Native or reduced pancreatic ribonuclease A having zero and eight thiol groups per mole, respectively, was treated with selenious acid to evaluate the role of thiol groups in the nonenzymic incorporation of inorganic selenite into proteins. Over the pH range of 2-7, no selenium was incorporated into native ribonuclease A, but reduced ribonuclease A treated with selenious acid at pH 2 and 4° showed rapid loss of thiol groups, spectral changes equivalent to selenotrisulfide (RSSeSR) formation, and the uptake of 2 moles of selenium/mole of ribonuclease A, in accord with the over-all reaction 4RSH + H2SeO3 RSSeSR + RSSR + 3H2O. At higher pH, elemental selenium was liberated during the reaction, decreasing the incorporation of selenium to 1.41 and 0.44 moles of Se per mole of ribonuclease A at pH 4.7 and 7, respectively, but pH 7 caused little or no release of selenium from the final reaction product. Gel filtration and sedimentation velocity studies of the pH 2 derivative indicate that it is homogeneous, monomeric, and more unfolded than native ribonuclease A. Spectral perturbations in the 280-290-mμ region likewise suggest that the degree of folding is intermediate between that of reduced and native ribonuclease A. The exchange of approximately 1 mole of selenium into ribonuclease A from the selenotrisulfide derivative of 2-mercaptoethanol occurred with reduced ribonuclease A at 4° at pH 2-7, with extensive liberation of elemental selenium, but none was exchanged into native ribonuclease A. The enzyme activity of the derivatives against yeast ribonucleic acid or 2′,- 3′-cyclic cytidylic acid equaled only a few per cent of that of native ribonuclease A. It is concluded that selenium was incorporated between two sulfur atoms to form an intramolecular selenotrisulfide linkage in place of a disulfide. Such derivatives may be useful for a variety of protein structure-function studies. © 1969, American Chemical Society. All rights reserved.
