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A two-step assay for trimethylamine-N-oxide aldolase (the target enzyme) is described in which the second step, the indicator reaction, is of the enzymatic end-point type. This indicator reaction is carried out at a slightly alkaline pH, outside the target enzyme's active pH range. An effective inactivation of the target enzyme is thus obtained solely by shifting the pH to that of the indicator reaction, thereby avoiding a deproteinisation step. By inserting a downward pH step as the stopping method, many samples may be collected and applied to the indicator reaction simultaneously. These features make the method very productive and easy to perform.

An enzyme‐specific staining method for trimethylamine oxide demethylase (TMAO‐ase) was developed. Direct visualization could be reached by coupling the reactions of the specific TMAO‐ase assay with another reaction step generating as final product a dark‐blue formazan. For these purposes 3‐(4,5‐dimethyl‐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 μm isoelectric 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 pI range where TMAO‐ase was located was 5.6—6.6 for extracts and 6.2—6.6 for partially purified TMAO‐ase. Specificity of stained TMAO‐ase bands was assessed by the preparation of staining solution without the substrate trimethylamine oxide (TMAO) and by extraction of TMAO‐ase from the gel and performance of the specific TMAO‐ase assay.

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