Abnormal tyrosine residues in subtilisin-modified ribonuclease.

Unfolding of the native conformation of pancreatic ribonuclease disrupts certain intramolecular associations of amino acids involving tyrosine residues (1) ; the subsequent absorbance changes serve as an index of the extent of denaturation of the enzyme (2). Of the 6 tyrosine residues of RNase A, 3 display distinct spectral properties which may be evaluated by determination of changes in ultraviolet absorbance and spectral maximum (3) or in the pK of phenolic hydroxyl ionization (4). Bigelow (5) has carefully evaluated the molar absorbance changes associated with complete or partial disruption by various denaturants of the conformation of the native protein. He has indicated that integral values may be assigned to exposure of each of the 3 abnormal tyrosine residues (at h = 287 rnp, molar absorbance changes of -1000, -700, and -1000 are associated with residues arbitrarily designated as A, B, and C, respectively) . With simple heat denaturation, only 2 of the 3 residues become solvent-accessible, indicating that some aspects of the normal conformation of the molecule persist after extensive heating (5). The spectral properties of RNase S, the fully active RNase derivative formed by cleavage of the peptide bond between residues 20 and 21, are similar to those of the native protein (6). RNase S-protein, the inactive derivative comprising residues 21 through 124 in the amino acid sequence, has alterations in ultraviolet absorption maximum and spectrophotometric titration which indicate that 1 or more of the tyrosine residues ordinarily shielded is solvent-accessible in this derivative prior to denaturation (6, 7). The relative stability of the three-dimensional structure of these proteins has previously been examined by a comparison of their optical rotatory and spectral changes during progressive denaturation by heat and urea (8). A detailed and quantitative comparison by difference spectroscopy of the absorbance changes of RNases A, S, and S-protein therefore seemed warranted (a) to determine whether integral absorbance changes similar in molar value to those described by Bigelow (5) for RNase A could be identified during the denaturation of RNases S and S-protein, (b) to determine whether the subtilisin derivatives retain 1 or more abnormal tyrosine residues after heat denaturation, and (c) to derive from a consideration of (a) and (b) further information about the similarities and differences in the conformation of 3 covalently related molecules.