Comparative 1H NMR studies on the structural looseness of the aged (A) and non-aged (N) bovine mercaptalbumin in the alkaline region.

Bovine serum albumin (BSA) and mercaptalbumin (BMA) exhibit the N-B transition in the alkaline region (pH 7.0-->9.0). BMA has 17 disulfide bonds and one SH group at Cys-34 which catalyzes the intramolecular SH, S-S exchange reaction at low ionic strength in the alkaline region (the N-A isomerization, where N and A indicate the non-aged and aged BMA, respectively). The N-A isomerization is a reversible first-order reaction of the type N<-->A. In the alkaline region, such as pH 8.6, (A)/(N) was approximately 1 in the absence of added salt and approximately 0 in 0.10 M NaCl, indicating the shift of the equilibrium from the A- to the N-forms. Using iodoacetamide-blocked BSA (IA-BSA) and aged BMA (IA-A-BMA), the 1H NMR cross-relaxation times (TIS), which reflect the structural looseness in proteins, were measured in the N-, B- (IA-BSA), N*- and B*-forms (IA-A-BMA), where the N*- and B*-forms indicate IA-A-BMA in the neutral (approximately pD 7) and alkaline regions (approximately pD 9), respectively. At pD 9.1-9.4 in the absence of added salt, the TIS values for the B- and B*-forms exhibited elongation, indicating the liberation of structural looseness resulting in (A)/(N) approximately 1. At pD 9.3 in 0.10 M NaCl, the TIS values for the B*-form exhibited elongation and those for the B-form did not, indicating the presence of structural looseness in only the B*-form resulting in (A)/(N) approximately 0.