Geographic variations, cloning, and functional analyses of the venom acidic phospholipases A 2 of Crotalus viridis viridis

Abstract

Geographic venom samples of Crotalus viridis viridis were obtained from South Dakota, Wyoming, Colorado, Oklahoma, Texas, New Mexico, and Arizona. From these samples, the phospholipases A 2 (PLA 2s) were purified and their N-terminal sequences, precise masses, and in vitro enzymatic activities were determined. We purified two to four distinct acidic PLA 2s from each sample; some of them displayed different inhibition specificities toward mammalian platelets. One of the acidic PLA 2s induced edema, but had no anti-platelet activity. There was also a common basic PLA 2 myotoxin in all the samples. We have cloned five acidic PLA 2s and several hybrid-like nonexpressing PLA 2s. Molecular masses and N-terminal sequences of the purified PLA 2s were matched with those deduced from the cDNA sequences, and the complete amino acid sequences of five novel acidic PLA 2s were thus solved. They share 78% or greater sequence identity, and a cladogram based on the sequences of many venom acidic PLA 2s of New World pit vipers revealed at least two subtypes. The results contribute to a better understanding of the ecogenetic adaptation of rattlesnakes and the structure–activity relationships and evolution of the acidic PLA 2s in pit viper venom.