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.
Anal Chem
; 82(9): 3883-9, 2010 May 01.
Article
em En
| MEDLINE
| ID: mdl-20361767
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Benzoxazóis
/
Análise Química do Sangue
/
Alanina
/
Mieloblastina
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Polônia