Primary structure and functional properties of cobra (Naja naja naja) venom Kunitz‐type trypsin inhibitor

Abstract

A trypsin inhibitor from the venom of the cobra Nuja nuju nuja has been isolated by a single step of reversephase high-performance liquid chromatography. The protein strongly inhibits trypsin (Ki = 3.5 pM). The primary structure was determined by peptide analysis of the [ ''C]carboxymethylated inhibitor. The 57-residue polypeptide chain belongs to the family of Kunitz-type inhibitors, and exhibits 42% residue identity with bovine pancreatic trypsin inhibitor. The structure shows only 70% identity with the corresponding peptide from the Capa cobra (Naju neviu), establishing that the inhibitor molecule exhibits extensive variations. Functionally, a basic residue at position P3' correlates with strong inhibition.Basic protease inhibitors of the serine proteases are abundant in nature and appear to be present in all forms of life [l]. Different types have been isolated and extensively studied. Based on different properties, they have been classified into at least seven families [2].Studies of snake venoms reveal the presence of several protease inhibitors, particularly from Elapidae and Viperidae snakes. These inhibitors belong to the protein family of pancreatic trypsin inhibitors of the Kunitz type [3]. The amino acid sequences of the venom inhibitors from Viperu russelli (Russell's viper) [4], Nuju neviu (Cape cobra) [5], Dendroaspis polylepis (black mamba) [6], Vipera ummodytes (long-nosed viper) [7] and Hemachatus haemuchutus (Ringhalls cobra) [5] have been characterized. These analyses, correlated with the known tertiary structure of the pancreatic inhibitor [S], have facilitated understanding of the structure/function relationships for the inhibitors. Comparisons show conservation of residues at both the site of the main contacts with serine proteases [9] and the region of weak contacts [S]. The snake venom inhibitors can inhibit the activities of serine proteases, including bovine trypsin, kallikrein and plasmin [7].During investigation of the venom of the nominate race of cobra, Naja naja naja (from Pakistan), we have isolated a highly potent trypsin inhibitor of this venom. In this work, we report a single-step purification of the inhibitor on reversephase HPLC, enzymatic parameters for the protease inhibition, and the primary structure of the inhibitor, explaining its potency and antitrypsin activity.

The transcriptome recipe for the venom cocktail of Tityus bahiensis scorpion

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Non-Lethal Polypeptide Components in Cobra Venom

“…Study of the distribution of proteinase inhibitor in snake venom has shown their presence in H. heamachatus, N. nivea and N. haje venom [113]. Later on the Kunitz-type inhibitors were isolated from the venoms of H. heamachatus and N. nivea [114], N. naja [115,116], O. hannah [117] as well as N. atra [118]. All these proteins share homology with bovine pancreatic trypsin inhibitor.…”

Common evolution of waprin and kunitz-like toxin families in Australian venomous snakes

“…They act as competitive antagonists binding to, and inhibiting, the active site of serine proteases such as trypsin and chymotrypsin [3]. Kunitz-type inhibitors have been isolated from the venom of a number of Elapidae and Viperidae snakes [4][5][6][7]. In addition to their ability to act as protease inhibitors, some snake venom kunitz-like homologs have evolved as neurotoxins by inhibiting calcium and potassium channels [8].…”