Visualization of the enzyme trimethylamine oxide demthylase in isoelectricfocusing gels by an enzyme-specific staining method

An enzyme-specific staining method for trimethylamine oxide demethylase (TM AO-ase) was developed. Direct visualization could be reached by coupling th e reactions of the specific TMAO-ase assay with another reaction step gener ating as final product a dark-blue formazan. For these purposes 3-(4,5-dime thyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium bromide (MTT) as tetrazolium salt and phenazine methosulfate (PMS) as electron transfer substance were used. Clear, dark-blue colored bands could be detected on 300 mu m isoelect ric focusing gels (IEF). Comparisons of enzyme-stained and protein-stained gels showed that diffusion could not be observed and that the band pattern of TMAO-ase could also be seen in the protein stain. The p/ range where TMA O-ase was located was 5.6-6.6 for extracts and 6.2-6.6 for partially purifi ed TMAO-ase. Specificity of stained TMAO-ase bands was assessed by the prep aration of staining solution without the substrate trimethylamine oxide (TM AO) and by extraction of TMAO-ase from the gel and performance of the speci fic TMAO-ase assay.