Skip to main content
SpringerLink
Log in

The use of hexazonium-p-rosanilin in the histochemical demonstration of peptidases

  • Published:
Histochemistry Aims and scope Submit manuscript

Summary

The suitability of hexazonium-p-rosanilin (HP) in the histochemical demonstration of peptidases was investigated. The detection was carried out in cold microtome sections adherent to slides or semipermeable membranes. Alanyl-1-naphthylamide, alanyl-2-naphthylamide, leucyl-2-naphthylamide, leucyl-4-methoxy-2-naphthylamide (all substrates in concentration of 0.4 mg/1 ml of citrate phosphate buffer pH 6.5), γ-L-glutamyl-1-naphthylamide, γ-L-glutamyl-2-naphthylamide (both substances in concentration of 0.24 mg/1 ml of acetate buffer pH 6.5) were used as the substrates. Results were compared with those obtained with Fast Blue B and Fast Garnet GBC.

In comparison with Fast Blue B and Fast Garnet GBC HP is a faster coupler, furnishes azodyes which are stable, amorphous (even without lipid extractions from sections), more substantive and in the case of 1-naphthylamine almost insoluble in ordinary lipid solvents used for the dehydration and clearing of sections before mounting. The molecular extinction coefficient of azodyes furnished by HP is 1.5x higher for 1-naphthylamine than for 2-naphthylamine. It is higher than that of Fast Garnet GBC, however, lower than that of Fast Blue B. The inhibitory influence of individual diazonium salts on enzyme activity (activities) splitting leucyl-2-naphthylamide amounts to 36% (Fast Garnet GBC), 37% (Fast Blue B), 52% (HP, 0.03 ml/1 ml) and 63% (HP, 0.09 ml/1 ml) at pH 6.5. For γ-glutamyl-transpeptidase the corresponding values are 50%, 59%, 62% and 67%. The higher inhibitory influence of HP is compensated by the possibility of its using in the technic of semipermeable membranes.

HP improves greatly the localization of peptidases in cold microtome sections from which lipids were not extracted. The best results are furnished by 1-naphthylamine dervatives. In the case of 4-methoxy-2-naphthylamine derivatives the localization is very sharp, however, the azodye is less distinct than that of 2-naphthylamine.

The localization as obtained with HP in combination with substrates derived of simple naphthylamines is similar or even better than with 4-methoxy-2-naphthylamine derivatives applied with Fast Blue B. Typical examples are shown.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Albert, Z., Szewczuk, A.: Histochemical and biochemical studies on Co++-activated acylase in rodent tissues. Acta histochem. (Jena) 42, 68–76 (1972a)

    Google Scholar 

  • Albert, Z., Szewczuk, A.: Pyrrolidonyl peptidase in some avian and rodent tissues. Histochemical localization and biochemical studies. Acta histochem. (Jena) 44, 98–105 (1972b)

    Google Scholar 

  • Albert, Z., Orlowski, M., Szewczuk, A.: Histochemical demonstration of gamma-glutamyl transpeptidase. Nature (Lond.) 191, 767–768 (1961)

    Google Scholar 

  • Albert, Z., Szewczuk, A., Mulczyk, M.: Histochemical and biochemical studies of glycyl peptidase and glycyl arylamidase. Acta histochem. (Jena) 39, 118–124 (1971)

    Google Scholar 

  • Albert, Z., Orlowska, J., Orlowski, M., Szewczuk, A.: Histochemical and biochemical investigations of gamma-glutamyl transpeptidase in the tissues of man and laboratory rodents. Acta histochem. (Jena) 18, 78–89 (1964)

    Google Scholar 

  • Albert, Z., Orlowski, M., Rzuczidlo, Z., Orlowska, J.: Studies on γ-glutamyl transpeptidase activity and its histochemical localization in the central nervous system of man and different animal species. Acta histochem. (Jena) 25, 312–320 (1966)

    Google Scholar 

  • Burstone, M. S.: Enzyme Histochemistry and Its Application in the Study of Neoplasms. New York and London: Academic Press 1962

    Google Scholar 

  • Burstone, M. S., Folk, J. E.: Histochemical demonstration of aminopeptidase. J. Histochem. Cytochem. 4, 217–226 (1956)

    Google Scholar 

  • Davis, B. J.: Histochemical demonstration of erythrocyte esterases. Proc. Soc. exp. Biol. (N.Y.) 101, 90–93 (1959)

    Google Scholar 

  • Davis, B. J., Ornstein, L.: High resolution enzyme localization with a new diazo reagent, “hexazonium pararosanilin”. J. Histochem. Cytochem. 7, 297–298 (1959)

    Google Scholar 

  • Glenner, G. G.: The preservation of peptidase activity localization using β-naphthylamide substrates. J. Histochem. Cytochem. 10, 257–258 (1962)

    Google Scholar 

  • Glenner, G. G., Folk, J. E.: Histochemical demonstration of a γ-glutamyl transpeptidase-like activity. Nature (Lond.) 192, 338–339 (1961)

