Feb 15, 2018
Spectrophotometric assay for measuring mannitol oxidase activity
- Alexandre Lobo-da-Cunha1,
- Vítor Costa1
- 1Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Portugal
External link: https://doi.org/10.1371/journal.pone.0193078
Protocol Citation: Alexandre Lobo-da-Cunha, Vítor Costa 2018. Spectrophotometric assay for measuring mannitol oxidase activity. protocols.io https://dx.doi.org/10.17504/protocols.io.naudaew
Manuscript citation:
Lobo-da-Cunha A, Amaral-de-Carvalho D, Oliveira E, Alves
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 15, 2018
Last Modified: March 28, 2018
Protocol Integer ID: 10292
Keywords: Mannitol oxidase
Abstract
Mannitol oxidase is an enzyme present in some gastropods, converting the polyalcohol mannitol into the sugar mannose. Molecular oxygen is the hydrogen acceptor and hydrogen peroxide is produced. Therefore, this enzymatic activity can be measured by monitoring H2O2 production using a horseradish peroxidase coupled assay adapted from previously published methods (Malik et al., 1987; Cablé et al., 1993; Rocha et al., 2003). This assay was used to evaluate mannitol oxidase activity in the digestive gland of gastropods, but in some slugs and snails this enzyme was also detected in digestive tract tissues (Vorhaben et al., 1984; Malik et al., 1987). This enzyme can also oxidase other polyalcohols (Large & Connock, 1994)
Materials
MATERIALS
Potter-Elvehjem homogenizer
Bath sonicator
Centrifuge
Double-bean UV-visible spectrophotometer, with temperature control in the cuvette compartment
Before start
Prepare the following solutions:
Homogenization medium for marine gastropods
(adapted from Stewart et al. 1994 and Moyers et al. 1991)
Sucrose - 500 mM
Potassium chloride - 150 mM
Ethylenediamine tetraacetic acid (EDTA) sodium salt - 1 mM
Tris-HCl 50 mM pH 7.4
This medium, without phenylmethylsulfonyl fluoride (PMSF), can be stored at -20º C in aliquots. To avoid the inactivation of PMSF in aqueous solution, add 5 µl of 200 mM PMSF (in absolute ethanol) per ml of medium just before use (final PMSF concentration 1 mM).
Good results were also obtained with freshwater and terrestrial gastropods using this high osmolarity medium.
Incubation medium
Phenol - 1.060 mM
4-aminoantipyrine - 0.082 mM
Horseradish peroxidase (» 200 U/mg) - 5 U/ml
Bovine serum albumin - 0.06 % (w/v)
Mannitol (or other polyalcohol) - 25 mM
Potassium phosphate 50 mM pH 7.4
This medium can be stored frozen (-20º C) in aliquots without loss of activity.
In control medium the substrate is omitted.
Sample preparation
Sample preparation
- Homogenize approximately 0.05-0.06 g of digestive gland tissue per ml of cold homogenization medium using a Potter-Elvehjem homogenizer at 1,000 rpm, keeping the tube in ice.
- Register the exact weight of digestive gland tissue used and the final homogenate volume.
- Sonicate the homogenate during 3 x 15 s using a bath sonicator, keeping the tube in an ice-water bath.
- Centrifuge the homogenate at 1,000 g for 5 min at 4º C, and use the supernatant to assess mannitol oxidase activity. Keep samples on ice and use immediately after preparation.
- In a tube, mix 960 µl of incubation medium with 40 µl of sample (sample dilution factor in the assay: 25), agitate with a vortex, and pour into the spectrophotometer cuvette.
- Measure the increase of absorbance at 500 nm, at 25º C, during 3-5 min (or longer for samples with very low enzyme activity). Make sure that activity is linear in time and proportional to sample concentration. If not, adjust sample dilution.
- For control, monitor non-specific increases of absorbance at 500 nm using medium without substrate, and subtract if necessary.
Construction of a calibration line with H2O2 standards
Construction of a calibration line with H2O2 standards
- Prepare a standard solution (≈1 mM) by diluting 1/10,000 a 30% H2O2 stock solution (≈10 M).
- Measure the absorbance at 240 nm, and calculate the exact concentration of the standard solution using the extinction coefficient of H2O2 at 240 nm (ε = 43.6 M-1. cm-1).
- Repeat the assay used for mannitol oxidase activity, using increasing amounts of H2O2 as indicated in the following table:
| µl per tube | |||||||
| Incubation medium | 900 | 900 | 900 | 900 | 900 | 900 | |
| H2O | 100 | 80 | 60 | 40 | 20 | 0 | |
| H2O2 standard solution | 0 | 20 | 40 | 60 | 80 | 100 | |
| Concentration of H2O2 in the assay (nmol . ml-1) | 0 | ||||||
| Absorbance at 500 nm | 0 |
Use the absorbance values and the corresponding H2O2 concentrations to obtain a calibration line and its equation.
Determination of enzyme activity
Determination of enzyme activity
Use the equation of the calibration line to calculate the production of H2O2 per min.
Activity (nmol . ml-1. min-1) = Δ [H2O2] per min x 25*
* sample dilution factor in the assay
Calculate the activity per g of tissue (nmol . g-1. min-1) or per mg of protein (nmol . mg-1. min-1).
References
References
Cablé S., Kedinger M. and Dauça M., 1993. Peroxisomes and peroxisomal enzymes along the crypt-villus axis of the rat intestine. Differentiation 54: 99–108.
Malik Z., Jones C.J.P. and Connock M.J., 1987. Assay and subcellular localization of H2O2 generating mannitol oxidase in the terrestrial slug Arion ater. J. Exp. Zool. 242: 9–15.
Moyes C.D., Suarez R.K., Brown G.S. and Hochachka P.W., 1991. Peroxisomal β-oxidation: insights from comparative biochemistry. J. Exp. Zool. 260: 267–273.
Large A.T. and Connock M.J., 1994. Centrifugal evidence for association of mannitol oxidase with distinct organelles ("mannosomes") in the digestive gland of several species of terrestrial gastropod mollusc. Comp. Bioch. Physiol. 107A: 621–629.
Rocha M.J., Rocha E., Resende A.D. and Lobo-da-Cunha A., 2003. Measurement of peroxisomal enzyme activities in the liver of brown trout (Salmo trutta), using spectrophotometric methods. BMC Biochem. 4 (2): 1-9.
Stewart J.M., Carlin R.C., Macdonald J.A. and Van Iderstine S., 1994. Fatty acid binding proteins and fatty acid catabolism in marine invertebrates: Peroxisomal β-oxidation. Inv. Rep. Dev. 25: 73–82.