US3947324A
Expired / Lapsed
US
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Method for isolating high purity peroxidase
Assignee: GD Searle LLC
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Filing: 1975-04-21
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Granted: 1976-03-30
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Expires: 1995-04-21
Abstract
Abstract The present invention encompasses a method for isolating peroxidase enzyme from plant tissue containing peroxidase. The invention has as its critical step treating an aqueous extract of said plant tissue having the pH adjusted to 6 - 9 with at least 2.7 × 10 - 3 moles per liter of zinc ion. Unexpectedly at this pH the zinc ion selectively precipitates contaminating impurities from the extract and thereby provides a substantially purified peroxidase solution from which peroxidase is isolated by a variety of conventional techniques. Thus the critical step of the present invention is used in conjunction with salt fractionation, solvent fractionation, dialysis, reverse osmosis, electrophoresis, column chromatography and other techniques for purifying protein. The peroxidase isolated by methods of the present invention is useful in analytical diagnostic kits and other applications in enzyme and substrate assays in conjunction with a chromogenic donor in assaying systems producing hydrogen peroxide.
Inventors
- 👤 Krishnaiyer Lakshminarayanan
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-27 22:59
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0065; Y; Y10; Y10S; Y10S435/00; Y10S435/814; Y10S435/815; Y10S435/816
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
62 words
A method for isolating peroxidase enzyme from plant tissue containing peroxidase enzyme comprising treating aqueous extracts of said plant tissue having a pH adjusted to 6 - 9 with at least 2.7 × 10 - 3 moles per liter of zinc ion to form a zinc ion-protein contaminant precipitate and supernate, removing the precipitate, and separating the peroxidase enzyme from the supernate.
Claim 2
Independent Claim
62 words
A method for isolating peroxidase enzyme from plant tissue containing peroxidase enzyme comprising treating aqueous extracts of said plant tissue having a pH adjusted to 6 - 9 with at least 2.7 × 10 - 3 moles per liter of zinc ion to form a zinc ion-protein contaminant precipitate and supernate, removing the precipitate, and separating the peroxidase enzyme from the supernate.
Claim 3
Dependent Claim
59 words
The method according to claim 1 for isolating peroxidase enzyme from horseradish root comprising treating aqueous extracts of horseradish roots having a pH adjusted 6 - 9 with at least 2.7 × 10.sup. -3 moles per liter of zinc to form a zinc ion-protein contaminant precipitate and supernate, removing the precipitate, and separating the peroxidase enzyme from the supernate.
Claim 4
Dependent Claim
31 words
The method according to claim 1 wherein the pH is 7.5 - 8.5 and the zinc ion concentration is 2.7 × 10.sup. -2 to 1.4 × 10.sup. -2 moles per liter.
Claim 5
Dependent Claim
74 words
In a method for isolating peroxidase enzyme from plant tissue containing peroxidase enzyme of type including extraction, salt fractionation, dialysis, and solvent fractionation, the improvement which comprises treating aqueous extracts of said plant tissue having a pH adjusted to 6 - 9 with at least 2.7 × 10.sup. -3 moles per liter of zinc ion to form a zinc ion protein contaminant precipitate, removing the precipitate and separating the peroxidase enzyme from the supernate.
Claim 6
Dependent Claim
74 words
In a method for isolating peroxidase enzyme from plant tissue containing peroxidase enzyme of type including extraction, salt fractionation, dialysis, and solvent fractionation, the improvement which comprises treating aqueous extracts of said plant tissue having a pH adjusted to 6 - 9 with at least 2.7 × 10.sup. -3 moles per liter of zinc ion to form a zinc ion protein contaminant precipitate, removing the precipitate and separating the peroxidase enzyme from the supernate.
Claim 7
Independent Claim
74 words
A method for further purifying substantially pure peroxidase having protein contaminant and having an RZ value of 0.1 to 1.3 comprising treating an aqueous solution of substantially pure peroxidase having a pH adjusted to 6 - 9 with at least 2.7 × 10.sup. -3 moles per liter of zinc ion thereby forming a zinc ion protein contaminant precipitate, separating the precipitate, and isolating the purified peroxidase having an RZ value from 1.0 - 3.0.
Claim 8
Independent Claim
74 words
A method for further purifying substantially pure peroxidase having protein contaminant and having an RZ value of 0.1 to 1.3 comprising treating an aqueous solution of substantially pure peroxidase having a pH adjusted to 6 - 9 with at least 2.7 × 10.sup. -3 moles per liter of zinc ion thereby forming a zinc ion protein contaminant precipitate, separating the precipitate, and isolating the purified peroxidase having an RZ value from 1.0 - 3.0.
Claim 9
Dependent Claim
15 words
A method as in claim 1 wherein the source of peroxidase is horseradish root washings.
Claim 10
Dependent Claim
24 words
A method as in claim 1 wherein the zinc ion concentration is 2.7 × 10.sup. -3 to 1 × 10.sup. -1 moles per liter.
Claim 11
Dependent Claim
17 words
A method as in claim 1 wherein the peroxidase is separated from the supernate by reverse osmosis.
Claim 12
Dependent Claim
20 words
A method as in claim 8 wherein the peroxidase is further separated from the supernate by absorption on carboxymethyl cellulose.
Claim 13
Dependent Claim
143 words
A method according to claim 1 for isolating peroxidase comprising: a. extracting horseradish root with water, b. adjusting the pH to 7.5 - 8.5, c. adding 2.7 × 10.sup. -3 to 1 × 10.sup. -1 moles per liter of zinc ion to form a zinc ion -- contaminant precipitate, d. removing the precipitate by filtration or centrifugation, e. removing zinc ion and low molecular weight organic material from the supernate by reverse osmosis to provide a concentrated supernate having a conductivity of 1 - 3 mMho, f. absorbing the concentrated supernate on carboxymethyl cellulose at a pH of 4 - 7, g. eluting peroxidase from the carboxymethyl cellulose with aqueous buffer having a pH of 7 - 9, h. precipitating the peroxidase form the aqueous buffer with methanol, ethanol, dioxane, propanol, isopropanol, acetone or mixtures thereof, and i. filtering and drying the precipitate.
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) | 1979-03-30 | 1979-09-29 | 1980-03-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1983-03-30 | 1983-09-29 | 1984-03-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1987-03-30 | 1987-09-29 | 1988-03-30 | $7700.00 | Window Closed / Lapsed |