Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles

Abstract

Galactosylated BSA (galBSA) and its conjugate to horseradish peroxidase (galBSA-HRP) enter the galactose-specific pathway of hepatocytes. 10 min after intravenous injection, structures containing either ligand sediment mostly between 33,000 and 3 X 10(6) g X min (LP fraction) and have an equilibrium density of 1.11-1.13 g/ml in sucrose gradients (Quintart, J., P. J. Courtoy, J. N. Limet, and P. Baudhuin, 1983, Eur. J. Biochem., 131:105-112). Such low density fractions, prepared from rats given galBSA-HRP, were incubated for 30 min at 25 degrees C in 5.5 mM 3,3'-diaminobenzidine (DAB) and 11 mM H2O2 in buffered sucrose. Upon equilibration in a second sucrose gradient, the galBSA-HRP distribution shifted towards higher (approximately 1.19 g/ml) density, but the bulk of protein remained at low density. In the absence of H2O2, galBSA-HRP distribution was also found at low density. As observed by electron microscopy, particles equilibrating at higher density after DAB cytochemistry were largely made of vesicles or tubules filled with DAB reaction product. The density shift of galBSA-HRP-containing organelles after incubation with DAB and H2O2 is attributed to the trapping of HRP-oxidized DAB inside the host organelles. If the low density fractions isolated from a rat injected with [3H]galBSA-HRP were mixed in vitro with similar fractions from another rat given [14C]galBSA, the 3H distribution shifted after DAB cytochemistry, but the 14C distribution was essentially unaffected. By contrast, if both derivatives were injected simultaneously, a concomitant density shift was observed. In conclusion, the DAB-induced density shift was specific to ligand-HRP-containing organelles. The potentials of the method include the purification of HRP-containing particles and the study of their association to ligands, fluid-phase tracers, or marker enzymes.