Experiment 2
EFFECT OF EXTRACTION CONDITIONS AND TITRIMETRIC ASSAY OF
ENZYME ACTIVITY
Some enzymes are present in extracellular fluids, secretions and excretions;
assay and purification of the enzyme may be done directly on those fluids. Other
enzymes are contained in cells either in the cytoplasm or in subcellular organelles.
The cellular and organelle membranes must be ruptured by grinding, blending,
pressure or ultrasonic oscillation in order to release the enzyme. The pH and ionic
strength, as well as dielectric constant, of the extracting fluid is often quite
important in releasing and stabilizing the enzymes. We will use isobutanol in
Experiment 11 to solubilize alkaline phosphatase from its adsorption on the fat
globules of milk. Plant cells concentrate acids in the vacuoles. When the cells and
vacuoles are ruptured the acids are released and will denature enzymes not stable at
lower pHs. Denaturation is prevented during extraction by use of a buffer to
maintain the pH in a range where the enzyme is stable. Enzymes often are
attached to membranes and cell walls and are released only at higher ionic
strengths. In this experiment we shall look at the effect of three extraction
conditions on the amount of pectinesterase activity recovered from the albedo
(white) of an orange.
During many enzyme reactions a proton is either a substrate or product of
the reaction. In the presence of a buffer with adequate capacity the pH is
maintained constant and one does not observe a change in the [H+]. If the buffer is
eliminated, or is in low amount, the pH will change in such reactions. If NaOH or
HCl is added to the reaction at a rate that keeps the pH constant, then the rate of
addition of titrant measures the rate of the enzyme reaction. All one needs to
obtain the rate of reaction is a pH meter, a burette filled with titrant of known
concentration, a timer and lots of patience. A pH stat, designed to automatically
add and record the amount of titrant needed to keep the pH constant, greatly
simplifies the experimental work.
Pectinesterase is found in most higher plants and is in particularly large
amounts in the albedo of the orange. Pectinesterase catalyzes the hydrolysis of the
methyl ester bond of pectin giving pectic acid and methanol as products (Eqn. 1).
Assuming a pK of 4 for the carboxyl group, above pH 6 an equivalent amount of
H+ will be liberated for each ester bond hydrolyzed.
O
O
O
COCH3
COCH3
CO-
O
O
O
OH
O
OH
O
OH
O
O
n
O
OH
OH
OH
Pectin
COO
2 H2O
O
OH
O
OH
Pectic acid
+ 2 CH3 OH + 2 H+
REAGENTS:
• Extraction fluids: The solutions have been prepared for you. Chill them in an
ice bucket. They are
0.5% NaCl
0.1 M sodium phosphate, pH 7.5
0.1 M sodium phosphate, 0.5% NaCl, pH 7.5
• Oranges (1 case for two laboratory sections)
• Crude pectinesterase: Peel two oranges so as to just remove the flavedo
(orange-colored skin). Then strip the albedo from the pulp of the orange. The
albedo will be used as described below.
• Pectin: 1% solution in 0.1 M NaCl. Pectin (high methoxy) or any good grade
purified pectin may be used. The pectin must be added slowly to the NaCl
solution, with stirring, to dissolve the pectin.
• NaOH. 0.02 or 0.1 N , standardized. The more dilute NaOH is used with
low enzyme activities.
PROCEDURE:
Extraction of enzyme. Weigh out 10.0 grams of albedo and add to it 50 mL of
cold 5% NaCl. Blend at top speed in a Waring blender for only 10 sec. Blending
too long will give time for partial enzymatic hydrolysis of the orange pectin to
pectic acid and the pectin will gel. Filter on a small Buchner funnel under low
vacuum. You will not need to filter all the material to obtain the amount of
enzyme needed for your experiments.
n
Repeat this extraction procedure with 0.1 M phosphate, pH 7.5, and 0.1 M
phosphate, pH 7.5, containing 5% NaCl as extracting fluids.
