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

Abstract

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.

Quantitative Relationship between Trimethylamine Oxide Aldolase Activity and Formaldehyde Accumulation in White Muscle from Gadiform Fish during Frozen Storage

“…The ability of the suggested model to describe the accumulation of formaldehyde, on the basis of in vitro TMAOase activities and storage time, is a strong indication that white muscle in situ TMAOase activity is the primary cause for formation of formaldehyde and DMA in gadiform fish. If isoenzymes exist as suggested by several authors (48)(49)(50), these must therefore either exhibit a common value of Q or their activities must be in constant proportions. The influence of variations in the in situ TMAOase reaction conditions also seems to be secondary to the enzyme concentration.…”