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Effects of Peroxidase Dilutions and Substrate Concentrations on Enzyme Reactions, Lab Reports of Biochemistry

Florida International University (FIU)Biochemistry

A multi-week lab experiment where students will extract and assay the peroxidase enzyme from plants. They will investigate the impact of enzyme and substrate concentration, ph, inhibitors, and temperature on the rate of a peroxidase-catalyzed reaction. Key concepts include substrate, active site, catalyst, e.c. 1.11.1.7, inhibition, inhibitors, lysosome, peroxisome, vmax, vo, and hydrogen peroxide. Students will use a dye like o-dianisidine to detect peroxidase and measure reaction rates.

Typology: Lab Reports

Pre 2010

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BCH3033L ENZYMES, Part I
Purpose: This multi-week lab will demonstrate how to extract and assay the enzyme peroxidase
from plants. You will use this knowledge to study the effect of enzyme and substrate concentration,
pH, inhibitors and temperature on the rate of a peroxidase catalyzed reaction.
Key Concepts and Terms:
[Enzyme]
[Substrate]
Active site
Catalyst
E.C. 1.11.1.7
Inhibition – reversible/non-reversible
inhibitors
Lysosome
Peroxisome
Vmax
Vo
Background:
Enzymes are usually proteins that act as catalysts in biochemical reactions. Catalysts cannot
initiate reactions that would not happen in their absence, but can, and do, radically affect reaction
rates with the result that the cell can carry out rapid and complex chemical activities at relatively
low temperatures. Most enzymes are highly specific. They tend to accelerate only one or a group of
related reactions. The result is that many different enzymes may be present in a cell and may act
simultaneously without mutual interferences. Here we demonstrate the characteristics of enzyme
catalyzed reactions by examining peroxidase (E.C. 1.11.1.7) from plants. What does the first “1” in
EC 1.11.1.7 mean?
Hydrogen peroxide (H2O2) is a common end product of oxidative metabolism and, being a
strong oxidizing agent, would be toxic if allowed to accumulate. To prevent this, eukaryotic cells
have enclosed the enzymes producing peroxides within a membrane-bound organelle, the
peroxisome, which is similar in size and appearance to a lysosome. Peroxisomes also contain high
concentrations of peroxidase – the enzyme that functions to reduce the peroxide to water, rendering
it harmless. A variety of electron donors can be used, including aromatic amines, phenols, and
enediols like ascorbic acid.
A dye like o-dianisidine can be used as the electron donor (colorless) to easily detect
peroxidase in vitro because its oxidized product is highly colored (Extinction coefficient is 11.3
mM-1cm-1). The rate of appearance of this colored pigment can be measured colorimetrically and is
equivalent to the rate of reaction.
H2O2+ Colorless Dye(reduced) peroxidase > H2O + Colored Dye(oxidized)
When first measuring enzyme activity from a tissue (in this case turnip or horseradish root)
one must first “range find” to determine the amount (extent) of enzyme activity in the tissue. So,
right after grinding up the root to make a crude enzyme extract, we will dilute the extract (enzyme)
and measure the rate of reaction. This will tell us what amount of enzyme to use.
In these assays, the tubes will be made up with buffer and substrates (completely mixed).
This tube is used to blank the spectrophotometer BEFORE adding enzyme dilution. Then the tube
is removed, the enzyme dilution is added and immediately returned to the cuvette chamber of the
spectrophotometer. OD readings are made immediately and then at 30 second intervals to get a rate
pf3
pf4

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Download Effects of Peroxidase Dilutions and Substrate Concentrations on Enzyme Reactions and more Lab Reports Biochemistry in PDF only on Docsity!

BCH3033L ENZYMES, Part I

Purpose: This multi-week lab will demonstrate how to extract and assay the enzyme peroxidase from plants. You will use this knowledge to study the effect of enzyme and substrate concentration, pH, inhibitors and temperature on the rate of a peroxidase catalyzed reaction.

Key Concepts and Terms: [Enzyme] [Substrate] Active site Catalyst E.C. 1.11.1. Inhibition – reversible/non-reversible

inhibitors Lysosome Peroxisome Vmax Vo

Background:

Enzymes are usually proteins that act as catalysts in biochemical reactions. Catalysts cannot initiate reactions that would not happen in their absence, but can, and do, radically affect reaction rates with the result that the cell can carry out rapid and complex chemical activities at relatively low temperatures. Most enzymes are highly specific. They tend to accelerate only one or a group of related reactions. The result is that many different enzymes may be present in a cell and may act simultaneously without mutual interferences. Here we demonstrate the characteristics of enzyme catalyzed reactions by examining peroxidase (E.C. 1.11.1.7) from plants. What does the first “1” in EC 1.11.1.7 mean? Hydrogen peroxide (H 2 O 2 ) is a common end product of oxidative metabolism and, being a strong oxidizing agent, would be toxic if allowed to accumulate. To prevent this, eukaryotic cells have enclosed the enzymes producing peroxides within a membrane-bound organelle, the peroxisome, which is similar in size and appearance to a lysosome. Peroxisomes also contain high concentrations of peroxidase – the enzyme that functions to reduce the peroxide to water, rendering it harmless. A variety of electron donors can be used, including aromatic amines, phenols, and enediols like ascorbic acid. A dye like o-dianisidine can be used as the electron donor (colorless) to easily detect peroxidase in vitro because its oxidized product is highly colored (Extinction coefficient is 11. mM-1cm-1). The rate of appearance of this colored pigment can be measured colorimetrically and is equivalent to the rate of reaction.

