A Biolchemical and Cytochemical Study of Peroxidase Activity in Roots of Pisum sativum: I. A COMPARISON OF DAB-PEROXIDASE AND GUAIACOL-PEROXIDASE WITH PARTICULAR EMPHASIS ON THE PROPERTIES OF CELL WALL ACTIVITY

Peroxidase activity in roots of Pisum sativum has been examinedusing both guaiacol and 3,3'-diaminobenzidine (DAB) as hydrogendonors. Biochemically, differences were observed between thetwo donors with respect to the pH optimum (6–9 and 4–0,respectively), and in response to added NaCl (guaiacol-peroxidasewas unaffected while the DAB-peroxidase was markedly inhibited).Both reactions showed highest specific activity in a high speedsupernatant fraction, and, of nine anionic bands demonstratedby gel electrophoresis with DAB, only six were visible withguaiacol. Histochemically, similar staining patterns were observedwith both donors. Cell wall fractions prepared by bead filtration contained 2%and 3.5% of the total peroxidase and acid phosphatase activitiesrespectively. 50% and 27% of these activities were ionicallybound, as indicated by salt treatment In addition, washing withsalt solutions produced a marked stimulation of peroxidase activityat high salt concentrations: this affect was not observed withthe supernatant peroxidase or with cell wall acid phosphatase.Possible functions of cell wall peroxidase are discussed     

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.     

Subcellular structure of bovine thyroid gland. The localization of the peroxidase activity in bovine thyroid.

1. After differential pelleting of bovine thyroid tissue the highest relative specific activities for plasma membrane markers are found in the L fraction whereas those for peroxidase activities (p-phenylenediamine, guaiacol and 3,3'-diaminobenizidine tetrachloride peroxidases) are found in the M fraction. 2. When M + L fractions were subjected to buoyant-density equilibration in a HS zonal rotor all peroxidases show different profiles. The guaiacol peroxidase activity always follows the distribution of glucose 6-phosphatase. 3. When a Sb fraction is subjected to Sepharose 2B chromatography three major peaks are obtained. The first, eluted at the void volume, consists of membranous material and contains most of the guaiacol peroxidase activity. Most of the protein (probably thyroglobulin) is eluted with the second peak. Solubilized enzymes are recovered in the third peak. 4. p-Phenylenediamine peroxidase activity penetrates into the gel on polyacrylamidegel electrophoresis, whereas guaiacol peroxidase activity remains at the sample zone. 5. DEAE-Sephadex A-50 chromatography resolves the peroxidase activities into two peaks, displaying different relative amounts of the different enzymic activities in each peak. 6. The peroxidase activities may be due to the presence of different proteins. A localization of guaiacol peroxidase in rough-endoplasmic-reticulum membranes (or in membranes related to them) seems very likely.     

Diaminobenzidine Induces Fluorescence in Nervous Tissue and Provides Intrinsic Counterstaining of Sections Prepared for Peroxidase Histochemistry

3,3'-Diaminobenzidine (DAB) is widely used as a chromogen for visualization of horseradish peroxidase activity in neuroanatomical tracing experiments and in immunohistochemistry. The product of the enzymatically catalyzed oxidation of DAB by hydrogen peroxide is brown and nonfluorescent. In frozen sections of formaldehyde fixed rat and mouse brain that had been exposed to DAB either alone or with hydrogen peroxide, we observed strong greenish fluorescence in myelinated nerve fibers and in the somata of some neurons. This fluorescence was not associated with brown coloration and was not due to endogenous peroxidase activity. Extractions, blocking reactions, and other histochemical tests indicate that the fluorescence resulted from the combination of DAB with aldehyde groups that were formed by oxidation of unsaturated linkages in lipids. DAB induced fluorescence provides a simple and useful demonstration of background anatomy in sections that also contain specifically localized deposits of peroxidase activity.     

Evidence of the presence of extraperoxisomal catalase in chloragogen cells of the earthworm, Lumbricus terrestris L

The DAB reactivity of the midintestine of the earthworm, consisting of epithelial layer, muscle layer, and chloragogen tissue, was examined electron microscopically. Besides the mitochondrial membranes of the examined cell types and the hemoglobin content of the blood vessels and chloragogen cells, a considerable DAB reactivity was found in the whole cytosol of the chloragocytes. The DAB reaction of the cytosol was more intensive when incubation medium for catalase, less intensive when incubation medium for peroxidase, was used and did not occur when H2O2 was omitted. Cytosol of the chloragogen cells was isolated and preliminary assay of catalase and peroxidase activities was made. Cytosol samples showed moderate peroxidase activity, but catalase activity measured by the decomposition of hydrogen peroxide showed a very high rate. Catalase and peroxidase activities of the cytosol were heat-sensitive and might have been inhibited by azide and cyanide, respectively. Results prove the assumption that the intensive DAB reactivity of the chloragocyte cytosol is caused by its extraperoxisomal catalase content.     

