The determination of various reaction constants yields the following assay for the photometric evaluation of
acid beta-galactosidase (measurement of the azoindoxyl
dye at 540 nm after extraction with
dimethylformamide or -
acetamide): 1.5 mM 5-Br-4-Cl-3-indolyl-beta-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
acid beta-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-roaniline 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
acid beta-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
acid beta-galactosidase: 7.5 mg (1.5 mM) 5-Br-4-Cl-3-indolyl-beta-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 acid beta-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
acid beta-galactosidase the azoindoxyl procedure is superior if fixed material is used; it is equivalent or inferior in connection with membrane technique. The biochemical azoindoxyl assay represents a useful method for combined qualitative and quantitative studies of
acid beta-galactosidase.