    Google Scholar 

  • Glenner, G. G., Folk, J. E., McMillan, P. J.: Histochemical demonstration of a gamma-glutamyl transpeptidase-like activity. J. Histochem. Cytochem. 10, 481–489 (1962)

    Google Scholar 

  • Gomori, G.: Chromogenic substrates for aminopeptidase. Proc. Soc. exp. Biol. (N.Y.) 87, 559–561 (1954)

    Google Scholar 

  • Hopsu-Havu, V. K., Eckfors, T. O.: Distribution of a dipeptide naphthylamidase in rat tissues and its localization by using diazo coupling and labelled antibody techniques. Histochemie 17, 30–38 (1969)

    Google Scholar 

  • Hopsu-Havu, V. K., Glenner, G. G.: A new dipeptide naphthylamidase hydrolysing glycylprolyl-β-naphthylamide, Histochemie 7, 197–201 (1966)

    Google Scholar 

  • Hopsu-Havu, V. K., Rintola, P., Glenner, G. G.: A hog kidney aminopeptidase liberating N-terminal dipeptides. Partial purification and characteristics. Acta chem. scand. 22, 299–308 (1968)

    Google Scholar 

  • Hopsu, V. K., Santti, R., Glenner, G. G.: Characterization of enzymes hydrolyzing acyl naphthylamides. III. Role of kynurenine formamidase. J. Histochem. Cytochem. 14, 653–657 (1966)

    Google Scholar 

  • Lojda, Z.: Demonstration of hydrolases. In: Lojda, Z., Papoušek, F.: Histochemical demonstration of enzymes. Brno: Institute for Postgraduate Education and Czechoslovak Society of Histo- and Cytochemistry 1970 [in Czech.]

    Google Scholar 

  • Lojda, Z.: Aktuelle Probleme der Cytochemie der lysosomalen Hydrolasen. Acta morph. Acad. Sci. hung. 20, 269–293 (1972)

    Google Scholar 

  • Lojda, Z.: Histochemical methods for acid β-galactosidase: Technics for semipermeable membranes. Histochemie 37, 375–378 (1973)

    Google Scholar 

  • Lojda, Z.: Topochemistry of β-glycosidases in the aortae and coronary arteries of rats, guinea-pigs and rabbits under normal conditions and after cholesterol feeding. Čs. Path. 10, 1–9 (1974) [in Czech.]

    Google Scholar 

  • Lojda, Z., Havránková, E.: The histochemical demonstration of aminopeptidase with bromoindolyl leucinamide. Histochemistry 43, 355–366 (1975)

    Google Scholar 

  • Lojda, Z., Večerek, B., Pelichová, H.: Some remarks concerning the histochemical detection of acid phosphatase by azocoupling reactions. Histochemie 3, 428–454 (1964)

    Google Scholar 

  • Monis, B., Wasserkrug, H., Seligman, A. M.: Comparison of fixatives and substrates for aminopeptidase. J. Histochem. Cytochem. 13, 503–509 (1965)

    Google Scholar 

  • Nachlas, M. M., Crawford, D. T., Seligman, A. M.: The histochemical demonstration of leucine aminopeptidase. J. Histochem. Cytochem. 5, 264–278 (1957)

    Google Scholar 

  • Nachlas, M. M., Monis, B., Rosenblatt, D., Seligman, A. M.: Improvement in the histochemical localization of leucine aminopeptidase with a new substrate, L-leucyl-4-methoxy-2-naphthylamide. J. biophys. biochem. Cytol. 7, 261–264 (1960)

    Google Scholar 

  • Pearse, A. G. E.: Histochemistry. Theoretical and Applied. 3rd ed. London: J & A. Churchill 1968

    Google Scholar 

  • Rutenburg, A. M., Kim, H., Fischbein, J. W., Hanker, J. S., Wasserkrug, H. L., Seligman, A. M.: Histochemical and ultrastructural demonstration of γ-glutamyl transpeptidase activity. J. Histochem. Cytochem. 17, 517–526 (1969)

    Google Scholar 

  • Stutte, H. J.: Hexazotiertes Triamino-tritolyl-methanchlorid (Neufuchsin) als Kupplungssalz in der Fermenthistochemie. Histochemie 8, 327–331 (1967)

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Research Laboratory of Angiology Faculty of General Medicine, Charles University, Praha, ČSSR

    Zdeněk Lojda

  2. 1st Department of Pathology, Faculty of General Medicine, Charles University, Praha, ČSSR

    Zdeněk Lojda

Authors
  1. Zdeněk Lojda
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Dedicated to the 60th birthday of Prof. MUDr. B. Bednář, DrSc., Head of the 1st Department of Pathology, Faculty of General Medicine, Charles University, Praha.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lojda, Z. The use of hexazonium-p-rosanilin in the histochemical demonstration of peptidases. Histochemistry 44, 323–335 (1975). https://doi.org/10.1007/BF00490369

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00490369

Keywords

Search

Navigation