Quantitative cytospectrophotometrical determination of the total protein thiols with "mercurochrom": optimization and calibration of the histochemical reaction.

Mercurochrom (2,7-dibromo-4-(hydroxymercuri)-fluoresceine-disodium salt) reacts histochemically not only with protein-SH-groups, but is also bound unspecifically to cellular proteins. The amount of the unspecifically staining approximately equals the specific SH-staining. Two methods are described to remove the unspecifically bound Mercurochrom, without influencing the specific reaction with the protein thiols. The first applies 0,1 m thioglycolate pH 4.0 (MT4-method), the other a special tris-cyanide-buffer pH 7.4 (MCN)-method). Aliquots from preparations of rat hepatocytes, Yoshida ascites tumor cells, Ehrlich ascites tumor cells, isolated nuclei of Ehrlich-cells and chicken thymocytes were investigated as well for the protein thiol content of the cells macroscopically with DTNB as microspectrophotometrically for the extinctions of the cells after staining with MT4- or MCN-method. A strong correlation was found between the macroscopically determined total-protein-SH-contents and the microphotometrically determined mean-total-extinctions of the cells. Additionally the molar absorptivities determined macroscopically by Schauenstein and Scheuringer (1980) coincide excellently with the values found microspectrometrically on MT4- and MCN-stained cells.