US4327178A
Expired / Lapsed
US
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Urinary kallikrein assay: specific substrates and assay method
Assignee: University of Miami
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Filing: 1981-03-31
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Granted: 1982-04-27
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Expires: 2001-03-31
Abstract
Abstract Substrates and method for the assay of urinary kallikrein are provided. The substrates have the general formula ##STR1## wherein R is H, acetyl, benzoyl, cyclopentylcarbonyl, succinyl, R 1 --Ser 1 or R 1 --Phe--Ser where R 1 is H, acetyl, benzoyl, cyclopentylcarbonyl or succinyl, X is H, tritium, 3-iodo or 4-iodo, and n is 0 or 1. Radioactive label may be incorporated in the anilide or benzylamide moiety. Hydrolysis catalyzed by urinary kallikrein yields labeled aniline or benzylamine as product. The assay method includes mixing the enzyme with substrate in a buffered solution, pH 7.5-10.5, the substrate being present preferably at a concentration substantially below K m . After incubating to allow the reaction to proceed, the reaction is terminated and the radioactive hydrolysis product is separated and counted. Also disclosed are compounds of the type ##STR2## wherein R, R 1 and n are as defined above. X is 3--OH or 4--OH. Y 1 is 3-iodo if X is 4--OH or 6-iodo if X is 3--OH. Y 2 may be H or 5-iodo if Y 1 is 3-iodo, or 4-iodo if Y 1 is 6-iodo.
Inventors
- 👤 James W. Ryan
- 👤 Alfred Chung
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 18:12
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/34; C12Q1/37; Y; Y10; Y10S; Y10S530/00; Y10S530/802
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
65 words
A composition for the assay of mammalian urinary kallikrein comprising a human urinary kallikrein substrate having the formula ##STR3## wherein R is hydrogen, acetyl, benzoyl, cyclopentanecarbonyl, succinyl, R 1 --L--Ser or R 1 --L-Phe-Ser where R 1 is hydrogen, acetyl, benzoyl, cyclopentanecarbonyl or succinyl; X is hydrogen, tritium, 3-iodo or 4-iodo; and, n is 0 or 1; and suitable buffer in the pH range 7.5-10.5.
Claim 2
Dependent Claim
65 words
A composition for the assay of mammalian urinary kallikrein comprising a human urinary kallikrein substrate having the formula ##STR3## wherein R is hydrogen, acetyl, benzoyl, cyclopentanecarbonyl, succinyl, R 1 --L--Ser or R 1 --L-Phe-Ser where R 1 is hydrogen, acetyl, benzoyl, cyclopentanecarbonyl or succinyl; X is hydrogen, tritium, 3-iodo or 4-iodo; and, n is 0 or 1; and suitable buffer in the pH range 7.5-10.5.
Claim 3
Dependent Claim
13 words
A composition according to claim 1 wherein the buffer pH range is 8.5-10.5.
Claim 4
Dependent Claim
13 words
A composition according to claim 1 wherein the buffer pH range is 9.0-10.0.
Claim 5
Dependent Claim
22 words
A composition according to claim 3 wherein R is hydrogen, L--Ser or L--Phe--L--Ser; X is hydrogen or tritium, and n is 0.
Claim 6
Dependent Claim
19 words
A composition according to claim 3 wherein R is H, X is H or tritium, and n is 0.
Claim 7
Dependent Claim
22 words
A composition according to claim 3 wherein R is hydrogen, L--Ser or L--Phe--L--Ser; X is hydrogen or tritium, and n is 1.
Claim 8
Dependent Claim
19 words
A composition according to claim 3 wherein R is H, X is H or tritium, and n is 1.
