Specific labelling of thiol groups in mammalian keratin suitable for electron microscope studies.

Abstract

SUMMARYQuantitative Polarographic studies of the reaction between methylmercuric iodide and variously reduced wool keratin indicate that reaction occurs specifically with the thiol groups from cystine. Since loss of protein from fibres is negligible, this organomercurial is preferred as a selective amino acid label in electron microscope studies. Similar reactions of wool with parachloromercuribenzoate (PCMB) result in extensive structural modifications.The mercury mercaptides were shown to be thermally unstable in the electron beam and cooling of the specimen between — 140°C and — 88°C coupled with low beam intensities was found necessary to study the original distribution of the mercurials in the samples. Since a direct relationship between the electron scattering sites of the mercurial and cystine within the keratin was established, it has been possible to demonstrate unambiguously for the first time that:(a) There exists a radial distribution of cystine within cuticular cells. Indeed a new sulphur‐rich component at the outer edge of the ‘a’ layer in peripheral cuticular cells has been identified.(b) Para‐cortical cells of intact keratin contain a higher proportion of cystine than ortho‐type cells.(c) A dense concentration of cystine labelled by the electron scattering centres (mercurials) corresponds closely to an intermicrofibrillar‐matrix system in the cortex.Furthermore, a method has been devised for determining the cystine content of the exocuticle using data obtained from electron micrographs.