The Heterogeneity of Bovine Albumin with Respect to Sulfhydryl and Dimer Content

Abstract

Abstract A relatively fresh bovine plasma albumin (BPA) preparation (0.65 sulfhydryl group per albumin molecule; 5 to 6% dimer) and an old bovine mercaptalbumin (BMA) preparation (0.72 sulfhydryl group per albumin molecule; 8 to 9% dimer) were fractionated in a phosphate gradient on DEAE-Sephadex A-50. Two dimer fractions were obtained. One dimer fraction had a relatively high sulfhydryl content and could not be converted to monomer by treatment with thioglycolate. The other dimer fraction was essentially sulfhydryl-free, and approximately 50% of this dimer could be converted to monomer on treatment with thioglycolate. The first type of dimer predominated in the BPA preparation, while the second type of dimer was the major dimer component of the BMA preparation. BPA monomer fractions of varying sulfhydryl content were obtained. The first major fraction to be eluted was mercaptalbumin. Non-mercaptalbumin fractions were retarded in their elution from the column, and it was not possible to achieve more than partial regeneration of the sulfhydryl group of the more retarded non-mercaptalbumin fractions by treatment with 0.05 m thioglycolate. These results agree with the findings of Hartley, Peterson, and Sober and of Andersson. The BMA monomer fractions were similar in their elution volume and sulfhydryl content to the BPA fractions; as with the BPA non-mercaptalbumin fractions, the more retarded the albumin monomer fraction, the lower the sulfhydryl content after treatment with 0.05 m thioglycolate. The similarities in the behavior of the BPA and BMA non-mercaptalbumin monomer fractions are somewhat surprising since the presence of these non-mercaptalbumin fractions in plasma albumin has been shown to be due to mixed disulfide formation between mercaptalbumin and plasma cysteine and glutathione. It is difficult to see how the BMA non-mercaptalbumin monomer fractions could be present owing to such mixed disulfide formation, since the BMA preparation originally contained one sulfhydryl group per albumin molecule and has not since been in contact with small molecule thiols. Some oxidative pathway other than disulfide formation must be operative in the formation of non-mercaptalbumin monomer.