Studies on the Binding between Acid Proteinases and Aliphatic Alcohols with Dyes as Probes

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Abstract
Binding between four kinds of acid proteinases (pepsin, acid proteinase from Cladosporium, and acid proteinases A and B from Aspergillus niger) and aliphatic alcohols, which are competitive inhibitors of acid proteinases, was studied in terms of the absorbance changes of zinc(Il)-pyridine-2-azo-p-dimethylaniline (Zn-PAD) complex, a catalytic site probe for acid proteinases. Binding between three of the enzymes (pepsin, acid proteinase from Cladosporium, and acid proteinase B from Aspergillus niger) and n-amyl alcohol was studied in terms of the fluorescence changes of 2-p-toluidinylnaphthalene-6-sulfonate (TNS), a probe for hydrophobic sites of proteins. It was found that five alcohols (methanol, ethanol, n-propanol, n-butanol, and n-amyl alcohol) affected the absorption spectra of the Zn-PAD-enzyme complexes, and n-amyl alcohol affected the fluorescence spectra of the enzyme-TNS complexes. The results are consistent with the view that the binding of alcohols with the enzymes in a molar ratio of 1:1 induces the spectral change. These results suggest that the acid proteinases studied have a hydrophobic binding site for alcohols near the catalytic site. The logarithm of the apparent dissociation constant between the acid proteinases and alcohols (logK1) decreased linearly with the increase of the chain length of the alcohols. The standard Gibbs free energy change for the binding between pepsin and methylene groups of alcohol was estimated to be about 2.5 kJ/mol (0.6 kcal/mol) per methylene group (at 25°C, pH 5.0).