Circular dichroic and 1 H‐NMR studies on the aged form of bovine plasma albumin

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Abstract
Bovine plasma albumin (BPA) has 17 disulfide bonds and approximately one SH group at Cys-34 which catalyzes the intramolecular SH, S-S exchange reaction in the alkaline region at low ionic strength, resulting in the formation of the aged form (A-form). 1) Fractions of α-helix (fα) and β-form (fβ) of iodoacetamide-blocked non-aged BPA (IA-BPA) at pH 6.5 (the N-form) and 9.0 (the B-form) in the absence of added salt were 0.70, 0.12 and 0.62, 0.18, respectively (Era et al. (1990)). However, there were no changes in fα and fβ of the iodoacetamide-blocked A-form (IA-A-form) over the pH range from 5.5 to 9.1 in the absence of added salt or in 0.10 m KCl(fα∼ 0.60, fβ∼ 0.20), indicating that the secondary structure of the IA-A-form might be similar to that of non-aged IA-BPA at pH 9.0 (B-form) in the absence of added salt, that is, the frozen B-form, stabilized covalently by the repairing of disulfide bonds. 2) The rigidity of the A- and IA-A-forms, as monitored by cross-relaxation times between irradiated and observed protein protons, was similar to or slightly higher than that of non-aged IA-BPA or BMA. However, spin-echo 1H-NMR spectra indicated that side chains of the IA-A-form, such as ɛ-CH2 of Lys, -CH3, in the absence of added salt are more mobile than those of non-aged IA-BPA. 3) The ratio of the hydrodynamic volume of the A-form to that of non-aged BPA at pH5.5 was approximately 1.1.