Activity determinations. I. Place 20 mL of 1% pectin solution containing 0.1 M
NaCl into a 50 mL beaker. Place a magnetic stirring bar in the beaker, and with
the beaker on a magnetic stirring motor lower the pH electrodes so that they are in
the liquid but do not touch the stirring bar. Turn on the stirrer. Adjust the pH to
about pH 6 by adding 0.1 M NaOH dropwise. Add 0.1 to 2.0 mL of one of the
enzyme solutions and immediately readjust the pH to 7.50. At this time start the
timer. Add NaOH from the 50 mL burette to maintain the pH essentially constant.
Take readings of the amount of NaOH added at approximately one min intervals
for 10 min. Be sure the pH reads exactly 7.50 when the reading is taken and record
the exact time of the reading. You can overshoot the titration slightly, then record
the time when the pH reaches 7.5. The amount of enzyme used should be sufficient
to cause a reasonable rate of ester hydrolysis but not so much as to cause the pH to
change faster than can be compensated for by NaOH addition.
Repeat the procedure for each of the enzyme preparations.
Record the temperature of the reaction as determined in the reaction vessel.
Definition of unit of activity and avtivity yield. One unit of activity is the
amount of enzyme that will hydrolyze one millimole of ester per min under the
conditions used. Activity yield is the number of units extracted per gram of albedo.
Activity determinations. II. A more convenient procedure to determine [H+]
produced by hydrolysis of the methyl ester is to use a pH indicator dye in an
unbuffered solution. We will use bromthymol blue (also called bromothymol blue),
which has a pKa of 7.10. It is blue above pH 7.6, and yellow below pH 6. We will
measure the decrease in the blue color by measuring the absorbance at 620 nm in
the spectrophotometer, which means that we will have a continuous assay, unlike
the titration described above. The system is calibrated by using a standard curve
obtained from samples containing bromthymol blue and several concentrations of
galacturonic acid.
The method is summarized in the following table:
A: Enzymatic Analysis
Tube
Compound
Kinetic Assays
A
B
C
All volumes are mLs
Water
2.00
2.00
2.00
1% Pectin pH 7.5/0.1M NACL
1.00
1.00
1.00
0.3% Bromthymol Blue pH 7.5
0.02
0.02
0.02
A: enzyme prep: 1M NACL
0.10
NA
NA
B: enzyme prep: 100 mM phosphate pH 7.5/1M NACL
NA
0.10
NA
C: enzyme prep: 100 mM phosphate pH 7.5
NA
NA
0.10
Note: Do not add enzyme prep until at spectrophotometer!
Kinetic Mode:
Program: Pectinesterase
Wavelengh: 620 nm
Cycle time=1sec
Total time=100 sec
Scale=0.5-1.0 Absorbance
Referance Blank is Type I H2O
B. Standard Curve
TUBE
Compound
Photometric Assay
1
2
3
4
All volumes are mLs
Type I Water
2.00
2.00
2.00
2.00
1% Pectin pH 7.5/0.1 M NACL
1.00
1.00
1.00
1.00
0.3% Bromthymol Blue pH 7.5
0.02
0.02
0.02
0.02
0
0.02
0.04
0.08
0.10
0.08
0.06
0.02
5 mM galacturonic acid
Type I Water
Photometric Mode:
Program: Galacturonic
wavelengh: 620 nm
Referance Blank is Type I H2O
QUESTIONS:
1. Describe some other variables that might be investigated in a more complete
experiment on factors affecting efficiency of extraction of an enzyme.
2. Suppose the titration version of the experiment had been performed at pH 5.0.
Show how the measured milliequivalents (millimoles) of NaOH uptake relate to
the milliequivalents of ester hydrolyzed.
3. Describe at least two other experimental procedures that might be used to
follow pectinesterase activity.
4. Calculate the ionic strength of each of the extracting fluids.
EXPERIMENT 2
Effect of Extraction Conditions and Titrimetric Assay of Enzyme Activity
Laboratory Report
Name:
Date Performed:__________________
A. Extraction
1. Record data on the exact amount of albedo, extracting fluid and blending
conditions used.
B. Assay of enzyme activity
1. Submit a graph in which millimoles of ester hydrolyzed (y axis) is plotted
versus time for each of the reactions. Indicate the temperature of the reaction
and the amount of extract used.
2. Determine initial velocities, units of activity and activity yield for each
preparation and report the values in a table. Discuss the reasons for the
different results obtained with the different extracting fluids.
3. Submit answers to the questions (previous page).