On the Non‐Essentiality of Two Specific Disulphide Bonds in Ribonuclease for its Biological Activity

The reaction of phosphorothioate with bovine pancreatic ribonuclease at pH 9.0 in the absence of urea led to a derivative, denoted 4PS‐ribonuclease, in which two of the four disulphide bonds in the molecule were opened. 4PS‐ribonuclease is a unique molecular species, and not a mixture of native and open‐chain ribonuclease; this is apparent from electrophoretic studies. By the use of the “diagonal map” technique it has been established unequivocally that the disulphide bonds opened were between half‐cystines 3–8 and 4–5, counting from the amino terminus along the ribonuclease chain. 4PS‐ribonuclease is fully active enzymically toward RNA, and more active than the native enzyme toward cytidine 2′,3′‐cyclic phosphate. It is indistinguishable from native ribonuclease in its immunological reaction with antiserum to ribonuclease, and in its spectrophotometric titration, which shows that three of the six tyrosine phenolic groups in 4PS‐ribonuclease titrate abnormally. Unlike native ribonuclease, 4PS‐ribonuclease is digested by trypsin, even though at a lower rate than open‐chain ribonuclease.