Sonoma State University
Department of Biology - Animal Physiology

Introduction: Chemical reactions in living organisms occur rapidly at moderate temperatures and under mild conditions primarily because of the catalytic action of specialized proteins called enzymes. Each step in a chain of biochemical reactions is usually catalyzed by a specific enzyme. The lack of even one enzyme can prove fatal. The actual catalytic site of an enzyme molecule is a small area where the enzyme's component amino acids are arranged to precisely "fit" its substrate, which is the substance acted upon by the enzyme and changed to the product. The rate of an enzyme-catalyzed reaction depends partly on how well the enzyme and substrate fit together. Thus an environmental factor (such as pH or high temperature) which might change the shape of either enzyme or substrate could alter the rate of reaction. In addition the concentration of active enzyme and substrate molecules would be expected to have important effects on the rate. The rate of an enzyme-catalyzed reaction may be measured by (1) the disappearance of substrate, or (2) the appearance of product. The enzyme and substrate are mixed and allowed to react for a certain time period; the amount of either the substrate or the product is then measured. This amount gives the rate of activity of the enzyme per unit of time. Alkaline phosphatase, the enzyme we will be studying this week, is important in recycling phosphate within living cells. It seems to be particularly prevalent in tissues which are transporting nutrients, including intestine and kidney. This enzyme catalyzes the cleavage of a phosphate group from a variety of compounds, including the "artificial" substrate used here, p-nitrophenyl phosphate. This substrate is colorless. However, one of the products, p-nitrophenol, is yellow in basic solutions. The appearance and intensity of yellow color thus indicates the degree to which the substrate has been acted upon by the enzyme.

Tips on Use of the Spectronic 20 Spectrophotometer: The amount of p-nitrophenol resulting from the enzyme- catalyzed hydrolysis of p-nitrophenyl phosphate is measured using a spectrophotometer, an instrument capable of detecting the amount of light absorbed by colored solutions. According to Beer's law, A = E X L X C where A is the measured absorbance, E is an empirical constant (the extinction coefficient), L is the length in centimeters of the light path through the sample solution, and C is the concentration of absorbing material in moles/liter. The amount of light absorbed (absorbence) is thus directly proportional to the concentration of absorbing material. Follow these steps to operate the spectrophotometer:

a. APPLY POWER -- Turn the power switch (left knob) clockwise to turn on the instrument. Allow the instrument at least five minutes to warm up, longer in older instruments. b. SELECT THE WAVELENGTH -- Turn the wavelength control (top knob) to the desired wavelength in nanometers (10^-9 meters). c. ADJUST DARK CURRENT -- With no sample in the cuvet holder, adjust the left knob so that the meter needle reads infinite absorbence marked at the left side of the meter scale. d. STANDARDIZE THE LIGHT CONTROL -- Add at least 3 milliliters (ml) of "blank" solution (may be water or buffer, depending on the experiment) to a clean cuvet (a special test tube). Wipe fingerprints from the outside of the cuvet, using a Kimwipe, and insert it all the way into the cuvet holder, aligning the mark on the cuvet with the mark on the holder. Adjust the light control (right knob on front) until the meter needle reads zero absorbence (right-most mark on meter). Remove the "blank" cuvet and set aside for later use. e. MAKE A SAMPLE MEASUREMENT -- Add at least 3 ml of the sample solution to be measured to a clean cuvet, wipe the outside of the cuvet, and insert the cuvet into the holder, aligning marks as before. Now read and record the absorbence, interpolating the third decimal point. Treat the cuvets with care to avoid scratching them.

