A new sensitive colorimetric assay for peroxidase using 3,3'-diaminobenzidine as hydrogen donor

A new simple colorimetric assay for measurement of peroxidase activity using 3,3′-diaminobenzidine tetrahydrochloride as hydrogen donor is described. The DAB is stable under the usual assay conditions, and its rate of auto-oxidation is negligible. Under optimal conditions, a linear relationship is found between peroxidase concentration and the rate of oxidation of 3,3′-diaminobenzidine tetrahydrochloride (ΔA_405nm/min). Using horseradish peroxidase, the DAB method appears more sensitive than the o-dianisidine and the guaiacol assays for peroxidase. This method can also be used for measurement of peroxidase activity in tissue fractions.     

CYTOCHEMICAL LOCALIZATION OF LACTIC DEHYDROGENASE IN WHITE SKELETAL MUSCLE

The limitations of the conventional histochemical methods for localization of lactic dehydrogenase (LDH) in white skeletal muscle have been analyzed quantitatively. It is demonstrated that more than 80 per cent of LDH diffuses into the incubation medium within the first 10 minutes of incubation. Furthermore, it is confirmed that the addition of phenazine methosulfate (PMS) to the ingredients of the histochemical reaction for LDH increases substantially the capacity of the white muscle extract to reduce Nitro-BT. Based on these observations, a modified method of cytochemical localization of LDH has been developed. This method prevents the leakage of LDH from tissue sections by the application of all the ingredients of the histochemical reaction to tissue sections in a thin gelatin film. The incubation mixture contains PMS so that the staining system is independent of tissue diaphorase. The application of this method to the adductor magnus muscle of the rabbit revealed a fine reticulum in the sarcoplasm of all muscle fibers, in addition to the staining of mitochondria. The distribution of the staining suggests that LDH is localized in the sarcoplasmic reticulum.     

Cytochemical detection of catalase with 3,3′-diaminobenzidine

Summary The influence of various parameters of fixation and incubation upon the oxidation of DAB by catalase have been analyzed. Crystalline beef liver catalase was fixed with different concentrations of glutaraldehyde and peroxidatic activity was determined spectrophotometrically using DAB as hydrogen donor. Although aldehyde fixation appeared to be important in elicitation of the peroxidatic activity of catalase, the final pigment production after 60 min incubation was optimal with the lowest concentration of glutaraldehyde (1%), after the shortest fixation period (30 min), and at the lowest temperature (5° C) tested. Similarly cytochemical studies with rat kidney sections incubated for 10 min confirmed that the staining of peroxisomes in proximal tubules was strongest after the mildest fixation conditions. The pH and the temperature of incubation were closely interrelated, so that at room temperature (25° C) the maximal pigment production was obtained at pH 10.5 but incubation at 45° C gave the strongest staining at pH 8.5. The production of pigment increased with higher DAB concentrations which required larger amounts of H₂O₂ in the incubation medium. Cytochemical studies on renal peroxisomes were in agreement with these biochemical findings. The observations indicate that there are several options for the localization of catalase depending on the fixation and incubation conditions. Hence, these conditions should be selected according to the tissue and the purpose of the study. Examples for such selective applications are presented.     

Immunocytochemical localization of a urate oxidase immunoreactive protein in the plasma membranes and membranes of the secretory/endocytic compartments of digestive gland cells of the mussel Mytilus galloprovincialis

The subcellular compartmentalization of urate oxidase (UOX) in the digestive glands of mussels, Mytilus galloprovincialis Lmk, was studied by means of immunoblotting and immunocytochemistry, using an antibody raised in rabbit against rat liver UOX. Western blot analysis of subcellular fractions revealed an immunoreactive polypeptide with a molecular weight similar to the corresponding mammalian hepatic protein. This crossreactive polypeptide of 32 kDa was particle-bound yet not peroxisome-associated. In paraffin sections the antiserum specifically labeled the plasma membrane of the digestive gland epithelial cells and discrete regions within the perinuclear and apical portions of the digestive tubules and duct cells. By electron microscopy gold particles representing antigenic sites were found on the microvilli and the lateral plasma membrane as well as the membranes of the secretory/ endocytic compartments, that is, the Golgi complex, secretory and some endocytic vesicle membranes. Since the peroxisomal UOX-antibody exhibits a comparable immunoreactivity towards a urate-transporter channel protein in rat kidney proximal tubules and has been used for its molecular cloning (Leal-Pinto et al., 1997, J. Biol. Chem. 272, 617-625), we suggest that the membrane protein identified in mussel digestive glands could represent a homologous urate-transporter protein.     

Luminometric determination of oxidase activity in peroxisomal fractions of rat liver: glycolate oxidase

The feasibility of using the H2O2-mediated chemiluminescence for determination of the activity of oxidases in peroxisomes of rat liver has been investigated. In an assay medium containing luminol, horseradish peroxidase, and azide with glycolate as substrate, a linear relationship is obtained between the amount of peroxisomal protein used and the luminescence signal. In comparison with other techniques available for measuring the activities of peroxisomal oxidases the luminometric approach described here is 5-10 times more sensitive than the spectrophotometric methods and 100 times more efficient than the polarographic determination of O2. Under the optimal assay conditions the glycolate oxidase activity can be determined in amounts as low as 0.5 micrograms peroxisomal protein.     

Decrease by glucagon in hepatic succinyl-CoA

Succinyl-CoA has been determined in freeze-clamped rat liver tissue to amount to 9.5 ± 0.7 nmoles/g fresh wt. (n=11). Intravenous injection of glucagon lowers this level within 10 min to 4.5 nmoles/g fresh wt. (n=11; P < 0.0005). Isolated hepatocytes also show a significant decrease in their succinyl-CoA content when incubated with glucagon or dibutyryl cyclic AMP. This effect appears to be fully expressed already 1 min after exposure of isolated hepatocytes to glucagon.     

Selective cytochemical localization of peroxidase,cytochrome oxidase and catalase in rat liver with 3,3′-diaminobenzidine

Summary In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specifity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15–30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylate buffer pH 6.5 and 0.02% H₂O₂. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) butffer at pH 10.5 and 0.15% H₂O₂. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.     

Peroxisomes in digestive gland cells of the mussel Mytilus galloprovincialis Lmk. Biochemical, ultrastructural and immunocytochemical characterization.

The present study was undertaken because of the paucity of information on peroxisomes in molluscs and the increasing importance of these organisms as sensitive indicators of environmental pollution. Peroxisomes were identified by electron microscopy in all three main cell types of the digestive gland of the bivalve mollusc Mytilus galloprovincialis Lmk. They stained weakly with the alkaline diaminobenzidine reaction but showed distinct immunolabeling with an antibody against mammalian catalase by the postembedding protein A-gold procedure. In addition, mussel digestive gland peroxisomes were isolated by differential and metrizamide-density gradient centrifugation, and a 30-fold enrichment of catalase and a 20-fold enrichment of palmitoyl-CoA oxidase was obtained over the initial homogenate. By Western blotting, isolated peroxisomes crossreacted with antibodies to catalase and, furthermore, specific and prominent labeling of isolated peroxisomes was also demonstrated in thin sections incubated with anti-catalase antibodies. These observations establish that peroxisomes in molluscan digestive gland contain the peroxisomal marker enzymes catalase and acyl-CoA oxidase and that they can be labeled by cytochemical and immunocytochemical techniques. Further studies of alterations of molluscan peroxisomes by environmentally relevant xenobiotics are warranted.