Question

Calculate the enzymatic activity (μmol/min/mL) of a protein. The kinetic assay was graphed absorbance vs. time (sec).

Linear regression provided the following equation: y=0.0135x+0.0072.

The kinetic assay protocol requires 0.2 mL of enzyme. Total volume of assay tube is 4 mL.

Molar absorptivity (ε) = 5300 M^-1 cm^-1; path length = 1 cm.

Answer 1

In the experiment to determine enzyme activity using spectrometry, the graph obtained by plotting Absorbance vs time, the given data was obtained:

y = 0.0135x + 0.0072 , where y = Absorbance measured, t = time, m = slope of the line = 0.0135

Now, Slope of the line = A = Relative absorbance = 0.0135

Now, according to Beer's Law,

, A = Relative absorbance, = Molar absorptivity coefficient = 5300 M^-1cm^-1

l = Path length = 1 cm, c = Concentration of enzyme, or (Activity of the enzyme = Concentration/time)

So,

  

We need to multiply this by the dilution factor to get the final answer

Now, total volume of assay tube = 4 mL , volume of enzyme = 0.2 mL

So, Dilution factor =

So, Enzymatic activity = 2.54 x 10^-3 x 20 = 5.08 x 10^-2 umol/min.mL = 0.0508 umol min^-1mL^-1