H 2 O 2 + Colorless Dye (reduced) peroxidase^ > H 2 O + Colored Dye (oxidized)

When first measuring enzyme activity from a tissue (in this case turnip or horseradish root) one must first “range find” to determine the amount (extent) of enzyme activity in the tissue. So, right after grinding up the root to make a crude enzyme extract, we will dilute the extract (enzyme) and measure the rate of reaction. This will tell us what amount of enzyme to use.

In these assays, the tubes will be made up with buffer and substrates (completely mixed). This tube is used to blank the spectrophotometer BEFORE adding enzyme dilution. Then the tube is removed, the enzyme dilution is added and immediately returned to the cuvette chamber of the spectrophotometer. OD readings are made immediately and then at 30 second intervals to get a rate

of reaction from a plot of the OD (ordinate or y-axis) against time (abscissa or x-axis). At low dilutions (highest amount of enzyme, the rate may go to fast to accurately measure, at high dilutions (lowest amount of enzyme) the rate may be too slow. Thus, the first range finding will allow each group to find a reasonable rate for future experiments: the amount of enzyme that will produce a change in OD of 0.4 to 0.7 in 5 minutes. Then using this amount each time, we can examine the effect of substrate concentration on this rate; that is do the classical Michaelis-Menten experiment.

Materials and Reagents:

Equipment Needed:

  • Spectronic 20 spectrophotometer set at 460 nm.
  • One box of spec 20 tubes per group.
  • P200 and P1000 pipets and tips.
  • Vortex mixers – one per group.
  • Blender.
  • Small Buchner funnel, Whatman #1 filter paper, single hole stopper.
  • 1 500 ml side arm flask.
  • Timers.
  • 13x100 glass test tubes & racks.
  • 1 500 ml Beaker for o-dianisidine waste

Reagents Needed:

  • Fresh turnip or horseradish root. 40 grams needed per group.
  • 1 liter 0.10M phosphate buffer, pH 7.
  • 200 ml each of 0.10M phosphate buffer, pH 2.0, 4.0, 5.0, 6.0, 8.0 and 10.0.
  • 200 ml of 8.8mM H 2 O 2 substrate one.
  • 50 ml 0.5% w/v o-Dianisidine dye (Sigma #D-3127) in methanol, substrate two. WARNING : O-Dianisidine is a proven carcinogen and toxic. Avoid contact with skin. Dispose of material as directed by TA. MW o-dianisidine (=3,3-dimethoxybenzidine) is
  • Acid-alcohol bath.

Procedures:

I. Extraction of Horseradish Peroxidase

  1. Peel, wash and cut ~40g fresh turnip or horseradish root (best to use) into ~1 inch cubes.
  2. Homogenize with 100 ml water in a blender.
  3. Filter with buchner funnel and Whatman filter paper by vacuum into a sidearm.
  4. Keep filtrate cool, on ice during the experiment.

II. Reaction Time Course for Enzyme Dilutions

these two dilutions. You could either do an intermediate dilution and use 50 μl of enzyme in the assay or use the 1:2 dilution at something around 20 μl.

Enzyme Kinetics with Substrate Concentration In this experiment, the enzyme reaction is run with different levels of substrate (H 2 O 2 ) to examine the effect of substrate concentration on enzyme kinetics.

  1. Prepare 10 ml of the appropriate dilution of your enzyme extract.
  2. In a clean set of Spec 20 tubes, prepare the following reaction mixtures. Remember: Do not add the enzyme extract until you are ready to do the reaction.

Tube Vol pH 7 buffer Vol Dye Vol Water Vol H 2 O 2 When ready to Run o-dianisidine Reaction, add Extract

1 2.4 ml 50 μl 0.48 ml 20 μl 50 μl or lower 2 2.4 ml 50 μl 0.45 ml 50 μl 50 μl or lower 3 2.4 ml 50 μl 0.40 ml 100 μl 50 μl or lower 4 2.4 ml 50 μl 0.30 ml 200 μl 50 μl or lower 5 2.4 ml 50 μl 0.20 ml 300 μl 50 μl or lower 6 2.4 ml 50 μl 0.10 ml 400 μl 50 μl or lower 7 2.4 ml 50 μl ----- 500 μl 50 μl or lower

  1. Blank the Spec 20 460 nm for each tube as before.
  2. Set up a watch or timer with 30 second intervals.
  3. Starting with tube #1, add 50 μl of the extract dilution indicated, vortex and quickly place the tube in the Spec 20. Read the absorbance every 30 seconds for 5 minutes. Remove the tube from the Spec 20 and set aside in a rack for disposal.
  4. Repeat with each of the tubes at a different substrate concentration. Warning: These tubes contain o-dianisidine.
  5. With the data collected, you will determine the relationship between the velocity (rate) of the reaction (v) and the substrate concentration (s) at the different substrate concentrations. To do this you have to convert your ∆OD/min to concentration/minute. For this, the extinction coefficient of the diansidine oxidized product is 11.3 mM-1cm-1. Remember the Beer-Lambert law: OD = Ε.^ c.^ l where Ε is the extinction coefficient, c is the concentration and “l” is the light path (here 1 cm). So this can be set up neatly to be: c = (∆OD)/Ε. Plot the data as both Michaelis-Menten and Lineweaver-Burke graphs. Calculate the Km and Vmax. Refer to Chapter 8 in Lehninger if you have questions about how to do this, then see the instructor.