Analysis of Wheat Germ Acid Phosphatase
Lab Week 6
An enzyme is a biological catalyst, made of protein. It works by speeding up reactions that otherwise might not happen. For instance, if you dissolve table sugar (sucrose) in water, the sucrose will remain stable for a very long time. However, add an enzyme, and the sucrose can be broken apart into its two monosaccharide components (glucose and fructose).
In an enzymatic reaction, substrate gets converted to product, and the enzyme remains unchanged, able to be recycled:
enzyme
substrate (S) ------ product (P)
and the rate of an enzymatic reaction can be measured in terms of the rate of disappearance of substrate, or rate of appearance of product:
rate = -[S]/t = [P]/t
Each enzyme has an optimal temperature and pH range; changing the temperature and pH can significantly affect the activity of the enzyme. In fact, deviations too far from optimum may cause denaturation and inactivation. Thus, when measuring the rate of an enzyme-catalyzed reaction, it is important to hold these two factors constant. Rate of an enzymatic reaction is proportional to substrate concentration up to a point; when all enzyme molecules have been consumed, adding additional substrate will not increase reaction rate, and the enzyme is said to be saturated.
To determine the rate of an enzymatic reaction, start with a fixed concentration of enzyme and substrate. During the early part of the reaction, the amount of product formed increases linearly with time. However, in the latter part of the reaction, the rate of product appearance diminishes to a point where product is no longer formed. This may occur for a number of reasons, including depletion of substrate or denaturation of enzyme. Therefore, the rate of an enzymatic reaction is determined during the early phase of the reaction – called the initial velocity.
Wheat germ acid phosphatase catalyzes the hydrolysis of phosphate groups from macromolecules and smaller molecules that are stored in the wheat seed. The growing wheat embryo uses the freed phosphate in germination and growth. In this experiment, you will measure the velocity of the reaction catalyzed by purified acid phosphatase. Nitrophenyl phosphate, a colorless compound, will be used as a substrate in the experiment. The hydrolysis products are nitrophenol and phosphate.
Under alkaline conditions, nitrophenol is converted to a nitrophenoxide ion, which is yellow. Using a spectrophotometer, the concentration of product (nitrophenol) can be measured by the increase in absorbance due to the yellow color.
The addition of base at the end of the reaction serves two purposes. First, it converts the product into a colored substance that we can use to measure concentration. Second, it stops the reaction by denaturing the enzyme. In this way, we can take samples out of the reaction mixture at various timepoints, and determine the amount of product that had been formed at each point.
There are two parts to this experiment. First, you will use acid phosphatase to catalyze the hydrolysis of nitrophenyl phosphate. The reaction will be stopped by the addition of base at various time intervals. The concentration of nitrophenol can be determined by measuring the absorbance and extrapolating the concentration from the standard curve.
Then, you will make your standard curve. You will prepare a series of test tubes containing increasing concentrations of nitrophenol. You will measure the absorbance of the different concentrations under alkaline conditions. The absorbance should vary linearly with concentration. If you plot the concentration of nitrophenol vs. absorbance, you can use this standard curve to determine the concentration of an unknown sample.
EQUIPMENT
stopwatchspectrophotometer
10 mL graduated cylindersmall beaker
small and large test tubestransfer pipet
test tube rack
CHEMICALS
1 mM nitrophenyl phosphate in 0.09 M citrate buffer, pH 4.8
0.3 M KOH solution
4 mM nitrophenol standard
wheat germ acid phosphatase
PROCEDURE
Part A: Enzyme Assay
1 Prepare a rack of 10 small test tubes that will fit in the spectrophotometer. Label the tubes from 1-10.
2. Use a 1-mL transfer pipet to place 1 mL of KOH solution into each tube.
3. Obtain approximately 15 mL of phosphatase substrate (nitrophenyl phosphate solution) in a small beaker.
4. Transfer 11 mL of the phosphatase substrate from the beaker into a large test tube.
5. Using a 1-mL transfer pipet, place 0.5 mL of wheat germ acid phosphatase in the large test tube (along with the substrate). Mix the tube by shaking gently. Immediately remove 1 mL and place in test tube 1 for your first time point! Begin timing!!
6. At the times indicated below, remove 1 mL of the solution from the large test tube. Place the solution into the tube corresponding to the time o the withdrawal.
Time Interval / Tube Number0 minutes / 1
1 minute / 2
2 minutes / 3
3 minutes / 4
4 minutes / 5
5 minutes / 6
7.5 minutes / 7
10 minutes / 8
15 minutes / 9
20 minutes / 10
7. Add 2 mL of distilled water to each test tube.
Part B: Preparing Serial Dilutions
8. Number another set of test tubes from 1-6.
9. Add 2 mL of distilled water to test tubes 1-5 and 3.8 mL of distilled water to test tube 6.
10. Add 0.2 mL nitrophenol standard (4 mM) to test tube 6 and mix the contents.
11. Transfer 2 mL of the contents of test tube 6 to test tube 5. Mix well.
12. Transfer 2 mL of the contents of test tube 5 to test tube 4. Mix well. Continue in this manner until you have transferred 2 mL of the solution from test tube 3 to test tube 2 and mixed the contents.
13. Remove 2 mL of the solution from test tube 2 and discard.
14. Add 2 mL of 0.3 M KOH to each of the 6 test tubes.
15. Number a corresponding set of test tubes from 1-6. Using a transfer pipet, place 2 mL of each of these standards into the corresponding tube.
16. Before the addition of base, these test tubes contained the following concentrations. Use the chart below when plotting your standard curve.
Tube # / Concentration1 / 0 nmol nitrophenol/mL
2 / 25 nmol nitrophenol/mL
3 / 50 nmol nitrophenol/mL
4 / 100 nmol nitrophenol/mL
5 / 200 nmol nitrophenol/mL
6 / 400 nmol nitrophenol/mL
Part C: Standard Curve and Concentration Measurement
17. Allow the Spec-20 to warm up at least 5 minutes prior to taking readings.
18. Set the wavelength to 410 nm. You will read the standard curve first. Use test tube 1 of your standard curve as the blank (set at zero absorbance). Adjust your spectrophotometer as instructed in lab.
19. Read and record the absorbance of each of the nitrophenol standards, test tubes 2-6.
20. Read and record the absorbance (A) of each time point tube (1-10) from your enzyme assay.
21. (the next two steps may be done at home) For your standard curve tubes, plot concentration on the x-axis vs. absorbance on the y axis.
22. Using your standard curve plot from step 21, determine the number of nanomoles of nitrophenol produced in each of your enzyme assay tubes.
PRELAB QUESTIONS
1. When you are measuring rate of an enzymatic reaction, holding concentration of enzyme constant while increasing concentration of substrate, the rate of the reaction eventually slows down despite the fact that you continue to add more substrate. Explain what is happening.
2. Why do we add base (KOH) to our reaction tubes?
POSTLAB QUESTIONS
1. In a table, show how many nanomoles of nitrophenol were produced in each of your enzyme assay tubes (step 22 of the experimental steps).
2. Draw a plot of nanomoles of nitrophenol produced vs. time in your enzyme reaction. (You may do this by hand or using Excel.) Is it a straight-line plot? (or close to, using a best-fit line) Does it slow down at any point?
3. Using initial rate data from the beginning of the curve, calculate a rate for the production of nitrophenol.
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