Bread & WineEnzyme Catalyzed Reactions

This page gives some theory about enzymes and the procedures needed to extract, dilute and use plant peroxidase. First, ensure that you have the materials and reagents needed for these assays. Specific procedures follow for studying the effect of [enzyme], [Substrate], pH, inhibitors or temperature on the rate of a peroxidase catalyzed reaction. You can also jump to other lab procedures in an appendix, my home page or the addresses at the bottom. 

Enzymes, as you know, are proteins which act as catalysts in biochemical reactions. A catalyst cannot initiate a reaction that would not happen in its absence, but it can, and does, radically affect the reaction rate 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).

Hydrogen peroxide (H2O2) is a common end product of oxidative metabolism and, being a strong oxidizing agent, could prove 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 whose function is to reduce the peroxide to water, rendering it harmless. A variety of electron donors are used, including aromatic amines, phenols, and enediols like ascorbic acid.

A dye like o-dianisidine can be used as the electron donor to detect peroxidase because its oxidized product is colored. 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)

You can go back to the top, to the enzyme dilution, reaction mixture preparation or assay procedure for the [Enzyme] section, or the closing from here. 

Materials and Reagents:

Equipment Needed:

Reagents Needed:
*CAUTION: This dye is a proven carcinogen and possibly toxic. Avoid inhalation or contact with powder or solution.
The substrate (reactant) solution used is made by diluting 0.10 mL of 30% H2O2 to 200 mL with water. Calculate the molarity of this solution! You will need this to calculate substrate concentrations when plotting your data. The molecular weight of H2O2 is 34 daltons, and therefore, 34 g of H2O2/ L of solution is a one molar concentration (= 1.0 M H2O2). A 30% (w/v) concentration is 30 g of H2O2/ 100 mL of solution.

Procedure: Extraction of Peroxidase

  1. Peel, wash, and cut a turnip (rutabaga, radish, horseradish root) into 1" cubes .
  2. Homogenize about 40 g in 200 mL of dH2O in a blender at high speed for 3-4 x 15 sec.
  3. Clarify the extract by centrifugation (10-15,000 rpm/ 10 min.) and/or suction filtration through Whatman #1 paper.
  4. The extract may be stored under toluene for at least a week at 4°C, if necessary.
Why would water, rather than the usual isotonic buffer, improve peroxidase extraction?

You can go back to the top, to the enzyme dilution, reaction mixture preparation or assay procedure for the [Enzyme] section, or the closing from here. 

The Effect of Enzyme Concentration
When developing an enzyme assay, one needs to adjust the enzyme concentration so the reaction rate will be easily and accurately detected. In addition, you must determine if the enzyme extract contains any unknown activators (such as organic or inorganic cofactors) or inhibitors, because these must be controlled or removed if further experiments are to be valid. Both of these requirements can be met by assaying various enzyme concentrations under standardized conditions and saturating levels of substrate, as given below.

Procedure: Enzyme dilution

Prepare a stock dilution of each enzyme concentration needed as follows:

Table 1. Preparation of Enzyme Dilutions
Tube #
Enzyme Added (mL) 
dH2O Added (mL) 
Total Dilution
Total ml
Left in Tube
1
2.0 mL of Extract
0 mL
None = 1/1
2.0
2
2.0 mL of Extract
2.0
1/2
2.0
3
2.0 of Tube #2
2.0
1/4
2.0
4
2.0 of Tube #3
2.0
1/8
2.0
5
2.0 of Tube #4
2.0
1/16
4.0

Serial Dilution Diagram

Procedure: Reaction Mixture preparation

In clean tubes, prepare 5 reaction mixtures and a reagent blank (to adjust the Spec 20 to 0 absorbance) as indicated in Table 2 (do not add the enzyme!).

