1. Measuring Enzyme Activity Using Spectrophotometry (Beer’s Law)

2. What is spectrophotometry?
Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.
Spectrophotometry deals with visible, near-ultraviolet, and near-infrared light.
A sample of monochromatic light is passed through a solution. A fraction of that light is absorbed by the substance(s) in the solution.
The amount of light that is absorbed is proportional to the concentration of the substance. This relationship is referred to as Beer’s Law.

3. Why is Spectrophotometry Important. Spectrophotometry is used to
Why is Spectrophotometry Important? Spectrophotometry is used to - identify solutes in mixtures - estimate concentrations of known compounds, especially in dilute solutions

4. The Spectrophotometer
Measures absorbance as a function of wavelength
The Spectronic 20 is the classic single-beam spectrophotometer, used by students since Here’s how a spectrophotometer works:

5. Components of a Spectrophotometer
monochromator
sample cell
detector
slit
diffraction grating
light source

6. Conceptual Basis of Beer’s Law
- Light of a particular wavelength enters the ‘sample’.
Light scatters from particles in solution, reducing light transmission
- Light is absorbed by molecules/particles (momentum
change) and remitted at different wavelengths, reducing light transmission

7. Spectrophotometers vs. Colorimeters
A spectrophotometer can measure absorbance at any wavelength between 300 and 900 nm (including ultraviolet and near infrared)
A colorimeter can only measure a handful of wavelengths.
The vernier colorimeter can has four pre-set wavelengths:
430 nm, 470 nm, 565 nm, 635 nm
For this lab, the 470 nm setting is used.
Spectrophotometer
Colorimeter

8. This relationship is given by the equation
The ratio between the intensity of the exiting light to the entering light is called transmittance
The amount of exiting light is highly dependent on the pathlength (shown as l on the figure)
This relationship is given by the equation
alpha is the molar absorptivity
l is the path length
c is the solute concentration

9. Beer’s Law is usually stated in a way to make certain quantities easy to compare and interpret.
A is referred to as the absorbance, which gives a linear plot against path length.

10. The College Board uses different symbols for molar absorbtivity (a) and path length (b)
A = abc
Molar absorbtivity (a) is unique for each wavelength for any substance. Concentrations of substances with high molar absorbtivities can be measured to a high degree of sensitivity.
The path length (b) is nearly always 1 cm.
At high solute concentrations, beer’s law breaks down due to increased scattering of light:
Rule of thumb: A<1

11. You might ask, why do we have to put that blank in every time we change wavelength? It’s a pain.
It has to do with the output of the light source. Here are some examples:
The light source is not uniform for all wavelengths. You have to set Io absorbance to zero every time because of this.
Professionals get around this by using a double beam spectrophotometer, where the light is split into two components and passes through the blank and solution simultaneously.

12. Enzyme Lab Objectives
To understand the relationship between enzyme structure and function.
To make some generalizations about enzymes by studying just one enzyme in particular.
To determine which factors can change the rate of an enzyme reaction.
To determine which factors that affect enzyme activity could be biologically important.

13. Peroxidase (Catalase)
Peroxidase is one of several enzymes that break down peroxide, a toxic metabolic waste product of aerobic respiration.
Catalase is a common enzyme found in nearly all living organisms exposed to oxygen.
It catalyzes the decomposition of hydrogen peroxide to water and oxygen. It is a very important enzyme in protecting the cell from oxidative damage by reactive oxygen species (ROS).

14. Catalase has one of the highest turnover numbers of all enzymes….
One catalase molecule can convert millions of molecules of hydrogen peroxide to water and oxygen each second!

15. The general reaction: The specific reaction:
Enzyme + Substrate --> Enzyme-Substrate Complex --> Enzyme + Product(s) + ∆G
The specific reaction:
Peroxidase + Hydrogen Peroxide --> Complex --> Peroxidase + Water + Oxygen
2H2O2 (aq)→ 2H2O (l) + O2 (g)

16. Enzymes are not consumed by reactions.
To determine the rate of an enzymatic reaction, you must measure a change in the amount of at least one specific substrate or product over time.
In a decomposition reaction of peroxide by peroxidase, the easiest molecule to measure would probably be oxygen, a final product.
This could be done by measuring the actual volume of oxygen gas released or by using an indicator. In this experiment, an indicator for oxygen will be used.

17. Guaiacol A yellowish aromatic oil
Usually derived from guaiacum or wood creosote.
Samples darken upon exposure to air and light.
Guaiacol is present in wood smoke.
The compound contributes to the flavor of many compounds, e.g. roasted coffee.

18. The molecular oxygen produced by peroxidase converts guaiacol to tetraguaiacol:

19. Safety Considerations
Wear proper footwear, safety goggles or glasses.
Use proper pipetting techniques, and use pipette pumps, syringes, or rubber bulbs. Never pipette by mouth!
Dispose of any broken glass in the proper container.
0.1% hydrogen peroxide and 0.3% guaiacol can be rinsed down a standard laboratory drain.
The concentrations used here are deemed to be safe by all chemical standards, but recall that any compound has the potentiality of being detrimental to living things and the environment.
When you develop your individual investigations you must always consider the toxicity of materials used.