INHIBITION, REACTIVATION, AND DETERMINATION OF METAL-IONS IN MEMBRANEMETALLOPROTEASES OF BACTERIAL ORIGIN USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY COUPLED ONLINE WITH INDUCTIVELY-COUPLED PLASMA-MASS SPECTROMETRY

High-performance liquid chromatography coupled on-line with inductivel y coupled plasma mass spectrometry (HPLC-ICP-MS) was used for the char acterization of metal ions in several metalloproteases of bacterial or igin. The different components of the bacterial extracts were separate d on a size-exclusion column. The eluent of the HPLC system was contin uously transported to the ICP-MS system for rapid, reproducible, and s ensitive analyses of trace elements in the metalloproteases. Two diffe rent membrane proteases from Bacillus cereus and Pseudomonas aeruginos a were characterized to be zinc metalloproteases using enzymological m ethods and HPLC-ICP-MS, The zinc content was determined to be three mo lecules of zinc per protein molecule for the B. cereus protease and on e molecule of zinc per protein molecule for the P. aeruginosa protease . For another purified protease, a periplasmic alanyl aminopeptidase o f P. aeruginosa, the lack of protein-bound metal ions could be clearly determined-a confirmation that this main aminopeptidase of P. aerugin osa belongs to the cysteine protease family. The presence of nonionic detergents can influence the distribution of trace elements during the HPLC separation. Therefore, the use of these substances should be avo ided during enzyme purification for metal analyses or they should be e xchanged later for zwitterionic and ionic detergents with more strongl y dissociating properties. (C) 1997 Academic Press.