Measurement of Enzyme Activity

what enzyme is used

peroxidase

what is the coenzyme

heme

what are the substrates of peroxidase

H2O2 (ALWAYS)

Other varies (guaiacol)

For the reaction to occur _______ substrates must be present

both

what happens in peroxidase-catalyzed reaction to substrates

substrate fits up against the coenzyme Heme within the enzyme’s active site

what happens in peroxidase-catalyzed reaction to phenolic compound

guiacol is oxidzed to quinone

H2O2 reduced to water

Quinones react to one another and form colored complexes

How is color product formed measured/quantified

spectroVis Plus colorimeter

What is the relationship between the concentration of colored product and percent transmittance

As concentration increases, the percent transmittance decreases

What is rate of reaction

the amount of color formed over time

How do we get the peroxidase enzyme

live turnip tissue

what are the 4 things we should look for in enzyme behavior

1. Will reaction continue without enzyme

2. what happens if we only have one substrate

3. Effect of substrate concentration on the reaction rate

4. Effect of enzyme concentration on the reaction rate

why is it important to keep the enzyme cold?

to ensure enzyme activity since they are removed from cells

why do we prepare 1 assay at a time with the enzyme?

enzyme is added and reaction begins immediately so you need to measure immediately

what are two possible sources of error in this lab?

1. water left in the class tube/not completely dried out

2. dont mix solutions well enough

When plugging in vernier spectral analysis, what is the set up and wavelength

%Transmittance vs Time

500 nm

what is included in teach assay tube

Guaiacol

di wate

H2O2

what do the %T values start at

80-90

What does %T not start at 100

Turnip has grit so might be some fragments

Enzyme is added and reaction immediatey begins so some color may have already fored

When do you click stop on vernier?

when it hits 48%

from what percentages do u calculate time

70-50 percent

What does it mean when percent transmittance begins to drop?

Less light is passing through so the color is darkening

Means enzyme is working

What are the 3 Enzyme properties?

1. effect of substrate concentration on reaction rate

2. effect of enzyme concentration on reaction rate

3. reproducibility of results

independendent and dependent variable

IND: H2O2 concentration

DEP: reaction rate

calculation of reaction rate

200/time

Over time, increasing substrate concentration no longer raises the rate. Why?

Enzyme is reacting at maximum rate, so eventually saturates and does not change

what did we change for assay 13-15? What did we keep constant

We changed enzyme concentrate (peroxidase)

We kept both substrates the same (H2O2, gluacicoal)

What happens when you increase enzyme concentration, while keeping everything else constant?

More enzyme→faster color change→shorter time→higher reaction rate

What did we do for assay 16-18?

kept it the same way as assay 5

if assay 16-18 were the same as 5, why may the results/reaction time be different?

experimental error

what is the true value of the repeated trials (16-18 and 5)

average of all repeated trials

What is C1 V1 and C2 V2

C1 - 0.1

V1 - Amount added (changes on chart)

C2- ?

V2 - 5mL