BIOLOGY 163 LABORATORY Amylase Activity in Hordeum and Mya (Reviewed Fall 2017)

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1 BILGY 163 LABRATRY Amylase Activity in ordeum and Mya (Reviewed Fall 2017) An understanding of the nature and control of enzymatic action is essential to understand energetics at the cellular and molecular level. Since enzymes are proteins, any factor that alters protein structure will influence the biological activity of the enzyme. Proteins are more than long chains of amino acids held together by peptide bonds. They have a complex and highly specific pattern of folding and coiling -- called the tertiary structure -- held in place by disulfide bridges and by weak non-covalent interactions (WNs) such as hydrogen bonds. Since many of the forces that hold a protein molecule in its specific configuration are much weaker than the covalent bonds between the amino acids, the shape is subject to alteration by temperature, acidity, ionic concentrations, and other factors. Anything that affects the folding pattern of the enzyme molecule is likely to influence the ability of the enzyme to perform its function. Thus a study of enzyme activity may give insight into the more general mechanisms of protein structure and function. Amylases are enzymes found in a wide variety of organisms, microbial, plant, and animal. Their function is to hydrolyze starch, a major form of stored energy. You will investigate some of the properties of amylases from two sources: the crystalline style from the salt-water clam (Mya), and barley (ordeum) kernels. The crystalline style is a rod-shaped structure in the stomach of the clam. ilia in the stomach continuously rotate the rod. As it rotates, the end of the rod wears away, releasing the amylase contained within it. In barley, cells in the outer layer of the endosperm (the aleurone) synthesize amylase to be released during seed germination. This allows the seed to break down nutrients stored in the endosperm to provide energy for the growing embryo. Although plant and animal amylases are fundamentally similar in function, their structural properties may differ. Enzymes are studied by measuring the rate of the chemical reaction that the enzyme catalyzes. nce a reliable measure, called an assay, is worked out, various factors that might affect enzyme action can be tested by comparing the reaction rate with a normal control reaction, usually done under optimal conditions. A number of methods are used to measure the rate of a chemical reaction. ne may measure the rate at which the substrate is used, or, alternatively, the rate at which the product is formed. Making quantitative determinations of small chemical changes often is difficult, but one convenient way to do so is by measuring a color change. You can measure color changes accurately with a spectrophotometer, an instrument widely used in biochemical analyses. The technique is so valuable that much effort has been spent in adapting it to situations where no obvious color change is involved in the reaction being studied. ften substances undergoing chemical change do have differences in their "color" but only in the ultraviolet range of the spectrum. Also, all molecules have characteristic absorption properties in the infrared end of the spectrum. Thus ultraviolet, visible, and infrared spectrophotometers are valuable tools of the biologist. In this experiment you will use an interesting trick to make a color change as the amylases digest the starch -- a reaction that normally does not produce any visible color. An artificial substrate for the enzyme is made by reacting the starch molecules with an intensely blue dye. The dye molecule attaches to the sugar units of the starch molecules in such a way that they do not interfere with the enzymatic reaction. In an undigested state starch is insoluble and, since the dye remains bound to the starch granules, none is in the solution. (You will find more about the nature of the starch molecule in Appendix I.) When the amylase enzymes split starch to form the soluble sugars, the dye also goes into the solution with the sugar, coloring the solution blue. After removing the undigested starch particles, you can measure the color quantitatively with a spectrophotometer. Since the amount of color in the solution is directly proportional to the amount of starch digested, you can make quantitative measurements of the enzyme activity. In other words, the more intense (dark) the blue is, the higher the rate of enzyme activity.

