Method for measuring the activity of proteases

摘要

A method for determining the activity of proteases is provided. Fluorogenic substrates from which the fluorogen 7-amino-4-trifluoromethylcoumarin is eliminated proved to be particularly advantageous for the activity measurement. These substrates make it possible for measurement in microtiter plates with a fluorescence reader and thus for the fluorimetric determination of such enzyme activities in blood serum.

主权项

1. A method for measuring the activity of cysteine protease selected from the group consisting of cathepsin B and cysteine proteases in general, in a fluid sample selected from the group consisting of blood plasma and blood serum, which contains the cysteine protease and at least one protease inhibitor corresponding to the cysteine protease, the method comprising the steps of:
contacting the fluid sample with a carrier, wherein an inhibitor binding substance is bound covalently or in an adsorptive manner to the carrier, the inhibitor binding substance having a higher affinity or binding strength to the protease inhibitor than the cysteine protease; separating the carrier together with the protease inhibitor bound to the carrier from the fluid sample to form a modified fluid sample; separating a first portion of the modified fluid sample from a second portion of the modified fluid sample; adding a synthetic inhibitor specific for the cysteine protease to the first portion while the second portion is maintained without the addition of a synthetic inhibitor specific for the cysteine protease; adding a substrate for the at least one cysteine protease to each of the first and second portions; fluorometrically recording the proteolytic reaction of the substrate with the cysteine protease in each of the first and second portions, wherein the substrate for the cysteine protease includes a di- or oligopeptide sequence having a C-Terminus to which a fluorogen is bound, the N-terminus of the di- or oligopeptide sequence having a protecting group and the fluorogen is 7-amino-4-trifluoromethylcoumarin (AFC), whereby the fluorogen is cleaved during a proteolytic reaction, and wherein the substrate with the fluorogen has a maximum fluorescence emission wavelength which differs from a maximum fluorescence emission wavelength of the fluorogen being cleaved by the cysteine protease in the proteolytic reaction by at least 20 nm; and calculating the difference between the measured activities of the cysteine protease in the first portion and the second portion to determine the activity of the cysteine protease in the fluid sample.