C - Chemistry – Metallurgy – 07 – K
Patent
C - Chemistry, Metallurgy
07
K
150/14, 530/5.04
C07K 5/04 (2006.01) C07K 7/06 (2006.01) C12Q 1/38 (1985.01)
Patent
CA 1240987
URINARY KALLIKREIN ASSAY: SPECIFIC SUBSTRATES AND ASSAY METHOD Abstract Substrates and method for the assay of urinary kallikrein are provided. The substrates have the general formula R-L-Pro-L-phe-L-Arg-NH-(CH2)n Image wherein R is H, acetyl, benzoyl, cyclopentanecarbonyl, succinyl, R1-L-Ser or R1-L-Phe-L-Ser where R1 is H, acetyl, benzoyl, cyclopentanecarbonyl or succinyl, X is H, tritium, 3-iodo or 4-iodo, and n is 0 or 1. Ratioactive 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 preferrably at a concentration substantially below Km. 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 R-L-Pro-L-Phe-L-Arg-NH-(CH2)n Image wherein R, R1 and n are as defined above. X is 3-OH or 4-OH. Y1 is 3-iodo if X is 4-OH or 6-iodo if X is 3-OH. Y2 may be H or 5-iodo if Y1 is 3-iodo, or 4-iodo if Y1 is 6-iodo.
351000
Chung Alfred
Ryan James W.
Sim & Mcburney
University Of Miami
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