Thermal stability studies of hyperimmune horse antivenoms

Ampoules of horse antivenoms raised against Bothrops spp and Crotalus durissus (final product) produced by Fundação Ezequiel Dias (FUNED) were fractionated on the molecular filtration chromatography (SUPEROSE 12) and the expected MW species of F(ab')2 fragments were observed. It has been known that high temperatures promote aggregation and formation of protein precipitates. Phenol is used in preparations of antivenoms as preservative; however, as thus is a hydrophobic substance, it can also induce an increase in protein denaturation. Sorbitol and glycerol were used as osmolyte (natural substance or organic compound capable of stabilizing proteins) and decreased the formation of protein precipitates in solutions of antibodies; as judged by a spectrophotometric assay (280 nm), by nephelometry or when tested by the ELISA. In 1.0 M concentration, the sorbitol presented the best results when compared with glycerol. Circular Dichroism (CD) was used to study the spectra of antibodies in the presence of PBS, glycerol or sorbitol. Up to 1.0 M concentration of the osmolyte, there is no significant perturbation of the antibodies spectra in the amide region, in control samples not incubated (kept at room temperature with no phenol added) or incubated at 62 degrees C in presence of phenol. Nephelometry and gel SDS-PAGE techniques were used in assays that demonstrated the effects of phenol (denaturing) and the osmolytes (stabilizing) in horse antivenoms in high temperatures.