Improved purification of the membrane-bound hydrogenase-sulfur-reductase complex from thermophilic archaea using epsilon-aminocaproic acid-containingchromatography buffers

A hydrogenase-sulfur reductase (SR) complex was purified from membrane prep arations of the extremely thermophilic, acidophilic archaeon Acidianus ambi valens using a combination of sucrose density gradient centrifugation and c olumn chromatography (FPLC). All chromatographic steps were performed in th e presence of 0.5% epsilon-aminocaproic acid resulting in the elution of th e SR complex as a sharp peak. In contrast, chromatography using buffers wit hout E-aminocaproic acid, or in the presence of detergents, were not succes sful. The purified A. ambivalens SR complex consisted of at least four subu nits with relative molecular masses of 110 000, 66 000, 39 000 and 29 000, respectively. A similar procedure was applied to purify the membrane-bound hydrogenase from Thermoproteus neutrophilus, a non-related extremely thermo philic but neutrophilic archaeon, which consisted of only two subunits with relative molecular masses of 66 000 and 39 000, respectively. (C) 2000 Els evier Science B.V. All rights reserved.