Stabilization of activated galactose oxidase enzyme

• US 4,220,503 A
• Filed: 04/28/1978
• Issued: 09/02/1980
• Est. Priority Date: 04/28/1978
• Status: Expired due to Term

• Current Assignee: YSI Incorporated (Xylem, Inc.)
• Original Assignee: The Yellow Springs Instrument Company Incorporated
• Inventors: Johnson, Jay M.
• Primary Examiner(s): Kaplan, G. L.
• Application Number:
  US05/901,144
• Time in Patent Office: 858 Days
• Field of Search: 204/1 T, 204/1 E, 204/1 P, 204/195 P, 204/195 B, 195/103.5 R, 195/103.5 C, 435/25, 435/26, 435/27, 435/28
• US Class Current: 205/778
• 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 1/004 mediator-assisted
• CPC Class Codes: C12Q 1/26 involving oxidoreductase

Abstract

Galactose oxidase enzyme can effectively be used for the determination of a number of different substances if activated and stabilized in accordance with the present invention. Activation is accomplished with a redox agent such as ferricyanide. Stabilization of the activated galactose oxidase is accomplished by addition of cupric ion. Upon stabilization, activated, immobilized galactose oxidase enzymes may be stored and reused for at least 25 days.

Claims

1. In a method for the quantitative determination of a substance which reacts with a galactose oxidase enzyme in a manner whereby a reactant or a product of said reaction may be analytically measured and wherein said substance is introduced for determination by being included in a buffer solution which is brought into contact with said enzyme, said buffer solution also containing a sufficient amount of a reduction-oxidation agent to activate said enzyme, the improvement comprising adding to the buffer solution which contacts said galactose oxidase enzyme a sufficient amount of cupric ion to stabilize said activated enzyme whereby the stabilized, activated galactose oxidase enzyme may be reused and stored for a period of time after use without loss of its enzymic activity.

2. The method of claim 1 wherein said reaction ultimately produces hydrogen peroxide.

3. The method of claim 2 wherein said reduction-oxidation agent is ferricyanide.

4. The method of claim 3 wherein said hydrogen peroxide production is measured colorimetrically.

5. The method of claim 3 wherein said hydrogen peroxide production is measured polarographically.

6. A method for the quantitative polarographic determination of a polarographically inactive material which reacts with galactose oxidase enzyme in a manner whereby a reactant or a product of said reaction may be polarographically measured, comprising:

(a) providing a polarographic cell including at least one electrode positioned behind a membrane permeable to the material being measured and in contact with an electrolyte,

(b) establishing a potential across said cell such that a current is produced which is proportional to the amount of hydrogen peroxide or oxygen present on the electrode side of said membrane,

(c) bringing said cell into contact with a quantity of material containing said polarographically inactive material in the presence of galactose oxidase enzyme and a buffer containing a reduction-oxidation agent

said reduction-oxidation agent being present in said buffer in a sufficient amount to activate the galactose-oxidase enzyme,

(d) adding to said buffer a sufficient amount of cupric ion to stabilize the activated enzyme, and

(e) determining the current flowing across said cell as an indication of the amount of said substance present in said quantity of material.

7. The method of claim 6 wherein said galactose oxidase enzyme is immobilized in or on said membrane.

8. The method of claim 7 wherein said reduction-oxidation agent is ferricyanide.

9. The method of claim 8 wherein said polarographically inactive material is galactose.

10. The method of claim 9 wherein the determination of current flowing across said cell is a function of the amount of hydrogen peroxide formed.

11. The method of claim 10 wherein said galactose oxidase enzyme is immobilized with glutaraldehyde between an inner and outer membrane layer relative to said electrode.

12. The method of claim 11 wherein said inner layer is cellulose acetate and said outer layer is a perforated polycarbonate film.

13. The method of claim 12 wherein said buffer contains potassium ferricyanide in the approximate amount of 10 milligrams per 100 ml of buffer and cupric chloride dihydrate in the approximate amount of 0.2 milligrams per 100 ml of buffer.