An unexpected side reaction in the guaiacol assay for peroxidase.

In routine guaiacol assays for thyroid peroxidase and lactoperoxidase employing a newly purchased bottle of guaiacol from Aldrich Chemical Co., we were surprised to find the formation of a blue color instead of the expected amber color classically associated with this assay. This was observed also with horseradish, myelo-, and cytochrome c peroxidase. The blue color (Amax approximately 650 nm) was not formed with guaiacol reagents obtained from two other chemical companies, nor was it seen with a bottle of old Aldrich guaiacol that had been in use in the laboratory for more than 10 years. In the present investigation we provide evidence that formation of the blue color is closely associated with the presence of a low concentration of catechol (approximately 0.5 mol%) in the new Aldrich guaiacol reagent. Catechol itself, even in much higher concentration, is a very weak donor for peroxidase, forming a light pink color. The blue color in Aldrich new guaiacol is not formed to the exclusion of 470-nm-absorbing product(s). Formation of the latter is, however, inhibited, and use of Aldrich new guaiacol for assay leads to low values for peroxidase activity. Other dihydroxyphenols (resorcinol and hydroquinone) do not mimic the action of catechol in formation of the blue color. Resorcinol is a very potent inhibitor of peroxidation of guaiacol. Possible schemes are proposed for formation of the products that may be associated with the amber and blue colors.     

Formation of heartwood substances in the stemwood of Robinia pseudoacacia L. II. Distribution of nonstructural carbohydrates and wood extractives across the trunk

Summary The distributions of reserve carbohydrates and of three dominant heartwood extractives were determined in the trunkwood of Robinia pseudoacacia L. The trees were cut at different times of the year (September, November, January, and April). With the exception of the tree felled in January, all trunks exhibited highest contents of nonstructural storage carbohydrates (glucose, fructose, sucrose, and starch) in the youngest, outermost sapwood zone. With increasing depth of the trunk, the levels of carbohydrates decreased. At the sapwood-heartwood transition zone, only trace amounts of nonstructural carbohydrates were present. The heartwood itself contained no storage material. The wood zones of different ages of the trees cut in September, November, and January exhibited glucose/fructose ratios of approximately 1. In April, however, there was a shift to glucose. In the youngest sapwood the amounts of soluble sugars were higher in the earlythan in the latewood. Older zones of the sapwood and the sap-wood-heartwood transition zone showed the opposite behaviour. Three main wood extractives of Robinia were characterized and quantified: the flavanonol dihydrorobinetin (DHR), the flavonol robinetin (ROB) and a hydroxycinnamic acid derivative (HCA). Only DHR was present — in very low amounts — in the younger sapwood of all trunks investigated. Higher amounts (>1 mol/g dry weight) of this compound and the HCA were present in the sapwood-heartwood transition zone. DHR augmented within the heartwood up to a more or less constant level. HCA increased towards the heartwood and decreased again in the inner heartwood parts. ROB appeared in the innermost parts of the sapwood-heartwood transition zone and reached maximum values in older parts of the heart-wood. The results indicate that starch is hydrolyzed at the sapwood-heartwood boundary and thus represents a primary major source of hydroxycinnamic acid and flavonoid synthesis.Dedicated to Prof. Meinhart H. Zenk on the occasion of his 60th birthday     

Evidence of the presence of extraperoxisomal catalase in chloragogen cells of the earthworm,Lumbricus terrestris L.

Summary The DAB reactivity of the midintestine of the earthworm, consisting of epithelial layer, muscle layer, and chloragogen tissue, was examined electron microscopically. Besides the mitochondrial membranes of the examined cell types and the hemoglobin content of the blood vessels and chloragogen cells, a considerable DAB reactivity was found in the whole cytosol of the chloragocytes. The DAB reaction of the cytosol was more intensive when incubation medium for catalase, less intensive when incubation medium for peroxidase, was used and did not occur when H₂O₂ was omitted.Cytosol of the chloragogen cells was isolated and preliminary assay of catalase and peroxidase activities was made. Cytosol samples showed moderate peroxidase activity, but catalase activity measured by the decomposition of hydrogen peroxide showed a very high rate. Catalase and peroxidase activities of the cytosol were heat-sensitive and might have been inhibited by azide and cyanide, respectively. Results prove the assumption that the intensive DAB reactivity of the chloragocyte cytosol is caused by its extraperoxisomal catalase content.     

