Purification and partial characterization of thiosulphate dehydrogenase from Thiobacillus acidophilus

Abstract

Thiosulphate dehydrogenase (EC 1.8.2.2; thiosulphate:acceptor oxidoreductase) was purified to apparent homogeneity from Thiobacillus acidophilus by a combination of ammomium sulphate precipitation, hydrophobic interaction chromatography, anion-exchange chromatography and gel filtration. The enzyme catalysed the oxidation of thiosulphate (S2O32-) to tetrathionate (S4O6-2) with potassium ferricyanide as an artificial electron acceptor. The molecular mass of the native enzyme, as determined by gel filtration, was 102 ± 4.2 kDa. The enzyme contained two different subunits with a molecular mass of 24 ± 0.9 and 20 ± 1.0 kDa (SDS-PAGE), respectively. Both subunits contained c553-type haem with absorption bands at 553, 524 and 416 nm. A 77 K spectrum of purified thiosulphate dehydrogenase revealed that the absorption at 553 nm is due to different haem groups. A cytochrome content of 5.3 mole c-type haem per mole of native enzyme was calculated. The pH optimum of the purified enzyme was 3. Apart from ferricyanide, Wurster's blue (the free radical of tetramethyl p-phenylenediamine) and horse heart cytochrome c could also serve as electron acceptors, though less effectively than ferricyanide. At pH 7.0, the K(m) for thiosulphate was 0.54 mM. The K(m) could not be determined at the pH optimum due to the chemical reactivity of thiosulphate at low pH values. Sulphite was a potent inhibitor of enzyme activity.