Extinction coefficient of polymerized diaminobenzidine complexed with cobalt as final reaction product of histochemical oxidase reactions

1995 ◽  
Vol 104 (6) ◽  
pp. 473-477 ◽  
Author(s):  
Wilma M. Frederiks ◽  
Klazina S. Bosch ◽  
Rosier J. M. Munckhof
Keyword(s):  
Extinction Coefficient ◽  
Final Reaction Product ◽  
Final Reaction

scholarly journals A quantitative histochemical study of xanthine oxidase activity in rat liver using the cerium capture method in the presence of polyvinyl alcohol.

1994 ◽  
Vol 42 (8) ◽  
pp. 1091-1096 ◽  
Author(s):  
W M Frederiks ◽  
K S Bosch ◽  
R J Van den Munckhof ◽  
C J Van Noorden
Keyword(s):  
Polyvinyl Alcohol ◽  
Xanthine Oxidase ◽  
Rat Liver ◽  
Incubation Medium ◽  
Oxidase Activity ◽  
Specific Inhibitor ◽  
Semipermeable Membrane ◽  
Final Reaction Product ◽  
Final Reaction ◽  
Xanthine Oxidase Activity

A recently developed histochemical technique to demonstrate xanthine oxidase activity in milk globules of bovine mammary gland and in epithelial cells of rat small intestine using cerium ions and a semipermeable membrane was slightly modified. The semipermeable membrane method was replaced by the addition of 10% (w/v) polyvinyl alcohol to the incubation medium. This technically more simple procedure enabled detection of xanthine oxidase activity in unfixed cryostat sections of rat liver. Both methods gave qualitatively and quantitatively similar results. Activity was found in sinusoidal cells and in liver parenchymal cells, with 50% higher activity in pericentral than in periportal areas. The specificity of the reaction was proven by the generation of only small amounts of final reaction product on incubation either in the absence of the substrates hypoxanthine or oxygen or in the presence of hypoxanthine and allopurinol. Allopurinol is a specific inhibitor of xanthine oxidase activity. The amount of final reaction product, as measured cytophotometrically in rat liver, increased linearly with incubation time (15-90 min) and with section thickness (up to 12 microns). By varying the hypoxanthine concentrations, a Km value of 0.05 mM was found. Addition of dithiothreitol to the incubation medium reduced the amount of final reaction product by 85%, which was caused by conversion of reversible xanthine oxidase into xanthine dehydrogenase. This histochemical method can be used for quantitative analysis of in situ xanthine oxidase activity.

Cinétique de la formation de la métalloporphyrine Cu(II)—dérivé tétra éthylènediamino de la protoporphyrine IX (ENP) en milieu aqueux

1979 ◽  
Vol 57 (22) ◽  
pp. 2916-2922 ◽  
Author(s):  
Guy Paquette ◽  
Miklos Zador
Keyword(s):  
New Species ◽  
Reaction Kinetics ◽  
Side Chains ◽  
Mixed Complex ◽  
Final Reaction Product ◽  
Final Reaction ◽  
Kinetics Of Formation ◽  
Degree Of Association ◽  
Kinetics Of ◽  
A New Species

The kinetics of formation of the metalloporphyrin Cu(II)–ENP is dependent on the pH due to the protonation of the pyrrol nitrogen atoms and the protonation of the diamino groups of the side chains. The degree of protonation of these side chains also influences the degree of association of the prophyrin and the metalloporphyrin in solution. The order of reaction with respect to the porphyrin is not unity, a consequence of an inhibition by the reaction product; this inhibition operates via the formation of a new species, a mixed complex of porphyrin–metalloporphyrin of low reactivity.The order of reaction with respect to Cu(II) is also not unity, a consequence of the chelation of Cu(II) by the diamino groups of the side chains. The presence of Cu(II) ligands influence both the reaction kinetics and the nature of the final reaction product. [Journal translation]

A quantitative histochemical study of D-amino acid oxidase activity in rat liver in relationship with feeding conditions.

