2.4.1 Measurement with crude Tmm
The reaction mixture was incubated for 15 min with 0.1 mM NADPH and
0.025 mM FMN and 20 µl of Tmm crude enzyme mix in the Tris-NaCl
buffer. The reaction starts after adding trimethylamine and is quenched
after 30 min by adding 0.1% TFA.
The amount of NADPH consumed from the reaction mix is calculated by
monitoring the absorbance at 340 nm. And the amount of trimethylamine
consumed was monitored by taking a 500 µl aliquot from the quenched
reaction using trimethylamine-picrate formation. In another 500 µl
aliquot, the amount of TMAO formed is calculated. From
Table 2, the observed activity in the crude
sample is higher in the case of NADPH+ assay. On
checking the activity after each purification step, the activity values
become comparable, proving that NADPH+-dependent
assays are unreliable for crude samples.
Table 2 : Tmm enzyme
assay and product quantification