Bio 242 ‐ Cellular and Molecular Biology Fall 2015 Bates College Determination of the pH that yields maximum ‐amylase activity In this first week of work with amylase, you will learn how to work with the enzyme by determining the approximate pH, or range of pH, over which it has greatest hydrolytic activity. In this first set of assays, you will do the first step: you will run a set of reactions to approximate the pH that yields maximum (but not necessarily the optimal pH) for the enzyme you are studying. You will react the enzyme with the substrate over a broad range of pH (whole pH unit increments) to locate the approximate pH where you find maximum activity (IT IS NOT THE OPTIMAL pH). Once the this pH is known, you will use that same pH buffer in subsequent experiment(s) of your own design next week as you seek to answer questions about other functional properties of the enzyme. NOTE: Carefully study your data sheet so that you will understand what measurements you are to make, and also how to calculate the final values for enzyme activity. You must record 1) the conditions in each reaction mixture, 2) the time (T0) at which the enzyme is added to each reaction mixture, 3) the time (TSTOP) the digestion is terminated by centrifuging the mixture (we consider the reaction to be stopped when the centrifuge reaches force (9820 xg), and, 4) the A620 reading from the Spec 20. NOTE: Optimal pH is that specific pH, or narrow range, at which an enzyme has its maximum activity under a given set of conditions; to either side of that pH, activity generally (but not always) falls off dramatically. Determination of true optimal pH is a two‐step process: first testing activity over large increments of pH (whole units) – this is what you will do this week; then, a second step in which you test fine increments of pH around the peak determined in the first step (we are not doing this). Unless you do the second step as your week two experiment, you should NOT refer to “optimal pH” in your paper when addressing your results. Procedure: Measurement of Enzyme Activity 1. Prepare one poisoned control (will be your BLANK)and four treatments in standard 15 ml centrifuge tubes as described in Tables 1, 2, or 3. BE SURE YOU MIX, BUT NOT VORTEX, THE AMYLOSE‐AZURE SOLUTION. All components may be added to the mixtures except the enzyme. DO NOT add enzyme until explicitly directed to do so by a TA or instructor. What is the rationale for the BLANK in this experiment? The hydrolysis of the substrate (amylose‐azure) will occur over a 10‐15 minute incubation period and the A620 will be measured only at the endpoint. Since the substrate is a non‐soluble starch, only dye attached to soluble sugar molecules will go into solution. All non‐hydrolyzed starch will be removed by centrifugation. To know how much azure went into solution (i.e., how much hydrolysis occurred), we’ll use an enzyme solution without substrate to zero the Spec 20. This effectively sets the A620 to zero for a condition of no reaction. Thus, by zeroing with the blank, the final A620 of the treatment reactions will provide a reasonable measure of the total amount of substrate hydrolyzed. 2. When instructed to do so, invert your enzyme extract briefly to mix uniformly. Add 500 µl (= 0.5 ml) of your enzyme solution to each reaction mixture and mix by inverting. Record the time the enzyme was added to each mixture. Allow the reaction to incubate at room temperature for ~10 minutes – at which time you will be told to load your mixtures into the centrifuge. During this time, gently invert the tubes at least 2‐3 times per minute to ensure the enzyme and substrate stay in close contact. 3. After 10 minutes, centrifuge the mixtures at high force (8,000 rpm = 9820 x g) for two or three minutes. Record the time at which the centrifuge reaches 8,000 rpm (= reaction stop time or TSTOP; this will be announced with great hoopla by the lab staff). 44 Bio 242 ‐ Cellular and Molecular Biology Laboratory Manual Bio 242 ‐ Cellular and Molecular Biology Fall 2015 Bates College What is the rationale for using centrifugation to stop the reactions? The substrate used in these experiments, amylose‐azure, is an insoluble starch molecule that will settle out quickly during the experiment unless kept in suspension by frequent gentle mixing by inversion of the reaction mixtures. Contact between enzyme and substrate will only occur if the substrate is suspended. If the substrate settles out completely (and this can happen fairly quickly), virtually no hydrolysis will occur (yes, students have demonstrated this conclusively). The hydrolysis reaction can be effectively shut down by centrifuging the mixtures at relatively moderate force to quickly pellet the substrate, removing it from contact with the enzyme. To have a definitive time that we can call the end of the reaction, the time at which the centrifuge reaches 8000 RPM (9820 x g) will be called “time stop” (TSTOP). This time will be broadcast to both lab rooms with great fanfare and hoopla. Huzzah! Huzzah! 