1991 ◽  
Vol 39 (1) ◽  
pp. 81-86 ◽  
Author(s):  
H R Patel ◽  
W M Frederiks ◽  
F Marx ◽  
A J Best ◽  
C J Van Noorden
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Oxidase Activity ◽  
Specific Inhibitor ◽  
Physiological Role ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Specific Test ◽  
Final Reaction Product ◽  
Final Reaction

The histochemical method for the demonstration of D-amino acid oxidase activity in rat liver, based on the use of cerium ions and the diaminobenzidine-cobalt-hydrogen peroxide procedure, was improved by the application of unfixed cryostat sections and a semipermeable membrane interposed between section and gelled incubation medium. The amount of final reaction product precipitated in a granular form was about four times higher with this technique in comparison with conventional procedures using fixed sections and aqueous incubation media. The specificity of the reaction was proven by the 70% reduction of the amount of final reaction product when incubating in the presence of substrate and D,L-beta-hydroxybutyrate, a specific inhibitor of D-amino acid oxidase activity. Cytophotometric analysis of liver sections revealed that the specific test minus control reaction was linear with incubation time and section thickness. The Km value of the enzyme of 10.3 +/- 2.7 mM, as determined in periportal areas, is about five times the value found with biochemical methods in liver cell homogenates. The enzyme activity in periportal areas is about five times the activity in pericentral areas. Fasting (24 and 48 hr) induced a significant decrease in D-amino acid activity in periportal and pericentral areas. The possible physiological role of the enzyme in liver is discussed.

KINETICS OF THE ENZYMICALLY-CATALYZED OXIDATION OF INDOLEACETIC ACID

1956 ◽  
Vol 34 (1) ◽  
pp. 1233-1250 ◽  
Author(s):  
G. A. Maclachlan ◽  
E. R. Waygood
Keyword(s):  
Indoleacetic Acid ◽  
Aryl Radical ◽  
Reaction Sequence ◽  
Final Reaction Product ◽  
Final Reaction ◽  
A Chain ◽  
Kinetics Of ◽  
Catalyzed Oxidation

A study of the kinetics of the enzymically-catalyzed decarboxylation and oxidation of indoleacetic acid has provided evidence that it is a chain autoxidation initiated and propagated by two enzyme-controlled peroxidations. The following reaction sequence occurs:[Formula: see text]where S—COOH = indoleacetic acid; S∙ = skatole radical; SO2∙ = oxidized skatole radical or indole peroxide; SO2H = final reaction product; ROH = phenolic cofactor, i.e., resorcinol; RO∙ = semiquinol or aryl radical.

The ethenoxy radical in the pyrolysis of acetaldehyde

1970 ◽  
Vol 48 (15) ◽  
pp. 2315-2319 ◽  
Author(s):  
H.-P. Schuchmann ◽  
K. J. Laidler
Keyword(s):  
Final Reaction Product ◽  
Final Reaction

In the pyrolysis of pure acetaldehyde at 450–525 °C it was found that ketene, although stable under these conditions, is not formed as a final reaction product. This result requires a modification of the mechanism of Liu and Laidler, according to whom the ethenoxy radical CH2CHO splits into CH2CO and H. It is concluded that instead of splitting unimolecularly, CH2CHO disappears mainly through the reaction[Formula: see text]and that it also undergoes the process[Formula: see text]

KINETICS OF THE ENZYMICALLY-CATALYZED OXIDATION OF INDOLEACETIC ACID

1956 ◽  
Vol 34 (6) ◽  
pp. 1233-1250 ◽  
Author(s):  
G. A. Maclachlan ◽  
E. R. Waygood
Keyword(s):  
Indoleacetic Acid ◽  
Aryl Radical ◽  
Reaction Sequence ◽  
Final Reaction Product ◽  
Final Reaction ◽  
A Chain ◽  
Kinetics Of ◽  
Catalyzed Oxidation

A study of the kinetics of the enzymically-catalyzed decarboxylation and oxidation of indoleacetic acid has provided evidence that it is a chain autoxidation initiated and propagated by two enzyme-controlled peroxidations. The following reaction sequence occurs:[Formula: see text]where S—COOH = indoleacetic acid; S∙ = skatole radical; SO2∙ = oxidized skatole radical or indole peroxide; SO2H = final reaction product; ROH = phenolic cofactor, i.e., resorcinol; RO∙ = semiquinol or aryl radical.

scholarly journals Flow cytofluorometric analysis of enzyme reactions based on quenching of fluorescence by the final reaction product: detection of glucose-6-phosphate dehydrogenase deficiency in human erythrocytes.

