US4294923A
Expired / Lapsed
US
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Substrates and method for determining enzymes
Assignee: Individual
•
Filing: 1979-04-23
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Granted: 1981-10-13
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Expires: 1999-04-23
Abstract
Abstract A method is disclosed for determining the presence of an enzyme in a biological fluid, which includes the steps of contacting the fluid with a synthetic chromogenic substrate, which is an amino acid derivative of 7-amino-4-trifluoromethylcoumarin; incubating the substrate-containing fluid to effect enzymatic hydrolysis; and fluorometrically determining the presence of the free 7-amino-4-trifluoromethylcoumarin chromophore in the hydrolyzate.
Inventors
- 👤 Robert E. Smith
- 👤 Eugene R. Bissell
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 12:02
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/34; C12Q1/37; C07; C07D; C07D311/00; C07D311/02; C07D311/04; C07D311/06; C07D311/08; C07D311/16; C07K; C07K5/00; C07K5/04; C07K5/06; C07K5/06191; C07K5/08; C07K5/0827; C12Q2337/00; C12Q2337/20; C12Q2337/24; Y; Y10; Y10S; Y10S930/00; Y10S930/01; Y10S930/28
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
104 words
A method for determining the presence of an enzyme in an enzyme-containing analyte, comprising: (a) contacting the analyte with a substrate which can be hydrolyzed by said enzyme to liberate 7-amino-4-trifluoromethylcoumarin, said substrate having the formula ##STR6## wherein R is an amino acid, a peptide, an amino acid which has been reacted with a blocking group, or a peptide, the terminal amino acid of which has been reacted with a blocking group, thereby forming an analyte-substrate mixture; (b) incubating the analyte-substrate mixture under enzyme-hydrolyzing conditions to form an enzyme hydrolyzate; and (c) fluorometrically or spectrophotometrically determining the presence of 7-amino-4-trifluoromethylcoumarin in the enzyme hydrolyzate.
Claim 2
Independent Claim
104 words
A method for determining the presence of an enzyme in an enzyme-containing analyte, comprising: (a) contacting the analyte with a substrate which can be hydrolyzed by said enzyme to liberate 7-amino-4-trifluoromethylcoumarin, said substrate having the formula ##STR6## wherein R is an amino acid, a peptide, an amino acid which has been reacted with a blocking group, or a peptide, the terminal amino acid of which has been reacted with a blocking group, thereby forming an analyte-substrate mixture; (b) incubating the analyte-substrate mixture under enzyme-hydrolyzing conditions to form an enzyme hydrolyzate; and (c) fluorometrically or spectrophotometrically determining the presence of 7-amino-4-trifluoromethylcoumarin in the enzyme hydrolyzate.
Claim 3
Dependent Claim
20 words
The method of claim 1, wherein R is an amino acid chain comprising from 1 to about 12 amino acids.
Claim 4
Dependent Claim
20 words
The method of claim 1, wherin R is an amino acid chain comprising from 1 to about 6 amino acids.
Claim 5
Dependent Claim
9 words
The method of claim 1, wherein R is gly-gly-arg-.
Claim 6
Dependent Claim
9 words
The method of claim 1, wherein R is d-ala-leu-lys-.
Claim 7
Dependent Claim
9 words
The method of claim 1, wherein R is leu-.
Claim 8
Dependent Claim
9 words
The method of claim 1, werein R is Cbz-Val-Lys-Lys-Arg-.
Claim 9
Independent Claim
27 words
The method of any one of claims 2, 3, 4, 5, 6, or 7, wherein said blocking group is carbobenzoxy, benzoyl, glutaryl, t-butyloxycarbonyl, d-proline, d-valine or d-alanine.
Claim 10
Dependent Claim
31 words
The method of claim 1, wherein said enzyme-hydrolyzing conditions include a pH of from about 4 to about 10 and a temperature of from about 15° C. to about 50° C.
Claim 11
Dependent Claim
31 words
The method of claim 1, wherein said enzyme-hydrolyzing conditions include a pH of from about 5 to about 8.5 and a temperature of from about 20° C. to about 40° C.
Claim 12
Dependent Claim
30 words
The method of claim 9, wherein the determination is a fluorometric rate determination and employs an excitation wavelength of about 400 nm and an emission wavelength of about 505 nm.
Claim 13
Dependent Claim
19 words
The method of claim 9 wherein the determination is spectrophotometric and absorbance measurements are made at about 380 nm.
Claim 14
Dependent Claim
23 words
The method of claim 9, 10, 11, or 12 wherein the analyte is a biological fluid and the enzyme is a proteinase enzyme.
Claim 15
Dependent Claim
16 words
The method of claim 13, wherein the biological fluid is the cytoplasm within a single cell.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1984-10-12 | 1985-04-13 | 1985-10-13 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1988-10-12 | 1989-04-13 | 1989-10-13 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1992-10-12 | 1993-04-13 | 1993-10-13 | $7700.00 | Window Closed / Lapsed |