The pH Jump Study of Enzyme Proteins

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Abstract
Rapid conformational changes due to pH jump were studied kinetically at 25° mainly by the stopped-flow method using liquefying α-amylase from Bacillus subtilis [EC 3.2. 1. 1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conforrnational reconstitution, as observed through the protonation of phenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accom panied by an optical rotation change and no appreciable reversibility was detected by these methods.

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