The Reoxidation of Reduced Ribonuclease Derivatives.

It now appears to be well established that the tertiary structure of a protein is determined primarily by its amino acid sequence. The principal support for this concept is the demonstration that several enzymes (particularly bovine pancreatic ribonuclease, egg white lysozyme, and Tska-Amylase A) spontaneously regain their native conformations after they have been unfolded and their disulfide bonds have been reduced in 8 M urea and /I-mercaptoethanol (l-4). Additional information about the influence of sequence on tertiary structure has been derived from the x-ray crystallographic and amino acid sequence studies on the hemoglobin-myoglobin family of proteins. The work has indicated that globular proteins have distinct surfaces and interiors, the former containing most of the hydrophilic side chains and the latter being composed mainly of hydrophobic residues (5). On the basis of these observations, the following hypothesis was recently advanced: in general, modifications involving groups on the surface of a molecule should not interfere with proper folding of the polypeptide chain, especially if one polar group is substituted for another and the change in surface charge is not too great. Conversely, many alterations of interior residues could adversely affect folding, particularly if steric hindrance results or if a polar residue is substituted for a nonpolar one (6). The reversible reduction and reoxidation of enzymes, especially of RNase, has provided a useful system for studying t’he effects of modifications of amino acid residues on the ability of the unfolded molecule to attain an active tertiary conformation. Early experiments in which long chains of poly-nn-alanine were attached to the exposed (i.e. surface) lysine t-amino groups of RNase and of trypsin demonstrated that such modifications did not prevent the reduced enzymes from regaining active conformations (7, 8). In the present report are presented the results of experiments involving the reduction and reosidation of several other types of RNase in which groups on the surface have been modified.