Subunit Dissociation, Unfolding, and Inactivation of Bothrojaracin, a C-Type Lectin-like Protein from Snake Venom

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Abstract
Snake venoms contain a large number of hemostatically active proteins that are structurally related to Ca2+-dependent animal lectins. These proteins, called C-type lectin-like proteins (CLPs), are generally found as heterodimers composed of two homologous subunits linked by a disulfide bond. Here, bothrojaracin (BJC), a CLP from Bothrops jararaca venom that is also a thrombin inhibitor, has been used as a model to study the subunit dissociation and unfolding of CLPs from snake venom. Dithiothreitol (DTT) up to 10 mM produces minor effects on the tertiary structure and activity of BJC. On the other hand, chromatographic studies and fluorescence polarization measurements indicate that the interchain disulfide bond is disrupted by DTT, although the dimeric association is maintained. Treatment of BJC with urea produces a progressive red shift in the emission spectra of the tryptophan residues, and circular dichroism measurements show that BJC retains significant secondary structure in the presence of 8 M urea, suggesting only partial unfolding. The effects of urea are fully reversible, as there is complete recovery of BJC activity after removal of the denaturing agent. Addition of DTT to a protein sample previously treated with 8 M urea produces a slightly larger spectral shift than that observed with urea alone. Furthermore, in this condition BJC loses its secondary structure, and its subunits are dissociated. After removal of urea and DTT, BJC is inactive toward thrombin, suggesting the irreversibility of their combined action. Altogether, our data show that (i) BJC is highly resistant to urea or DTT effects, requiring the simultaneous action of both agents to fully denature the protein, and (ii) BJC monomers are tightly associated, and the presence of DTT combined with high urea concentrations is necessary to disrupt them. On the basis of these results we propose the first denaturation model for a CLP from snake venom.

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