Synopsis
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.
Similar content being viewed by others
References
Bachmann, R. &Seitz, H. M. (1961). Zur histochemischen Darstellung des Histidin mit Diazoniumsalzen.Histochemie 2, 307–12.
Barnard, E. A. (1961).General Cytochemical Methods, Vol. 2 (ed. J. F. Danielli), p.227. New York & London: Academic Press.
Barnard, E. A. &Ramel, A. (1962). Studies on the active centre of hexokinase.Biochem. J. 84, 72–3P.
Barnard, E. A. &Stein, W. D. (1959). The histidine residue in the active centre of ribonuclease. I. A specific reaction with bromoacetic acid.J. molec. Biol. 1, 339–49.
Cohen, L. (1966). Presence of lipids in keratohyalin granules of human gingiva.J. dent. Res. 46, 630.
Cohen, L. (1967). The histochemical localization of phospholipids in human oral epithelium.J. Periodontol. 38, 500–2.
Deitch, A. D. (1966).Introduction to Quantitative Cytochemistry (ed. G. L. Wied), pp. 347, 454–6. New York & London: Academic Press.
Deitch, A. D. &Terner, J. Y. (1965). Effect of acetylation on acid dye binding and the Sakaguchi reaction.J. Histochem. Cytochem. 13, 15.
Eastoe, J. E. (1963). The amino acid composition of proteins from the oral tissues. I. A comparison of human oral epithelium, epidermis and nail proteins.Archs oral Biol. 8, 449–58.
Gorbunoff, M. J. (1969). Exposure of tyrosine residues in proteins. III. The reaction of cyanuric fluoride andN-acetylimidazole with ovalbumin, chymotrypsinogen, and trypsinogen.Biochemistry 8, 2591–8.
Horinishi, H., Hachimori, Y., Kurihara, K. &Shibata, K. (1964). States of amino acid residues in proteins. III. Histidine residues in insulin, lysozyme, albumin and proteinases as determined with a new reagent of diazo-1-H-tetrazole.Biochim. biophys. Acta 86, 477–89.
Kasai, S. &Tanimoto, Y. (1964). Changes of gingival tissues of rats treated with short administration of Dilantin sodium.Shikagaku ho (J. Tokyo dental Coll.) 65, 328–32. (In Japanese with English summary.)
Landing, B. H. &Hall, H. E. (1956). Selective demonstration of histidine.Stain Technol. 31, 197–200.
Lazarus, N. R., Derechin, M. &Barnard, E. A. (1968). Yast hexokinase. III. Sulfhydryl groups and protein dissociation.Biochemistry 7, 2390–400.
Maddy, A. H. (1961)General Cytochemical Methods, Vol. 2 (ed. J. F. Danielli). p. 273. New York & London: Academic Press.
McHugh, W. D. (1964). The keratinization of gingival epithelium.J. Periodontol. 35, 338–47.
Pearse, A. G. E. (1960).Histochemistry: theoretical and applied, 2nd Ed., Appendix 5. Boston: Little Brown.
Pearse, A. G. E. (1968).Histochemistry: theoretical and applied, 3rd Ed., Vol. 1, p. 407. Boston: Little Brown.
Porter, R. R. (1948). The unreactive amino groups of proteins.Biochim. biophys. Acta 2, 105–12.
Ramachandran, L. D. (1956). Protein-iodine interaction.Chem. Rev. 56, 199–218.
Reaven, E. P. &Cox, A. J. (1963). The histochemical localization of histidine in the human epidermis and its relationship to zinc binding.J. Histochem. Cytochem. 11, 782–90.
Sober, H. A. (1968). (ed.)Handbook of Biochemistry. Selected Data for Molecular Biochemistry, p. C-164. Cleveland, Ohio: Chemical Rubber Co.
Staple, P. H. (1969a). Critical electrolyte concentration in staining of gingival tissue sections by anionic dyes.Internat. Assoc. dental Res. 47th Gen. Meeting Houston, Texas, Abstr. No. 649.
Staple, P. H. (1969b). Side effects of reaction media in histochemical blocking procedures.Histochem. J. 1, 377–81.
Staple, P. H. (1969c). Staining of tissue polycations by anionic dyes in salt solutions.J. Histochem. Cytochem. 17, 189.
Takahashi, K. (1968). The reaction of phenylglyoxal with arginine residues in proteins.J. biol. Chem. 243, 6171–9.
Takenaka, O., Horinishi, H. &Shibata, K. (1967). Three types of tyrosine residues in ribonuclease.J. Biochem. 62, 501–3.
Takenaka, A., Suzuki, T., Takenaka, O., Horinishi, H. &Shibata, K. (1969). States of amino acid residues in proteins. XVIII. A revised way of using diazonium-1-H-tetrazole for reactivity examination of histidine and tyrosine residues.Biochim. biophys. Acta 194, 293–300.
Toi, K., Bynum, E., Norris, E. &Itano, A. (1967). Studies on the chemical modification of arginine. I. The reaction of 1,2-cyclohexanedione with arginine and arginyl residues of proteins.J. Biol Chem. 242, 1036–43.
Voorhees, J. J., Chakrabarti, S. G. &Bernstein, I. A. (1968). The metabolism of ‘histidinerich’ protein in normal and psoriatic keratinization.J. invest. Derm. 51, 344–54.
Zerlotti, E. &Engel, M. B. (1962). The reactivity of proteins of some connective tissues and epithelial structures with 2,4-dinitrofluorobenzene.J. Histochem. Cytochem. 14, 147–58.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Staple, P.H. Observations on the use of diazo-1-H-tetrazole and phenylglyoxal in protein histochemistry as applied to human gingiva. Histochem J 2, 109–121 (1970). https://doi.org/10.1007/BF01003538
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01003538