Peroxidase Enzyme Lab

Transcription

1 Peroxidase Enzyme Lab An enzyme is a type of protein known as a biological catalyst. A catalyst speeds up chemical reactions by lowering the activation energy required. Enzymes, biological catalysts carry out the thousands of chemical reactions that occur in living cells. They are generally large proteins made up of several hundred amino acids. In an enzyme-catalyzed reaction, the substance to be acted upon, or substrate, binds to the active site, or business end, of the enzyme. The enzyme and substrate are held together in what is known as an enzyme-substrate complex. The enzyme then converts the substrate into a product. As is true of any catalyst, the enzyme is not used up as it carries out the reaction but is recycled over and over. One enzyme molecule can carry out thousands of reaction cycles every minute. Each enzyme is specific for a specific substrate and a specific reaction because its unique amino acid sequence causes it to have a unique three-dimensional structure. The active site also has a specific shape so that only one or a few of the thousands of compounds present in the cell can interact with it. Any substance that blocks or changes the shape of the active site will interfere with the activity and efficiency of the enzyme. If these changes are large enough, the enzyme can no longer act, and is said to be denatured. There are several factors that are especially important in determining the enzyme's shape, and these are closely regulated both in the living organism and in laboratory experiments to give the optimum or most efficient enzyme activity. Some of these factors include.. 1. Salt concentration. If the salt concentration is very low or zero, the charged amino acid side chains of the enzyme molecules will stick together. The enzyme will denature and form inactive enzyme. If, on the other hand, the salt concentration is very high, normal interaction of charged groups will be blocked, new interactions will occur, and again the enzyme be unable to function optimally. 2. ph. ph is a logarithmic scale that measures the acidity or H+ concentration in a solution. The scale runs from 0 to 14 with 0 being highest in acidity and 14 lowest. When the ph is in the range of 0-7, a solution is said to be acidic; if the ph is in the range of 7-14, the solution is basic. Likewise, as the ph is raised,

2 the enzyme will lose H+ ions and eventually lose its active shape. Many enzymes have an optimum in the neutral ph range and are denatured at either extremely high or low ph. 3. Temperature. All chemical reactions speed up as the temperature increases; more of the reacting molecules have enough kinetic energy to undergo the reaction. Since enzymes are catalysts for chemical reactions, enzyme reactions also tend to go faster with increasing temperature. However, if the temperature of an enzyme catalyzed reaction is raised still further, a temperature optimum is reached: Above this point the kinetic energy of the enzyme and water molecules is so great that the structure of the enzyme molecules starts to be disrupted. The positive effect of speeding up the reaction is now more than offset by the negative effect of denaturing more and more enzyme molecules. Many proteins are denatured by temperatures around o C, but some are still active at o C, and a few even withstand being boiled. 4. Inhibitors and Activators. Many small molecules other than the substrate may interact with an enzyme. If such a molecule increases the rate of the reaction it is an activator, and if it decreases the reaction rate it is an inhibitor. The cell can use these modulators to regulate how fast the enzyme acts. Any substance that tends to unfold the enzyme, such as an organic solvent or detergent, will also act as an inhibitor. II. Laboratory Exercise: Measuring the activity of turnip peroxidase In this experiment you will study the enzyme peroxidase. Peroxidases are enzymes that are widely distributed in plant and animal cells and catalyze the breakdown of hydrogen peroxide (H 2 O2). Any cell using molecular oxygen in its metabolism will produce small amounts of H2O2 as a highly toxic byproduct. A Peroxide has a very reactive structure, so it is critical that it be quickly removed by enzymes such as peroxidase before it can do damage to the cell. In order to follow the reaction as it proceeds, you will be using a substrate and a substance called guaiacol. Guaiacol is an organic chemical produced by the guaiac tree of Central America. In the this experiment, guaiacol will change from clear to a brown color in response to peroxidase activity. The intensity of the color change is measured by a spectrophotometer at a wavelength of 500 nm.

