Isolation and characterization of monodeamidated derivatives of bovine pancreatic Ribonuclease A

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Abstract
The isolation and characterization of the initial intermediates formed during the irreversible acid denaturation of enzyme RNA are described. The products obtained when RNase A is maintained in 0.5 M HCl at 30.degree. C for periods up to 20 h were analyzed by ion-exchange chromatography on Amberlite XE-64. Four distinct components eluted earlier to RNase A; these were designated RNase Aa2, Aa1c, Aa1b and Aa1a in order of their elution. With the exception of RNase Aa2, the other components are nearly as active as RNase A. Polyacrylamide gel electrophoresis at near-neutral pH indicated that RNase Aa1a, Aa1b and Aa1c are monodeamidated derivatives of RNase A; RNase Aa1c contains, in addition, a small amount of a dideamidated component. RNase Aa2, which has 75% enzymic activity as compared to RNase A, consists of dideamidated and higher deamidated derivatives of RNase A. Except for differences in the proteolytic susceptibilities at an elevated temperature or acidic pH, the monodeamidated derivatives had very nearly the same enzymic activity and the compact folded structure as the native enzyme. Fingerprint analyses of the tryptic peptides of monodeamidated derivatives showed that the deamidations are restricted to an amide cluster in the region 67-74 of the polypeptide chain. The initial acid-catalyzed deamidation occurs in and around the 65-72 disulfide loop giving rise to at least 3 distinct monodeamidated derivatives of RNase A without an appreciable change in the catalytic activity and conformation of the RNase molecule. Significance of this specific deamidation occurring in highly acidic conditions and the biological implications of the physiological deamidation reactions of proteins are discussed.