Method of forming stabilized urease solutions

• US 4,378,430 A
• Filed: 12/12/1980
• Issued: 03/29/1983
• Est. Priority Date: 09/11/1979
• Status: Expired due to Term

• Current Assignee: Bank Of America NA (Bank of America Corp.)
• Original Assignee: Ivan E. Modrovich
• Inventors: Modrovich, Ivan E.
• Primary Examiner(s): Castel, Benoit
• Application Number:
  US06/215,622
• Time in Patent Office: 837 Days
• Field of Search: 435/12, 435/188, 435/810, 435/7, 23/230 B, 252/408 R
• US Class Current: 435/12
• CPC Class Codes: C12N 9/80 acting on amide bonds in linear amides (3.5.1)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/58 involving urea or urease

Abstract

A stabilized urease solution is formulated by blending water, buffering agents, a bacteriostat, chelating agent, polyhydroxy organic compound and urease. The preferred buffering agents are selected from low-conductivity zwitterionic buffer salts, such as N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid, triethanolamine, tris(hydroxymethyl)aminomethane, diethanolamine, aminomethyl propanol and mixtures thereof.

Claims

1. A process for forming a stabilized urease solution comprising the steps of:

(a) mixing a stabilizing amount of N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid and water which is substantially free of metal ions;

(b) adding a bacteriostat to the mixture, which bacteriostat comprises an alcohol which is nondegradative to urease and is present in an amount sufficient to inhibit bacteria growth;

(c) adding a chelating agent to the mixture for complexing any metal ions present in the mixture;

(d) buffering the mixture to a pH range of from about 5 to about 9 by adding a buffering agent comprising a zwitterionic, low-conductivity compound selected from the group consisting of N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid, triethanolamine, tris(hydroxymethyl)aminomethane, diethanolamine, aminomethyl propanol and mixtures thereof, the amount of buffering agent being sufficient to maintain pH in the presence of ammonia formed from urea;

(e) admixing with the mixture a polyhydroxy organic compound selected from the group consisting of glycerol, ethylene glycol, sorbitol, and propylene glycol; and

(f) adding urease enzyme to the mixture to form a solution wherein the urease is effectively stabilized by the presence of the N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid, bacteriostat and the polyhydroxy organic compound.

2. A process as recited in claim 1 further comprising the step of diluting the urease solution with an aqueous solution of 2,4-dichlorophenol.

3. A process as recited in claim 1 wherein N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid is mixed with water in an amount which comprises about 0.5 to about 5% by weight of the water.

4. A process as recited in claim 1 wherein the bacteriostat comprises 2,4-dichlorophenol.

5. A process as recited in claim 4 wherein the 2,4-dichlorophenol is added in an amount from about 0.1 to about 1.0% by weight of the solution.

6. A process as recited in claim 1 wherein the chelating agent comprises ethylenediaminetetraacetic acid.

7. A process as recited in claim 6 wherein the ethylenediaminetetraacetic acid is added in an amount up to about 1% by weight of the solution.

8. A process as recited in claim 1 wherein the buffering agent provides a pH in the range from about 6 to about 8.

9. A process as recited in claim 1 wherein the buffering agent comprises triethanolamine.

10. A process as recited in claim 1 wherein the polyhydroxy organic compound is added in an amount of about 50% by volume of the solution.

11. A process as recited in claim 1 wherein the polyhydroxy organic compound comprises glycerol.

12. A process for forming a stabilized urease solution comprising the steps of:

(a) mixing a stabilizing amount of N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid and water which is substantially free of metal ions;

(b) adding 2,4-dichlorophenol to the mixture in an amount sufficient to inhibit bacteria growth;

(c) adding ethylenediaminetetraacetic acid to the mixture;

(d) buffering the mixture to a pH of from about 6 to about 8 by adding triethanolamine, the total amount of buffer being sufficient to maintain pH in the presence of ammonia formed from urea;

(e) admixing glycerol with the mixture; and

(f) adding urease enzyme to the mixture to form a solution wherein the urease is effectively stabilized by the presence of N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid, 2,4-dichlorophenol and glycerol.

13. A process for forming a stabilized urease solution comprising the steps of:

(a) mixing a stabilizing amount of N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid and water which is substantially free of metal ions;

(b) adding a bacteriostat which is nondegradative to urease to the mixture in an amount sufficient to inhibit bacteria growth;

(c) adding a chelating agent to the mixture for complexing any metal ions present in the mixture;

(d) buffering the mixture to a pH range of from about 5 to about 9 by adding a buffering agent comprising at least one zwitterionic, low-conductivity compound, the amount of buffering agent present being sufficient to maintai pH in the presence of ammonia formed from urea;

(e) admixing with the mixture a polyhydroxy organic compound; and

(f) adding urease enzyme to the mixture to form a solution wherein the urease enzyme is effectively stabilized by the presence of N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid, bacteriostat and polyhydroxy organic compound.

14. A stabilized urease solution for analysis of urea which comprises an admixture formed by urease enzyme addition to an aqueous solution of a stabilizing amount of N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid, a bacteriostat non-degradable to urease enzyme present to inhibit bacteria growth, a chelating agent for complexing metal ions in the aqueous solution, at least one zwitterionic, low-conductivity compound, the amount of zwitterionic compound present being sufficient to provide a solution having a pH of from about 5 to about 9 and sufficient to maintain with the N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid a solution of pH in the presence of ammonia formed from urea, and a stabilizing amount of at least one polyhydroxy organic compound, said aqueous solution being substantially free of free metal ions.

15. A stabilized urease solution for urea analysis which comprises an admixture formed by urease enzyme addition to an aqueous solution of a stabilizing amount of N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid, 2,4-dichlorophenol to inhibit bacteria growth, ethylenediaminetetraacetic acid, triethanolamine in an amount sufficient to provide a solution pH of from about 6 to about 8 and sufficient to maintain pH in the presence of ammonia formed from urea, and a stabilizing amount of glycerol, said solution being substantially free of free metal ions.

16. A stabilized urease solution kit which comprises:

(a) a reagent concentrate solution comprising distilled water, N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid, a chelating agent for heavy metal ions, a bacteriostat to inhibit bacteria growth, at least one zwitterionic low-conductivity buffering compound, a polyhydroxy compound, and urease; and

(b) a diluent solution selected from distilled water and a solution of bacteriostat in distilled water, the reagent concentrate and diluent solution when combined containing sufficient buffering compound to provide a solution pH of from about 5 to about 9 in the presence of ammonia formed from urea, the N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid, bacteriostat and polyhydroxy compound cooperating to stabilize the urease, both in the reagent solution and when the reagent and diluent solutions are combined.

17. A stabilized urease solution kit which comprises:

(a) a reagent concentration solution comprising distilled water, N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid, 2,4-dichlorophenol in an amount sufficient to inhibit bacteria growth, ethylenediaminetetraacetic acid, triethanolamine, glycerol, and urease; and

(b) a diluent solution selected from distilled water and a solution of 2,4-dichlorophenol and distilled water,

the reagent solution and diluent which when combined contain sufficient N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid and triethanolamine to provide a solution pH of from about 6 to about 8 which is maintainable in the presence of ammonia formed from urea, the N-2-hydroxyethyl-piperazine-N'"'"'-2-ethanesulfonic acid, 2,4-dichlorophenol and glycerol cooperating to stabilize urease, both in the reagent solution and when the reagent and diluent solutions are combined.