Table 2. Preparation of Reaction Mixtures
Tube # 
mL, pH 7 Buffer
+ mL of Dyea
mL of H2O2
+ mL of 
Enzymeb
from Tube #c
Dilution
1
4.0
0.10
1.0
|| 0.10 
1
1/1
2
4.0
0.10
1.0
|| 0.10 
2
1/2
3
4.0
0.10
1.0
|| 0.10 
3
1/4
4
4.0
0.10
1.0
|| 0.10 
4
1/8
5
4.0
0.10
1.0
|| 0.10 
5
1/16
6d
4.0
0.10
1.0 of dH2O 
|| 0.10 
1
1/1
aCaution: o-dianisidine dye is carcinogenic and toxic. Avoid contact with your skin (wash thoroughly after use) and notify your instructor immediately if a spill occurs.
bDo Not add the enzyme until you are ready to start the reaction. This is time = 0!
cThese numbers refer to the enzyme dilutions in Table 1.
dThe blank contains all the reagents used in the assay except that distilled or deionized H2O is substituted for H2O2 to prevent any reaction.


You can go back to the top, to the enzyme dilution or reaction mixture preparation for the [Enzyme] section, or the closing from here. 

Procedure: Reaction Rates with Enzyme Concentrations

Test how the reaction rate varies as you change the concentration of enzyme in each reaction as follows:

  1. Adjust the spectrophotometer to 0 and maximum absorbance (see appendix).
    1. Set the wavelength to 460 nm.
    2. With the front-left knob, set the needle to infinite absorbance with the sample chamber empty.
    3. Add enzyme to the cuvette and pour in reaction mixture #6. Cover with film and mix gently by inversion.
    4. Insert the cuvette with the blank solution (#6) into the chamber, close the lid, and adjust the needle to 0 absorbance with the front-right knob.
    5. Recheck both settings before starting the assays.
    6. Save the blank solution in tube #6 and rinse the cuvette for use with the assays; or use a matched cuvette (How?) and save the blank in its own cuvette.
  2. Start the reaction by adding 0.10 mL (100 µL) of enzyme dilution from tube #5 (= 1/16) into the cuvette and adding reaction mix #5 - this is time = 0!
  3. Cover the cuvette mouth with Parafilm®, invert several times to mix, and insert into the sample chamber to read the absorbance at 30 second intervals.
    4. Diagram of Rmix preparation
  4. Read the absorbance every 30 seconds thereafter until the absorbance exceeds 1.0, or 8 minutes have passed.
  5. Discard the mixture immediately (it will be orange), and thoroughly clean the cuvette.
  6. Repeat with the other reaction mixtures and the different enzyme dilutions (that is #4, then #3, etc.). Any reaction mixture that turns orange before the enzyme is added will have to be remade, after the tube is thoroughly washed.
Analysis of Data:
What shape do you expect, and what do you obtain, for these lines? How are they arranged relative to each other? Explain!
What shape is the line obtained for this plot? What does it mean?

A major conclusion about the mechanism of enzyme catalysis is that the substrates bind to a specific region of the enzyme, the active site, forming an enzyme-substrate complex prior to the reaction. This means that since there must be a finite number of enzyme molecules, and consequently active sites, available at any given [enzyme], substrate molecules, if present in enough concentration, should be able to saturate these binding sites. At this point the initial rate of the reaction (V0) will be at a maximum (Vmax) and will not increase when the [substrate] is increased. These principles are derived in large part from the shape of the graph of an experiment like that below.

Procedure: Rmix Preparation for V0 vs. [S]

  1. Prepare 7 reaction mixtures and a reagent blank as given in Table 3.
  2. Set the Spec 20 to 0 and infinite absorbance using the blank mixture (#7) as above.
  3. Add the H2O2 and mix just before starting each reaction.
  4. Add 0.10 mL of the enzyme dilution that gives change inA/min = 0.6 units to the cuvette and pour in the reaction mixture. This is time = 0!
  5. Mix and read the absorbance at 30 s and 1 minute only.
  6. Rinse the cuvette!
  7. Repeat steps 3.-6. for each reaction mix.
Table 3. Reaction Mixtures for V0 vs. [S]
Tube #
mL, pH 7 Buffer 
+ mL of Dyea
+ mL of dH2O
+ mL of H2O2 
|| + mL of Enzymeb
1
4.0
0.10
0.90
0.10
|| 0.10
2
4.0
0.10
0.80
0.20
|| 0.10 
3
4.0
0.10
0.60
0.40
|| 0.10 
4
4.0
0.10
0.40
0.60
|| 0.10 
5
4.0
0.10
0.20
0.80
|| 0.10 
6
4.0
0.10
---- 
1.0
|| 0.10 
7c
4.0
0.10
1.0
---- 
|| 0.10 
aCaution: o-dianisidine dye is carcinogenic and toxic. Avoid contact with your skin (wash thoroughly after use) and notify your instructor immediately if a spill occurs.
bDo Not add the diluted enzyme until you are ready to start the reaction. This is time = 0!
cThis is the new reagent blank. 
Analysis of Data:
Why is the curve shaped the way it is? What does this demonstrate? 