Determination of the Absorbence Maximum:

The first task will be to construct an "absorbence spectrum" to determine the wavelength at which the colored substance (in this case p-nitrophenol) demonstrates maximum absorbence. This wavelength will then be used in all subsequent measurements to optimize sensitivity of the spectrophotometer. To the "blank" cuvet, add 3 ml of a solution of 0.04 M sodium hydroxide (NaOH). Starting with a wavelength of 360 nm, use the blank cuvet to standardize the light control as described above. To the "sample" cuvet, add 1.0 ml of a solution of 0.5 mM p-nitrophenol to 2.0 ml of 0.04 M NaOH. Using the vortex mixer, mix thoroughly. Then take an absorbence measurement of the sample at 360 nm wavelength. Record the wavelength and absorbence value. Adjust the wavelength to 370 nm and repeat steps c, d, and e above. Increase the wavelength in steps of 10 nm and repeat your measurements until you are satisfied that you have found the absorbence maximum. You could instead place the cuvette with p-nitroanaline in the Spectronic 20, start at 360 nm and slowly turn the dial to change wavelength until you find the maximum absorption. Usually the best results are at a wavelength of near 460 nm.

Construction of a Standard Curve:

Using large test tubes, set up a standard dilution series in duplicate (two tubes of each) as follows, pipetting as carefully as possible:

0.04 M/l NaOH	0.5 mM/l p-Nitrophenol in 0.04M/l NaOH
4.0 ml	1.0 ml
3.0	2.0
2.0	3.0
1.0	4.0
0.0	5.0

Mix thoroughly by careful vortexing. Using the wavelength producing maximum absorbence, standardize the light control using your "blank" cuvet. Then measure the absorbence of each of the standard dilutions by pouring the contents of each test tube into a cuvet and reading the absorbence, rinsing with distilled water between each reading. Be sure to wipe fingerprints between each reading and align the marks properly.

Experiment:

We will measure the effects of substrate concentration on the functioning of the enzyme, alkaline phosphatase. The results we obtain will tell us much about the properties of the enzyme we are studying and in fact can tell us about the functioning of the tissue from which the enzyme was prepared. By varying the substrate concentration and measuring the rate of the enzyme catalyzed reaction, we will be able to construct a "Michaelis-Menten plot", graphing rate (in micromoles p-nitrophenol/minute) on the Y-axis against substrate concentration (in moles p-nitrophenylphosphate/liter) on the X-axis. The maximum rate will be designated as V_max, and one-half that value will be calculated. The Michaelis constant, K_m, is defined as the substrate concentration at one-half V_max. This K_m value is a very useful diagnostic estimate of the affinity between enzyme and substrate; the lower the K_m the higher the affinity and the more efficient the enzyme can be.

Procedure: Effect of Substrate Concentration on Enzyme Activity Set up thirteen (13) glass test tubes as follows. Volumes are given in microliters, not milliliters. Be sure to wipe the pipette tips after filling, using a clean Kimwipe. Add only the first four ingredients, then place the test tubes into a 37^oC water bath. The buffer should be 0.1 M 2 amino-2-methyl-1-propanol in 0.001 M MgSO4-7H2O, pH 10.0. The substrate is 0.01 M p-nitrophenylphosphate.

Where: buff = buffer, PNP-p, paranitrophenolphosphate = substrate, DW = deionized water, PNP = p-nitrophenol, Enz = the enzyme alkaline phosphatase, AB = absorbence.

When your tubes are ready, add enzyme (homogenate) at 20-second intervals, vortexing each tube and placing it back in the 37^oC water bath. Exactly 20 minutes after the first addition of enzyme, add 4.0 ml 0.04 M NaOH to stop the reaction and vortex to mix. Continue adding NaOH to each tube in succession at 20-second intervals so that each incubation lasts exactly 20 minutes. Set your spectrophotometer at the optimum wavelength for p- nitrophenol. With the cuvet holder empty, adjust the needle to the left mark on the scale (infinite absorbence) using the left-hand knob. Add at least 3 ml of tube B to a cuvet and insert it into the cuvet holder matching lines on tube and holder. Adjust the needle to the right mark on the scale (zero absorbence), using the right-hand knob. Remove and set aside the cuvet containing the B (blank) solution. Pour at least 3 ml of incubation tube 1 into a clean cuvet, wipe the outside of the cuvet and insert it into the cuvet holder. Without touching the knobs on the spectrophotometer, read the absorbence to three decimal places. Empty this cuvet into the waste beaker, pour contents of tube 2 into the empty cuvet, and read the absorbence in the spectrophotometer. Proceed with each incubation tube in succession, pouring the waste into the large beaker provided. Record your data in your lab notebook.

