Improved purification of the membrane-bound hydrogenase–sulfur-reductase complex from thermophilic archaea using ϵ-aminocaproic acid-containing chromatography buffers

A hydrogenase–sulfur reductase (SR) complex was purified from membrane preparations of the extremely thermophilic, acidophilic archaeon Acidianus ambivalens using a combination of sucrose density gradient centrifugation and column chromatography (FPLC). All chromatographic steps were performed in the presence of 0.5% ϵ-aminocaproic acid resulting in the elution of the SR complex as a sharp peak. In contrast, chromatography using buffers without ϵ-aminocaproic acid, or in the presence of detergents, were not successful. The purified A. ambivalens SR complex consisted of at least four subunits 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 thermophilic but neutrophilic archaeon, which consisted of only two subunits with relative molecular masses of 66 000 and 39 000, respectively.