You can go back to the top, to the [Enzyme], [Substrate], pH, or temperature sections, or the closing from here. 

The Effect of Inhibitors and Activators

Toxins and inhibitors affect everyone's daily life, and have since organisms first began using chemical warfare to gain evolutionary advantage over their competitors. Those that inhibit enzymes can be classed as either irreversible, in which case active enzyme cannot be recovered once it has been inhibited, or reversible, where the inhibitor can be physically removed leaving the enzyme active again. This class can be further subdivided into competitive inhibitors, in which the inhibitor binds to the active site of the enzyme, blocking the substrate's access, or noncompetitive inhibitors, in which the inhibitor binds elsewhere on the enzyme, changing its shape and thus its activity. If the substrate concentration is increased relative to inhibitor, a competitive inhibition will be overcome, while a noncompetitive will not.

p-Aminobenzoic acid, sodium azide (NaN3), cyanide, cyclopropanone, L-cystine, dichromate, ethylenethiourea, hydroxylamine, sulfide, sulfite, vanadate and the divalent anions of Cd, Co, Cu, Fe, Mn, Ni and Pb are reported to inhibit peroxidase (Horseradish; see http://www.sigma.sial.com/sigma/proddata/p8000.htm). In addition any salts or detergents that may be introduced during extraction or purification could potentially inhibit or activate the enzyme. Can you design an experiment to tell which type of inhibitor each is? You will need to determine the effective concentrations required or find them in the literature.

Procedure:

  1. Prepare two sets of reaction mixtures as in Table 3. above, but add 0.10 mL of the chosen inhibitor to each tube (CAUTION - toxic!) in one set.
  2. Do the analysis and collect the data on both sets as in the experiment above.
  3. Plot the change in A/min vs. [Substrate] on the same graph.
How has the line changed due to the inhibitor? Which type is it? 


You can go back to the top, to the [Enzyme], [Substrate], inhibitor or temperature sections, or the closing from here. 

The Effect of pH

All enzymes display a characteristic range of pH at which they are most active. This "pH optimum" may be due to several factors involving the structure and ionic state of the enzyme, substrate, or cofactors (see your text). In many cases it reflects the pH of the organelle in which the enzyme is active. What is the pH optimum for peroxidase, and the probable pH inside the peroxisome?

Procedure:

  1. Prepare a set of reaction mixtures as in Table 2., but substitute buffers of varying pH in all but one tube and the blank. Use pH 2, 4, 5, 6, 7, 8, & 10 if possible.
  2. Start each reaction with the [Enzyme] that gives change in A/min = 0.6 units only.
  3. Plot V0 vs. pH (x-axis).
At what pH is peroxidase most active? Are you sure?

Did you notice anything unexpected? Reevaluate your conclusions.

The Effect of Temperature

At low initial temperatures, increasing it increases the rates of all reactions, whether catalyzed or not. At higher temperatures, however, proteins denature (as in "to cook"). Since most enzymes are proteins, temperature changes will produce something like a temperature optimum, although at this point the protein is already being denatured.

Can you plan a properly controlled test of the effects of temperature on the peroxidase assay? Try! 

That's all for now. Again, you can jump to the beginning, to my home page or the biochemistry or non-major's chemistry pages.
If you have questions or comments, write the:

  Author of this page: Terry Helser - helsertl@oneonta.edu
    Web Coordinator: Philip S. Bidwell - bidwelps@oneonta.edu
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  Last Modified on 7/6/06  Made on a MacMac OS