2 amylase 2 EXPERIMENTAL PREDURES (Work as sides of tables, using either clam or barley, as directed by your instructor.) A. ENZYME PREPARATIN (Keep materials cold during enzyme preparations!) 1. RYSTALLINE STYLE Remove one valve of the clam by cutting the large adductor muscles and carefully slicing the tissue away from the upper shell. Pierce the stomach with a set of sharp forceps and poke around until the end of the crystalline style slips out (your instructor will likely demo this for you). Remove the style with the forceps and place it in seawater. You will need five styles for the enzyme preparation. a. Blot your five styles on filter paper to remove excess water. Weigh the styles together and record the total weight on the data sheet at the end of this handout. b. Place the styles in a mortar with a small pinch of sand and grind them into a dry paste. d. Add 5 ml seawater to the mortar and mix the style paste in thoroughly. Pour the mixture into a conical centrifuge tube. e. Add another 5 ml seawater to rinse the mortar and pestle. Add the rinse to your style suspension already in the conical centrifuge tube. f. hill the suspension on ice for 10 minutes. g. entrifuge the suspension at ~ 300 X g for 2 minutes. h. arefully decant the supernatant (solution above the pellet) into a test tube, and place on ice. TIS IS YUR RYSTALLINE STYLE ENZYME PREPARATIN. 2. BARLEY ALEURNE ENZYME a. btain a dish of germinated Barley seeds (about 130 seeds) and remove all roots and shoots. b. Weigh the seeds (minus roots and shoots). Record the weight (probably about 6-7g) on the data sheet at the end of this handout. c. Place the barley into a mortar with a small pinch of sand and grind to a paste. d. Add 10ml distilled water to the paste, and mix thoroughly to create a thick, milky suspension. e. Scrape the suspension into a small beaker and place it on ice for 10 minutes. f. Filter the suspension through miracloth into a large (40-50 ml) centrifuge tube. g. Your instructor will assist you in centrifuging your sample in a refrigerated, high-speed centrifuge at 15,000-18,000 rpm (~ 31,000 X g) for 10 minutes. h. Immediately upon removing your sample from the centrifuge, carefully decant the supernatant (solution above the pellet) into a test tube, and place on ice. TIS IS YUR BARLEY ENZYME PREPARATIN.

3 amylase 3 B. ASSAY PREDURES 1. PREPARATIN Prepare 5 beakers as indicated in the table below. Since no digestion takes place without the enzyme, you can pipette all reagents, EXEPT TE ENZYME, into the beakers at any time. Beaker Number Reagent ontrol p 5 p 6 p 7 p 8 Starch-Azure Substrate 2.5 ml 2.5 ml 2.5 ml 2.5 ml 2.5 ml Buffer, p ml Buffer, p ml Buffer, p ml 2.5 ml Buffer, p ml 2. DIGESTIN Study the procedure before you begin. Work carefully but quickly; you want to add the water (Beaker 1) and enzyme (Beakers 2-5) to their respective beakers at as close to the same time as possible! Use a 500µl (0.5ml) micropipettor for step 1. This will improve both the speed and accuracy of your pipetting. If you are unsure how to use a micropipettor, ask your instructor for a demonstration. 1. Use a 500µl (0.5ml) micropipettor to add 0.5 ml of DISTILLED 2 to your NTRL beaker (Number 1) and 0.5 ml of the ENZYME PREPARATIN (crystalline style or barley) to each of the remaining four beakers (Numbers 2-5). Record the exact time or start the timer as soon as you complete this step! 2. Swirl each beaker briefly to mix the contents, and swirl again every minute thereafter. 3. After approximately 10 minutes, pour the reaction mixture from each beaker into an appropriately labeled centrifuge tube. entrifuge the tubes at ~ 1100 X g for 4 minutes. (The digestion effectively ends at the beginning of centrifugation since the starch granules immediately go to the bottom of the tube where they have little contact with the enzyme.) Record the exact time or stop the timer as soon as you begin the centrifugation! 4. At the end of the centrifugation, carefully transfer the supernatant from each centrifuge tube into a clean spectrophotometer cuvette. This may be accomplished with careful decanting or with a clean transfer pipette. owever, note that you must not stir up any undigested starch from the bottom of the centrifuge tube! (If you do, you will need to recentrifuge that tube and try again.) 5. Measure the color intensity, comparing the value for each tube with that of your control (see Part 3 below for method for spectrophotometer readings). As a qualitative check, also record the relative intensity of color for each tube as perceived by the naked eye.

