Bovine pancreatic ribonuclease was found to react with the sulfhydryl reagents dithioerythritol or dithiothreitol in two steps: a fast step followed by a much slower one. The product at the end of the fast step was carboxymethylated and digested by pepsin, and the digest was analyzed by paper electrophoresis and chromatography. Only cystine bond IV-V was reduced during the fast reduction step. The partially reduced protein obtained at the end of the fast step reacts with either 1 or 2 moles of mercuric ions, yielding derivatives of the type -S-Hg-S- or -SHg+, respectively. The monomercury derivative was crystallized in a form which is very nearly isomorphous with the monoclinic crystalline form of native ribonuclease. The mono- and dimercury derivatives, as well as the carboxymethyl derivative of ribonuclease reduced at cystine IV-V, are enzymically as active as the native protein and are resistant to digestion by trypsin. Similarly, the mercury derivatives exhibit an optical rotatory dispersion which is identical with that of native ribonuclease. Three of their tyrosine side chains titrate abnormally as in native ribonuclease. The cystine bridge IV-V in bovine pancreatic ribonuclease thus seems to be completely unnecessary for enzymic activity or for maintaining the native macromolecular conformation of ribonuclease. © 1969, American Chemical Society. All rights reserved.
