Enzymes - BSC 2010L ENZYME KINETICS ENZYME In this lab, we...

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Unformatted text preview: BSC 2010L ENZYME KINETICS ENZYME In this lab, we will study the influence of In temperature and pH on the rate of an enzymatic reaction using spectrophotometry. spectrophotometry. Spectrophotometry Review Proteins Proteins A. A protein's function is due entirely to its A. overall shape = conformation. overall B. Conformation is determined by 1˚, 2˚, B. 3˚, and perhaps 4˚ structure of the protein. protein. The primary structure of a protein The The secondary structure of a protein The The tertiary structure of a protein The The quaternary structure of proteins The Enzymes A. Enzymes are protein catalysts. A. Enzymes B. Lower the activation energy necessary for a B. chemical reaction to occur. chemical C. Act on a substrate, form an enzymeC. substrate form enzymesubstrate complex. Ultimately results in a Ultimately product. product D. We are using barley amylase enzyme on a D. barley starch substrate. starch Enzymes lower the activation energy of a reaction Enzymes Formation of an enzyme-substrate complex Formation Environmental factors affecting enzyme activity Environmental Experiments: Experiments: A. Work in groups of two to four. A. You will perform one run from the pH You experiment or one from temperature experiment. BASIC PROCEDURE BASIC A. Set spectrophotometers to the λ max we determined A. for the starch/I2KI solution (560 nm). for B. In an erlenmeyer, add 35 ml starch + 35 ml H2O. B. In O. HAVE STUDENTS THOROUGHLY MIX STARCH BEFORE DRAWING VOLUME. BEFORE C. Put the I2KI indicator (0.1 ml = "100" on eppendorf) C. Put KI in each of 12 cuvettes. in D. Make the blank: 5 mls of water (5 ml glass pipette) D. into one of these cuvettes. into E. Take the initial reading: pipette 5 mls from E. initial reading pipette erlenmeyer (5 ml glass pipette) into a cuvette. Take an absorbance reading and record it on the data sheet. sheet. BASIC PROCEDURE BASIC pH experiment pH 1. Add 35 ml of buffer to 35 ml of starch solution. 1. Blank is 1:1 dH2O:buffer (+ iodine) 2. 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 - 2 minute 4.0, intervals for 20 minutes intervals Table 5-4. Absorbance readings over time of reactions at different pHs. TIME (min) 8 10 pH 4.0 4.5 5.0 5.5 6.0 6.5 0 2 4 6 12 14 16 18 20 Temperature experiment Temperature Mix as in basic procedure. The reaction flask is kept in Mix a water bath. NOTE: Very cold and very hot solutions might need to sit before being read. to 15, 30, 45, 55, 60, 70 degrees - 1 minute for 8 minutes, 15, then 10 and 20 minute readings. then Table 5-3. Absorbance readings over time of reactions at different temperatures. TIME (min) 4 5 Temp.(¡ C) 15 30 45 55 60 70 0 1 2 3 6 7 8 10 20 CALIBRATING SPECS: CALIBRATING 1. Set wavelength to ______ nm. 2. Chamber empty: set T = 0 with left Chamber knob. knob. 3. Blank in chamber: set A = 0 with Blank right knob. right pH procedure: pH 1. Rxn Flask: 35 mL starch + 35 mL buffer. 2. Blank cuvette: 2.5 mL water + 2.5 mL buffer + 0.1 mL iodine. 2.5 Calibrate spec. Calibrate 3. Experimental cuvettes: put 0.1 mL iodine into each of 11 cuvettes. 4. Time = 0: add 5 mL from Rxn Flask into one exp cuvette. Take add reading. reading. 5. Start Reaction: add 1 mL enzyme to Rxn Flask. Mix. Start Timing!!! add Start 6. Timed Readings: 5 mL from Rxn Flask into one exp cuvette at a mL time. Take reading. Repeat every 2 min up to 20 min. time. Temperature procedure: Temperature 1. Rxn Flask: 35 mL starch + 35 mL water. Put it in water bath and 35 leave it there!! leave 2. Blank cuvette: 5 mL water + 0.1 mL iodine. Calibrate spec. 3. Experimental cuvettes: put 0.1 mL iodine into each of 11 cuvettes. 4. Time = 0: add 5 mL from Rxn Flask into one exp cuvette. Take add reading. reading. 5. Start Reaction: add 1 mL enzyme to Rxn Flask. Mix. Start Timing!!! add Start 6. Timed Readings: 5 mL from Rxn Flask into one exp cuvette. Take mL reading. Repeat every 1 min up to 8 min, then do mins 10 and 20. reading. 1. Youwill prepare a data sheet (Enzyme Kinetics – 15 1. Enzyme pts.) in lab. pts.) A. Youmust provide the absorbance data collected by your Youmust group (p. 57). This will consist of one set of data on either temperature or pH. temperature B. Plot those data points in a properly constructed figure. Plot (Graph paper provided in manual.) (Graph C. Draw a best-fit curve to these data. D. Calculate a reaction rate for that line (pp. 52-54). E. You will share this reaction rate with the rest of the class. You F. You must then take these data and prepare two figures (one F. You for wavelength and one for light intensity) of the reaction rates. G. You should indicate on the figure the optimal conditions for G. You the enzyme reaction. the H. COMPLETE THIS DATASHEET IN LAB, BUT YOU CAN COMPLETE TAKE IT HOME TO HELP COMPLETE THE POSTLAB EXERCISE (p. 59) . EXERCISE Reaction Rate (pp. 52-54) Reaction = 1/2 change in absorbance / time 1/2 for this change to occur. Ai = 2.6 ∆A = 2.2 Ai­½∆A = 2.6 – 1.1 = 1.5 ½∆A = 1.1* R.R = 1.1/4.75 = 0.23/min T = 4.75 Af = 0.4 ENZYME KINETICS Reaction Rate Calculation Sheet Temp.(¡ C) 15 30 45 55 60 70 Ai Af ÆA * 1/2Æ A Ai-1/2ÆA TAi-1/2Æ A R.r. (*/ ) 1. Before the following lab, you must complete the 1. Before Enzyme Kinetics postlab exercise (p. 59). Enzyme 2. You must also complete the Bacterial Transformation You prelab assignment before the next lab. prelab The lab must be clean before you leave. ...
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This note was uploaded on 04/24/2011 for the course BSC 2010L taught by Professor Herrerabaerbolker during the Spring '08 term at University of Florida.

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