期刊
ANALYTICAL BIOCHEMISTRY
卷 442, 期 1, 页码 75-82出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2013.07.028
关键词
Neutrophil serine proteases; Intermolecular quenched substrate; Proteinase 3; Combinatorial chemistry
资金
- National Council of Science (NCN) [UMO-2012/07/N/ST5/00178]
We report the synthesis and enzymatic studies on a new proteinase 3 intermolecular quenched substrate with enhanced selectivity over neutrophil elastase. Using combinatorial chemistry methods, we were able to synthesize the hexapeptide library with the general formula ABZ-Tyr-Tyr-Abu-X-1'-X-2'-X-3'-Tyr(3-NO2)-NH2 using the mix and split method. The iterative deconvolution of such a library allowed us to obtain the sequence ABZ-Tyr-Tyr-Abu-Asn-Glu-Pro-Tyr(3-NO2)-NH2 with a high specificity constant (k(cat)/K-M = 1534 x 10(3) M-1 s(-1)) and superior selectivity over neutrophil elastase and other neutrophil-derived serine proteases. Moreover, using the obtained substrate, we were able to detect a picomolar concentration of proteinase 3 (PR3). Incubation of the above-mentioned substrate with neutrophil lysate resulted in a strong fluorescent signal that was significantly reduced in the presence of a PR3 selective inhibitor. (C) 2013 Elsevier Inc. All rights reserved.
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