US4268631A
Expired / Lapsed
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Apoglucose oxidase preparation
Assignee: Bayer Corp
•
Filing: 1979-06-04
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Granted: 1981-05-19
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Expires: 1999-06-04
Abstract
Abstract An apoglucose oxidase preparation having a residual glucose oxidase activity of less than 0.05%, and preferably less than 0.005%. The preparation is obtained by incubation of glucose oxidase in aqueous solution at a pH of less than about 2 and in the presence of about 20 to about 40, preferably about 30, percent by volume glycerol, and separating resulting dissociated prosthetic group, flavin adenine dinucleotide, from apoglucose oxidase by column chromatography, preferably on cross-linked dextran gel. The purified apoglucose oxidase is useful as a reagent in specific binding assays employing flavin adenine dinucleotide as a label.
Inventors
- 👤 Paul B. Ellis
- 👤 David L. Morris
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 21:55
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0006; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/536; G01N33/542; Y; Y10; Y10S; Y10S435/00; Y10S435/814; Y10S435/815
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
98 words
A method for preparing apoglucose oxidase comprising the steps of: (a) incubating an aqueous solution of glucose oxidase containing between about 20 and about 40 percent by volume glycerol and having a pH of less than about 2, (b) separating resulting dissociated flavin adenine dinucleotide from apoglucose oxidase in said incubated solution by column chromatography in an aqueous environment containing between about 20 and about 40 percent by volume glycerol and at a pH of less than about 2, and (c) collecting effluent containing apoglucose oxidase and adjusting the pH thereof to between about 6.0 and about 7.5.
Claim 2
Independent Claim
98 words
A method for preparing apoglucose oxidase comprising the steps of: (a) incubating an aqueous solution of glucose oxidase containing between about 20 and about 40 percent by volume glycerol and having a pH of less than about 2, (b) separating resulting dissociated flavin adenine dinucleotide from apoglucose oxidase in said incubated solution by column chromatography in an aqueous environment containing between about 20 and about 40 percent by volume glycerol and at a pH of less than about 2, and (c) collecting effluent containing apoglucose oxidase and adjusting the pH thereof to between about 6.0 and about 7.5.
Claim 3
Dependent Claim
24 words
The method of claim 1 wherein the percent by volume of glycerol in the aqueous solution referred to in step (a) is about 30%.
Claim 4
Dependent Claim
26 words
The method of claim 1 or 2 wherein the percent by volume of glycerol in the aqueous environment referred to in step (b) is about 30%.
Claim 5
Dependent Claim
20 words
The method of claim 1 wherein the pH of the aqueous solution referred to in step (a) is about 1.4.
Claim 6
Dependent Claim
53 words
The method of claim 1 wherein step (b) is accomplished by passing the incubated solution through a column of cross-linked dextran gel equilibrated with an aqueous solution containing between about 20 and about 40 percent by volume glycerol and having a pH of less than about 2, and collecting the effluent containing protein.
Claim 7
Independent Claim
123 words
A method for preparing apoglucose oxidase comprising the steps of: (a) incubating an aqueous solution of glucose oxidase containing between about 20 and about 40 percent by volume glycerol and having a pH of less that about 2, (b) passing the incubated solution through a chromatography column equilibrated with an aqueous solution containing between about 20 and about 40 percent by volume glycerol and having a pH of less than about 2, (c) collecting the effluent fractions containing protein, (d) adding an adsorbent for flavin adenine dinucleotide to the combined protein-containing effluent fractions and adjusting the pH of the resulting aqueous mixture to between about 6.0 and about 7.5, and (e) separating the adsorbent from the aqueous mixture, leaving an apoglucose oxidase solution.
Claim 8
Independent Claim
123 words
A method for preparing apoglucose oxidase comprising the steps of: (a) incubating an aqueous solution of glucose oxidase containing between about 20 and about 40 percent by volume glycerol and having a pH of less that about 2, (b) passing the incubated solution through a chromatography column equilibrated with an aqueous solution containing between about 20 and about 40 percent by volume glycerol and having a pH of less than about 2, (c) collecting the effluent fractions containing protein, (d) adding an adsorbent for flavin adenine dinucleotide to the combined protein-containing effluent fractions and adjusting the pH of the resulting aqueous mixture to between about 6.0 and about 7.5, and (e) separating the adsorbent from the aqueous mixture, leaving an apoglucose oxidase solution.
Claim 9
Dependent Claim
26 words
The method of claim 6 wherein the percent by volume of glycerol in the aqueous solutions referred to in steps (a) and (b) is about 30%.
Claim 10
Dependent Claim
22 words
The method of claim 6 wherein the pH of the aqueous solutions referred to in steps (a) and (b) is about 1.4.
Claim 11
Dependent Claim
30 words
The method of claim 6 wherein said aqueous glucose oxidase solution is incubated at between about -10 and about 10° C. and for between about 1 and about 3 hours.
Claim 12
Dependent Claim
19 words
The method of claim 9 wherein said incubation is carried out at about 4° C. for about 2 hours.
Claim 13
Dependent Claim
26 words
The method of claim 6 wherein said adsorbent added in step (d) is in a finely divided form and is separated in step (e) by filtration.
Claim 14
Dependent Claim
13 words
The method of claim 6 wherein said adsorbent is finely divided coated charcoal.
Claim 15
Dependent Claim
20 words
The method of claim 6 wherein the pH of the resulting solution in step (d) is adjusted to about 7.0.
Claim 16
Dependent Claim
19 words
The method of claim 6 wherein the glucose oxidase used has been dialyzed to remove low molecular weight materials.
Claim 17
Dependent Claim
15 words
An apoglucose oxidase preparation characterized by a residual glucose oxidase activity of less than 0.05%.
Claim 18
Dependent Claim
15 words
An apoglucose oxidase preparation characterized by a residual glucose oxidase activity of less than 0.05%.
Claim 19
Dependent Claim
15 words
An apoglucose oxidase preparation characterized by a residual glucose oxidase activity of less than 0.005%.
Claim 20
Dependent Claim
15 words
An apoglucose oxidase preparation characterized by a residual glucose oxidase activity of less than 0.005%.
Claim 21
Dependent Claim
25 words
The preparation of claim 15 or 16 in the form of an aqueous solution containing between about 20 and about 40 percent by volume glycerol.
Claim 22
Dependent Claim
19 words
The preparation of claim 17 wherein the percent by volume of glycerol in the aqueous solution is about 30%.
Claim 23
Dependent Claim
11 words
The preparation of claims 15 or 16 in a dry form.
Claim 24
Dependent Claim
9 words
The preparation of claim 19 in a lyophilized form.
Claim 25
Dependent Claim
16 words
The method of claim 6 wherein said chromatography column is a column of cross-linked dextran gel.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1984-05-18 | 1984-11-17 | 1985-05-19 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1988-05-18 | 1988-11-17 | 1989-05-19 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1992-05-18 | 1992-11-17 | 1993-05-19 | $7700.00 | Window Closed / Lapsed |