The interaction between heparin and Lys49 phospholipase A2 reveals the natural binding of heparin on the enzyme.
We have studied at a molecular level the interaction of heparins on bothropstoxin-I (BthTx-I), a phospholipase A2 toxin. The protein was monitored using gel filtration chromatography, dynamic light scattering (DLS), circular dichroism (CD), attenuated total reflectance Fourier transform infrared (ATR-FTIR) and intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. The elution profile of the protein presents a displacement of the protein peak to larger complexes when interacting with higher concentration of heparin. The DLS results shows two Rh at a molar ratio of 1, one to the distribution of the protein and the second for the action of heparin on BthTx-I structures, and a large distribution with the increase of protein. The interaction is accompanied by significant changes in the CD spectra, showing two common features: a decrease in signal at 208 nm (3 and 6 kDa heparins) and an isodichroic point near 226 nm (3 kDa heparin). FTIR spectra indicate that only a few amino acid residues are involved in this interaction. Alterations in the ITFE by binding heparins suggest that the initial binding occurs on the ventral face of BthTx-I. Together, these results add an experimental and structural basis on the action mechanism of the heparins over the phospholipases A2 and provide a molecular model to elucidate the interaction of the enzyme-heparin complex at a molecular level.
Bugs
Milton Roque
MR
Department of Physics, IBILCE/UNESP, Rua Cristóvão Colombo, 2265, CEP 15054-000 São José do Rio Preto, SP, Brazil. bugs@df.ibilce.unesp.br
Bortoleto-Bugs
Raquel Kely
RK
Cornélio
Marinônio Lopes
ML
D000818:Animals; D001688:Biological Products / Q000737:chemistry; D017837:Bothrops; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003435:Crotalid Venoms / Q000737:chemistry; D006493:Heparin / Q000737:chemistry*; D008027:Light; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D011485:Protein Binding; D011487:Protein Conformation; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared; D055672:Static Electricity; D013552:Swine; D014364:Tryptophan / Q000737:chemistry
D001688:Biological Products; D003435:Crotalid Venoms; C078097:bothropstoxin; D014364:Tryptophan; D006493:Heparin; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine