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Azoindoxylverfahren zum Hydrolasennachweis

Azoindoxyl methods for the investigation of hydrolases

II. Biochemische und histochemische Untersuchung

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Summary

The determination of various reaction constants yields the following assay for the photometric evaluation of acidβ-galactosidase (measurement of the azoindoxyl dye at 540 nm after extraction with dimethylformamide or-acetamide): 1.5 mM 5-Br-4-Cl-3-indolyl-β-d-galactoside (1 mg dissolved in 0.05 ml dimethylformamide) and 0.01–0.015 ml hexazotized p-rosaniline/ml in 0.1 M citric acid-phosphate buffer, pH 4. By means of this procedure it becomes evident that the activity of the enzyme differs considerably in various rat organs; NaCl does not influence acidβ-galactosidase. — Similar results were obtained with the indigogenic method; indigo can be dissolved and measured photometrically as the azoindoxyl dye.

The enzyme is suppressed by high concentrations of hexazotized p-rosaniline to 50%; low concentrations do not inhibit; the same is true for ferricyanide-ferrocyanide employed in the indigogenic media. — The effect of glutar-and formaldehyde on acidβ-galactosidase cannot be investigated with the azoindoxyl reaction since the azoindoxyl dye partially withstands extraction from fixed blocks of tissue.

On the basis of the biochemical findings the azoindoxyl technique can be recommended for the histochemical demonstration of acidβ-galactosidase: 7.5 mg (1.5 mM) 5-Br-4-Cl-3-indolyl-β-d-galactoside (dissolved in 0.25 ml dimethylformamide) and 0.05–0.15 ml hexazonium-p-rosaniline in 10 ml 0.1 M citric acid-phosphate buffer, pH 4. After incubation the sections can be treated with osmium tetroxide followed by dehydration and mounting in resins or can be mounted without prior osmification of the azoindoxyl dye in glycerin jelly. The osmium chelate resists treatment with organic solvents; the stability of the chelate depends on the concentration of hexazotized p-rosaniline.

After fixation in glutaraldehyde or in a mixture of form- and glutaraldehyde acidβ-galactosidase can be exactly localized in the lysosomes of many rat organs.

In comparison with the indigogenic, the metal precipitation and the simultaneous azocoupling reactions for the in situ detection of acidβ-galactosidase the azoindoxyl procedure is superior if fixed material is used; it is equivalent or inferior in connection with the membrane technique. The biochemical azoindoxyl assay represents a useful method for combined qualitative and quantitative studies of acidβ-galactosidase.

Zusammenfassung

Nach Ermittlung verschiedener Reaktionskonstanten resultiert folgender Ansatz zur photometrischen Bestimmung der saurenβ-Galactosidase (Messung des Azoindoxylfarbstoffs bei 540 nm nach Extraktion mit Dimethylformamid oder-acetamid): 1,5 mM 5-Br-4-Cl-3-Indolyl-β-d-galactosid (1 mg gelöst in 0,05 ml Dimethylformamid) und 0,01–0,015 ml Hexazonium-p-rosanilin/ml in 0,1 M Citronensäure-Phosphat-Puffer, pH 4. Die damit untersuchten Rattenorgane besitzen unterschiedliche Aktivitäten an saurerβ-Galactosidase; NaCl läßt die Enzymaktivität unbeeinflußt. — ähnliche Resultate liefert das Indigogen-Verfahren; Indigo kann wie der Azoindoxylfarbstoff gelöst und gemessen werden.

Hexazotiertes p-Rosanilin in höheren Konzentrationen hemmt die saureβ-Galactosidase zu etwa 50%; niedrige sowie Ferricyanid-Ferrocyanid inhibieren nicht. Die Hemmwirkung von Glutar- und Formaldehyd läßt sich mit dem Azoindoxylverfahren nicht messen, da der Farbstoff unvollständig aus fixiertem Material extrahiert wird.

Auf Grund der biochemischen Befunde ergibt sich nachstehendes histochemisches Medium zur Darstellung der saurenβ-Galactosidase: 7,5 (1,5 mM) 5-Br-4-Cl-3-Indolyl-β-d-galactosid (gelöst in 0,25 ml Dimethylformamid) und 0,05–0,15 ml Hexazonium-p-rosanilin in 10 ml 0,1 M Citronensäure-Phosphat-Puffer, pH 4. Nach der Inkubation können die Schnitte osmiert, dehydriert und in Kunstharz oder ohne Osmierung in Glycerin-Gelatine eingedeckt werden. Das Osmiumchelat ist in organischen Solventien unlöslich; seine Stabilität hängt von der Hexazonium-p-rosanilin-Konzentration ab.

Mit dieser Methode läßt sich die saureβ-Galactosidase nach Fixation in Glutaraldehyd oder einem Glutaraldehyd-Formaldehyd-Gemisch exakt in den Lysosomen zahlreicher Rattenorgane erfassen.

Verglichen mit dem Indigogen- und Metallsalzverfahren und den simultanen Azokupplungsreaktionen zum histochemischen Nachweis der saurenβ-Galactosidase ist die Azoindoxyltechnik überlegen oder gleichwertig; der biochemische Ansatz leistet Nützliches für kombinierte quantitativ-qualitative Studien des Enzyms.

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Gossrau, R. Azoindoxylverfahren zum Hydrolasennachweis. Histochemistry 51, 219–237 (1977). https://doi.org/10.1007/BF00567226

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