THE HISTOCHEMICAL VISUALIZATION OF PROTEIN-BOUND SULFHYDRYL GROUPS WITH AN AZOMERCURIAL REAGENT

MILTON B. ENGEL ¹ and EUGENIO ZERLOTTI ¹

¹ University of Illinois, College of Dentistry, Department of Orthodontics

An azomercurial reagent, 4-(p-dimethylamino benzene azo) phenyl mercuric acetate has been adapted for the histochemical demonstration of protein bound sulfhydryl groups. Sections were made from freeze-dried tissues including various keratinized structures, muscle and some parenchyma. They were stained directly in an alcoholic glycine buffered solution of the azomercurial. High resolution and good contrast were insured by viewing the sections with monochromatic light at 430 mµ.

The method, which is based on the work of Horowitz and Klotz, appears to be highly specific. Tissues which are very low in cysteine or virtually cysteine-free, do not react, and pretreatment of thiol containing sections with N-ethylmaleimide, p-chloromercuribenzoate or H₂O₂ prevents staining. Visualization of disulfide linkages following reduction was only partially successful due mainly to loss of soluble materials. Preliminary microphotometric studies suggest that the method could be used for semi-quantitative determinations. In many keratinizing tissues, cells and intercellular cement underlying the keratinized structures contain a high concentration of protein-bound thiol dispersed homogeneously as granules, or in fibrous form.

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