Methyl jasmonate promotes the transient reduction of the levels of 2-Cys peroxiredoxin in Ricinus communis plants.

Jasmonates are signaling molecules that play a key role in the regulation of metabolic processes, reproduction and defense against insects and pathogens. This study investigated the effects of methyl jasmonate on the protein pattern of Ricinus communis plants and the activity of guaiacol peroxidase, an antioxidant enzyme. Methyl jasmonate treatment caused a transient reduction in guaiacol peroxidase activity. A similar response was observed for the levels of 2-Cys peroxiredoxin protein. Moreover, the levels of the small and large chains of Rubisco were also reduced. The transient reduction of the levels and activity of antioxidant enzymes could account for the increase in the levels of H2O2, an important signaling molecule in plant defense.     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland     

In situ oxidative catalysis by neurofibrillary tangles and senile plaques in Alzheimer's disease: a central role for bound transition metals

There is a great deal of evidence to support a pathogenic role of oxidative stress in Alzheimer's disease (AD), but the sources of reactive oxygen species have not been directly demonstrated. In this study, using a novel in situ detection system, we show that neurofibrillary tangles and senile plaques are major sites for catalytic redox reactivity. Pretreatment with deferoxamine or diethylenetriaminepentaacetic acid abolishes the ability of the lesions to catalyze the H2O2-dependent oxidation of 3,3'-diaminobenzidine (DAB), strongly suggesting the involvement of associated transition metal ions. Indeed, following chelated removal of metals, incubation with iron or copper salts reestablished lesion-dependent catalytic redox reactivity. Although DAB oxidation can also detect peroxidase activity, this was inactivated by H2O2 pretreatment before use of DAB, as shown by a specific peroxidase detection method. Model studies confirmed the ability of certain copper and iron coordination complexes to catalyze the H2O2-dependent oxidation of DAB. Also, the microtubule-associated protein tau, as an in vitro model for proteins relevant to AD pathology, was found capable of adventitious binding of copper and iron in a redox-competent manner. Our findings suggest that neurofibrillary tangles and senile plaques contain redox-active transition metals and may thereby exert prooxidant or possibly antioxidant activities, depending on the balance among cellular reductants and oxidants in the local microenvironment.     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland This revised version was published online in November 2006 with corrections to the Cover Date.     

Kinetic studies on the oxidation of nitrite by horseradish peroxidase and lactoperoxidase

The reaction of nitrite (NO2-) with horseradish peroxidase and lactoperoxidase was studied. Sequential mixing stopped-flow measurements gave the following values for the rate constants of the reaction of nitrite with compounds II (oxoferryl heme intermediates) of horseradish peroxidase and lactoperoxidase at pH 7.0, 13.3 +/- 0.07 mol(-1) dm3 s(-1) and 3.5 +/- 0.05 x 10(4) mol(-1) dm3 s(-1), respectively. Nitrite, at neutral pH, influenced measurements of activity of lactoperoxidase with typical substrates like 2,2'-azino-bis[ethyl-benzothiazoline-(6)-sulphonic acid] (ABTS), guaiacol or thiocyanate (SCN-). The rate of ABTS and guaiacol oxidation increased linearly with nitrite concentration up to 2.5-5 mmol dm(-3). On the other hand, two-electron SCN- oxidation was inhibited in the presence of nitrite. Thus, nitrite competed with the investigated substrates of lactoperoxidase. The intermediate, most probably nitrogen dioxide (*NO2), reacted more rapidly with ABTS or guaiacol than did lactoperoxidase compound II. It did not, however, effectively oxidize SCN- to OSCN-. NO2- did not influence the activity measurements of horseradish peroxidase by ABTS or guaiacol method.     

Apoplastic Peroxidases and Lignification in Needles of Norway Spruce (Picea abies L.)

