[Quantitative determination of sulfhydryl groups with "mercurochrome" (author's transl)].
Dibrommercuryfluoresceine (DBMF) reacts stoichiometrically and quantitatively with the thiol group of cysteine, glutathione and thioglycolic acid respectively, at pH 7.0. Polarographical and spectrometrical titrations clearly show that in the spectra of the investigated mercaptides the wave length of the first absorption maximum of DMBF (507 nm) remains unchanged but the molar extinction coefficient increases by approximately 20%. Serum albumin, ovalbumin, beta-lactoglobulin and glyceraldehydephosphatedihydrogenase, after incubation with DBMF, form adducts with the dye from which the pure mercaptide complexes were separated by means of column chromatogrphy. These complexes were separated by means of column chromatography. These complexes show a bathochromic shift (520 nm) of the dye band which is decreased now by 50%. The molar extinction coefficient epsilon 520 has been determined from 32,000 to 33,850. On the basis of these values SH-contents of the four proteins were obtained which are in good accordance with data previously published in the literature. No selective reaction, f.i. with more accessible or/and reactive SH-groups was observed. After 30 min incubation with DBMF and washing with isotonic phosphate buffer, native animal tumor cells show in the main absorption band the bathochromically shifted dye maximum. A first temptative estimation of the protein SH-groups yielded 1.7-2.1 X 10(-14) mole SH/single cell. This result lies between the SH-content determined microspectrometrically on cells stained with DDD-Fast Blue B (1.1-1.55 X 10(-14)) and macroscopically on cell homogenates with DTNB (3.1 X 10(-14)). Up to now, no certain information can be given whether or to what extent unspecific absorption effects possibly might be involved in the data obtained with DBMF treated cells, but interaction with nucleic acids can be excluded with certainty on the basis of relevant model experiments.
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000032:analysis; D005452:Fluoresceins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis; D007782:Lactoglobulins / Q000032:analysis; D008622:Merbromin; D051379:Mice; D010047:Ovalbumin / Q000032:analysis; D011048:Polarography; D012510:Sarcoma 180 / Q000032:analysis; D012709:Serum Albumin / Q000032:analysis; D013057:Spectrum Analysis; D013438:Sulfhydryl Compounds / Q000032:analysis*
D005452:Fluoresceins; D007782:Lactoglobulins; D012709:Serum Albumin; D013438:Sulfhydryl Compounds; D010047:Ovalbumin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D008622:Merbromin
Quantitative Bestimmung von Sulfhydryl gruppen mit "Mercurochrom".