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