1989 ◽  
Vol 37 (9) ◽  
pp. 1313-1318 ◽  
Author(s):  
C J Van Noorden ◽  
F Dolbeare ◽  
J Aten
Keyword(s):  
Human Erythrocytes ◽  
Dependent Manner ◽  
Reaction Products ◽  
Final Reaction Product ◽  
Final Reaction ◽  
Enzyme Reactions ◽  
Quenching Of Fluorescence ◽  
Normal Individuals ◽  
Microscopic Inspection ◽  
Glucose 6 Phosphate Dehydrogenase

We developed a method for accurate cytofluorometric analysis of the final reaction product of enzyme reactions in individual cells. Glucose-6-phosphate dehydrogenase (G6PD) activity in human erythrocytes was demonstrated cytochemically, and the amount of final reaction product (formazan) per cell was detected indirectly by quenching of autofluorescence generated by glutaraldehyde fixation. Formazan quenches fluorescence in a dose-dependent manner. The method has been used for detection of G6PD deficiency. Heterozygous and homo(hemi)zygous deficiency could easily be established, even in cases of extreme "Lyonization" where microscopic inspection failed to discriminate between either normal individuals and heterozygously deficient patients or heterozygously and homozygously deficient patients. The principle of quenching of fluorescence by final reaction products of enzymes can be applied to flow cytofluorometric analysis of enzyme activity in individual cells in general.

scholarly journals A histochemical procedure for light microscopic demonstration of xanthine oxidase activity in unfixed cryostat sections using cerium ions and a semipermeable membrane technique.

1993 ◽  
Vol 41 (5) ◽  
pp. 667-670 ◽  
Author(s):  
W M Frederiks ◽  
F Marx
Keyword(s):  
Xanthine Oxidase ◽  
Oxidase Activity ◽  
Semipermeable Membrane ◽  
Myeloperoxidase Activity ◽  
Final Reaction Product ◽  
Final Reaction ◽  
Xanthine Oxidase Activity ◽  
Membrane Technique ◽  
Cryostat Sections ◽  
Unfixed Cryostat

Xanthine oxidoreductase exists in two functionally distinct forms. Under normal conditions, the larger part of the enzyme occurs as an NAD(+)-dependent dehydrogenase form which produces NADH and urate. The dehydrogenase can be transformed under various (patho)physiological conditions to an oxygen-dependent oxidase form which produces oxygen radicals and/or hydrogen peroxide and urate. Tetrazolium salts are used to demonstrate the total activity of both the dehydrogenase and the oxidase form of the enzyme. We have developed a procedure to detect the oxidase form only in unfixed cryostat sections with the use of cerium on the basis of the semipermeable membrane technique. The incubation medium contained hypoxanthine as substrate, cerium ions, and sodium azide to inhibit catalase and peroxidase activity. In a second-step reaction, diaminobenzidine was polymerized in the presence of cobalt ions by decomposition of cerium perhydroxide. Large amounts of final reaction product were found in milk droplets in the acini of lactating bovine mammary gland, whereas milk-secreting epithelial cells contained hardly any final reaction product. In rat duodenum, enzyme activity was found in the cytoplasm of enterocytes and goblet cells but not in the mucus. Control reactions performed in the absence of substrate or in the presence of substrate and allopurinol, a specific inhibitor of xanthine oxidase, were completely negative in both tissues, with the exception of polymorphonuclear leukocytes in the lamina propria of duodenum. The positive nonspecific reaction in these cells was caused by myeloperoxidase activity. We conclude that the present method is specific for the detection of xanthine oxidase activity. Moreover, conversion of the dehydrogenase form into the oxidase form can be prevented by omission of chemical fixation of the tissue in the present procedure.

Reactions of chromocene with diols and some their derivatives

1985 ◽  
Vol 50 (4) ◽  
pp. 828-833 ◽  
Author(s):  
Jaroslava Kalousová ◽  
Ludvík Beneš ◽  
Jiří Votinský ◽  
Milan Nádvorník
Keyword(s):  
Hydroxyl Groups ◽  
Oh Groups ◽  
Final Reaction Product ◽  
Final Reaction ◽  
Oxidation Reduction

Chromocene reacts with diols having OH groups at 1,3 position or at a greater distance to give chromium(II) diolates. Only the chromium(II) diolates obtained from reactions with 1,2-diols give the chromium(III) diolates with excess of the reagent. Alkylation of one of these hydroxyl groups, however, prevents the oxidation-reduction course. The reaction of chromocene with 2-chloroethanol gives chromium(II) 2-chloroethanolate in the first step, chromium(III) 2-chloroethanolate being the final reaction product.

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