DEAMIDATED ACTIVE INTERMEDIATES IN THE IRREVERSIBLE ACID DENATURATION OF RIBONUCLEASE-A

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Abstract
A study was made on the changes in the enzymatic activity of RNase-A exposed to highly acidic (pH < 1) aqueous environment. Irreversible alterations of activity were observed when the protein was exposed to an acidic medium for a long period (20-60 h). Prior to these changes in activity RNase-A formed intermediates which had very nearly the same activity of the native protein. The primary process in the acid denaturation of RNase-A was deamidation of the protein leading to the formation of active chromatographically distinct derivatives. The initial product of deamidation, a monodeamidated derivative, was isolated by chromatography on Amberlite XE-64. This initial deamidation reaction was highly specific. The subsequent deamidation reaction is comparatively slower, so that nearly 50% of the native protein could be converted to this derivative before any subsequent deamidation took place. This monodeamidated derivative was designated RNase-Aa1. The conversion of RNase-A to RNase-Aa-1 was not accompanied by any changes in the primary structure other than the observed deamidation. Apart from the differences in chromatographic and electrophoretic mobilities, RNase-Aa1 had very nearly the same activity and physicochemical properties as the native enzyme. Significance of this specific and faster deamidation of RNase-A in this denaturing medium and the biological significance of such deamidation reactions of proteins are discussed.

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