|CHEN 4800/5800||University of Colorado|
|Bioprocess Engineering||Dhinakar Kompala|
|Spring 1998||Jennifer Shull|
|Lab Exercise 1: Enzyme Kinetics|
In this laboratory exercise, you will perform an enzyme catalyzed reaction to characterize the effects of different concentrations of the substrate, the enzyme and an inhibitor on the reaction kinetics. The goals of the experim ent are to observe the effect of enzyme concentration on vmax, the effects of the inhibitor concentration on Km and vmax, and to determine whether the inhibitor is competitive or non-competitive.
ß-Galactosidase catalyzes the hydrolysis of ß-galactoside bonds in glycoproteins, polysaccharides, and disaccharides. This enzyme is one of the best characterized enzymes, with a number of chromogenic substrates, s uch as ortho- and para-nitrophenyl-ß-D-galactosides (ONPG and PNPG) available to follow the course of reaction by the appearance of a yellow color due to the formation of nitrophenyl anions. The reaction is shown below:
o-Nitrophenol can be detected readily by measuring its absorbance at 420 nm using a spectrophotometer. The rate of enzymatic reaction can be measured by the rate of product formation, given by the slope of the optical density with time .
Competitive inhibitors bear close resemblance to the normal substrate. Thus these inhibitors have the key to fit into the enzyme active site, but the fit is not identical so chemical reaction does not occur. Isopropyl-1-thio-b-D galactopyranoside (IPTG) is an analog of the substrate ONPG. In this experiment, you will investigate the effect of the addition of various concentrations of IPTG on the enzymatic cleavage of ONP G and determine if the inhibited reaction kinetics follow the classical competitive or non-competititve or mixed competitive mechanisms..
In this exercise, the different concentrations of the three chemicals to be used are :
1) the enzymeb-gal concentration [E]: 0.125 and 0.5 units/ml (1 unit (U) hydrolyzes 1 µmole ONPG to o-nitrophenol and D-galactose per min at pH 7.3 and 37šC)
2) the substrate ONPG concentration [S]: 0.4%, 0.3%, 0.2%, 0.1% and 0.05% w/w.
3) the inhibitor IPTG concentration [I]: 0.4%, 0.2%, 0.15%, 0.1% and 0% w/w.
Each group of two students will perform the enzyme kinetic reaction between the five different substrate concentrations and the two enzyme concentrations for one particular concentration of inhibitor.
1) Z buffer: 60 mM Na2HPO4.7H20, 40 mM Na2HPO4.H20, 10 mM KCl, 10 mM MgSO4 .7H 20, 40 mM b-mercaptoethanol pH adjusted to 7.0.
2) ONPG stock solutions in Z buffer (0.4%, 0.3%, 0.2%, 0.1% and 0.05% w/w).
3) IPTG stock solutions in Z buffer (0.4%, 0.2%, 0.15%, 0.1% and 0% w/w).
4) Twob-gal enzyme stock solutions (5 units/ml, 20 units/ml)
5) Disposable cuvettes with 10 mm light path.
6) Temperature controlled spectrophotometer, micropipettors and pipette-tips.
Laboratory Procedures: Kinetics of 5 reactions can be monitored simultaneously by the spectrophotometer. Each group will perform 2 sets of 5 reactions. Results obtained by each group will shared with the whole class for the fi nal report.
a) Take 5 cuvettes and label each of them on the ground glass side (which will not be in the light path).
b) Add 1.4 ml Z buffer to each cuvette. Cover the cuvette with parafilm.
c) Add 0.2 ml of an ONPG stock solution to each cuvette. To each cuvette you will be adding a different stock solution of ONPG.
d) Add 0.2 ml of an IPTG stock solution to each cuvette. Each group will be working with only one concentration of IPTG.
ADD ENZYME LAST..MAKE SURE THE SPECTROPHOTOMETER IS READY TO GO BEFORE ADDING THE ENZYME. ONCE THE ENZYME IS ADDED THE REACTION WILL START IMMEDIATELY, THEREFORE IT IS IMPORTANT TO TRANSFER THE CUVETTES INTO THE SPECTROPHOTOMETER A S SOON AS ENZYME IS ADDED AND START TAKING THE READINGS IMMEDIATELY.
Running the spectrophotometer. Press "Prog" and Program 2: BGAL will appear. Press "R/S". You do want to calibrate, so next press "0" and then "Enter". Insert the "blank" cuvette into the cuvette chamber in position 1 and pre ss "R/S". After a short time for calibration, the spec will "ask" you to insert the samples and then press "R/S" to read.
e) Add 0.2 ml ofb-gal enzyme stock to each cuvette. To the first five cuvettes add the first enzyme stock solution provided and to the next five cuvettes add the second en zyme concentration (from the second stock solution).
f) Place the cuvettes in the sample chamber of the temperature-controlled Beckman DU-50 spectrophotometer (temperature will be controlled at 37šC) in positions 2-6. Leave the "blank" cuvette in position 1.
g) Press the "R/S" button which will start recording the absorbance at 420 nm over a period of 8 minutes. In this period of 8 mins the spectrophotometer will print out the absorbance in each cuvette at 25 second time intervals. A fter the end of the 8 minutes the spectrophotometer will plot the absorbance values for each cell over time. After the spec. has plotted the OD vs time graph it will ask you whether you need to calculate the slopes.
h) Theb-galactosidase activity is calculated from the rate of change of the optical density for each sample and is expressed as U/ml by comparing the slope (r ate of change of absorbance) for the sample to the slope for a b-galactosidase standard. The spec is programmed to calculate the slope of your sample; you need to press the following buttons bef ore you will get these data.
1. After your samples have run for 8 minutes, the spec will "ask" for a cell number. Press "2" and "Enter".
2. The display will read "blank"; press "Enter" for the default setting.
3. The display will read "Time 1"; press "3" and then press the "Enter" button. We will only take the data from say, 3 minutes to 8 minutes for our assay; the first 3 minutes may be needed to allow the cuvettes to warm up to 3 7šC in the sample chamber.
IN SOME CASES ESPECIALLY FOR THE HIGH ENZYME CONCENTRATION AND NO IPTG CASE THE REACTION OCCURS VERY FAST AND SO THE TIME INTERVAL THAT YOU INPUT IN THE COMPUTER IN THE REGION WHERE PLOT OF OD VS. TIME IS LINEAR (SO BEFORE ENTERING THE TIME1 AND TIME2 VALUES CHECK THE GRAPH TO MAKE SURE THAT THE TIME INTERVAL IS IN THE LINEAR RANGE).
4. The display will read "Time 2"; press "Enter" for the default setting (8 minutes).
5. The display will read "Factor"; press "Enter" for the default setting.
In a few moments the data will appear at the bottom of your printout. Repeat steps 1-5 for cell numbers 3-6. After this is done press "0" and "Enter" in order to exit the program. Remove your 5 sample cuvettes and the blank from the spec.
The last column of data for each cell (chamber) is "Result"; this is the slopeof the absorbance readings for your sample from TIME1 to TIME2 minutes. This is (v) the ra te of reaction in each cuvette.
Report (due February 3, 1998):
Analyze the collected data set from all the students (the full data set will be e-mailed to you on Thursday evening if you leave your e-mail address with the lab co-ordinator) to determine the effects of enzyme and inhibitor concent rations on the rate parameters and the nature of inhibition (competitive vs. non-competitive vs. mixed). Report should be less than 5 pages, with attached graphs and/or tables as needed in your analysis.