Enzyme kinetics lab

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					                                                            Student Procedure Protein Week I

Enzyme Kinetics Lab
Objective: to measure the hydrolysis of benzoyl DL-arginine p-nitroanilide
hydrochloride (DL-BAPA) activity (kCAT and KM) of trypsin.
To measure the trypsin activity you will be measuring initial rates of the enzyme-
catalyzed reaction over a range of substrate concentrations from 0.1KM < S < 10KM
You measure the production of the product, p-nitroaniline, by the absorbance at 410 nm
with the UV/Vis spectrophotometer. The extinction coefficient of p-nitroaniline is
8,800 lit/mol/cm at 410 nm between pH of 5 and 10.5. Review the tutorial books on
UV/Vis spectrophotometry so you know how to convert the absorbance into enzyme rate
units. You can report rates in terms of grams of enzyme powder. DL-BAPA does not
hydrolyze in the absence of enzyme, so you can measure the background once for
reference.
Tris-HCl buffer is made by preparing the appropriate weight of Tris in water then
adding HCl to drop the pH to the desired value. Prepare 200 ml each of 40 mM
Tris-HCl pH 7.5, 8.0 and 8.5 with 50 mM CaCl2.
       1. In the real world, you would not know the kcat or KM of the enzyme, and the
process to find it is to
         Find the concentration of enzyme at which you can measure the kinetics
           such that you have a linear rate of reaction with time. Obviously, this depends
           on the concentration of the enzyme relative to the kcat and the frequency of
           measurement of your instrument.
         Find the concentration range of substrate that enables you to fit a
           Michaelis Menten model to the data. If you measure with concentrations
           only BELOW the KM then the data will appear linear and the Michaelis
           Menten model will not fit accurately. If you measure with concentrations only
           ABOVE the KM then the data will again appear linear. Hence, if you knew in
           advance the value of the KM you would chose concentrations over a range of
           about 10 fold both below and above the KM.
Since there is not adequate time in the lab to make all these measurements, this
procedure will give you the range of substrate concentrations you should use and also
the best concentration of the enzyme, and your goal will be to demonstrate the kinetic
measurements that can verify the kcat and KM.
        The substrate stock solution is prepared by dissolving 43.5 mg of substrate
          in 5 ml of dimethyl-sulfoxide. You can do this in a polypropylene culture
          tube.

       Do this immediately when you come
       to the laboratory.

       Vortex briefly, then place your eppendorf
       tube on the rotating shaker as shown. Spin
       rapidly.




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                                                              Student Procedure Protein Week I

After 10 minutes, you should be able to see no more undissolved crystals of the
substrate.
       2. You will be adding substrate (Na-Benzoyl-DL-arginine-4-nitroanilide
hydrochloride, mol. wt. 434.89) to solutions of Tris-HCl over the range of 0.1 mM to 4
mM. The solutions you prepare of the substrate should be identical in every way except
the concentration of the substrate. Because your substrate is in DMSO, you will need to
add pure DMSO to some of the samples so that your DMSO is constant in the samples.
       Prepare samples into spectrophotometer cuvettes according to the following table
of substrate concentrations.

                         Volume Substrate                         Volume of
 Volume of Tris buffer    Stock Solution    Volume of DMSO         enzyme        [substrate]
     (microliters)         (microliters)      (microliters)      (microliters)      mM
         700                     0                200                100              0
         700                    20                180                100             0.4
         700                    40                160                100             0.8
         700                    60                140                100             1.2
         700                    80                120                100             1.6
         700                   100                100                100              2
         700                   120                 80                100             2.4
         700                   140                 60                100             2.8
         700                   160                 40                100             3.2
         700                   180                 20                100             3.6
         700                   200                 0                 100              4

IMPORTANT: Once you add the enzyme, the reaction will start, so you will need to
prepare your sample vials with all but the enzyme and then add the enzyme just prior
to making the spectrophotometric measurement.

       3. For the enzyme stock solution, start with 45 mg of enzyme in 100 ml of cold
0.001 N HCl. Dissolve the enzyme by vigorous vortexing then keep the solution on ice
to prevent autohydrolysis. It will hydrolyze, so you can make fresh solution over the
course of the experiment. Prepare the enzyme solution when you are all ready
to begin making measurements. You will need to go as quickly as possible
to prevent significant measurements errors due to enzyme autohydrolysis.

       4. Now you are ready to begin the kinetic measurements. Pay attention to the
order of the following steps:
        Prepare your culture tubes with the tris and pure DMSO and leave them.
        Familiarize yourselves with the spectrophotometer. Test a run with an empty
          cuvette to be sure that you are getting the proper data. You should use the
          kinetic method, set the wavelength to 410 and your sample time is
          60 seconds with a cycle time of 2 seconds. Each measurement can be
          saved as a separate *.kd file which you will export as a *.csv file when you have
          finished all the measurements. If you can't use a spec that has a kinetics
          program, you can write the absorbance value every 10 seconds until you have 7
          values or so and then input these by hand later.
        Prepare the enzyme stock solution and keep on ice.

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                                                     Student Procedure Protein Week I

 Now you can prepare your first sample. You should randomize the order
  of the samples to reduce bias error. Take the tube of substrate and add
  the appropriate volume to your cuvet. Then add 100 μl of your enzyme stock
  solution. Using the pipette from the enzyme addition, gently pipette up and
  down a few times in the cuvette until it all appears to be well mixed.
 Place the cuvet in the spectrophotometer and take the measurement.
 The cuvettes should be reused so that you do not consume more than 30
  cuvettes during the period of the lab. Rinse the cuvettes 3 times with tap water
  then again 2 times with distilled water and leave them upside down when
  you are finished.
 Be sure to shut off the spectrophotometer lamp before you leave the
  lab.
 Export all your data files and create an excel spread sheet with the data. Now
  you are ready to calculate the kcat and KM.




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