Enzyme Isolation and Characterization
Potatoes store large quantities of starch. Several enzymes are involved in construction of starch molecules from glucose and then their break down when needed for energy and growth. One of the major enzymes involved in this process is starch phosphorylase. This enzyme catalyzes the following reaction:
An enzyme with similar activity, glycogen phosphorylase, helps to regulate sugar and glycogen levels in humans and other animals. In this lab you will isolate starch phosphorylase by taking advantage of differences in its physical characteristics including heat stability and solubility in salt solutions.We will then look at the rate of this reaction in both directions, starting first with the breakdown of starch and then with the production of starch.
Procedure:
Enzyme isolation
1Peel and cube approximately 400g of potatoes.
2Place in a blender with 100 ml of ice cold water.
3Homogenize until a thick slurry is obtained.
4Filter homogenate through cheese cloth then a Watman No. 1 filter using a Buchner funnel and suction.
5Place the filtrate in a 50 oC water bath for 5 min to inactivate amylase and other enzymes (this will not inactivate starch phosphorylase).
6Cool then add 20 g of solid (NH4)2SO4 for each 100 ml of filtrate.
7Centrifuge to remove precipitated proteins.
8Decant and save the supernatant.
9To precipitate phosphorylase, add 15 more grams of (NH4)2SO4 for every 100 ml of supernatant.
10Centrifuge to precipitate phosphorylase.
11Remove and discard supernatatant.
12Resuspend the pellet in 10 ml of KOH malate buffer pH 6.5.
Construction of a standard curve
1Starting with a 5 mg/ml starch solution, make solutions with the following concentrations: 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.5, and 0.0 mg/ml.
2Place 5 ml of each solution into a cuvette then add 20 micro liters of IKI solution.
3Mix then read the absorbance of each solution at 660 nm using a Spec 20.
4Make a plot of absorbance versus concentration.
Measurement of the rate of starch breakdown
1Place 6 ml of starch solution into 5 cuvettes along with 1 ml of 5 mg/l KH2PO4 solution.
2Number tubes 0, 15, 30, 45, and 60 seconds.
3Get ready to start timing.
4As quickly as possible, add 2 ml of the enzyme containing solution to each tube.
5Start timing, and add 20 microliters of IKI to tube 0 (this will stop the reaction).
6After 15 sec add IKI to tube 15, after 30 sec to tube 30 and so on until you get to tube 60.
7Measure and record the absorbance at 660 nm of each tube.
8Calculate the starch concentration based on the standard curve constructed in the previous section.
Measurement of the rate of starch synthesis
1Place 2 ml of glucose-1-phosphate solution into 5 cuvettes.
2Number tubes 0, 15, 30, 45, and 60 seconds.
3Get ready to start timing.
4As quickly as possible, add 2 ml of the enzyme containing solution to each tube.
5Start timing, and add 20 microliters of IKI to tube 0 (this will stop the reaction).
6After 15 sec add IKI to tube 15, after 30 sec to tube 30 and so on until you get to tube 60.
7Measure and record the absorbance at 660 nm of each tube.
8Calculate the starch concentrations based on the standard curve constructed in the previous section.
Determination of Km
Km is a constant that allows comparison of the rate at which enzymes speed up reactions. The velocity of a reaction changes as equilibrium is approached, therefore, the maximum velocity is at the start of the reaction when there are no products, only substrates (V0). When larger quantities of substrate are added, at the start of the reaction, the velocity increases up to a point. After a while, all the enzyme molecules are busy catalyzing the reaction, so they can’t make the reaction go any faster. This situation is referred to as the maximum velocity (Vmax) of a reaction. Of course, it is possible to speed thing up even more if you add more enzyme, but by keeping the enzyme concentration constant, it is possible to compare different enzymes by comparing Vmax. Km is defined as the substrate concentration at which velocity is half Vmax.
1Calculate velocities in terms of moles or grams/min using the data collected earlier. Plot the velocity (on the Y axis) versus concentration. You will note that it is not easy to get Vmax from this data directly as it plots a curve.
2Take the reciprocal of both your velocity and concentration data. This data can be used to make a Lineweaver-Burk plot. Plot the reciprocals, each on the same axis used previously, and something approaching a straight line should result. The point at which this line intercepts the Y axis (1/V) is 1/Vmax. The point at which the line intercepts the X axis is 1/Km.
Supplies:
Potatoes
Ammonium sulfate (NH4)2SO4 crystals
0.1 M KOH malate buffer pH 6.5
Glucose-1-phosphate solution 30 mg/ml
IKI solution
Starch solution 5 mg/L
Potassium phosphate KH2PO4 5 mg/ml