Heterogeneity of catalase staining in human hepatocellular peroxisomes

Hepatocellular peroxisomes stained for catalase activity have different electron densities. When measured by scanning transmission electron microscopy, density is inversely linear to diameter. We investigated whether this phenomenon is the result of a staining artifact that reflects more efficient diffusion of substrate into smaller peroxisomes (higher surface-to-volume ratio), or of differences in endogenous enzymatic activity. Measurements of optical density show that the amount of reaction product is proportional to the diaminobenzidine concentration in the medium; this is not the case for H2O2. Modifying the concentration of both substrates does not alter the heterogeneous staining pattern. Heterogeneity persists when the reaction is slowed by inhibitors or when diffusion takes place before the reaction, and in preparations that have not been subjected to cytochemical staining. These data show that catalase activity is different in individual peroxisomes and that the staining differences are not a consequence of variations in substrate diffusion. Some implications of this conclusion are discussed.