Isolation and characterisation of the initial intermediates in the acid denaturation of bovine pancretic ribonuclease a

Abstract

A study has been made on the isolation and characterisation of the initial products formed when RNase A is maintained in highly acidic aqueous solutions. When the enzyme is maintained in 0.5 M HCl at 30°C for periods up to 20 hours, it undergoes deamidation of the side-chain amide functions, yielding enzymatically active mono- and higher deamidated derivatives. The present thesis describes this investigation on deamidated derivatives.

RNase A has been maintained at 1 mM concentration in 0.5 M HCl at 30°C for 5, 10, 15, and 20 hours and brought back to neutral pH at the end of each period. The products obtained have been analysed by column chromatography on various cation-exchanger resins. The products obtained at various periods of acid treatment contained, in addition to RNase A, four other distinct components eluting earlier than RNase A. These components were found to be highly active and have been designated RNase Aa2, Aa1c, Aa1b, and Aa1a in order of their elution from Amberlite XE-64. There was a certain amount of overlap in the chromatographic resolution of these components. However, they have been isolated by rechromatography on Amberlite XE-64.

RNase Aa1a was as active as RNase A against yeast RNA. RNase Aa1b, Aa1c, and Aa2 had 95%, 90%, and 75%, respectively, of the enzymic activity of RNase A against yeast RNA. The UV spectra and the sedimentation coefficients of the derivatives were nearly the same as those of RNase A.

Polyacrylamide gel electrophoresis showed that both RNase Aa1a and Aa1b were electrophoretically homogeneous, having the same net charge as RNase B, which is known to have one net positive charge less than RNase A. RNase Aa2 was heterogeneous. It consisted of a major component having the same mobility as RNase B and a minor component having a mobility corresponding to two net positive charges less than RNase A. It was evident, therefore, that RNase Aa1a, Aa1b, and Aa2 are three different monodeamidated derivatives of RNase A, with RNase Aa2 containing a small amount of a dideamidated derivative as well.

The primary structures of the derivatives have been studied by fingerprint analysis of the tryptic peptides of their performic acid-oxidised derivatives. The difference in the peptide patterns of RNase Aa1a and Aa1b compared to RNase A was limited to the peptide O-Tryp 2, which represents the 67–85 region of ribonuclease A. This region contains four amide groups, namely Asn 67, Gln 69, Asn 71, and Gln 74. O-Tryp 2 from both RNase Aa1a and Aa1b was found to have the same higher anodic mobility, indicating that deamidation in both these derivatives is of the same magnitude and occurs in the 67–85 region. Further, hydroxylamine treatment of performic acid-oxidised RNase Aa1a and Aa1b indicated that Asn 67 is not deamidated in these derivatives.

Peptide analysis of the tryptic peptides of performic acid-oxidised RNase Aa1c showed that two of its peptides had higher anodic mobilities. In this case also, O-Tryp 2 had the same mobility as that of O-Tryp 2 from RNase Aa1a and Aa1b. In addition, O-Tryp 14, representing the 92–98 region of RNase A, also exhibited higher anodic mobility. Further, hydroxylamine treatment of performic acid-oxidised RNase Aa1c indicated that Asn 67 is not deamidated. From these results, it could be assessed that RNase Aa2, which is a mixture of mono- and di-deamidated RNase A, consisted of a monodeamidated component having its deamidation in the O-Tryp 2 region and a dideamidated component having its deamidation in the O-Tryp 2 and O-Tryp 14 regions (Asn 94). It is apparent, therefore, that the initial products of acid treatment of RNase A yield three monodeamidated derivatives, the location of deamidation being Gln 69, Asn 71, and Gln 74. However, the identification of the location of deamidation in the individual monodeamidated derivatives has not been carried out.

The derivatives were found to be resistant to the action of trypsin and chymotrypsin at neutral pH and room temperature (25°C) but were susceptible to proteolytic attack at 60°C. The derivatives were susceptible to extensive proteolytic hydrolysis by pepsin. This action was significantly different from the action of pepsin on RNase A. Thus, the conformation of the derivatives at pH 1.8 and 25°C appears to be different from that of RNase A. Circular Dichroism (CD) studies also support this conclusion.

Air oxidation of reduced RNase Aa1b and Aa1c was found to regenerate nearly 15–30% of the enzymic activity of native RNase A. The specific elution positions of the reduced-reoxidised derivatives indicate that the respective derivatives are regenerated. The UV spectra and the gel electrophoretic patterns of the reduced-reoxidised derivatives were similar to those obtained for the respective derivatives. The regain of enzymic activity in the case of the derivatives was comparatively slower than RNase A, the order being A > Aa1a > Aa1b > Aa2.

The present studies show that the initial deamidation of RNase A on treatment in highly acidic aqueous solutions at 30°C gives three monodeamidated products, the deamidation being restricted to the Gln 69, Asn 71, and Gln 74 residues. The overall structure, the catalytic function, as well as the refolding of the polypeptide are not markedly affected as a result of the deamidations in these residues. Nevertheless, small changes in all these parameters occur from this subtle alteration of the primary structure.