Isolation and biochemical characterization of a γ-type phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum

Toxicon. 2014 Apr:81:58-66. doi: 10.1016/j.toxicon.2014.01.012. Epub 2014 Feb 8.

Abstract

In the present work, we describe the isolation and partial structural and biochemical characterization of the first phospholipase A2 inhibitor (γPLI) from Crotalus durissus collilineatus (Cdc) snake serum. Initially, the Cdc serum was subjected to a Q-Sepharose ion exchange column, producing six peaks at 280 nm absorbance (Q1-Q6). Subsequently, Q4 fraction was submitted to affinity chromatography with immobilized PLA2 BnSP-7, a step that resulted in two fractions (NHS-1 and NHS-2). The latter contained the inhibitor, denominated γCdcPLI. The molecular mass of γCdcPLI, determined by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), was 22,340 Da. Partial sequences obtained by Edman degradation and by mass spectrometry (MALDI-TOF/TOF), showed similarity, as expected, to other related inhibitors. Circular dichroism (CD) analysis showed the presence of approximately 22% alpha helices and 29% beta sheets in the protein secondary structure. Additionally, CD studies also indicated no significant changes in the secondary structure of γCdcPLI when it is complexed to BpPLA2-TXI. On the other hand, dynamic light scattering (DLS) assays showed a temperature-dependent oligomerization behavior for this inhibitor. Biochemical analyses showed γCdcPLI was able to inhibit the enzymatic, cytotoxic and myotoxic activities of PLA2s. Structural and functional studies performed on this inhibitor may elucidate the action mechanisms of PLA2 inhibitors. In addition, we hope this study may contribute to investigating the potential use of these inhibitors for the treatment of snakebite or inflammatory diseases in which PLA2s may be involved.

Keywords: Crotalus durissus collilineatus; Inhibitor γ-type; Phospholipase A(2); Phospholipase A(2) inhibitors; Snake.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bothrops
  • Crotalid Venoms / chemistry
  • Crotalus / blood*
  • Glycoproteins / chemistry*
  • Glycoproteins / isolation & purification
  • Mice, Inbred BALB C
  • Molecular Sequence Data
  • Phospholipase A2 Inhibitors / chemistry*
  • Phospholipase A2 Inhibitors / isolation & purification
  • Phospholipases A2 / isolation & purification
  • Reptilian Proteins / chemistry*
  • Reptilian Proteins / isolation & purification
  • Sequence Alignment
  • Sequence Analysis, Protein
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Crotalid Venoms
  • Glycoproteins
  • Phospholipase A2 Inhibitors
  • Reptilian Proteins
  • phospholipase A2 inhibitor protein, Crotalus
  • Phospholipases A2