An evaluation of chromogenic substrates for characterization of serine protease produced by pathogenic Vibrio alginolyticus.
Four chromogenic substrates for characterizing serine protease of Vibrio alginolyticus were evaluated. The protease activity of bacterial extracellular products, or the fractions of 33 kD protease purified by the AKTA purifier system with various columns, was completely inhibited by ethylenediamine tetra-acetic acid, ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA), antipain and phenylmethylsulphonyl fluoride (PMSF) using water-soluble substrates (azoalbumin and azocasein). It was only completely inhibited by antipain and PMSF using water-insoluble substrates (azocoll and hide powder azure). The protease activity was not, or only partially, inhibited by 1,10-phenanthroline and sodium dodecyl sulphate (SDS) using all four substrates. Since chelating agents and 1,10-phenanthroline are commonly employed as inhibitors to identify metalloprotease, the two water-soluble substrates may not be appropriate for this purpose, except for using 1,10-phenanthroline as an inhibitor. Chelating agents may be still applicable as inhibitors using water-insoluble substrates and 1,10-phenanthroline is highly recommended in the characterization for metalloprotease to avoid confusion. In the present study, the 33 kD protease was further confirmed as an SDS-resistant serine protease and not a metalloprotease.
Chen
F R
FR
Department of Aquaculture, National Taiwan Ocean University, Republic of China.
Liu
P C
PC
Lee
K K
KK
D000818:Animals; D002863:Chromogenic Compounds / Q000378:metabolism*; D033364:Decapoda; D004492:Edetic Acid; D004533:Egtazic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D010664:Phenylmethylsulfonyl Fluoride; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors; D012967:Sodium Dodecyl Sulfate; D014733:Vibrio / Q000201:enzymology*
D002863:Chromogenic Compounds; D015842:Serine Proteinase Inhibitors; D012967:Sodium Dodecyl Sulfate; D004533:Egtazic Acid; D010664:Phenylmethylsulfonyl Fluoride; D004492:Edetic Acid; D012697:Serine Endopeptidases