Purification and some properties of a carboxylesterase from ovine liver.

Carboxylesterase ESB3 was extracted from ovine liver and purified to homogeneity by ammonium sulphate fractionation, hydrophobic interaction chromatography on Phenyl Sepharose, ion exchange chromatography on Mono-Q Sepharose and size exclusion chromatography on Superose 6. The enzyme is free of carboxylesterase ESB2 activity. The molecular mass of the enzyme is estimated 182 kDa as judged by size exclusion chromatography. Isoelectric focusing indicates the presence of six isoforms of pI 5.50-5.77 with three main isoforms of pI 5.55-5.65. The enzyme is active towards the substrates p-nitrophenyl acetate and the aliphatic substrates ethyl acetate, ethyl propionate, ethyl butyrate, and ethyl valerate. Of the ethyl esters the affinity is lowest towards acetate and highest towards ethyl butyrate. The enzyme is totally inhibited by phenylmethylsulphonyl fluoride (PMSF) and mercuric chloride but not affected by eserine or cupric chloride. The pH optimum of the enzyme is 7.5 and it is stable at 55 degrees C for 20 min.