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.
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.
Wysocka
Magdalena
M
Faculty of Chemistry, University of Gdanìsk, Poland.
Lesner
Adam
A
Guzow
Katarzyna
K
Kulczycka
Julia
J
Łegowska
Anna
A
Wiczk
Wiesław
W
Rolka
Krzysztof
K
D000409:Alanine / Q000737:chemistry*; D001583:Benzoxazoles / Q000737:chemistry*; D001774:Blood Chemical Analysis / Q000379:methods*; D019937:Diagnostic Techniques and Procedures / Q000191:economics / Q000592:standards; D006801:Humans; D015394:Molecular Structure; D053146:Myeloblastin / Q000097:blood*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D001583:Benzoxazoles; D053146:Myeloblastin; D000409:Alanine