The objective of the present study was to investigate the correlation of soluble apoplastic peroxidase activity with lignification in needles of field-grown Norway spruce (Picea abies L.) trees. Apoplastic peroxidases (EC 1.11.1.7) were obtained by vacuum infiltration of needles. The lignin content of isolated cell walls was determined by the acetyl bromide method. Accumulation of lignin and seasonal variations of apoplastic peroxidase activities were studied in the first year of needle development. The major phase of lignification started after bud break and was terminated about 4 weeks later. This phase correlated with a transient increase in apoplastic guaiacol and coniferyl alcohol peroxidase activity. NADH oxidase activity, which is thought to sustain peroxidase activity by production of H2O2, peaked sharply after bud break and decreased during the lignification period. Histochemical localization of peroxidase with guaiacol indicated that high activities were present in lignifying cell walls. In mature needles, lignin was localized in walls of most needle tissues including mesophyll cells, and corresponded to 80 to 130 [mu]mol lignin monomers/g needle dry weight. Isoelectric focusing of apoplastic washing fluids and activity staining with guaiacol showed the presence of strongly alkaline peroxidases (isoelectric point [greater than or equal to] 9) in all developmental stages investigated. New isozymes with isoelectric points of 7.1 and 8.1 appeared during the major phase of lignification. These isozymes disappeared after lignification was terminated. A strong increase in peroxidase activity in autumn was associated with the appearance of acidic peroxidases (isoelectric point [less than or equal to] 3). These results suggest that soluble alkaline apoplastic peroxidases participate in lignin formation. Soluble acidic apoplastic peroxidases were apparently unrelated to developmentally regulated lignification in spruce needles.     

Localization and Characterization of Peroxidases in the Mitochondria of Chilling-Acclimated Maize Seedlings

We present evidence of two peroxidases in maize (Zea mays L.) mitochondria. One of these uses guaiacol and the other uses cytochrome c as the electron donor. Treatments of fresh mitochondria with protease(s) indicate that ascorbate and glutathione peroxidases are likely bound to the mitochondria as cytosolic contaminants, whereas guaiacol and cytochrome peroxidases are localized within the mitochondria. These two mitochondrial peroxidases are distinct from contaminant peroxidases and mitochondrial electron transport enzymes. Cytochrome peroxidase is present within the mitochondrial membranes, whereas guaiacol peroxidase is loosely bound to the mitochondrial envelope. Unlike other cellular guaiacol peroxidases, mitochondrial guaiacol peroxidase is not glycosylated. Digestion of lysed mitochondria with trypsin activated mitochondrial guaiacol peroxidase but inhibited cytochrome peroxidase. Isoelectric focusing gel analysis indicated guaiacol peroxidase as a major isozyme (isoelectric point 6.8) that is also activated by trypsin. No change in the mobility of guaiacol peroxidase due to trypsin treatment on native polyacrylamide gel electrophoresis was observed. Although both peroxidases are induced by chilling acclimation treatments (14[deg]C), only cytochrome peroxidase is also induced by chilling (4[deg]C). Because chilling induces oxidative stress in the maize seedlings and the mitochondria are a target for oxidative stress injury, we suggest that mitochondrial peroxidases play a role similar to catalase in protecting mitochondria from oxidative damage.     

An improved assay method for thyroid peroxidase applicable for a few milligrams of abnormal human thyroid tissues

A peroxidase assay method (Mini assay method) which is applicable for a minute amount (as small as a few mg) of thyroid tissue was developed, employing guaiacol or iodide as the second substrate. This method is a modification of the previous one (Ordinary assay method): the volume of the reaction mixture was reduced to about one-tenth with prior solubilization of the enzyme. The correlation between the Mini assay and Ordinary assay methods, and between the guaiacol and iodide assays by both methods were satisfactorily good, but the iodine content of thyroglobulin was found to be not directly correlated to the peroxidase activities. Protein-based specific activities of peroxidase from normal human thyroid tissue were about 0.030 guaiacol units/mg protein and 0.0066 iodide units/mg protein, which were slightly higher than those of porcine thyroid tissue. The Mini assay method developed in the present study was used for the determination of peroxidase activity in a small amount (1-8 mg) of thyroid tissue obtained by means of a needle biopsy from patients with thyroid disorders. One specimen (goitrous cretinism) showed no peroxidase activity in both the guaiacol and iodide assays, and three specimens (two chronic thyroiditis, one familial nontoxic goiter) possessed no ability to catalyze the oxidation of iodide in spite of the high reactivity towards guaiacol, suggesting the presence of an abnormal peroxidase in these tissues.     

A novel tetrazolium method for peroxidase histochemistry and immunohistochemistry.

We developed a new method for the histochemical demonstration of peroxidase. This method, which has a novel reaction mechanism, is based on the oxidation of phenol by peroxidase and coupling of this reaction to the reduction of a tetrazolium salt, with the deposition of an insoluble formazan at sites of enzyme activity. This new method was compared with an established diaminobenzidine (DAB) technique for peroxidase histochemistry and immunohistochemistry. Although both methods identified peroxidase activity in myeloid cells of bone marrow biopsy specimens, there was no interference from red cell pseudoperoxidase activity with the phenol-tetrazolium method, in contrast to the diaminobenzidine method. The detection of cytokeratin using an indirect immunoperoxidase technique was compared with both methods for demonstrating peroxidase activity. The phenol-tetrazolium method gave results similar to that obtained with DAB and appeared to be at least as sensitive as DAB in detecting low amounts of antigen. In addition, the production of a formazan as the final reaction product means that the phenol-tetrazolium method is ideally suited for quantitative peroxidase histochemistry. Therefore, the phenol-tetrazolium method represents a useful alternative method to DAB and for certain applications offers significant advantages over DAB.     

