Biochemistry Lab: a Kinetic Study of Beta-Galactosidase

A Kinetic Study of Beta-Galactosidase

Introduction:

In the assay “A Kinetic Study of Beta-Galactosidase”, the enzyme beta-galactosidase is measured to show how readily the enzyme produces a product with the substrate o-nitrophenyl-galactoside(ONPG). This experiment will measure the product produced, which is o-nitrophenolate(ONP), under alkaline conditions. During the assay many different sets of data will be collected to quantitatively show how readily beta-galactosidase will cleave ONPG to produce ONP. Also, an unknown inhibitor will be introduced to the reaction to determine the type of inhibition. Using the Michaelis-Menten, Lineweaver-Burke, and Eadie-Hofstee graphs the type of inhibition will be determined. This experiement should show how inhibitors affect enzymes and create an understanding on how beta-galactosidase acts on the substrate.

Results:

Part 1: A standard curve of the product ONP was created using a 169µMolar ONP solution. The following data was collected at an absorbance of 420nm:

Part 1 420m

Tube # mL Buffer mL ONP mL 2% Na2CO3 Absorbance

0 5 0 1 0

1 4.5 0.5 1 0.07

2 4 1 1 0.13

3 3 2 1 0.2

4 2 3 1 0.37

5 1 4 1 0.48

6 0 5 1 0.6

To find nmoles of ONP the following calculations where made:

Tube 1 Tube 2

Tube 3 Tube 4

Tube 5 Tube 6

Part 2: In this step the enzyme and substrate are measured to determine the rate of the reaction for the enzyme.

Part2 stagger @ 30s 420nm

Tube # mL Buffer mL 2.5mM ONPG mL Beta-Galactosidase mL 2% Na2CO3 Absorbance

0 4 1 0 1 0

1 3 1 1 1 0.04

2 2 1 2 1 0.1

3 1 1 3 1 0.15

4 0 1 4 1 0.22

The calculations to find velocity (nmol/minutes) and mg of beta-galactosidase:

Mg Beta-Galactosidase(0.5mg/mL)

Tube 1 Tube 2

Tube 3 Tube 4

Finding Velocity (nmol ONP/minutes), nmol of ONP found from the standard curve from part 1:

Tube 1 85nmole ONP/5min. =17nmol/min.

Tube 2 160nmole ONP/5min. =32nmol/min.

Tube 3 220nmole ONP/5min. =44nmol/min.

Tube 4...