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Wysocka M.,University of Gdanisk | Lesner A.,University of Gdanisk | Guzow K.,University of Gdanisk | Kulczycka J.,Medical University of Gdansk | And 3 more authors.
Analytical Chemistry | Year: 2010

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-NH2 that serves as a fluorescence acceptor. Five substrates with general formula X-Tyr-Tyr-Abu-ANB-NH2 were synthesized, and their kinetic parameters against proteinase 3 were determined. The highest kcat/KM value, 1.5 × 106 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. © 2010 American Chemical Society.

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