Conformational Changes of Aspergillus sojae Alkaline Proteinase with Denaturing Reagents

Abstract

The conformational changes of the alkaline proteinase of Aspergillus sojae were analyzed, after the treatments with urea and guanidine hydrochloride, by the optical rotatory dispersion, Cotton effects, ultraviolet difference spectra, and viscosity measurements. The results are as follows; (1) The treatment with 8M urea (pH 7.0) seemed to cause at least two different conformational changes assumed from the changes in the Moffitt parameter, a₀. One is a rapid and reversible change and the other is a gradual and irreversible change. During the process of the later change, it was observed that the complete disruption of the α-helices assumed from the b₀ value and the Cotton trough at 233mμ proceeded in parallel with the loss of enzymatic activity. (2) The conformational change after the treatment with 1.6M guanidine hydrochloride (pH 7.0) was considered to be rather small, judging from the changes in the a₀ value and intrinsic viscosity.
However, by this treatment, the two tryptophan residues which are buried in the interior of the molecule were exposed to the solvent, the α-helices were completely disrupted, and the enzymatic activity was completely lost.