1. Exercise 4:
Enzymes

2. Announcements
Post Lab 4 and Pre Lab 5 are due by the time your lab meets next.
LNA Enzymes is assigned today, and due next week within the first 5 minutes of your lab period.

3. Exercise 4A: Spectrophotometry
Goals:
Understand the process by which spectrophotometry can be used to quantify experimental results.
Develop skills taking measurements using a spectrophotometer.
Read Slide

4. Measurement of light absorption or transmission through a solution
Spectrophotometry
Measurement of light absorption or transmission through a solution

5. Types of photometers:
Colorimeters and spectrophotometers measure the amount of light absorbed by solutions.
Turbidimeters and nephelometers measure the light scattered by suspensions.
Fluorimeters measure the fluorescence produced by absorbed light.

6. Light through a solution:
Example ONP (o-nitrophenol)
Absorbs blue light and allows yellow light to pass through.
Solution therefore appears yellow.
Proteins do no absorb visible light and therefore appear to be uncolored. However, proteins strongly absorb specific wavelengths of UV light

7. Absorption spectrum of ONP
A plot of the relative amount of light absorbed by a compound as a function of the wavelength
Here is the absorption spectrum for ONP. Note that the optimal wavelength for a molecule can be determine from an absorption spectrum. Here 420 nm is the wavelength absorbed maximally by ONP
Can anyone think of reason the light absorbed by a molecule in a solution could be complicated to determine? What if the molecule is dissolved in a solvent that also absorbs light? Here we use a “BLANK”
Absorption spectrum of ONP

8. Components of a Typical Spectrophotometer
Tungsten lamp emits white light
Light is focused on a mirror and then passes through a monochromator that divides the light into different wavelengths
The desired wavelength is selected and passed through the sample chamber
The solution is placed in a clear tube called a cuvette
The photoelectric detector measures the light that passes through the solutiona nd converts the signal to an electric current that is translated to an optical density reading on the meter
The greater the amont of light absorbed or scatterd by the solution the greater the optical density recorded

9. 4A Techniques:
Using dyed water samples, take measurements using a spectrophotometer.
Keep cuvettes free from fingerprints.
Align cuvette the same each time you take a measurement.

10. 4B: Enzymes Goals: Describe the principles of enzymatic reactions
Use the principes of spectrophotometry to determine the concentration of the product of an enzyme- catalyzed reaction.
Determine the effect of ß-galactosidase concentration on the rate of cleavage of ONPG.

11. Introduction:
Enzymes increase the rate of reactions, but do not allow reactions to occur that could not occur otherwise.

13. There are two ways to increase the rate of a chemical reaction
Increase the average kinetic energy by raising the temperature, or
Lower the activation energy by adding a catalyst (enzyme)

14. Catalyzed Reaction Reach a Maximum Rate
Life8e-Fig RL.jpg
Because there is usually less enzyme than substrate present, the reaction rate levels off when the enzyme becomes saturated.
An enzyme speeds up the rxn. At the maximum rxn. Rate, all enzyme molecules are occupied with substrate molecules.
With no enzyme present, the rxn. Rate increases steadily as substrate concentrations increase.

15. Enzyme Regulation

16. O-nitrophenyl--D-galactopyranoside (ONPG)
-galactosidase
O-nitrophenyl--D-galactopyranoside (ONPG)
The enzyme B-gal catalyzed the hydrolysis of lactose to glucose and galactose.
In addition to lactose B-gal can also catalyze analogs of lactose (today in lab the analog we will use is ONPG)
So if the initial rate of the rxn is measured, the amont of product (o-nitorphenol) produced is propotional to the amount of B-gal and the time of the rxn.
When we plot OD420 versus time we can show the rate of the appearance of o-nitrophenol and therefore the rate of the rxn.
Zero order rxn because the reaction is independent of substrate concentration.
So today you will take samples of this reaction at different time points and stop the reaction by inactivating the enzyme (changing the pH with Na2CO3)

17. Hypothesis Generation
Identify one characteristic you expect to change as you add ONPG, buffer, and enzyme
A yellow solution is produced as o- nitrophenolate is produced

18. Rephrase your speculation to the if, then format
If ONPG is catalyzed by -galactosidase, and I add the enzyme in various amounts, the products of o-nitrophenolate (yellow color) will differ as well in readings on the spectrophotometer.

19. Amount of -galactosidase
Independent Variable
Amount of -galactosidase

20. OD420 reading of o-nitrophenolate produced
Dependent Variable
OD420 reading of o-nitrophenolate produced

21. Controls
ONPG + Buffer, but no enzyme added and Buffer + enzyme, but no ONPG added

22. The Experiment:
Take measurements of various amounts of ONPG catalyzed by -galactosidase

23. Assignment Reminders
Post Lab (LON-CAPA) 4 is due by your next lab period.
Pre Lab (LON-CAPA) 5 is due by your next lab period.
LNA (lab notebook assignment) Exercise 4 is due within the first 5 minutes of your next lab period.
LNA Exercise 5: pre lab write up (purpose/protocol) are due at the start of your next lab period.