Structural and functional characterization of complex formation between two Kunitz-type serine protease inhibitors from Russell's Viper venom

Biochimie. Sep-Oct 2016;128-129:138-47. doi: 10.1016/j.biochi.2016.08.005. Epub 2016 Aug 12.

Abstract

Snake venom Kunitz-type serine protease inhibitors (KSPIs) exhibit various biological functions including anticoagulant activity. This study elucidates the occurrence and subunit stoichiometry of a putative complex formed between two KSPIs (Rusvikunin and Rusvikunin-II) purified from the native Rusvikunin complex of Pakistan Russell's Viper (Daboia russelii russelii) venom (RVV). The protein components of the Rusvikunin complex were identified by LC-MS/MS analysis. The non-covalent interaction between two major components of the complex (Rusvikunin and Rusvikunin-II) at 1:2 stoichiometric ratio to form a stable complex was demonstrated by biophysical techniques such as spectrofluorometric, classical gel-filtration, equilibrium gel-filtration, circular dichroism (CD), dynamic light scattering (DLS), RP-HPLC and SDS-PAGE analyses. CD measurement showed that interaction between Rusvikunin and Rusvikunin-II did not change their overall secondary structure; however, the protein complex exhibited enhanced hydrodynamic diameter and anticoagulant activity as compared to the individual components of the complex. This study may lay the foundation for understanding the basis of protein complexes in snake venoms and their role in pathophysiology of snakebite.

Keywords: Kunitz-type serine protease inhibitor; Phospholipase A(2); Protease; Protein-protein interaction; Snake venom complex.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Aprotinin / chemistry
  • Aprotinin / metabolism
  • Cattle
  • Chromatography, High Pressure Liquid
  • Circular Dichroism
  • Electrophoresis, Polyacrylamide Gel
  • Fibrin / metabolism
  • Fibrinogen / metabolism
  • Humans
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / metabolism*
  • Protein Binding
  • Proteolysis
  • Russell's Viper / metabolism*
  • Serine Proteinase Inhibitors / chemistry
  • Serine Proteinase Inhibitors / metabolism*
  • Serum Albumin, Bovine / metabolism
  • Spectrophotometry
  • Tandem Mass Spectrometry
  • Viper Venoms / chemistry
  • Viper Venoms / metabolism*

Substances

  • Multiprotein Complexes
  • Serine Proteinase Inhibitors
  • Viper Venoms
  • Serum Albumin, Bovine
  • Fibrin
  • Fibrinogen
  • Aprotinin