Announcements  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: 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: Spectrophotometry  Goals:  Understand the process by which spectrophotometry can be used to quantify experimental results.  Develop skills taking measurements using a spectrophotometer.

Spectrophotometry Measurement of light absorption or transmission through a solution

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:  Example ONP (o-nitrophenol)  Absorbs blue light and allows yellow light to pass through.  Solution therefore appears yellow.

Absorption 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:  Using colored water, take measurements using a spectrophotometer.  Keep cuvettes free from fingerprints.  Align cuvette correctly each time you take a measurement.

3B: 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:  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 a 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 Regulation

 -galactosidase O-nitrophenyl-  -D-galactopyranoside (ONPG)

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

 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 Variable Amount of  -galactosidase

Dependent Variable OD 420 reading of o-nitrophenolate produced

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

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