidic amino acid or a salt thereof is sodium glutamate.

27. A composition according to claim 21, wherein the acidic amino acid or a salt thereof is sodium aspartate.

28. A composition according to claim 21 in which the acidic amino acid is selected from the group consisting of glutamic acid, aspartic acid, alpha-aminoadipic acid and salts of these acidic amino acids.