US4220503A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Stabilization of activated galactose oxidase enzyme
Assignee: YSI Inc
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Filing: 1978-04-28
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Granted: 1980-09-02
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Expires: 1998-04-28
Abstract
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.
Inventors
- 👤 Jay M. Johnson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 17:27
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0006; C12N9/96; C12Q; C12Q1/00; C12Q1/001; C12Q1/004; C12Q1/26
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
122 words
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.
Claim 2
Dependent Claim
122 words
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.
Claim 3
Dependent Claim
12 words
The method of claim 1 wherein said reaction ultimately produces hydrogen peroxide.
Claim 4
Dependent Claim
11 words
The method of claim 2 wherein said reduction-oxidation agent is ferricyanide.
Claim 5
Dependent Claim
13 words
The method of claim 3 wherein said hydrogen peroxide production is measured colorimetrically.
Claim 6
Dependent Claim
13 words
The method of claim 3 wherein said hydrogen peroxide production is measured polarographically.
Claim 7
Independent Claim
180 words
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.
Claim 8
Independent Claim
180 words
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.
Claim 9
Dependent Claim
17 words
The method of claim 6 wherein said galactose oxidase enzyme is immobilized in or on said membrane.
Claim 10
Dependent Claim
11 words
The method of claim 7 wherein said reduction-oxidation agent is ferricyanide.
Claim 11
Dependent Claim
12 words
The method of claim 8 wherein said polarographically inactive material is galactose.
Claim 12
Dependent Claim
24 words
The method of claim 9 wherein the determination of current flowing across said cell is a function of the amount of hydrogen peroxide formed.
Claim 13
Dependent Claim
25 words
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.
Claim 14
Dependent Claim
21 words
The method of claim 11 wherein said inner layer is cellulose acetate and said outer layer is a perforated polycarbonate film.
Claim 15
Dependent Claim
39 words
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.
References Cited (Backward Citations)
US3539455A
Cited
US3886045A
Cited
GB1442303A
Cited
US3947328A
Cited
US3979274A
Cited
US4073713A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1983-09-02 | 1984-03-03 | 1984-09-02 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1987-09-02 | 1988-03-03 | 1988-09-02 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1991-09-02 | 1992-03-03 | 1992-09-02 | $7700.00 | Window Closed / Lapsed |