Purification and preliminary characterisation of praelongin phospholipases, antiplatelet agents from the snake venom of Acanthophis praelongus

Abstract

Three praelongin phospholipases were chromatographically purified from the snake venom of Acanthophis praelongus. The purity and homogeneity of the praelongins were assessed by RP-HPLC, HPCE and mass spectrometry. The purified enzymes, praelongins 2bIII, 2cII and 2cIV were found to have phospholipase A2 activities with specific activities of 31.4 ± 0.4, 326.1 ± 10.2 and 362.5 ± 12.0 U/mg, respectively. Mass spectrometry studies showed the molecular mass of praelongin 2bIII to be 12,782,9 ± 2.6 and praelongins 2cII and 2clV to have very similar molecular mass values, 12,971.4 ± 4.5 and 12,971.9 ± 3.6, respectively. However, platelet aggregation studies showed the praelongins to display different IC50 values, 180μM for praelongin 2cII and 55 μM for praelongin 2cIV; praelongin 2bIII was found to be a more potent antiplatelet agent, having an IC50 of 0.65 μM. Praelongins 2bIII, 2cIV and 2cII were found to have pI values of 10.3 ± 0.3, 9.6 ± 0.6 and 9.4 ± 0.6 as determined by HPCE. The antiplatelet potencies do not correspond to their in vitro phospholipase catalytic potencies, but appear to be related to the enzyme isoelectric points.