4. At the end of centrifugation, use a Pasteur pipette to carefully draw off approximately 4.0 ml of the supernatant from each tube and deliver it in a corresponding SPEC 20 cuvette. Be gentle so as not to disturb the pellet. You need enough volume to get a reading in the spec 20. 5. Seal each tube with Parafilm and vortex briefly. 6. Set the SPEC 20 to measure absorbance at 620 nm wavelength. Use the POISONED CONTROL to blank the SPEC 20—to give us A620 at the To point, before hydrolysis. Then measure the A620 of each treatment. Thus, at TSTOP, A620 = total change in absorbance (A620) over that time. 7. Calculate and record the A620 /min for each reaction and interpret your results. Calculate the working, or final, concentrations of all reaction mixture components For each set of reaction mixtures, calculate the working concentration of each component using the units of concentration of the stock solutions. Reaction mixture formulations should be reported in your Methods section using these working concentrations. More information on doing such calculations can be found on the Resources website on the Making Simple and Solutions and Dilutions page. Report these values in the Methods of your paper. Table 1. Reaction mixture formulations for the corn ‐amylase pH experiment. Treatment Deionized Water pH buffer2 (ml) Amylose‐azure3 (ml) Stock= 2mg/ml pH 4 ‐‐‐‐‐ 0.5 ml 2 ml pH 4 buffer 2.5 ml pH 5 ‐‐‐‐‐ 0.5 ml 2 ml pH 5 buffer 2.5 ml pH 6 ‐‐‐‐‐ 0.5 ml 2 ml pH 6 buffer 2.5 ml pH 7 ‐‐‐‐‐ 0.5 ml 2 ml pH 7 buffer 2.5 ml BLANK pH 7 ____ 3.0 ml 2 ml pH 7 buffer ‐‐‐‐‐‐ 1
Citric acid (stock = 0.1 M C6H8O7) – disodium phosphate buffer (stock = 0.2 M Na2PO4). The buffer is made by combining different volumes of the two solutions to obtain the approximate pH, and then adjusted with HCl. Therefore, specific molarities of citric acid and disodium phosphate vary by pH 2
Stock amylose‐ azure = 2 mg/ml; THE STOCK SHOULD APPEAR VERY BLUE AND SHOULD NOT BE CLEAR. The final working concentration depends on the final dilution in your reaction mixes. Bio 242 ‐ Cellular and Molecular Biology Laboratory Manual 45
Bio 242 ‐ Cellular and Molecular Biology Fall 2015 Bates College Table 2. Reaction mixture formulations for the clam crystalline style ‐amylase pH experiment. Treatment 3.5% NaCl pH buffer2 (ml) Amylose‐azure3 (ml) pH 6 ‐‐‐‐‐ 0.5 ml 2 ml pH 6 buffer 2.5 ml pH 7 ‐‐‐‐‐ 0.5 ml 2 ml pH 7 buffer 2.5 ml pH 8 ‐‐‐‐‐ 0.5 ml 2 ml pH 8 buffer 2.5 ml pH 9 ‐‐‐‐‐ 0.5 ml 2 ml pH 9 buffer 2.5 ml BLANK pH 7 ___ 3.0 ml 2 ml pH 7 buffer ‐‐‐‐‐‐‐ 1
Citric acid (stock = 0.1 M C6H8O7) – disodium phosphate buffer (stock = 0.2 M Na2PO4). The buffer is made by combining different volumes of the two solutions to obtain the approximate pH, and then adjusted with HCl. Therefore, specific molarities of citric acid and disodium phosphate vary by pH 2
Stock amylose‐ azure = 2 mg/ml; THE STOCK SHOULD APPEAR VERY BLUE AND SHOULD NOT BE CLEAR. The final working concentration depends on the final dilution in your reaction mixes. Table 3. Reaction mixture formulations for the human salivary ‐amylase pH experiment. Treatment 0.2 M HgCl2 1 (ml) 0.9 % NaCl pH buffer2 (ml) Amylose‐azure3 (ml) pH 6 ‐‐‐‐‐ 0.5 ml 2 ml pH 6 buffer 2.5 ml pH 7 ‐‐‐‐‐ 0.5 ml 2 ml pH 7 buffer 2.5 ml pH 8 ‐‐‐‐‐ 0.5 ml 2 ml pH 8 buffer 2.5 ml pH 9 ‐‐‐‐‐ 0.5 ml 2 ml pH 9 buffer 2.5 ml BLANK pH 7 ‐‐‐‐‐‐ 3.0 ml 2 ml pH 7 buffer ‐‐‐‐‐‐ 1
Citric acid (stock = 0.1 M C6H8O7) – disodium phosphate buffer (stock = 0.2 M Na2PO4). The buffer is made by combining different volumes of the two solutions to obtain the approximate pH, and then adjusted with HCl. Therefore, specific molarities of citric acid and disodium phosphate vary by pH 2
Stock amylose‐ azure = 2 mg/ml; THE STOCK SHOULD APPEAR VERY BLUE AND SHOULD NOT BE CLEAR. The final working concentration depends on the final dilution in your reaction mixes. EXPENDABLES AND CHEMICAL WASTE DISPOSAL All chemical wastes should be disposed of in the SAA in the hood in your lab room. 
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All tubes should be emptied into the amylose‐azure receptacle. Pipette tips and plastic centrifuge tubes go in the WASTE container near your bench; and then in the regular trash at the end of lab. Emptied Spec 20 glass tubes are disposed of in the tub marked GLASS WASTE. 46 Bio 242 ‐ Cellular and Molecular Biology Laboratory Manual