The reaction between thioglycollate and wool proteins

Abstract

Proteins extracted from wool by alkaline thioglycollate containing ³⁵S-thioglycollic acid were radioactive even after exhaustive dialysis. The amount of thioglycollate binding was dependent to a certain extent on the method of preparation of the proteins. The thioglycollate content of S-carboxymethyl kerateine 2 (SCMK 2), a soluble protein fraction prepared from a pH 12.3 extract of wool which had previously been extracted five times at pH 10.5, was about 10 μmoles/g protein. By contrast, another soluble protein fraction designated S-carboxymethyl kerateine and prepared from a pH 11 0 extract of wool, contained from 59 to 97 μmoles thioglycollate/g protein, depending on the final stage of preparation. Possible reasons for these variations are discussed. The strength of binding of the thioglycollate was tested by attempts to displace it with a number of reagents. Several of these were ineffective in removing radioactivity from the wool proteins, whereas disulphide-breaking agents resulted in the removal of all but the equivalent of 3 and 13 μmoles thioglycollate/g SCMK 2 and SCMK respectively. It is postulated that the bound thioglycollate is present partly in the form of a mixed disulphide composed of half-cystine and half-dithiodiglycollic acid (β-carboxy-β-aminoethyl carboxymethyl disulphide) and partly in an unknown form. The former may arise as an intermediate in the reaction between wool protein and thioglycollic acid. The possible nature of the bound sulphur in the unknown form is discussed. The thioglycollate content of wool proteins as determined by isotopic means was equal to or less than the thiol+disulphide content measured by amperometric titration with mercurials. It was concluded that SCMK preparations contain up to 19 μmoles cystine and/or cysteine/g protein in addition to any half-cystine residues involved in the mixed disulphide. This conclusion was qualitatively supported by polarographic experiments using the catalytic cobalt wave for the detection of certain sulphur containing compounds. The importance of the mixed disulphide with regard to heterogeneity and other problems associated with soluble wool proteins is discussed.