Purification and Physicochemical Properties of Rhodanese from Liver of Goat, Capra aegagrus hircus

Abstract

Rhodanese from the liver of domestic goat (Capra aegagrus hircus) was extracted, purified and the catalytic as well as physicochemical properties determined in order to gain an insight into how domestic goats are able to feed and thrive successfully on cyanogenic plants such as sorghum, millet and cassava.

A domestic goat was purchased from a freehold rearing place in Ile-Ife, Osun State, Nigeria, where it was slaughtered and the liver excised. It was rinsed with normal saline (0.9 % NaCl, pH 7.4) to remove blood and other impurities. Approximately 95 g of the liver was homogenized in 3 volumes of 0.1 M acetate glycine buffer, pH 7.8 containing 1 mM ԑ-amino-n-caprioc acid and 10 mM sodium thiosulphate in a blender, after which it was centrifuged for 15 min at 12,000 rpm at 4⁰C.

A specific activity of 1.55 micromole thiocyanate formed per millilitre per minute (Rhodanese Unit; RU) per mg of protein (U/mg) with a purification fold of 1.88 and 36 % yield was obtained from the rhodanese extracted from the liver of goat rhodanese after CM-Sephadex, Sephacryl S-400 and Reactive Blue 2- agarose column chromatography. An apparent molecular weight of 36 kDa was obtained on Sephacryl S-400 and a subunit molecular weight of 19 kDa was obtained from Sodium dodecyl sulphate polyacylamide gel electrophoresis (SDS-PAGE). A K_m value of 0.034±0.007 mM and V_max of 16.70±3.36 units/ml was obtained for Na₂S₂O₃ while a K_m value of 0.038±0.004 mM and V_max of 18.77±1.58 units/ml was obtained for KCN. An optimal pH of 8.5, 9.5 and 7.0 were obtained using citrate-phosphate-borate, Tris-HCl and citrate-phosphate buffers respectively. Goat liver rhodanese showed an optimal temperature of 40⁰C. All the chloride salts used which included KCl, MgCl₂, CaCl₂, MnCl₂, AlCl₃ and NH₄Cl inhibited rhodanese activity, with the highest inhibition was observed with MnCl₂ and least inhibition observed with KCl. Goat liver rhodanese was inhibited by all active site inhibitors, with the the highest inhibition observed with arsenic acid.

It can therefore be concluded that domestic goats (Capara aegagrus hircus) are able to feed and survive on cyanogenic plants due to the presence of the cyanide detoxyfying enzyme, rhodanese, present in their liver at high activity with suitable kinetic properties.