H-NMR and CD studies on the structural transition of serum albumin in the acidic region--the N-->F transition.

Helical content (f(alpha)) of bovine mercaptalbumin (BMA) showed the characteristic two-step decrease in the acidic region, one corresponding to the N-->F transition (pH 4.40-->3.75; f(alpha), 0.68-->0.58) and the other to the F-->E transition (the acid-expansion) (pH 3.60-->2.90; falpha, 0.58-->0.48). However, falpha of human serum albumin (HSA) mainly decreased in the N-->F transition (N-->F, pH 4.6-->3.4; falpha, 0.70-->-0.55 and F-->E, below pH 3.0; falpha, 0.55-->0.52). The difference in pH-profile of f(alpha) between BMA and HSA might be due to the microheterogeneity. The 1H-NMR spectra and cross-relaxation times (T(IS)) from irradiated to observed protein protons, which reflect the structural fluctuation and/or mobilability in proteins, were measured on the N-, F-, E-forms of HSA and BMA, and the N*-form (8.23 M urea, neutral pD) of iodoacetamide-blocked HSA (IA-HSA) and bovine serum albumin (IA-BSA). The 1H-NMR spectra and elongations of T(IS) values for the F- and E-forms of HSA and the E-form of BMA were quite similar to those for the N*-form of IA-HSA and IA-BSA, indicating the liberation of the intramolecular motion in the F- and E-forms. Those for the F-form of BMA were intermediate between the N- and E-form. The present results together with the reported data on hydrodynamic radii and D-H exchange reaction, indicate that the F-form of HSA and presumably BMA has a native-like globule form with a highly helical state and fluctuating tertiary structure. Thus, all of the present findings on the F-form of serum albumin seem to be in accord with the structural features for the F-form suggested by Foster's group (1-3, 19, 20, 22, 23) and the molten globule state demonstrated by Dolgikh et al. (40), and Ohgushi and Wada (36, 37).