Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers

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Abstract

A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.

Section snippets

Materials and methods

Reagents. All solvents were of HPLC grade unless otherwise stated and purchased from Sigma (Poole, Dorset, UK) except acetic acid, which was obtained from BDH (Poole, Dorset, UK).

Acquisition of venoms. All venoms were collected from authenticated snake species housed at the Alistair Reid Venom Unit of the Liverpool School of Tropical Medicine, University of Liverpool (Liverpool, UK), and were supplied in a lyophilized form.

Gel permeation chromatography. Lyophilized snake venoms (9–10 mg dry

Chromatographic fractionation of snake venoms

Thirty snake venoms were fractionated using Sephadex G50 gel permeation chromatography (data not shown). Three of these venoms, from A. bilineatus (Mexican moccasin), C. v. viridis (prairie rattlesnake), and L. muta (bushmaster), displayed striking similarities in their gel permeation profiles (Fig. 1). These all displayed a penultimate peak of absorbance that was larger than the final, total volume peak of absorbance (Fig. 1). Pooled fractions representing this penultimate peak from the three

Discussion

In this study, the low molecular weight peptides present in gel permeation chromatographic fractions of the venoms from three species of New World pit vipers from the subfamily, Crotalinae, that is the Mexican moccasin (A. bilineatus), the prairie rattlesnake (C. v. viridis), and the South American bushmaster (L. muta), were characterized using HPLC, MALDI-TOF mass spectrometry, and Edman sequencing. This strategy led to the identification of a novel peptide entity that we named bradykinin

Acknowledgment

We thank the Department of Education Northern Ireland for funding the PhD studentship of Robert Leslie Graham.

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