An Enzymatic Method for Methanol Quantification in Methanol/Ethanol Mixtures with a Microtiter Plate Fluorometer
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An Enzymatic Method for Methanol Quantification in Methanol/Ethanol Mixtures with a Microtiter Plate Fluorometer Igor Kučera 1 & Vojtěch Sedláček 1
Received: 19 August 2016 / Accepted: 10 October 2016 # Springer Science+Business Media New York 2016
Abstract In response to the need for a rapid, high-throughput screening of methanol contamination in spirits, a new microplate-based assay was developed. In this assay, alcohol oxidase first oxidizes methanol to formaldehyde, which is further oxidized to formate by formaldehyde dehydrogenase while reducing NAD+ to NADH. The latter product then reacts with resazurin under catalysis by FerB, a diaphorase-type enzyme, to give the highly fluorescent resorufin. These reactions are run simultaneously in 200 μL final volume in a 96well plate and quantified using a plate reader and fluorescence detector. It is shown that the rate of fluorescence change is related to methanol and ethanol concentrations according to the rate law for two competing substrates. Quantification of methanol in real samples is carried out by applying the standard additions technique with four different spiking concentrations of the methanol standard; methanol content in the sample is calculated from the x-intercept of the fitted line. The high activity of FerB with resazurin and low rate of further conversion of resorufin to non-fluorescent dihydroresorufin indicate that FerB may be advantageous over commercially available diaphorases for use in fluorescence enzyme assays.
Keywords Methanol . Alcohol oxidase . Formaldehyde dehydrogenase . Diaphorase . Resazurin . Fluorescence
* Igor Kučera [email protected]
1
Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic
Introduction The recent methanol mass poisoning in the Czech Republic caused by adulterated alcoholic beverages (Zakharov et al. 2014) sparked our interest in developing methods for rapid determination of methanol in a large number of samples containing a high fraction of ethanol. In enzymatic methods of analysis of methanol alone or in combination with ethanol, alcohol oxidase is usually used to convert the alcohols to their corresponding aldehydes (Anthon and Barrett 2004; Demaria et al. 1995; Giles et al. 1993; Mizgunova et al. 1996; Rodionov et al. 2002; Sekine et al. 1993; Vinet 1987; Vinet 1988). The reaction requires oxygen and produces hydrogen peroxide: RCH2 OH þ O2 →RCHO þ H2 O2 ðR ¼ H or CH3 Þ
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Formaldehyde is more reactive than acetaldehyde and can be determined either chemically or enzymatically with a relatively high degree of specificity. Chemical methods that have been applied to determine formaldehyde include its condensation with acetylacetone and ammonia (Anthon and Barrett 2004; Rodionov et al. 2002), 4-amino-3-hydrazino-5mercapto-1,2,4-triazole (purpald) (Anthon and Barrett 2004; Jendral et al. 2011), or N-methylbenzothiazolinone-2hydrazone (MBTH) (Anthon and Barrett 2004). An enzymatic procedure relies upon the oxidation of formaldehyde to formic acid by the action of formal
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