Reversible Denaturation of Human Serum Albumin by pH, Temperature, and Guanidine Hydrochloride Followed by Optical Rotation

The pH-dependent transitions and the thermal unfolding of defatted human serum albumin in 0.2 m KCl as followed by polarimetry at 233 nm are found to be independent processes. The apparent net loss of structure (α helix) in the unfolding between pH 7 and 9 is 2.5%, in the N-F transformation it is 8% and in the acid expansion 1%. The two last mentioned transitions seem to be independent of each other. There is a temperature of maximum stability of the N stage in the N-F transformation at 18°. The thermal unfolding and the unfolding by guanidine hydrochloride at pH 2.6 both are continuous processes of apparently low cooperativity with no distinct intermediates. The temperature of maximum stability (Tmax) of human serum albumin is at all pH values below 0°, furthermore it is found to vary continuously with the concentration of guanidine hydrochloride, and in 0.2 m KCl at pH 2.6, Tmax can be extrapolated to -6°. From the transition profiles for thermal denaturation, the three dimensional structure of human serum albumin might be pictured to consist of segments of helical configuration with differential stabilities toward denaturation which impart the ability to unfold simultaneously, but more or less independently.