Physicochemical and kinetic characteristics of rhodanese from the liver of African catfish Clarias gariepinus Burchell in Asejire lake

Abstract

Two forms of rhodanese were purified from the liver of Clarias gariepinus Burchell, designated catfish rhodanese I (cRHD I) and rhodanese II (cRHD II), by ion-exchange chromatography on a CM-Sepharose CL-6B column and gel filtration through a Sephadex G-75 column. The apparent molecular weight obtained for cRHD I and cRHD II was 34,500 +/- 707 and 36,800 +/- 283 Da, respectively. The subunit molecular weight determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was 33,200 +/- 283 and 35,100 +/- 141 Da for cRHD I and cRHD II, respectively. Atomic absorption spectrophotometric analysis revealed that cRHD II contained a high level of iron (Fe), which presumably was responsible for the brownish colour of the preparation. In contrast, no Fe was identified in cRHD I, and its preparation was colourless. Further characterization of cRHD II gave true Michaelis-Menten constant (K(m)) values of 25.40 +/- 1.70 and 18.60 +/- 1.68 mM for KCN and Na(2)S(2)O(3), respectively, an optimum pH of 6.5 and an optimum temperature of 40 degrees C. The Arrhenius plot of the effects of temperature on the reaction rate consisted of two linear segments with a break occurring at 40 degrees C. The apparent activation energy values from these slopes were 7.3 and 72.9 kcal/mol. Inhibition studies on the cRHD II enzyme showed that the activity of the enzyme was not affected by Mn(2+), Co(2+), Sn(2+), Ni(2+) and NH(4) (+), but Zn(2+) inhibited the enzyme considerably.