Highly Specific Substrates of Proteinase 3 Containing 3-(2-Benzoxazol-5-yl)-L-alanine and Their Application for Detection of This Enzyme in Human Serum

摘要

A set of benzoxazolyl-L-alanine derivates along with the MCA moiety (donors of fluorescence) were introduced into a proteinase 3 (PR3) substrate with a C-terminal ANB-NH_(2) that serves as a fluorescence acceptor. Five substrates with general formula X-Tyr-Tyr-Abu-ANB-NH_(2) were synthesized, and their kinetic parameters against proteinase 3 were determined. The highest k_(cat)/K_(M) value, 1.5 X 10~(6) M~(-1) s~(-1), was obtained for (Pyr) Box-Ala-Tyr-Tyr-Abu-NH_(2) where (Pyr)Box-Ala stands for N-methylpyrrole benzoxazole-L-alanine. Titration of this peptide with proteinase 3 resulted in measurable fluorescence at an enzyme amount equal to 29 pmol. This substrate was selected used to detect quantifiable levels of proteinase 3 in serum samples, including those of normal subjects. For all c-ANCA-positive samples (diagnosed Wegener granulomatosis), a significant increase of PR3 concentration was observed. Wegener granulomatosis is a severe autoimmune disease causing inflammation of the blood vessels. Our results clearly show that this substrate can be used for the construction of a very reliable, inexpensive, and easy to use diagnostic test for PR3 determination.