Light scattering and viscosity of proteins in anhydrous formic acid

The use of anhydrous formic acid as a solvent for proteins has been investigated with a view to its application to proteins which are aggregated or insoluble in aqueous media. Five proteins of known molecular weight–bovine serum albumin, ovalbumin, lysozyme, β‐lactoglobulin, and insulin–have been studied in this solvent by light scattering and viscosity techniques. Bovine serum albumin and ovalbumin showed time‐dependent changes in viscosity which complicated the interpretation of viscosity measurements. These changes in viscosity with time were not due to hydrolysis or aggregation of the protein. It was necessary to correct the light scattering data for fluorescence of the protein in formic acid. The molecular weights determined in formic acid and in the presence of neutral salt indicate that β‐lactoglobulin and insulin are largely dissociated to the monomeric form; the other proteins gave slightly higher molecular weights in formic acid than in aqueous solution. In the absence of salt the concentration dependence of both properties measured is typical of highly charged polyelectrolytes. The KC/R 90 vs. C plots show a very steep rise from zero concentration and level off at higher concentrations. The η sp / C vs. C plots for bovine serum albumin, ovalbumin, and β‐lactoglobulin exhibit maxima at protein concentrations between 0.1 and 0.2%. On addition of neutral salt or water to the formic acid, the light scattering and viscosity show a normal dependence on concentration. The difference in polyelectrolyte behavior between the proteins is attributed to differences in molecular weight, varying degrees of intramolecular crosslinking by disulfide bonds, and different distributions of positively charged side‐chains along the polypeptide backbone. The viscosity measurements show that in formic acid the proteins are in a highly swollen state. Optical rotation measurements indicate that the proteins are denatured in formic acid.