Enzymes & Spectrophotometers Introduction: Enzymes as Biological Catalysts: Enzyme-Substrate Complex reversibly

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1 Enzymes & Spectrophotometers Introduction: In today s lab you will do two different procedures. 1) You will investigate an enzymatically-controlled.chemical reaction at various phs. ) You will learn how to use a spectrophotometer and use it to make three different absorption spectra. In your next lab you will use the spectrophotometer to investigate a different chemical reaction. Enzymes as Biological Catalysts: As I have already stated in class, your physical being as well as all cells and all other organisms made from cells can be described as compartmentalized biochemical soup. For that biochemical soup to function not only must many chemical reactions occur, they must occur at the correct time and in the correct place. For example the biochemical reaction that produces hair does not happen in all cells. Virtually every chemical reaction that takes place in biological organisms is catalyzed by an enzyme. This is very fortunate because total chaos would result if all reactions could occur spontaneously. Therefore enzymes not only allow reactions to occur, but are used to determine when and where reactions take place. Enzymes are necessary because they allow chemical reactions to start and continue at rates that maintain life. All chemical reactions have to surmount an energy barrier to begin. This is\ called the activation energy barrier. One way to surmount that energy barrier is to add in energy. This is what you do when we light a fire with a match we add energy to get over the energy barrier. Cells are killed when too much energy is added to start a reaction. The other way to begin a chemical reaction is to lower the activation energy barrier. This is what enzymes do. This is described nicely in your text and will be discussed in detail in class. The specificity of which reaction will take place is due to the close relationship between the shape of the enzyme and its active site and the fit of the substrate. Most enzymes are extremely specific though some can catalyze several similar types of reactions. The active site of an enzyme is the region that can interact with the substrate. The Enzyme-Substrate Complex is formed when the substrate binds to the active site. The binding of the substrate can induce a change in the shape of the enzyme, and the substrate itself may also change in a way that distorts its chemical bonds and helps the reaction proceed. When enzyme and substrate molecules combine they react reversibly at the active site to form an enzyme-substrate complex. Once the substrate is converted to product, the active site returns to its normal configuration and the enzyme can be used again. Since once the product is freed from the enzyme, it can catalyze another reaction a small amount of enzyme can cause a dramatic increase in the reaction rate. The majority of enzymes are made up of proteins (they may have other compounds attached.). Their activity depends on their shape, which of course depends on their tertiary (or quaternary) structure. Enzymes ability to function then depends on having the correct environmental conditions so that the proteins are not denatured. Enzymes generally work best under certain narrowly defined conditions of temperature, ph, and salinity. Any change in these optimal conditions will adversely affect an enzymes activity because it will change the enzymes shape. When the protein that makes up the enzymes are denatured enzymes lose their catalytic activity because the substrates can no longer bind to the active site.

2 Enzymes & Spectrophotometer Procedure for Enzyme Lab: Catalase & H O The effects of ph can be investigated with catalase, an enzyme in plants and animals that speeds up the breakdown of hydrogen peroxide (H O ), which is toxic to the cell. Hydrogen peroxide is broken down by catalase to release water and oxygen gas. Catalase H O H O + O 1. Grind up a piece of meat about an inch in diameter in the mortar & pestle with about 5 ml of d-h O.. Strain the mixture through cheesecloth. The enzyme catalase is in the solution from this mixture. 3. Label three test tubes as follows: A B C 4. Add 3 ml of catalase (the solution from the meat mixture) to each tube. 5. Add ml ph-7 buffer to the ph-7 tube B. 6. Add 1 ml of d-h O to tubes A and C. 7. Add 1 ml (or 0 drops) of 1.0 M HCl to tube A. 8. Add 1 ml (or 0 drops) of 1.0 M NaOH to tube C. 9. Test the B tube first, then the other two, by adding 1mL of 3% H O and watching how many oxygen bubbles are produced. 10. Record the amount of bubbling on a scale of 1-5, with 5 being the amount generated in the tube B. 11. Determine the actual ph of the three tubes by dropping several drops from the tube onto ph paper. Record the ph underneath each Tube 1. Plot a graph of O Produced (using the scale of 1 to 5) vs. ph for the three tubes on graph on page 7. Do Not need to label the graph with A, B and C. What we are measuring is the amount of oxygen production in response to ph. Results of ph Tests Tube and ph Oxygen Production (1-5) A ph: B ph: C ph:

