You run an enzyme assay designed to determine the -glucosidase activity after 1

Question

You run an enzyme assay designed to determine the -glucosidase activity after 1 hour (60 min) by determining the concentration of p-nitrophenol. The results you collected are presented below.

Part 1

Enzyme Dry Weight Absorbance at 580 nm : 0.190

The Required dilution factor: 55

Part 2

p-nitrophenol absorbance at 400 nm : 0.962

You now need to analyze the results and calculate the following: (Answer each question to 3 significant figures)

Part 1 Calculate the Dry Weight (DW) of your enzyme using the following equation.

DWenzyme = 0.35 · A580 · DF = ???g

If the molar absorptivity of the p-nitrophenol is 7.28 mM1·cm1 and the path length is 1.0 cm, what is the concentration of p-nitrophenol in the sample? ????mM

Using the formula below, what is the activity of your enzyme?

Activity = A400 * 1000 0.00728 * DWenzyme * 60 = ???(µmol/min.g)

Explanation / Answer

Solution:

DW of enzyme = 0.35 * 0.190 * 55 = 3.6575 gm

p-nitro phenol Concentration = Absorbance / (molar absorptivity * path length)

                                          = 0.962 / (7.28 E-03 * 1)

                                         = 0.1321 mM

Activity = 0.962 * 10000.00728* 3.6575 * 60

           = 2.11 E06 µmol/min.g

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