Observations on the use of diazo-I-H-tetrazole and phenylglyoxal in protein histochemistry as applied to human gingiva.

Abstract

A solution of diazo-1-H-tetrazole, freshly prepared by the diazotization of 5-amino-1-H-tetrazole under conditions to avoid explosion, was adjusted to pH 8.8, diluted (1∶1 or 1∶9) with 0.67 M bicarbonate buffer, pH 8.8, and used immediately as a histochemical reagent for demonstrating histidine, tryptophan, and tyrosine residues in deparaffinized sections of frozen-dried human gingiva, rat abdominal skin, and mouse larynx fixed in modified Newcomer's solution. Diazo-1-H-tetrazole reacted histochemically like other diazonium coupling reagents in common use, except that in sections pretreated with bromoacetic acid at pH 7, diazo-1-H-tetrazole staining was increased, rather than decreased as expected. Pretreatment with bromoacetic acid also increased staining in gingival sections exposed to an acetic anhydride-pyridine mixture and then reacted with diazo-1-H-tetrazole. Similarly, pretreatment with bromoacetic acid increased the intensity of Millon's reaction in gingival sections. Sections of human gingiva or mouse larynx pretreated with diazo-1-H-tetrazole stained less intensely with Biebrich Scarlet used respectively at pH 2.62 and 6.50. In test-tube experiments to check the specificity of diazo-1-H-tetrazole for amino acids, only histidine, tryptophan, and tyrosine gave solutions with colours visually distinguishable from the buffer blank. In similar tests a solution of ribonuclease A gave a colour like that given by histidine and tyrosine. Whereas pyridine failed to yield a colour with undiluted diazo-1-H-tetrazole reagent in test-tube experiments, gingival sections exposed to pyridine for 24 hr stained more intensely with diazo-1-H-tetrazole, but diazo-1-H-tetrazole staining of abdominal skin sections was not altered by prior treatment with pyridine. Phenylglyoxal, used as a 1.5% w/v solution inN-ethylmorpholine-acetate buffer (0.2 M acetate) pH 8, blocked the Sakaguchi reaction in human gingival sections. Pretreatment with phenylglyoxal also led to a reduction in their staining by Biebrich Scarlet at pH 2.62, dinitrofluorobenzene, or diazo-1-H-tetrazole. In addition the dimethylaminobenzaldehyde nitrite reaction for tryptophan was reduced. Phenylglyoxal blockade of arginine residues in gingival sections was labile to 1% acetic acid containing 0.05 M choline chloride after 60 min; but in test-tube experiments extending over 320 min, di(phenylglyoxal)-l-arginine hydrochloride was more stable in this acetic acid-choline solution than in water. It is suggested that after treatment of gingival sections with bromoacetic acid at pH 7.0, additional tyrosine residues become available for reaction with diazo-1-H-tetrazole. Pyridine is thought to remove ‘bound lipid’ from gingival epithelium, thereby exposing protein residues reactive with diazo-1-H-tetrazole. The use of diazo-1-H-tetrazole and phenylglyoxal for characterizing amino acid residues of gingival proteins responsible for anionic dye binding is discussed.