AnnouncementsAnnouncements
Post Lab 3 and Pre Lab 4 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.
Exercise 2B:Exercise 2B:
1. Experimental Plates: Examine your plates and observe the type of bacteria or fungal growth that appears on each.
2. Streak Plates: Examine the colonies; TA will be around to assign your “Skills” score
3. Complete your LNA: Exercise 2 and turn it in as directed by your TA.
Exercise 3A: Exercise 3A: SpectrophotometrySpectrophotometry
Goals: Understand the process by which spectrophotometry
can be used to quantify experimental results. Develop skills taking measurements using a
spectrophotometer.
SpectrophotometrySpectrophotometry
Measurement of light absorption or transmission through a solution
Types of photometers: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.
Light through a solution:Light through a solution:
Example ONP (o-nitrophenol) Absorbs blue light and allows yellow light to pass
through. Solution therefore appears yellow.
Absorption SpectrumAbsorption Spectrum
A plot of the relative amount of light absorbed by a compound as a function of the wavelength
Absorption spectrum of ONP
Components of a Typical SpectrophotometerComponents of a Typical Spectrophotometer
3A Techniques:3A Techniques:
Using colored water, take measurements using a spectrophotometer.
Keep cuvettes free from fingerprints.
Align cuvette correctly each time you take a measurement.
3B: Enzymes3B: 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.
Introduction:Introduction:
Enzymes increase the rate of reactions, but do not allow reactions to occur that could not occur otherwise.
There are two ways to increase the rate of There are two ways to increase the rate of a chemical reactiona chemical reaction
1. Increase the average kinetic energy by raising the temperature, or
2. Lower the activation energy by adding a catalyst
Catalyzed Reaction Reach a Maximum Rate
Enzyme RegulationEnzyme Regulation
-galactosidase-galactosidase
O-nitrophenyl--D-galactopyranoside (ONPG)
Hypothesis GenerationHypothesis 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
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
Independent VariableIndependent Variable
Amount of -galactosidase
Dependent VariableDependent Variable
OD420 reading of o-nitrophenolate produced
ControlsControls
ONPG + Buffer, but no enzyme added
and
Buffer + enzyme, but no ONPG added
The Experiment:The Experiment:
Take measurements of various amounts of ONPG catalyzed by -galactosidase