Proteases are classified on the basis of their catalytic mechanism. Hartley pointed out that four distinct High-performance liquid chromatography coupled types of catalytic mechanism were used by peptidases, on-line with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) was used for the character-and this observation has been developed into a practiization of metal ions in several metalloproteases of cal system for classification (1). The serine-type peptibacterial origin. The different components of the bacdases (EC 3.4.21) have an active center serine involved terial extracts were separated on a size-exclusion colin the catalytic process, the cysteine-type peptidases umn. The eluent of the HPLC system was continuously (EC 3.4.22) have a cysteine residue in the active center, transported to the ICP-MS system for rapid, reproducthe aspartic-type peptidases (EC 3.4.23) depend on two ible, and sensitive analyses of trace elements in the aspartic acid residues for their catalytic activity, and metalloproteases. Two different membrane proteases the metallopeptidases (EC 3.4.24) use a metal ion (comfrom Bacillus cereus and Pseudomonas aeruginosa monly zinc) in the catalytic mechanism (2). The methiowere characterized to be zinc metalloproteases using nine aminopeptidases of various origins (3-5), first isoenzymological methods and HPLC-ICP-MS. The zinc lated from Escherichia coli (5), bearing two cobalt content was determined to be three molecules of zinc atoms instead of zinc in its active center, are the single per protein molecule for the B. cereus protease and known exceptions among peptidases in this regard. The one molecule of zinc per protein molecule for the P. effects of inhibitors give the most reliable information aeruginosa protease. For another purified protease, a about the catalytic type of a peptidase, although few of periplasmic alanyl aminopeptidase of P. aeruginosa, the available inhibitors are perfect diagnostic reagents the lack of protein-bound metal ions could be clearly and false-negative or false-positive reactions do occur. determined-a confirmation that this main aminopep-Metallopeptidases are the most diverse of the catatidase of P. aeruginosa belongs to the cysteine protelytic types of peptidases, approximately 30 families bease family. The presence of nonionic detergents can ing recognized. Most of the known zinc-dependent metinfluence the distribution of trace elements during the allopeptidases (with the notable exception of the HPLC separation. Therefore, the use of these subcarboxypeptidases) share a common pattern of the stances should be avoided during enzyme purification primary structure (6). About half of the families comfor metal analyses or they should be exchanged later prise enzymes containing the His-Glu-Xaa-Xaa-His (or for zwitterionic and ionic detergents with more HEXXH) motif that has been shown by X-ray crystalstrongly dissociating properties.