RESUMEN
Lactate dehydrogenase (LDH) enzyme is a major component of aspartate aminotransferase (AST) and alanineaminotransferase (ALT) diagnosis kits. In this work, the LDH enzyme was purified and characterized from buffalo liverfor direct application in the preparation of AST and ALT diagnosis kits. One major LDH (BLLDH) isoform and twoother secondary LDH peaks were analyzed for buffalo liver by diethylaminoethyl (DEAE) cellulose chromatography.BLLDH was obtained by ammonium sulfate sedimentation and chromatographically separated on ion exchange andsize-exclusion matrices. The isolated BLLDH has a specific activity of 17.6 units/mg proteins represented 16 foldsand 32% recovery. BLLDH was manifested homogeneous on native and SDS gels with 35 kDa native mass. OptimumpH of BLLDH was displayed at pH 7.6. BLLDH activity was diminished by FeCl2 and SDS. The produced BLLDH isutilized in constructing of AST and ALT diagnosis kits that were sensible and analogous to trade ready kits.
RESUMEN
Xanthine oxidase (XO) is an important enzyme with broad medical applications as detecting reagent in many diagnostic kits. In this study, buffalo liver xanthine oxidase (BLXO) was purified to homogeneity by acetone precipitation and chromatography on DEAE-cellulose and Sephacryl S-300 columns with a specific activity of 7.2 units / mg protein which represent 31.3 folds. The native molecular weight of the purified enzyme is 200 kDa and its subunit molecular weight was determined by SDS-PAGE to be 67 kDa. The isoelectric point (pI) value of BLXO isoenzyme is at pH 6.0 – 6.2. It displayed its pH optima at 7.6 and the Km value is 1.1 mM xanthine. FeCl2 increased the activity of BLXO while CuCl2, MnCl2 and ZnCl2 were found to be inhibitors of the purified enzyme. Allopurinol inhibits BLXO competitively and has one binding site on it with Ki value of 0.025mM. Abbreviations: BSA, Bovine serum albumin, XO, Xanthine oxidase, NBT, Nitroblue tetrazolium, PAGE, Polyacrylamide gel electrophoresis, PMS, Phenazine methosulphate, BLXO, Buffalo liver xanthine oxidase