3 Enzymes & Spectrophotometer Spectrophotometers:. A spectrophotometer is an instrument that measures light intensity. A spectrophotometer works by passing a light through a sample and measuring how much light reaches a detector. The difference between how much light was originally transmitted and how much arrives at the detector is the amount of light that the sample absorbed. When using a spectrophotometer, measurements can be made in transmittance (how much light passed through) or absorbance. Spectrophotometers are among the more useful instruments in many fields of science including biology, chemistry and physics. I will briefly discuss only two applications of spectrometry. All molecules absorb different quantities of different colors of light. Any color that is absorbed disappears and what we see are the colors that are reflected or transmitted. Therefore if a pigment is red that means it has absorbed all other colors than red. By measuring how much light is transmitted or absorbed at each color of light, i.e., wavelength you can determine which molecule is in a sample. An absorbance spectrum is a graph of a chemical or solution s light absorption at different colors of light. Another commonly used application of a spectrophotometer is to measure the quantity of a substance in a solution. There is a direct relationship between absorbance and the quantity of a molecule in solution as long as you measure absorbance using the color of light that the molecule absorbs the most. For this part of the lab you will measure the absorbance spectrum of three different solutions and complete the table and graphs for Absorbance vs. Wavelength. Some Important Notes on Using a Spectrophotometer:.Cuvette: These resemble a small test tube. Each cuvette is marked at the top and must be positioned toward the front of the spectrophotometer when taking measurements. Handle these tubes with extreme care to keep both the inside and outside surfaces clean and free of scratches. 1. NEVER use a brush to clean the inside of the cuvette.. Rinse the tube with distilled water several times. 3. Wipe the outside of the cuvette with a soft tissue (Chemwipe) to remove any moisture or fingerprints from the outside surface before placing the cuvetter into the spectrohotomer. Procedure: 1) Fill three different cuvettes ¾ full with the appropriate solutions, One cuvette will have the red dye, one will have the blue dye, and one will be filled with the chlorophyll solution. You do NOT need to label the cuvettes. ) Make two different blanks. A blank is a cuvette which is filled with the solvent that was used to dissolve/dilute the solute of interest. The red and blue dyes are both in water, while the chlorophyll was extracted from a chloroplast using acetone. Therefore one blank will be a cuvette filled ¾ with distilled water and one blank will be filled with acetone. The purpose of the blank is to subtract out the amount of absorbance of the solvent so that you are only measuring the absorbance of the compound of interest and not the solution. 3

4 Taking Readings using the Spectrophotometer.: 1. Turn on the Power Switch and allow 15-0 minutes for the instrument to warm up.. Set the desired wavelength with the Wavelength control knob. You will start your readings at 400nm. 3. Set the filter lever to the appropriate position for the selected wavelength. 4. Adjust the display to 0% Transmittance with the Power Knob (knob on your left). 5. Place the water blank cuvette in the sample compartment and align the guide mark on the cuvette with the guide mark at the front of the sample compartment. Press the tube firmly into the sample compartment and close the cover. 6. Adjust the display to 0 absorbance (100% Transmittance) with the Transmittance/Absorbance control. 7. Remove the blank cuvette from the sample compartment. 8. Insert the the cuvette with the red dye into the sample compartment, read and record absorbance (Table on page 5) 9. Insert the the cuvette with the blue dye into the sample compartment, read and record absorbance. 10. Change the wavelength to 440 and again place the water blank into the sample compartment and adjust the display to 0 absorbance. 11. Insert the cuvettes with the red and blue dyes (one at a time obviously) read, and record absorbance. 1. Continue until you have made measurements of absorbance between 400 and 70 nm. REMEMBER to BLANK EVERY TIME YOU CHANGE THE WAVELENGTH. Also, if your machine has a filter lever, make sure it is set correctly for the wavelength you are measuring. 13. Repeat the protocol above with the acetone blank and the chlorophyll solution. Record your results as you read them on page 5 Write-Up The only write-up for this lab will be the graphs and tables.. All graphs and tables should be handed in one per group of two. Make sure both of your names are on your graphs and tables. Next lab we will use the spectrophotomer for a much more involved lab, so you should become very familiar with their usage 4

5 Names: Lab Section: List the absorbance readings at the following wavelengths for each of the solutions. Wavelength Chlorophyll Color of Solution Color of Solution 400 nm 440 nm 480 nm 50 nm 560 nm 600 nm 640 nm 680 nm 70 nm Chlorophyll Absorbance Absorbance vs Wavelength Wavelength (nm) 5

6 Color of Solution: Absorbance Absorbance vs Wavelength Wavelength (nm) Color of Solution: Absorbance Absorbance vs Wavelength Wavelength (nm) 6

7 ph 7