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Two phospholipase A2s (PLA2s), Asp49 (D49) and Lys49 (K49), were purified by one-step reverse-phase high performance liquid chromatography (RP-HPLC) from the venom of each of the three subspecies of cottonmouth snake, Western cottonmouth (Agkistrodon piscivorus leucostoma; Apl), Eastern cottonmouth (Agkistrodon piscivorus piscivorus; App) and Florida cottonmouth (Agkistrodon piscivorus conanti; Apc). Venom protein profiles and PLA2s elution pattern of the three cottonmouth snakes were remarkably similar displaying four similar sharp and two wide peaks; in all cases K49 PLA2 eluted first followed by D49 PLA2. The yields of K49 and D49 PLA2s were, respectively, 13.2 and 17.5 mg/g venom from the Western cottonmouth, 16.8 and 19.2 mg/g from the Eastern cottonmouth, and 17.3 and 22.7 mg/g from the Florida cottonmouth. Biochemical and enzymatic techniques were used to characterize the purified PLA2. The amino acid sequences of all three K49PLA2s were identical; App-D49 and Apc-D49 were also identical but displayed a single amino acid difference with Apl-D49. To our knowledge, this is the first report on the amino acid sequence and molecular mass of Apc-D49 and Apc-K49 PLA2s from Florida cottonmouth venom. As expected, PLA2 enzymatic analysis revealed that D49 PLA2s from all three venoms hydrolyze phospholipids to a similar extent, whereas no phospholipid hydrolysis was detectable by any of the K49 PLA2s purified. Cottonmouth snake venoms contain abundant PLA2 isoforms, thus the identification of PLA2s in these venoms is of interest to facilitate the purification of specific PLA2 from rich sources of subspecies venom for future biological and biomedical research. Results of this study also contribute towards the understanding of venom protein profiles, variation, and evolution in subspecies of venomous snakes.We purified two different PLA2s from the venom of each of the three cottonmouth snakes by one-step reverse phase HPLC.The amino acid sequence of each PLA2 was analyzed by either LC-MS/MS or Edman degradation.All Asp49 PLA2s hydrolyze phospholipids, whereas no phospholipid hydrolysis was detectable by all Lys49 PLA2s.