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Analysis of serine proteases from marine sponges by 2‐D zymography
Author(s) -
Wilkesman Jeff G.,
Schröder Heinz C.
Publication year - 2007
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200600332
Subject(s) - zymography , proteases , protease , serine protease , biochemistry , serine , biology , enzyme , pmsf , molecular mass , microbiology and biotechnology
Proteolytic activities isolated from the marine demosponges Geodia cydonium and Suberites domuncula were analyzed by 2‐D zymography, a technique that combines IEF and zymography. After purification, a 200 kDa proteolytically active protein band was obtained from G. cydonium when analyzed in gelatin copolymerized 1‐D zymograms. The enzymatic activity was quantified using α‐ N ‐benzoyl‐ D ‐arginine p ‐nitroanilide (BAPNA) as a substrate and corresponded to a serine protease. The protease activity was resistant to urea and SDS. DTT and 2‐mercaptoethanol (2‐ME) did not significantly change the protease activity, but induced a shift in molecular mass of the proteolytic band to lower M r values as detected by zymography. Under mild denaturing conditions, lower M r bands (<200 kDa) were identified in 1‐D zymograms, suggesting that the protease is composed of subunits which retain the catalytic activity. After 2‐D zymography, the protease from G. cydonium revealed a p I of 8.0 and an M r shift from 200 to 66 kDa. To contrast these results, a cytosolic sample from S. domuncula was analyzed. The proteolytic activity of this sponge after 2‐D zymography corresponded to an M r of 40 kDa and a p I of 4.0. The biological function of both sponge proteases is not yet known. This study demonstrates that mild denaturing conditions required for IEF may alter the interpretation of the 2‐D zymography, and care must be taken during sample preparation.