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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 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. Groups pH 4.0 4.5 5.0 5.5 6.0 Groups Temp 15 30 45 55 60 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 = 100μl = “010" on C. Put “010" eppendorf) in each of 12 cuvettes. eppendorf) 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 - 2 minute intervals for 4.0, 20 minutes 20
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 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 2.5 iodine. Calibrate spec. iodine. 3. Experimental cuvettes: put 0.1 mL iodine into each of put 11 cuvettes. 11 4. Time = 0: add 5 mL from Rxn Flask into one exp add cuvette. Take reading. cuvette. 5. Start Reaction: add 1 mL enzyme to Rxn Flask. Mix. add Start Timing!!! Start 6. Timed Readings: 5 mL from Rxn Flask into one exp mL cuvette at a time. Take reading. Repeat every 2 min up to 20 min. to Temperature procedure: Temperature
1. Rxn Flask: 35 mL starch + 35 mL water. Put it in water 35 bath and leave it there!! bath 2. Blank cuvette: 5 mL water + 0.1 mL iodine. Calibrate mL spec. spec. 3. Experimental cuvettes: put 0.1 mL iodine into each of put 11 cuvettes. 11 4. Time = 0: add 5 mL from Rxn Flask into one exp add cuvette. Take reading. cuvette. 5. Start Reaction: add 1 mL enzyme to Rxn Flask. Mix. add Start Timing!!! Start 6. Timed Readings: 5 mL from Rxn Flask into one exp mL cuvette. Take reading. Repeat every 1 min up to 8 min, then do mins 10 and 20. then • Groups pH
1. Rxn Flask: 35 mL starch + 35 mL buffer. 1. Blank cuvette: 2.5 mL water + 2.5 mL buffer + 0.1 mL 2.5 iodine. Calibrate spec. iodine. • Groups Temp
1. Rxn Flask: 35 mL starch + 35 mL water. Put it in water 35 bath and leave it there!! bath 2. Blank cuvette: 5 mL water + 0.1 mL iodine. Calibrate mL spec. spec. 1. You will prepare a data sheet (Enzyme Kinetics – 15 1. Enzyme pts.) in lab. pts.)
A. You must provide the absorbance data collected by your You group (p. 55). 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 (p. 59). E. You will share this reaction rate with the rest of the class. You F. Each student must then take these data and prepare two F. Each figures (one 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 Reaction Rate (pp. 52-54) Reaction
= 1/2 change in absorbance / time 1/2 for this change to occur.
A. Worksheet at the end of the chapter to help calculate reaction rate (p. 59). help B. Hand this in with data sheet. B. 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
* 1/2∆A † Ai-1/2∆A TAi-1/2∆A R.r. (*/†) Temp.(°C) 15 30 45 55 60 70 Ai Af ∆A 1. Before the following lab, you must complete the 1. Before Enzyme Kinetics postlab exercise (p. 61). Enzyme 2. You must also complete the Bacterial Transformation You prelab assignment before the next lab in Sakai. prelab 3. In the Tests and Quizzes tool in Sakai READ THE FOLLOWING DOCUMENT FIRST READ IN YOUR RESOURCE FOLDER: IN
– Transformations_StudentSheet.pdf – dna_restriction_analysis_student_guide.pdf The lab must be clean before you leave. ...
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