Fluorescence malate dehydrogenase

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. 

Tsukatani, T. and K. Matsumoto. 2000. Quantification of L-tartrate in wine by stopped-flow injection analysis using immobilized D-malate dehydrogenase and fluorescence detection. Anal. Sci. 16(3) 265-268. 

FIGURE 12.4 (a) Enzyme distribution in native chitosan scaffolds stained with fluorescein (green) combined with fluorescent (Alexa Fluor 546)-stained malate dehydrogenase (purple), (b) Enzyme distribution in butyl-modified chitosan scaffolds (same staining), (c) Enzyme distribution in ALA-modified chitosan scaffolds (same staining). (Reproduced with permission from Ref. [27]. Copyright 2009, American Chemical Society.)... 

Fluorescence is an excellent tool to probe chemical microenvironments. For example, in 2010, Martin et al. used a specific shift in fluorescence emission spectra of polar sensitive fluorophores, for example, acrylodan, to characterize the polarity immediately surrounding immobilized enzymes [34]. Cytoplasmic malate dehydrogenase (cMDH) and mitochondrial malate dehydrogenase (mMDH), for example, were tagged with fluorescent probes and then entrapped within macroporous three-dimensional chitosan scaffolds. A blue shift in the acrylodan emission maximum was taken to indicate a polar shift in the chemical microenvironment directly surrounding enzymes immobilized within the scaffold (Figme 12.5). The emission... 

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