Bio Lab 6 - Measurement of Enzyme Activity

peroxidase

enzyme used a model to generalize all enzymes

one member of a group of enzymes found in all life

eliminates toxic H2O2, usually produced in metabolic rxns in cells

H2O2

byproduct of metabolic rxns in cells that is eliminated by enzymes immediately

suffix -ase

signifies an enzyme

heme

porphyrin molecule with an iron ion sitting at its center (like hemoglobin)

coenzyme tucked into peroxidase active site

found in hemoglobin

Peroxidase substrates

Has two:

1) filled with H2O2

2) second varies

guaiacol

phenolic compound used as second substrate in peroxidase

phenolic compound

6 carbon ring with at least one hydroxyl group

Reaction catalyzed by peroxidase

guaiacol gets oxidized to quinone

H2O2 gets reduced to water

methanol is by product

what do quinones do

spontaneously react with each other or other molecules to form colored complexes

How do we measure amount of colored product?

colorimeter set to 500nm wavelength

rate of reaction

Amount of colored product measured by Spectro Vis plus over time (product/time)

concentration of colored product and transmittance relationship

As concentration of colored product increases the percent transmittance decreases

Inversely related

More product stops light from passing through

Preparation of lab

• put ice to 1/3 beaker full

• add tap water until 2/3 beaker full

• Leave test tube containing enzyme in bath (enzymes denature after being removed from cells but low temps delays this)

• Cut up onion into ¼ inch pieces and add 10g and 200mL DI water to liquify in food processor

• Strain extract through cheesecloth and keep in ice bath

Assay

Do the test one time:

combine volumes of different solutions than measure change in %T with colorimeter

Sources of error

• water left over after washing test tube before moving onto next assay

• inaccurate pipette measurements

• Not mixing solutions well enough

What is assay #1 used for

It is the blank used to calibrate the colorimeter

Accounts for water, substrate, and cuvette but there is no enzyme

Calibration of colorimeter

• plug in and allow colorimeter to heat up

• have blank (assay #1) ready

• Select %transmittance vs. time

• place cuvette inside

• click finish calibration

• Set wavelength at top to 500nm and click done

Usage of colorimeter for other assays

• pipette specified volumes of guaiacol, water, and H2O2 into test tube and swirl

• Swirl enzyme extract and pipette into separate test tube

• Pour first test tube into the one with enzyme extract and mix well

• transfer to cuvette and place in colorimeter

• Click collect and %T will start around 90%-80%

• %T will start decreasing so stop at 48%

• Measure time between 70%-50%

For assay #12 how long will the reading take to drop from 70% to 50% transmittance?

Won’t drop because no H2O2 (substrate) is present so the guaiacol has nothing to react with meaning no quinones will be present to produce colored products.

What do we know about the reaction rate if the reading is not changing and there are no products being made?

The reaction rate is not being catalyzed and is very slow

assay concentration differences

• All assays are 5.0mL

• Guaiacol is always 2.0mL (constant, so no difference in effect on rxn)

• Enzyme is always 0.2mL (constant)

• H2O2 varies (independent variable)

• water volume adjusts to H2O2 volume to keep constant 5.0mL solution

Why change only volume of H2O2 in assays?

To see how the effect of the H2O2 substrate has on reaction rate

Effect of higher H2O2 conc

More substrate increases product formation per unit of time (faster drop from 70 to 50 percent transmittance)

rate of enzyme activity

amount of product formed per unit of time or amount of substrate consumed per unit of time

(As product is formed substrate is consumed)

What is the time needed to drop from 70% to 50% dependent of

substrate (H2O2) concentration. (higher concentration, faster it drops)

What is the amount of colored product that corresponds to drop from 70% to 50%

200 micromoles

How to calculate rate of rxn

200 micromoles / time to drop 70% to 50%

What is the independent and dependent variables on the graph

Independent (X-axis) - Substrate (H2O2) concentration

Dependent (Y-axis) - rate of reaction

Michaelis-Mentin curve correlation to this lab

Origin will be assay #12 (0,0) then going backwards (11, 10, 9) curve will rise and level off at highest substrate conc

Saturation

Highest conc of substrate where reaction rate levels off (no more substrate can be added, enzyme is working at max rate)

Change in assay #13-15

Substrate concentration is held constant but enzyme concentration varies

What will the trend in rate be when enzyme conc differs (#13-15)?

More enzymes increases rate (More enzyme means more active sites to catalyze rxn)

assays #16-18 and possible error

Same concentrations as assay 5 which should yield same times. Variation among them is due to errors such as, pipetting, differences in temp, improper mixing, human reaction time, instrumental error from colorimeter.

True value (16-18)

The average of the repeated trials