Blood-sucking arthropods have different types of anticoagulants to allow the ingestion of a blood meal from their hosts. In this study, five anticoagulants prolonging the activated partial thromboplastin time were resolved from the salivary gland crude extract of the camel tick Hyalomma dromedarii by chromatography on diethylaminoethyl (DEAE)-cellulose column. They were designated P1, P2, P3, P4 and P5 according to their elution order. P5 was found to be a potent thrombin inhibitor and purified by ultrafiltration through two centrifugal concentrators of 50 and 30 kDa molecular weight cut-off (MWCO), respectively. The camel tick salivary gland thrombin inhibitor was purified 60.6 folds with a specific activity of 564 units/mg protein. It turned out to be homogenous on native-PAGE with molecular weight of 36 kDa as detected on 12% SDS-PAGE. It inhibits bovine thrombin competitively with K value of 0.55 μM. A task for the future will be the elucidation of this thrombin inhibitor structure to allow its application in thrombosis treatment.

Pullulanase (EC 3.2.1.41) has been isolated and purified from white edible mushrooms by ammonium sulphate precipitation (20-70%) followed by ion exchange chromatography (DEAE-cellulose) and gel filtration (Sephadex G 75-120), with final yield (20%) and purification fold (17.8). The molecular mass of pullulanase enzyme was 112 kDa as estimated by SDS-PAGE and the pI value was 6.2. The apparent Km and Vmax values for purified pullulanse on pulluan were 0.27 mM and 0.74 μM min -1 respectively. The activity was optimum at 40 ○ C and pH 6. Pullulanase showed moderate thermo-stability. A relative substrate specificity for hydrolysis of soluble starch, amylopectin and glycogen was 80, 60 and 30% respectively. Enzyme activity was highly activated by Fe +2 , Mn +2 and Ca +2 ions, while the activity was inhibited by Hg +2 and Ag + ions. Ethylenediaminetetraacetic acid (EDTA) and Dithiothreitol (DTT) were activated the enzyme activity. On contarary iodoacetate and sodium fluoride were inhibited the activity. HPTLC (High Performance Thin Layer Chromatography) plate showed that the purified pullulanase caused the complete hydrolysis of pullulan to maltotriose.

Background Honey bee venom contains various enzymes with wide medical and pharmaceutical applications. Results The phospholipase A2 (PLA2) has been apparently purified from the venom of Egyptian honey bee (Apis mellifera lamarckii) 8.9-fold to a very high specific activity of 6033 U/mg protein using DEAE–cellulose and Sephacryl S-300 columns. The purified bee venom PLA2 is monomeric 16 kDa protein and has isoelectric point (pI) of 5.9. The optimal activity of bee venom PLA2 was attained at pH 8 and 45 °C. Cu2+, Ni2+, Fe2+, Ca2+, and Co2+ exhibited a complete activating effect on it, while Zn2+, Mn2+, NaN3, PMSF, N-Methylmaleimide, and EDTA have inhibitory effect. Conclusions The purified bee venom PLA2 exhibited anti-platelet aggregation and anti-coagulation activities which makes it promising agent for developing novel anti-clot formation drugs in future.

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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.

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Xanthine oxidase is a commercially important enzyme with wide area of medical applications to develop diagnostic kits. Xanthine oxidase was extracted, purified and characterized from sheep liver (SLXO). The purification procedure involved acetone precipitation and chromatography on DEAE-cellulose and Sephacryl S-300 columns. The sheep liver xanthine oxidase was homogeneously purified 31.8 folds with 3.5 U/mg specific activity and 24.1% recovery. SLXO native molecular weight was 150 kDa and on SDS-PAGE appeared as single major band of 75 kDa representing a homodimer protein. Isoelectric focusing of the purified SLXO resolved into two closely related isoforms with pI values of 5.6 and 5.8. The apparent Km for xanthine oxidase at optimum pH 7.6 was found to be 0.9 mM xanthine. FeCl2 and NiCl2 increased the activity of SLXO, while CuCl2 and ZnCl2 were found to be potent inhibitors of the purified enzyme. Allopurinol inhibits SLXO competitively with one binding site on the purified molecule and Ki value of 0.06 mM.