Inhibition, reactivation, and determination of metal ions in membrane metalloproteases of bacterial origin using high-performance liquid chromatography coupled on-line with inductively coupled plasma mass spectrometry.
High-performance liquid chromatography coupled on-line with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) was used for the characterization of metal ions in several metalloproteases of bacterial origin. The different components of the bacterial extracts were separated on a size-exclusion column. The eluent of the HPLC system was continuously transported to the ICP-MS system for rapid, reproducible, and sensitive analyses of trace elements in the metalloproteases. Two different membrane proteases from Bacillus cereus and Pseudomonas aeruginosa were characterized to be zinc metalloproteases using enzymological methods and HPLC-ICP-MS. The zinc content was determined to be three molecules of zinc per protein molecule for the B. cereus protease and one molecule of zinc per protein molecule for the P. aeruginosa protease. For another purified protease, a periplasmic alanyl aminopeptidase of P. aeruginosa, the lack of protein-bound metal ions could be clearly determined-a confirmation that this main aminopeptidase of P. aeruginosa 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 avoided during enzyme purification for metal analyses or they should be exchanged later for zwitterionic and ionic detergents with more strongly dissociating properties.
Leopold
I
I
Department of Stress and Developmental Biology, Institute of Plant Biochemistry, Weinberg 3, Halle, 06120, Germany.
Fricke
B
B
D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D018826:CD13 Antigens / Q000037:antagonists & inhibitors / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D002614:Chelating Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D004492:Edetic Acid / Q000494:pharmacology; D007854:Lead / Q000032:analysis; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D008970:Molecular Weight; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D014131:Trace Elements / Q000032:analysis*; D015032:Zinc / Q000032:analysis
D002614:Chelating Agents; D003902:Detergents; D010618:Phenanthrolines; D011480:Protease Inhibitors; D014131:Trace Elements; D007854:Lead; D004492:Edetic Acid; D018826:CD13 Antigens; D008666:Metalloendopeptidases; D015032:Zinc; C025205:1,10-phenanthroline