Data Analysis: Ask for a demonstration of Excel software. You may use either Excel, Lotus or SlideWrite to produce the graphs you will need, but you should learn enough about the software to do the calculations. It's wonderful, actually! Calculate the alkaline phosphatase activity for each of your incubation tubes as follows: Calculate the mean absorbence of tubes 1 and 2 from the substrate run. These tubes contain 0.1 ml of a solution of 0.5 micromoles/ml p-nitrophenol. How many micromoles are represented? Now divide the average absorbence by the calculated micromoles to give absorbence/micromole. This value represents the slope of the standard curve for p-nitrophenol, calculated using two points: 0,0 and the absorbence and micromoles for tubes 1 and 2 from the substrate run. Now divide each of the absorbences for tubes 1-10 by the calculated absorbence/micromole value, to give micromoles of p-nitrophenol produced by your enzyme over the 20 minute incubation. To get micromoles p- nitrophenol/minute, divide by 20. This is the "activity" of alkaline phosphatase measured under the conditions defined by the experiment. In the substrate experiment, you will need to calculate the actual concentration of substrate in moles per liter for each incubation. You know that the original concentration of substrate is 0.01 moles/liter. All incubation tubes will contain an incubation volume of 1.0 ml (1000 µl). For tubes containing 100 µl of substrate solution, the concentration of substrate will thus be diluted by 10, and will equal 0.001 moles/liter. You should be able to calculate substrate concentrations for the remaining tubes. Plot alkaline phosphatase activity ( moles p-nitrophenol produced per minute) on the Y-axis against substrate concentration (moles/liter) on the X-axis. Manually determine Vmax, calculate one-half that value, and read K_m on the substrate scale (in units of moles/liter). You should also use an alternative method of calculating K_m by means of a double-reciprocal (Lineweaver-Burke) plot, where 1/activity is plotted against 1/substrate concentration. Here, the Y-intercept will be 1/ V_max, the X-intercept will be -1/K_m, and the slope of the straight line will be K_m/V_max. How does the result of this method to calculate K_m compare with the first method?

Questions:

1) What can you say about the functioning of alkaline phosphatase in the particular tissue chosen?

2) Does a freshwater organism like the crayfish show the same levels or properties of alkaline phosphatase as the blue crab? If you homogenize crayfish gills and test for alkaline phosphatase, would you expect to see similar levels of activity of the enzyme?

NOTES Chemicals, glassware and equipment needed Chemicals

6 X 100 ml 0.04 M/l NaOH
PNP 6 X 30 ml 0.5 mM/l p-nitrophenol in 0.04 M NaOH
Buff 6 X 30 ml 0.1 M/l 2-amino-2-methyl-1-propanol in .001 M MgSO₄ . 7H₂O
Sub 6 X 10 ml 0.01 M/l p-nitrophenylphosphate in propanol buffer
put any extra substrates and buffer in the lab.
Enz 10 ml of 0.5 units/ml alkaline phosphatase in buffer

 

Glassware

12 cuvets for Spectronic 20
90+ test tubes that hold about 10 ml
12 50 ml beakers

Supplies

6 Kimwipes
Parafilm
20 sheets arithmetic graph paper
1 bag large (5 ml) pipette tips
1 bag small (200 microliter) pipette tips

Equipment

6 Spectronic 20
2 water baths @ 37^o
6 vortex - in lab
6 test tube racks

Animals

1000 units of alkaline phosphatase