I recently bought lactate oxidase from abroad and received it after 3 months. How can I check for enzyme activity? I know that for some enzymes such as glucose oxidase there are tests but is there any tests for LOx?!
Thanks
Hi there,
As the reaction produce hydrogen peroxyde you can make a coupled assay with HRP.
To Elham Asadian and Dominique Liger
PLease to formulate the lactate oxidase reaction. Generally we talk about lactate dehydrogenase, an oxidoreductase enzyme.
Thank you in advance
Yes, Lactate Oxidase is a different enzyme than Lactate Dehydrogenase. Dominiques answer is the best solution although it does not give you any details or guidance about how to set up the coupled assay.
Effectively, lactate oxidase reaction on lactate produces hydrogen peroxide, H2O2. For how you measure hydrogen peroxide in a coupled assay, the following references are helpful to you:
Allain et al 1974, Clinical Chemistry, 20, 470-475
Kreit et al. 1992, Lipids, 27, 458-465
Joseph Kreit
As far as I understand, the best way is to detect H2O2 produced in the reaction. Can I use UV-Vis technique?
The H2O2 absorption coefficient in the UV is weak and impractical in your case
Using HRP, there are a wide variety of chromogenic (generate color) and fluorogenic (generate fluorescence) substrates which give a response proportional to the amount of H2O2 formed. One of the most common is ADHP (10-Acetyl-3,7-dihydroxyphenoxazine) which forms a colored and fluorescent product. One important point to consider is that particularly with the fluorescent assay, the reaction pH needs to be over 7, preferably 7.5 since the fluorescence is dependent upon pH. Since the enzyme has a maximum activity at lower pH, you are sacrificing max enzyme activity to get max fluorescence.
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