Hi everyone,
I am measuring the activity of some mitochondrial enzymes, by measuring the oxidation/reduction of relevant substrate using spectrophotometer. However, after analysis, my enzymatic activity (nmol/min/mg) becomes a negative value (not always, but most of times).
Is this because my analytes are being used (thus decreased) so their absorbances decrease?
Another question; if the extinction cofficient of my analyte in 340 nm is 6.22 mM-1.cm-1, and the length of cuvette is 0.6 cm, and the concentration of my analyte is 0.1 mM, how should I calculate my enzymatic activity in nmol/min/mg?
Big thanks in advance
If you are measuring the conversion of NAD(P)H to NAD(P)+ at 340 nm, the absorbance decreases as the reaction progresses. You should perform your calculation using the absolute value (i.e. positive value) of the negative absorbance change.
Use Beer's Law to calculate the change in concentration of the analyte based on the absorbance change, where the absolute value of the absorbance decrease divided by the path length (0.6 cm) and divided by the extinction coefficient (6.22 mM-1cm-1), give the concentration change of the analyte in mM units.
The calculation of specific activity from this result requires knowing the volume of the sample that was measured and the concentration of protein that was present during the reaction.
specific activity = nanomolar concentration of product formed or substrate consumed divided by concentration of protein in mg/liter and divided by time of reaction.
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