Regeneration of native secondary and tertiary structures by air oxidation of reduced ribonuclease.

Although there is an abundance of evidence in support of the template hypothesis of protein biosynthesis (I), there appears to have been no satisfactory experimental basis for deciding whether the role of the template includes the coiling and folding of the protein chain to produce the secondary and tertiary structures’ or is restricted to the formation of amino acid sequence. It has been reported (4) that fully reduced and enzymatically inactive bovine pancreatic ribonuclease can be oxidized in vitro by atmospheric oxygen to produce a soluble protein in high yield with a specific activity approaching that of native ribonuclease. The reduced molecule is apparently devoid of the native secondary and tertiary structures (5). Intramolecular oxidation of this form of ribonuclease to yield four disulfide bonds per mole from the eight sulfhydryl groups per mole would, with every conceivable combination of half-cystine residues, result in 105 isomers (6). However, the high regeneration of enzymatic activity suggests either that this oxidative process entails the preferential reformation of the disulfide bonds and other features of the secondary and tertiary structures that exist in native ribonuclease, or that there is more than one three-dimensional configuration of ribonuclease that would permit the expression of enzymatic activity. The results of comparative studies on the main chromatographic components of native and fully reduced, air-reoxidized ribonucleases indicate that these enzymes possess identical secondary and tertiary structures. This evidence suggests that all of the information needed for establishing these structural features resides in the primary structure and therefore that the template may be concerned only with determination of amino acid sequence in the protein chain.