Ultrastructural cytochemistry of the diaminobenzidine reaction in the aquatic fungus Entophlyctis variabilis

Ultrastructural localization of peroxidatic activity was investigated in the chytrid Entophlyctis variabilis with the 3,3-diaminobenzidine (DAB) cytochemical prodedure. The subcellular distribution of reaction product varied with changes in pH of the DAB medium and with the developmental stage of the fungus. Incubations in the DAB reaction medium at pH 9.2 produced an electron dense reaction product within single membrane bounded organelles which resembled microbodies but which varied in shapes from elongate to oval. At this pH the cell wall also stained darkly. When the pH of the DAB medium was lowered to pH 8.2 or 7.0, DAB oxidation product was localized within mitochondrial cristae as well as in microbodies and zoosporangial walls. As soon as zoospores were completely cleaved out of the zoosporangial cytoplasm, endoplasmic reticulum (ER) also stained. When the wall appeared around the encysted zoospore, ER staining was no longer found. The influence of the catalase inhibitor, aminotriazole, and the inhibitors of heme enzymes, sodium azide and sodium cyanide, on the staining patterns within cells incubated in the DAB media indicates that microbody staining is due to both catalase and peroxidase, mitochondrial staining is due to cytochrome c, and ER staining is due to peroxidase.Abbreviations DAB 3,3-diaminobenzidine-HCl - ER endoplasmic reticulum     

Peroxidases in Tobacco Abscission Zone Tissue: II. Time Course Studies of Peroxidase Activity during Ethylene-induced Abscission

Ethylene-induced abscission in flower pedicels of Nicotiana tabacum L. cv. Little Turkish causes a progressive increase in peroxidase activity during the first 4 hours of a 5-hour time course ethylene treatment period, with decrease in peroxidase activity occurring between 4 hours and 5 hours, when the supernatant extracts of abscission zone segments are tested spectrophotometrically for peroxidase activity, using guaiacol and hydrogen peroxide. Nonethylene-treated tissue has a much lower level of peroxidase activity over the same time course period. In ethylene-treated tissue the decline in break-strength correlates with the beginning of increase in peroxidase activity (3 hours). When the abscission zone area of the pedicel is further divided into proximal, abscission zone, and distal portions, respectively, the ethylene-treated tissue has the highest peroxidase activity in the abscission zone portion, with the maximum peak occurring at 4 hours and decreasing between 4 hours and 5 hours. Acrylamide gel electrophoresis of enzyme breis from ethylene-treated aand nonethylene-treated plants reveals that no new peroxidase isozymes are formed in response to ethylene, indicating an increase in the amount of one or in both of the two already existing isozyme banding patterns. The measurement of protein in the proximal, abscission zone, and distal segments, over a 5-hour ethylene treatment period, indicates that it is being translocated in a distal to proximal direction in the abscission zone pedicel. The possible participatory role for peroxidase in ethylene-induced tobacco flower pedicel abscission are discussed.     

Natural wood colouring process in Juglans sp. (J. nigra, J. regia and hybrid J. nigra 23 ×J. regia) depends on native phenolic compounds accumulated in the transition zone between sapwood and heartwood

 Radial distribution of soluble phenolics was investigated at different heights in stems of Juglans nigra, J. regia and hybrids J. nigra 23 × J. regia. Four major phenolic compounds were studied: hydrojuglone glucoside (HJG), quercitrin (QUER) and two unknown compounds characterized as two ellagic acid derivatives E1 and E2. HJG and E1 content increased gradually in the sapwood, peaked in the sapwood-heartwood transition zone, and decreased drastically in the heartwood. QUER was accumulated preferentially around the transition zone, and its content was relatively low in the outer part of the sapwood and in the inner part of the heartwood. E2 content was low in the sapwood and increased in the heartwood. The heartwood formation was marked by the accumulation of new soluble compounds. The relationship between wood extractives and wood colour were evaluated and discussed. HJG was considered to be a major precursor of heartwood colour providing chromophores through hydrolysis (deglucosylation), oxidation and polymerization processes. Received: 2 September 1997 / Accepted: 23 November 1997