4 amylase 4 3. SPETRPTMETER READINGS The spectrophotometer you use has a variable light source that can be set to any wavelength in the visible spectrum. For maximum sensitivity to the blue dye used in this experiment, the light is set to the wavelength corresponding to the complementary color. 1. Turn on the Spec-200 (switch is in back). When prompted, press Enter (unlabeled round button) and wait for initializing to complete. Make sure Spec 200 Modern Interface is selected, and press Enter. 2. Make sure the Application is set to Live Display and the Measurement Mode is set to Abs (Absorbance). Use the down arrow to select G and press Enter. 3. Set the wavelength (λ) to 620 nm using either the forward/back arrow keys, or the λ knob. (Note: for fine control, press down on the knob while turning.) 4. pen the sample compartment and place the cuvette containing the control solution completely into the LEFT sample holder. NTE: BE SURE T ALIGN TE UVETTE S TAT TE LEAR SIDES FAE TWARD TE LIGT! lose the lid completely and press the 0.00 button. (By doing this, your experimental readings are made relative to the absorbance of the control tube. In effect, the control absorbance is subtracted automatically from all experimental readings, simplifying the arithmetic!) 5. After zeroing is complete, place each experimental tube in the sample holder (again paying attention to the alignment), close the lid and record the absorbance value. Do not press any additional buttons during this step! 4. ALULATING ENZYME ATIVITY The enzyme activities you measured are for 0.5 ml of your enzyme preparation. Since the weights of individual styles are not uniform from one clam to another and the amount of enzyme in the barley may be quite different from that in the style, the quantity of enzyme in your 0.5 ml samples may vary considerably from that in someone else's preparation. It is also important to consider the elapsed time during which the starch is exposed to the enzyme, as this may vary from assay to assay. Enzyme was not added to the NTRL sample. Since we know there is no enzymatic activity in the control, we use it to set the absorbance scale of the spectrophotometer to zero. This effectively subtracts from the experimental measurements the effects of light absorption that may occur from sources other than starch digestion. Any enzymatic activity that takes place in the experimental samples will result in a color change and a corresponding increase in the measured absorbance of the sample. Therefore, we are able to quantify enzyme activity by measuring absorbance over a given period of time. In order to facilitate comparisons among samples, absorbance measurements will be standardized for time and tissue weight. A few simple calculations yields absorbance/minute/gram of tissue a useable measure of enzyme activity. Pay careful attention to the directions in the data sheet, as these will guide you through the required calculations!

5 amylase 5 APPENDIX I: Starch Digestion Starches are polymers comprised of sugar molecules cells produce them as molecules for energy storage (poly = many, -mer = parts). Amylose is a form of starch consisting of glucose molecules joined in a chain by amyl bonds (amyl = the type of bond, the suffix -ose denotes a sugar). The digestive enzyme that splits these bonds is therefore known as amylase (the suffix ase denotes an enzyme). When amylase splits the amyl bond, a molecule of water is ionized and added to the product. ne end of the split chain accepts a hydrogen ( + ) while the other end receives an -. Splitting a large molecule into fragments by adding a water molecule is called hydrolysis. Therefore amylase (like all other digestive enzymes) is a hydrolytic enzyme. As the starch chain is split into progressively shorter fragments, the chemical and physical properties of the polymer change. The small pieces of partially hydrolyzed starch are dextrans. (The glue often used on postage stamps is dextran--it has a mildly sweet taste, and is edible.) The dextrans are broken down finally to two and three unit sugars called maltose and malto-triose. These are quite soluble and are acted upon by a different enzyme for the final digestion to glucose. In this experiment, we use a form of amylose that incorporate an attached blue dye azure. The amylose itself is insoluble it (and its attached azure dye) exist only in suspension, and therefore do not contribute any color to the solution. owever, the sugars that result from digestion with amylase are soluble. When they dissolve they take the dye with them, imparting a blue color to the solution that is proportional to the amount of starch digested (Figure 1). 2 * Figure 1. Amylose-azure hydrolysis. Amylase cleaves the insoluble starch (amylose) into smaller soluble sugars. As the sugars dissolve, they take the attached azure dye (represented by asterisks) with them into the solution, which turns blue as a result. 2 * APPENDIX II: p and Buffers A buffer is something that lessens or absorbs the shock of an impact. In biological and chemical terminology, a buffer is a chemical that shields or protects a reaction system from sudden changes in acidity or alkalinity (p). If you wish to study a reaction at a particular p, a buffer is used to establish and maintain the desired p. In this experiment, you will use a citrate buffer. This is a mixture of citric acid and disodium phosphate in the appropriate proportions required to give the desired p. In solution, these two chemicals are able to "absorb" either added acid (hydrogen ions, + ) or added base (hydroxyl ions, - ) by reacting with these ions and effectively neutralizing them. Similar buffering systems occur in living organisms and serve to protect the internal body fluids of the organism from sudden or drastic changes in p.

