Glucose-6-phosphate dehydrogenase from camel liver was purified to homogeneity by ammonium sulfate precipitation and a combination of DEAE-cellulose, Sephacryl S-300 gel filtration, and 2′, 5′ ADP Sepharose 4B affinity chromatography columns. The specific activity of camel liver G6PD is increased to 1.80438 units/mg proteins with 63-fold purification. It turned out to be homogenous on both native PAGE and 12% SDS PAGE, with a molecular weight of 64 kDa. The molecular weight of the native form of camel liver G6PD was determined to be 194 kDa by gel filtration indicating a trimeric protein. The K m value was found to be 0.081 mM of NADP+. Camel liver G6PD displayed its optimum activity at pH 7.8 with an isoelectric point (pI) of pH 6.6–6.8. The divalent cations MgCl2, MnCl2, and CoCl2 act as activators; on the other hand, CaCl2 and NiCl2 act as moderate inhibitors, while FeCl2, CuCl2, and ZnCl2 are potent inhibitors of camel liver G6PD activity. NADPH inhibited camel liver G6PD competitively with K i value of 0.035 mM. One binding site was deduced for NADPH on the enzyme molecule. This study presents a simple and reproducible purification procedure of G6PD from the camel liver.

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Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme involved in the pentose phosphate pathway. This works represents purification of two buffalo liver glucose-6-phosphate dehydrogenases (BLG6PD1 and BLG6PD2) using combination of ammonium sulfate precipitation and several chromatographic columns. Both enzymes (BLG6PD1 and BLG6PD2) were homogenous on both native PAGE as well as 12% SDS PAGE with molecular weights of 28 and 66 kDa. The molecular weight of BLG6PD1 and BLG6PD2 native forms were determined to be 28 and 66 kDa by gel filtration; indicating monomeric proteins. The K(m) values for BLG6PD1 and BLG6PD2 estimated to be 0.059 and 0.06 mM of β-nicotinamide adenine dinucleotide phosphate. The optimum activity of BLG6PD1 and BLG6PD2 were displayed at pH 8.0 and 8.2 with an isoelectric point (pI) of pH 7.7-7.9 and 5.7-5.9. The divalent cations MgCl2, and CoCl2 act as activators, on the other hand, FeCl2, CuCl2 and ZnCl2 are potent inhibitors of BLG6PD1 and BLG6PD2 activity. NADPH inhibited both isoenzymes competitively with Ki values of 0.012 and 0.030 mM. This study describes a reproducible purification scheme of G6PD from the liver of buffalo as a rich source.

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Two forms of the nymphal thrombin inhibitors (NTI) 3.2 kDa and 14.9 kDa were purified by chromatography on CM-cellulose. Sephacryl S-300 and Sephadex G-50 columns and designated NTI- 1 and NTI-2 respectively. The NTI-2 turned out to be homogenous monomeric protein in both native-PAGE and denatured SDS-PAGE with M(r) value of 14.9 kDa approximately and its pI value ranged from 7.2 to 7.5. The NTI-1 and NTI-2 displayed anticoagulant activity since they prolonged both the activated partial thromboplastin time (APTT) and the prothrombin time (PT) of the camel plasma in a concentration-dependent manner. The potency of NTI-I toward thrombin was 5-fold higher than that toward FXa, while NTI-2 was 3-fold active toward FXa than thrombin. However, both of them did not inhibit any of the other examined proteases. The types of inhibition of thrombin by NTI-1 and NTI-2 were non-competitive and competitive with inhibition constants (Ki) values of 11.7 microM and 211 nM respectively. One binding site was deduced on thrombin for each inhibitor.

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