3 III. INTRODUCTION TO THE SPECTROPHOTOMETER A spectrophotometer is an instrument that measures the amount of light of a selected wavelength that passes through colored or turbid (cloudy) solutions. Figure 1: The spectrophotometer The spectrophotometer is usually used to achieve one of the following goals: a) To determine the concentration of a solution. This is possible because concentration is related to the amount of light absorbed or scattered by the solution. b) To determine the identity of an unknown substance(s) in the solution by observing which wavelengths of light are absorbed by the solution. INSTRUCTIONS FOR OPERATING A. For Initial Readings: 1. Turn the instrument on by rotating the Power Switch/Zero Control (lower left knob) clockwise. Allow 15 minutes for warm-up.

4 2. Set zero: Close the cover of the Sample Well and adjust the meter needle to on the Absorbance scale (Optical Density) by turning the Power Switch/Zero Control knob. 3. Set to desired wavelength the Wavelength Control knob. For the turnip peroxidase/guaiacol experiment, set the wavelength to 500nm. 4. Insert a cuvet containing your "Blank" solution in to the Sample Holder and close the lid. 5. Set full scale, i.e., wait until the needle stops moving and then adjust the meter reading to 0 Absorbance, i.e., 100% Transmittance (O.D.), with the 100% T Control knob. 6. Insert unknown: Remove the Blank, insert the tube containing your unknown, and close the Sample Holder lid. 7. Read Absorbance (check your manual) of your unknowns. IV. Experimental Procedure: Turnip Extract will be provided by your instructor. The turnip extract contains the enzyme peroxidase. The activity of the turnip extract will vary from day to day, depending on the size and age of the turnip and the extent of blending. 1. Read the entire procedure before beginning. Pay special attention to the requirements for mixing the contents of the substrate and enzyme tubes and timing the reaction. Use a dedicated serological pipet for each solution. Precise volume measurements and accurate timing are crucial for rate studies. 2. Turn on the spectrophotometer, adjust the wavelength setting to 500 nm, and allow the instrument to warm up for minutes. 3. Prepare a "blank" by combining 4 ml ph 5 buffer, 2 ml 0.02% hydrogen peroxide, 1 ml 0.2% guaiacol, and 2 ml phosphate buffer in a 13 x 100 mm test tube.

5 4. Zero the spectrophotometer (zero absorbance, 100% transmittance) at 500 nm using the blank solution. 5. Prepare three series of separate 13 x 100 mm test tubes containing substrates (tube S) and the enzyme (tube E) with buffers as shown in the table below. Note that the enzyme concentration is varied in Trials 1-3 but that the concentration of substrate and the total volumes are held constant. The ph 5 buffer provides ph control, while the presence of the phosphate extraction buffer in tube E makes it possible to vary the enzyme concentration while maintaining the overall buffer composition constant. 5a. Make a hypothesis: 6. When ready to begin Trial 1 for the kinetics run, carefully pour the contents of tube Si into tube El and immediately start timing. Pour the combined contents back into tube Si, wipe the outside of the tube with lab tissue, and place the test tube in the spectrophotometer tube holder. 7. Measure and record the absorbance as a function of time every 20 seconds over a total period of seconds. Since initial rate data corresponding to the first 10% of reaction completion is more accurate than longer term rate data, it is crucial to obtain accurate measurements as early as possible. The elapsed time between mixing the tubes and recording the first absorbance measurement should be no greater than seconds! 8. Repeat steps 5 and 6 for the remaining two trials with different concentrations of the enzyme.

6 9. Graph absorbance versus time for each trial to determine the rate of the reaction (three line graph). Consider the enzyme concentration in Trial 1 to be a "normalized" concentration and compare the enzyme concentration and rate of reaction for each trial to that of Trial 1. Analysis Questions: 1. Analyze your graph. Based on your data, in which trial and which enzyme concentration was peroxidase most active in breaking down H 2 O2? 2. Why would an increased concentration of enzyme lead to more reactions (breakdown of H 2 O2) (What is physically happening within the solution?) 3. If you were run three additional trials keeping enzyme concentrations equal in each but substrate concentrations in each trail, what results would you expect? 4. If you were to test a variable other than enzyme concentration or substrate concentration, what variable would you test and how would you design such an experiment?