The synthesis of ribonuclease A.

A protected linear polypeptide of 124 amino acid residues with the sequence of bovine pancreatic ribonuclease A was synthesized by the solid phase method. The polypeptide was removed from the solid support and pursed, and the four disulfide bonds were closed by air oxidation of the reduced form. The synthetic enzyme was fractionated by gel filtration and ion exchange chromatography, and the material that eluted at the same position as natural reduced-reoxidized RNase A was isolated. The product was further pursed by incubation with trypsin and removal of the enzymically degraded components. A fractional precipitation with (NH4)2.S04 gave a purified synthetic RNase A with a specific activity of 78 %. The synthetic RNase A was indistinguishable from natural bovine pancreatic RNase A by gel filtration on Sephadex G-75, by chromatography on carboxymethylcellulose, and by electrophoresis. Amino acid analyses, peptide maps of tryptic digests, and the Michaelis constant agreed well with those of the natural enzyme. The synthetic enzyme showed the high substrate specificity to be expected of RNase A. It was highly active against yeast RNA and 2’,3’-cyclic cytidine phosphate and was completely inactive toward DNA, 2’,3’cyclic guanosine phosphate, and the dinucleotides 5’-(3’guanylyl)-cytidylic acid and 5’-(3’-adenylyl)-adenylic acid. During the synthesis, samples were removed after 99 and 104 residues had been coupled and the corresponding polypeptides, des-(Zl-25)S-protein and S-protein, were isolated. They were then reduced and reoxidized in the presence of natural or synthetic S-peptide to give RNase S and des(21-25)RNase S. The specific activities of the two products were approximately equal, thus indicating that the five NH2-terminal residues of S-protein are not required for the protein to oxidize and fold in the presence of S-peptide to give an active enzyme.