Claim 9
Dependent Claim
75 words
An inhibitor composition of human urinary kallikrein comprising a compound having the formula ##STR4## wherein R is hydrogen, acetyl, benzoyl, cyclopentanecarbonyl, succinyl, R 1 --L--Ser or R 1 --L--Phe--L--Ser where R 1 is hydrogen, acetyl, benzoyl, cyclopentanecarbonyl or succinyl; X is hydrogen, tritium, 3-iodo or 4-iodo; and, n is 0 or 1; said compound being present with the kallikrein at a concentration greater than 10 μM in a suitable buffer in the pH range 7.0-10.5.
Claim 10
Dependent Claim
75 words
An inhibitor composition of human urinary kallikrein comprising a compound having the formula ##STR4## wherein R is hydrogen, acetyl, benzoyl, cyclopentanecarbonyl, succinyl, R 1 --L--Ser or R 1 --L--Phe--L--Ser where R 1 is hydrogen, acetyl, benzoyl, cyclopentanecarbonyl or succinyl; X is hydrogen, tritium, 3-iodo or 4-iodo; and, n is 0 or 1; said compound being present with the kallikrein at a concentration greater than 10 μM in a suitable buffer in the pH range 7.0-10.5.
Claim 11
Independent Claim
172 words
A method for the assay of human urinary kallikrein comprising: mixing said kallikrein with a substrate compound having the formula ##STR5## wherein R is hydrogen, acetyl, benzoyl, cyclopentanecarbonly, succinyl, R 1 --Ser, or R 1 --Phe-Ser where R 1 is hydrogen, acetyl, benzoyl, cyclopentanecarbonyl or succinyl; X is hydrogen, tritium, 3-iodo or 4-iodo; and, n is 0 or 1; at least a portion of the molecules of said substrate having a [ 3 H], [ 14 C] or [ 125 I]]substituted for the C 1 , H or I atoms in that portion of the substrate connected by amide linkage to the carboxy group of the arginine moiety, said substrate being present at a concentration less than 10 μm in a buffer having a pH in the range 7.5-10.5; incubating the kallikrein with the substrate to permit kallikrein-catalyzed hydrolysis thereof; terminating the kallikrein-catalyzed hydrolysis; separating the hydrolysis product from the substrate; and, measuring the amount of product formed by counting the radioactivity thereof, whereby an assay of the kallikrein activity is obtained.
Claim 12
Independent Claim
172 words
A method for the assay of human urinary kallikrein comprising: mixing said kallikrein with a substrate compound having the formula ##STR5## wherein R is hydrogen, acetyl, benzoyl, cyclopentanecarbonly, succinyl, R 1 --Ser, or R 1 --Phe-Ser where R 1 is hydrogen, acetyl, benzoyl, cyclopentanecarbonyl or succinyl; X is hydrogen, tritium, 3-iodo or 4-iodo; and, n is 0 or 1; at least a portion of the molecules of said substrate having a [ 3 H], [ 14 C] or [ 125 I]]substituted for the C 1 , H or I atoms in that portion of the substrate connected by amide linkage to the carboxy group of the arginine moiety, said substrate being present at a concentration less than 10 μm in a buffer having a pH in the range 7.5-10.5; incubating the kallikrein with the substrate to permit kallikrein-catalyzed hydrolysis thereof; terminating the kallikrein-catalyzed hydrolysis; separating the hydrolysis product from the substrate; and, measuring the amount of product formed by counting the radioactivity thereof, whereby an assay of the kallikrein activity is obtained.
Claim 13
Dependent Claim
15 words
The method of claim 9 wherein the buffer has a pH in the range 8.5-10.5.
Claim 14
Dependent Claim
19 words
The method of claim 9 wherein the hydrolysis is terminated by addition of an excess of 0.1 N NaOH.
Claim 15
Dependent Claim
20 words
The method of claim 9 wherein the hydrolysis product is separated by extracting the reaction mixture with an aprotic solvent.
Claim 16
Dependent Claim
33 words
The method of claim 12 wherein the buffer has a pH in the range 9.0-10.0, the hydrolysis is terminated by adding an excess of 0.1 N NaOH and the aprotic solvent is toluene.
