Quantitative determination of free thiol and disulphide groups by a fluorescent maleimide procedure

Abstract

To determine the quantitative reliability of thiol histofluorometry, the distribution of protein thiol and disulphide groups was reinvestigated in normal human epidermis, labelled with DACM-3 after cryocutting. Microscopical observations roughly confirmed, that living keratinocytes and inner stratum corneum are the main sites of free thiol groups, while disulphide crosslinks are almost exclusively found in cornified cells. Histofluorometric quantitation led to free--SH, --S--S-- or combined --SH and --S--S-profiles through the different strata, that were not compatible with previous absorption-histophotometric studies and lacked internal consistency. The difficulties reside mainly at the methodological level and may partially be resolved--at least in keratinocytes--by extraction of nonstructural SH-groups in a prerinsing step. Permanent mounting further contribute to the realization of normal fluorescence behaviour, as visualized by the decay curves. DACM-saturation of structural protein reactive groups was only reached after prolonged staining.