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Visualization of the enzyme trimethylamine oxide demthylase in isoelectric focusing gels by an enzyme‐specific staining method
Author(s) -
Havemeister Wiebke,
Rehbein Hartmut,
Steinhart Hans,
GonzalesSotelo Carmen,
KrogsgaardNielsen Michael,
Jørgensen Bo
Publication year - 1999
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(19990701)20:10<1934::aid-elps1934>3.0.co;2-b
Subject(s) - chemistry , trimethylamine , isoelectric focusing , formazan , chromatography , staining , trimethylamine n oxide , isoelectric point , substrate (aquarium) , enzyme , biochemistry , biology , genetics , ecology
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 p I 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.