Malate dehydrogenase methods

A free energy study of malate dehydrogenase [29] using semiempirical QM-MM methods has also been reported, and that shidy also attributes many of the benefits to simulation of enzyme reactions found in the BPTP shidy. 

Students will isolate intact mitochondria from beef heart and fractionate them to prepare submitochondrial particles. Each fraction will be characterized by protein estimation by the biuret method and measurement of malate dehydrogenase and monoamine oxidase activity. 

Malate dehydrogenase from H. marismortui (AMDH) is the halophilic protein that has been studied most by solution structure methods. A molar mass of 87 kg/mol was determined for the native enzyme. It is stable at high concentrations of NaCl or KC1 and unfolds and dissociates below 2.5 M salt. Pundak and Eisenberg (1981) first measured values for the solvent interactions of AMDH and found that, in contrast to nonhalophilic globular proteins in similar conditions (Bi 0.2—0.3 g/g,B3 0.01 g/g),the halophilic protein bound... 

Fluorimetric methods involving six dehydrogenases have been used to determine the concentrations of 21 of the more common organic acids (58). Sensitivity of the methods range from 0.02 fxg/m for determination of D-isocitric acid with isocitrate dehydrogenase to 200 /xg/ml for determination of D-tartaric acid with malate dehydrogenase. 

Product inhibition is a cause of nonlinearity of reaction progress curves during fixed-time methods of enzyme assay. For example, oxaloacetate produced by the action of aspartate aminotransferase inhibits the enzyme, particularly the mitochondrial isoenzyme. The inhibitory product may be removed as it is formed by a coupled enzymatic reaction malate dehydrogenase converts the oxaloacetate to malate and at the same time oxidizes NADH to NADL... 

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. 

Enzyme reactions may be followed continuously, or sampled at fixed time intervals. Either the product of the reaction, or the residual substrate can be measured, although in a two-stage reaction in which an intermediate forms, only substrate disappearance gives a true measure of the reaction rate. Continuous ultraviolet (u.v.) recording methods are easily performed with enzymes that utilise NAD or NADP as co-enzyme. The reduced forms of both these substances exhibit strong absorption peaks at 334 nm and 366 nm. The formation or disappearance of the reduced form of either co-enzyme is thus readily followed in an enzyme reaction mixture which does not contain additional ultraviolet absorbing material, e.g. malate dehydrogenase. 

This enzyme is a pyridoxal protein which is present in excessive amounts in blood serum during diseases of the liver or cardiac muscle. It may be assayed by an ultra-violet method using malate dehydrogenase in an indicator reaction [274]. Alternately, the oxalacetate formed is decomposed to pyruvate which is treated with DNP to form pyruvate-dinitrophenylhydrazone. In the presence of sodium hydroxide, an intense brown colour is produced with an absorption maximum at 505 nm [275]. 

PEP carboxylase methods. Treatment of the plasma with an alkaline buffer results in the conversion of the dissolved CO2, bicarbonate and carbonic acid to the bicarbonate form. The bicarbonate is made to react with a phos-phoenolpyruvate (PEP) carboxylase/malate dehydrogenase coupled enzyme system as follows ... 

This reaction was exploited by Tsukatani and Matsumoto (2000) in a stopped-flow FIA method. An immobilized D-malate dehydrogenase enzyme reactor was employed and the reduced enzymatic cofactor NADH that was formed was monitored fluorometrically (Xex = 340 nm = 460 nm). Due to the slow reaction rate, the flow was stopped with the sample in the reactor to increase reaction time. The intrinsic sample fluorescence was also assessed using a parallel blank reactor without immobilized enzyme. The method was validated through the analysis of red and white wine samples. The enzyme reactor stability was also evaluated and it was found that the sensitivity (evaluated as amplitude of response at a constant concentration of the analyte) gradually decreased to 60% within a week but then remained stable for a month. As D-malate cannot be present in naturally fermented wines (except for fraudulent addition), the interference of this primary substrate can be considered negligible. 

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