Visualization of the enzyme trimethylamine oxide demethylase in isoelectric focusing gels by an enzyme-specific staining method.
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 microm 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.
Havemeister
W
W
Federal Research Center for Fisheries, Institute of Biochemistry and Technology, Hamburg, Germany.
Rehbein
H
H
Steinhart
H
H
Gonzales-Sotelo
C
C
Krogsgaard-Nielsen
M
M
Jørgensen
B
B
D000446:Aldehyde-Lyases / Q000032:analysis*; D004396:Coloring Agents; D005396:Fish Products / Q000032:analysis*; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008773:Methylphenazonium Methosulfate; D013194:Staining and Labeling / Q000379:methods*; D013778:Tetrazolium Salts; D013844:Thiazoles
D004396:Coloring Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; D008773:Methylphenazonium Methosulfate; D000446:Aldehyde-Lyases; C023188:trimethylamine-oxide aldolase; C022616:thiazolyl blue