Urea denaturation of bovine serum albumin at pH 9

The action of 5 m urea on bovine serum albumin has been studied at pH 9.0 and 25°C. Analysis by the acrylamide gel electrophoresis revealed the presence of a few components 1, 1′, 2, 3, 4 and 5. The components 1 and 1′ are monomers, component 2 is a dimer, and components 3, 4 and 5 are aggregates. In presence of SH blocking reagent, bovine serum albumin gave only the zone 1, indicating that the components 1′-5 were formed by the SH to S-S exchange reactions. Component 1′ was formed by the intramolecular SH to S-S exchange reaction, and components 2–5 were formed by the intermolecular exchange reaction. Addition of cysteine either to bovine serum albumin or to the SH-blocked bovine serum albumin increased the percent of zone 1′, indicating that a complex bovine serum albumin-cysteine was formed or that the SH-catalyzed structural alteration occurred in bovine serum albumin. Components 1, 1′, 2 and 3 were isolated separately by the preparative disc gel electrophoresis. The sedimentation coefficients 1 and 1′ differed slightly indicating that they were different monomers, and values were slightly smaller than the normal value of bovine serum albumin, indicating that these components were in slightly expanded state. Isolated component 1 was exposed to 5 m urea again, but no further change occurred. This supports the concept of microheterogeneity of bovine serum albumin.