Principles of Enzyme Assays

Question 1

What does the Michaelis-Menton curve show?

Answer 1

Shows how velocity/rate of reaction (y-axis) changes with [substrate] (x-axis)

Question 2

Name 3 conditions that must be controlled when performing activity measurements.

Answer 2

1. Temperature (Q10: relative reaction rates at two temps differing by 10 degrees)
2. pH - optimum varies from 1.5-10
3. Precise [substrate] and ionic strength of buffers

Question 3

Describe the 2 main units of enzyme activity in usage.

Answer 3

International unit (IU):

• 1 Unit catalyses the conversion of 1 μmol of substrate per minute (temp not defined)

Katal:

• 1 Katal converts 1 mol of substrate per second

Question 4

Name 6 methods of continuously monitoring enzyme-catalysed reactions.

Answer 4

1. Absorbance
2. Fluorescence
3. Optical rotation
4. Conductivity
5. Viscosity
6. Radiochemical

Question 5

What is the Lambert-Beer law?

Answer 5

A = ecl

where:

• A = absorbance
• e = exctinction coefficient
• c = molar concentration
• l = light path length (cm)