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
I. Leopold et B. Fricke, 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, Analytical biochemistry, 252(2), 1997, pp. 277-285
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.