An enzyme catalyzed reaction produces a product that has a m

An enzyme catalyzed reaction produces a product that has a maximum absorbance at 412 nm. The extinction coefficient is 4000 M^-1 cm^-1. A 10 to 1 dilution of the enzyme is made and 20 microL of the dilution are put into a cuvette with 80 microL of water and 1.9 mL of reaction cocktail. The absorbance change per minute is 0.05.

a) How many units are in the cuvette if U=1 micromol/min?

b) How many units are in the cuvette if U= 1nmol/min?

c) What is the relative activity of the undiluted enzyme (1U = 1 micromol/min)?

*Please show all work/calculations! Thank you

Solution

a)

Using Beer-Lambert\'s law,

A= ecl; Given e = 4000 M^-1 cm^-1 = 4000* 10^-6 micromol^-1cm^-1

c = 1/10 * 20 = 2 µl/molecular weight of enzyme in µg; stock solution is 10X; working solution is 1X; So, working solution of enzyme has 1/10 of 20 microliter.

l = 1cm

A = 4000* 10-6* 2 *1= 0.008 (Let molecular weight of enzyme be 1microgm)

0.05 absorbance change per minute

0.008 Absorbance will change in 0.008/0.05 = 0.16 micromoles/minutes = 0.16 units are there in cuvette.

b)

If U = 1nmol/min

Then, 0.16 * 10³ U is present.

c)

For calculating relative activity you need to know the O.D