Claim 17
Dependent Claim
77 words
A composition for the assay of mammalian urinary kallikrein comprising a urinary kallikrein having the formula ##STR6## wherein R is hydrogen, L--Ser or L--Phe-L--Ser; n is 0 or 1; x is 3-hydroxy or 4-hydroxy; Y 1 is 3-iodo if X is 4-hydroxy or 6-iodo if X is 3-hydroxy; and, Y 2 is hydrogen or 5-iodo if Y 1 is 3-iodo or 4-iodo if Y 1 is 6-iodo; and a suitable buffer in the pH range of 7.5-10.5.
Claim 18
Dependent Claim
77 words
A composition for the assay of mammalian urinary kallikrein comprising a urinary kallikrein having the formula ##STR6## wherein R is hydrogen, L--Ser or L--Phe-L--Ser; n is 0 or 1; x is 3-hydroxy or 4-hydroxy; Y 1 is 3-iodo if X is 4-hydroxy or 6-iodo if X is 3-hydroxy; and, Y 2 is hydrogen or 5-iodo if Y 1 is 3-iodo or 4-iodo if Y 1 is 6-iodo; and a suitable buffer in the pH range of 7.5-10.5.
Claim 19
Dependent Claim
30 words
The composition of claim 14 wherein at least a portion of said iodo-substituents are [ 125 I] or [ 131 I] and the buffer in the pH range of 8.0-10.0.
Claim 20
Independent Claim
165 words
A method for the assay of urinary kallikrein comprising: mixing said kallikrein with a substrate having the formula ##STR7## wherein R is hydrogen, L--Ser or L--Phe-L--Ser; n is 0 or 1; X is 3-hydroxy or 4-hydroxy; Y 1 is 3-iodo if X is 4-hydroxy or 6-iodo if X is 3-hydroxy; and, Y 2 is hydrogen or 5-iodo if Y 1 is 3-iodo and 4-iodo if Y 1 is 6-iodo; at least a portion of the molecules of said substrate having a [ 125 I] or [ 131 I] substituted for the I atoms in that portion of the substrate connected by amide linkage to the carboxy group of the arginine moiety, said substrate being present at a concentration less than 10 uM in a buffer having a pH in the range of 7.5-10.5; terminating the kallikrein-catalyzed hydrolysis; separating the hydrolysis product from the substrate; and, measuring the amount of product formed by counting the radioactivity thereof, whereby an assay of the kallikrein activity is obtained.
Claim 21
Independent Claim
165 words
A method for the assay of urinary kallikrein comprising: mixing said kallikrein with a substrate having the formula ##STR7## wherein R is hydrogen, L--Ser or L--Phe-L--Ser; n is 0 or 1; X is 3-hydroxy or 4-hydroxy; Y 1 is 3-iodo if X is 4-hydroxy or 6-iodo if X is 3-hydroxy; and, Y 2 is hydrogen or 5-iodo if Y 1 is 3-iodo and 4-iodo if Y 1 is 6-iodo; at least a portion of the molecules of said substrate having a [ 125 I] or [ 131 I] substituted for the I atoms in that portion of the substrate connected by amide linkage to the carboxy group of the arginine moiety, said substrate being present at a concentration less than 10 uM in a buffer having a pH in the range of 7.5-10.5; terminating the kallikrein-catalyzed hydrolysis; separating the hydrolysis product from the substrate; and, measuring the amount of product formed by counting the radioactivity thereof, whereby an assay of the kallikrein activity is obtained.
Claim 22
Dependent Claim
44 words
The method of claim 16 wherein the hydrolysis is terminated by addition of an excess of 0.1 N NaOH, the hydrolysis product is separated by extracting the reaction mixture with an aprotic solvent, and the buffer has a pH in the range of 9.0-10.0.
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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) | 1985-04-26 | 1985-10-26 | 1986-04-27 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1989-04-26 | 1989-10-26 | 1990-04-27 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1993-04-26 | 1993-10-26 | 1994-04-27 | $7700.00 | Window Closed / Lapsed |