University of Texas, DallasBIOL 3380Lab 9 Stud Protocol - Report F16 Lab 9 Stud Protocol

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BIOL 3380 Name:_____________________________________ Circle Session: T-AM T-PM W-AM W-PM R-AM R-PM F-AM F-PM Experiment 9 – Pre-lab Homework Enzyme Kinetics of LDH This pre-lab homework assignme... nt is due at the beginning of your lab session. You are provided with the following portion of a protocol:  Determine concentration of enzyme stock solution, if unknown, by taking an A280 nm reading of a 1:100 dilution (in water). Use a total volume of 1 ml in the cuvette.  Dilute some of the enzyme stock with buffer A to make a 4 mg/ml solution.  Serially dilute the 4 mg/ml solution with buffer A to make working solutions of 400 µg/ml and 40 µg/ml.  Prepare 30 µl of each working solution for every sample The PI of the lab gives you a tube of enzyme and tells you the following before disappearing into the office to write more grant proposals:  There is 50 µl of enzyme stock solution. The enzyme is expensive to purify, so follow the protocol exactly, using as little of the stock solution as possible.  The concentration of the stock solution is currently not known, but a 1 mg/ml concentration of the pure enzyme has an A280 nm of 2.0.  You’ll be performing the assay on 12 samples.  Make enough of each working solution so that you have at least 400 ul to work with when you do the assay (to cover any waste and/or inefficiencies in pippetting). Using the spectrophotometer to read the absorbance at 280 nm, you get a reading of 0.784. 1. (2 pts) What is the concentration of the solution in the cuvette? What is the concentration of the stock solution? (Show your work, box your answers.) 2. (1 pt) Calculate the minimum amount of each working solution would you need in order to do the number of assays requested. How much excess has the PI instructed you to make? 9-13. (6 pts) Figure out your plan for making each solution required in the protocol. Be certain you FINISH with the required volumes of EACH solution! a. Calculate what is needed to make the 4 mg/ml solution b. Calculate what is needed to make the 400 ug/ml working solution c. Calculate what is needed to make the 40 ug/ml working solution 4. (1 pt) You compare the data from your assay to data obtained on a previous day by the lab’s star graduate student. You recorded a reading of 0.020 ΔA340/5 secs. The grad student recorded a reading of 0.160 ΔA340/min. Whose sample had more LDH activity? (Show your work for credit.) 5. (1pt) Looking at the “Reaction Rate as a Function of Enzyme Concentration” section in the lab protocol, which assay would you predict to have the highest LDH activity and why? 6. (1pt) Looking at the “Reaction Rate as a Function of Substrate Concentration” section in the lab protocol, which assay would you predict to have the highest LDH activity and why? 9-2Experiment 9 Enzyme Kinetics of LDH You are in a position to characterize the LDH enzyme as a catalyst. This is done by measuring the kinetics of enzyme reactions. (kinetics means the rate of change, or reaction rate). First, a review of enzyme basics: I. Definitions Enzymes increase the rate at which a reaction occurs. Enzymes are not consumed by the reaction. They do not shift the equilibrium of the reaction. Substrates are substances acted upon by an enzyme. II. Properties of Enzymes A. The majority of known enzymes are proteins (some nucleic acids with catalytic function have been identified). This means that those factors that alter protein [Show More]

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