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 96-well 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.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Anthon GE, Barrett DM (2004) Comparison of three colorimetric reagents in the determination of methanol with alcohol oxidase. Application to the assay of pectin methylesterase J Agric Food Chem 52:3749–3753. doi:10.1021/jf035284w
Boyaci IH, Genis HE, Guven B, Tamer U, Alper N (2012) A novel method for quantification of ethanol and methanol in distilled alcoholic beverages using Raman spectroscopy. J Raman Spectrosc 43:1171–1176. doi:10.1002/jrs.3159
Bueno C, Villegas ML, Bertolotti SG, Previtali CM, Neumann MG, Encinas MV (2002) The excited-state interaction of resazurin and resorufin with amines in aqueous solutions. Photophysics and photochemical reaction. Photochem Photobiol 76:385–390
Cha S (1968) Kinetics of enzyme reactions with competing alternative substrates. Mol Pharmacol 4:621–629
Commission Regulation (EC) No. 2870/2000 of 19 (2000) Laying down community reference methods for the analysis of spirits drinks. Off J Europ Union L 333:20–46
Couderc R, Baratti J (1980) Oxidation of methanol by the yeast, Pichia pastoris. Purification and properties of the alcohol oxidase. Agric Biol Chem Tokyo 44:2279–2289
Demaria CG, Manzano T, Duarte R, Alonso A (1995) Selective flow-injection determination of methanol using immobilized enzyme reactors. Anal Chim Acta 309:241–250. doi:10.1016/0003-2670(95)00040-7
Giles HG, Hirst M, Hoffmann E, Kapur BM (1993) A color test for methanol. Clin Chem 39:693–693
Guilbault GG, Kramer DN (1964) New direct fluorometric method for measuring dehydrogenase activity. Anal Chem 36:2497–2498. doi:10.1021/Ac60219a001
Ho MH, Richards RA (1990) Enzymatic method for the determination of formaldehyde. Environ Sci Technol 24:201–204. doi:10.1021/Es00072a007
Jendral JA, Monakhova YB, Lachenmeier, DW (2011) Formaldehyde in alcoholic beverages: large chemical survey using purpald screening followed by chromotropic acid spectrophotometry with multivariate curve resolution. Int J Anal Chem 2011: Article ID 797604. doi:10.1155/2011/797604
Kurokawa J, Asano M, Nomoto S, Makino Y, Itoh N (2010) Gene cloning and characterization of dihydrolipoamide dehydrogenase from Microbacterium luteolum: a useful enzymatic regeneration system of NAD+ from NADH. J Biosci Bioeng 109:218–223. doi:10.1016/j.jbiosc.2009.09.040
Lachenmeier DW (2007) Rapid quality control of spirit drinks and beer using multivariate data analysis of Fourier transform infrared spectra. Food Chem 101:825–832. doi:10.1016/j.foodchem.2005.12.032
Mazoch J, Tesarik R, Sedlacek V, Kucera I, Turanek J (2004) Isolation and biochemical characterization of two soluble iron(III) reductases from Paracoccus denitrificans. Eur J Biochem 271:553–562. doi:10.1046/j.1432-1033.2003.03957.x
Miller JN, Miller JC (2000) Statistics and chemometrics for analytical chemistry. 4th edn. Prentice Hall, Harlow, England; New York.
Mizgunova UM, Zolotova GA, Dolmanova IF (1996) Enzymic method for the determination of ethanol and methanol with spectrophotometric detection of the rate of the process. Analyst 121:431–433. doi:10.1039/An9962100431
Ogushi S, Ando M, Tsuru D (1984) Substrate specificity of formaldehyde dehydrogenase from Pseudomonas putida. Agric Biol Chem Tokyo 48:597–601
Regulation (EC) No 110/2008 of the European Parliament and of the Council of 15 (2008) On the definition, description, presentation, labelling and the protection of geographical indications of spirit drinks and repealing council regulation (EEC) no 1576/89. Off J Europ Union L39:16–54
Rodionov YV, Sukhacheva MV, Keppen OI (2002) Photometric assay for methanol in the presence of ethanol. Appl Biochem Microbiol 38:607–609. doi:10.1023/A:1020747215826
Sedlacek V, Kucera I (2010) Chromate reductase activity of the Paracoccus denitrificans ferric reductase B (FerB) protein and its physiological relevance. Arch Microbiol 192:919–926. doi:10.1007/s00203-010-0622-4
Sedlacek V, van Spanning RJM, Kucera I (2009) Characterization of the quinone reductase activity of the ferric reductase B protein from Paracoccus denitrificans. Arch Biochem Biophys 483:29–36. doi:10.1016/j.abb.2008.12.016
Sedlacek V, Klumpler T, Marek J, Kucera I (2014) The structural and functional basis of catalysis mediated by NAD(P)H:acceptor oxidoreductase (FerB) of Paracoccus denitrificans. PLoS One 9:e96262. doi:10.1371/journal.pone.0096262
Sedlacek V, Ptackova N, Rejmontova P, Kucera I (2015) The flavoprotein FerB of Paracoccus denitrificans binds to membranes, reduces ubiquinone and superoxide, and acts as an in vivo antioxidant. FEBS J 282:283–296. doi:10.1111/febs.13126
Sekine Y, Suzuki M, Takeuchi T, Tamiya E, Karube I (1993) Selective flow-injection determination of methanol in the presence of ethanol based on a multienzyme system with chemiluminescence detection. Anal Chim Acta 280:179–184. doi:10.1016/0003-2670(93)85119-5
Tesarik R, Sedlacek V, Plockova J, Wimmerova M, Turanek J, Kucera I (2009) Heterologous expression and molecular characterization of the NAD(P)H:acceptor oxidoreductase (FerB) of Paracoccus denitrificans. Protein Expres Purif 68:233–238. doi:10.1016/j.pep.2009.07.014
Vinet B (1987) An enzymic assay for the specific determination of methanol in serum. Clin Chem 33:2204–2208
Vinet B (1988) Enzymic methanol determination—toxic concentrations of ethanol may give positive values. Clin Chem 34:1944–1944
Weng JL, Ho MH (1990) Fluorometric enzymatic method for determination of formaldehyde. Anal Lett 23:2155–2174
Zakharov S et al (2014) Czech mass methanol outbreak 2012: epidemiology, challenges and clinical features. Clin Toxicol 52:1013–1024. doi:10.3109/15563650.2014.974106
The authors are grateful to Helena Zavadilová from the Department of Chemistry for performing the gas chromatography analyses. Thanks are also due to Marcela Hrnčířová for excellent technical assistance.
This work was supported by the Czech Science Foundation Project No. GAP503/12/0369 to Igor Kučera.
Conflict of Interest
Igor Kučera declares that he has no conflict of interest. Vojtěch Sedláček declares that he has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
About this article
Cite this article
Kučera, I., Sedláček, V. An Enzymatic Method for Methanol Quantification in Methanol/Ethanol Mixtures with a Microtiter Plate Fluorometer. Food Anal. Methods 10, 1301–1307 (2017). https://doi.org/10.1007/s12161-016-0692-2