6 amylase 6 Amylase Activity in ordeum and Mya DATA SEET alculate Amylase Activity (ΔA 620 /min/g) according to the following formula: A B B / A = / D*** = E Beaker Number Exper. ond. Qual. Visual bs.* Initial Time Final Total Elapsed Time (minutes)** Absorbance (A 620 ) for Total Elapsed Time Δ A 620 / Minute Δ A 620 / Minute / Gram of Tissue Barley 1 ontrol 2 p 5 3 p 6 4 p 7 5 p 8 Style 1 ontrol 2 p 5 3 p 6 4 p 7 5 p 8 * Qualitatively rank the depth of color of each sample as observed with the naked eye. ** To facilitate calculations, convert minutes and seconds to minutes and tenths of minutes using the following formula: number of seconds / 60 = tenths of minutes (e.g., 9:30 = 9.5 minutes) *** D = Grams Tissue per 0.5 ml Enzyme Preparation as follows: Barley Kernels ( g barley / ml original liquid) x 0.5 = gm barley per 0.5 ml enzyme preparation = D lam Style ( g style / ml original liquid) x 0.5 = gm style per 0.5 ml enzyme preparation = D

7 amylase 7 ASSIGNMENT Building on previous assignments, you will present this study in the form of a partial scientific paper including graphical results and a full discussion. You may find the Guide to Writing Scientific Papers and Working with Statistics helpful. Use the following guidelines to focus your efforts. Results (figure(s) only) Using group data provided by your instructor, graphically present the results of the study in a meaningful and informative way. TIP: Large differences in amylase activity between groups (barley vs. clam) may necessitate creative approaches to your presentation so that smaller values don t become lost among larger values. Keep in mind that while quantitative comparisons are meaningful within groups, they are not particularly important when making comparisons between groups. In this case it s the differences in the overall trends that will tell the story! Discussion (two or three well-developed paragraphs) A full discussion not only states the conclusions drawn from the results, but also interprets those conclusions in a larger biological context. Your discussion should address each of the following questions thoroughly but concisely. (INT: Again, keep the structure-function relationship of enzymes in mind when developing your responses!) onclusions (answer the experimental questions based on the results) Does p affect amylase activity? If so, does p affect barley amylase the same as it does clam amylase? Interpretations (explain your conclusions based on your broader biological knowledge) TIP: Keep the structure-function relationship of enzymes in mind when developing your responses! What is p a measurement of? ow could changes in p change the activity of an enzyme or other protein? ow do you explain differences in the way p affects the activity of the same enzyme from different sources? Would significant changes in p within a living organism be undesirable? Explain. Acknowledgements If you discussed this assignment with someone else, you must acknowledge that collaboration here. Literature ited If you utilized outside sources in developing your discussion, you must cite those sources appropriately and include a full bibliographic reference here. Your instructor may provide additional detailed instructions.