Phenol oxidases assay

Radiolabeled products were separated from substrates by chromatography on a Merck Qg column (5 /an). The mobile phase contained 0.1 M sodium acetate, 0.1 M citric acid, 0.1 mAf sodium octylsulfate, 0.15 mAf EDTA, and 0.2 mAf dibutylamine in 10% methanol (v/v). The pH was 4 for the monoamine oxidase assay and 3.7 for phenol sulfotransferase. A flow-through radioisotope detector was used to quantitate the amount of radioactivity in the eluted peaks. 

Potato Phenol Oxidase. The studies of Kubowitz on the copper oxidase of potatoes were part of the efforts of Warburg s institute to find enzyme systems that could catalyze oxygen consumption coupled with substrate oxidation. The enzyme was purified on the basis of an assay involving transfer of electrons from pyridine nucleotides to o-quinone and from the resulting catechol to oxygen (I). The reduction of catalytic quantities of quinone was probably catalyzed by a flavoprotein present in the Zwischenferment preparation used to reduce TPN. In the presence of... 

Cholesterol assay solution. The stock reagent contains pancreatic cholesterol esterase, microbial cholesterol oxidase, horseradish peroxidase, 4-aminoantipyrine, and phenol. Your instructor will reconstitute the stock reagent by addition of water. 

It should also be noted that peroxidases and mixed-function oxidases can also bring about the oxidation of phenolic substrates this can be checked by appropriate controls (e.g., addition of H202 to the assay system will stimulate peroxidase activity). 

Polarographic and Spectrophotometric Assay of Diphenol Oxidases Key References Machiex et al., 1990. See above. A comprehensive review of all aspects of the chemistry and biochemistry phenolic compounds in fruits. Mayer, A.M. 1987. Polyphenol oxidases in plants—recent progress. Phytochemistry 26 11-20. A useful review of plant DPOs. 

The assay for monoamine oxidase contained in a final volume of 100 pL to 30 pL of homogenate and 50 pAf [7-14C] dopamine (0.93 mCi/mmol) or [7-,4C] tyramine (3.11 mCi/mmol) in 0.5 M phosphate buffer (pH 7.4). The assay for phenol sulfotransferase was also initiated by adding 30 pL of homogenate. The mixture contained 1.7 pAf [35S] 3 -phosphoadenosine-5 -phosphosulfate (1.51 Ci/mmol) and 50 pAf dopamine, 3,4-dihydroxyphenylacetic acid, or phenol in 10 mAf phosphate buffer (pH 6.4). After various incubation periods, the activity of either enzyme was stopped by addition of 30 pL of 2 N HC1. The resulting mixtures were centrifuged or filtered before analysis by HPLC. 

The main advantage of the proposed wine analysis is its selectivity because only primary amines can be detected using this method. Also, byproducts do not interfere with phenols or thiols. The quality of the wine and its organoleptic characteristics are well defined considering the effects of the malolactic fermentation process. The electrometric methods assure reliable results for the 1-malic and 1-lactic acids assay. The biosensors construction for 1-malic and 1-lactic acids assay in wine are based on malate dehydrogenase and lactate oxidase enzymes.117 The reproducibility of the results as well as the selectivity make it reliable for establishing the quality of the wine. 

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