41
Experimental Section
The Influence of pH on Electrophoretic Mobility, A
Laboratory Investigation
S FRISCIA l, S L TURCHI 2 and C E HEPFER 1
Departments of Biology I and Chemistry 2
Millersville University of Pennsylvania
Millersville, PA 17551, USA
Introduction
Electrophoresis is often used to separate and identify com-
ponents of a mixture of biological molecules. The rate and
direction of movement for a specific molecule in an electrical
field depends upon its size and charge. 1 The magnitude and sign
of the charge depends on the particular molecule, the pH and the
solvent. 2 When placed in a pH gradient, a biological molecule
will migrate in an electric field until it reaches a position within
the gradient at which it has no net charge. The pH at this
position is referred to as the isoelectric point or pI for that
molecule.
In undergraduate biochemistry laboratories, SDS-polyacryl-
amide gel electrophoresis (PAGE) is routinely employed to
demonstrate the principles of molecular separation. This de-
naturing technique allows separation on the basis of size alone.
Non-denaturing P A G E may be used to provide students with
some information about the effects of charge, but molecular size
still significantly influences mobility. Despite its importance, the
influence that pH has on the rate and direction of movement of
biological molecules is rarely demonstrated due to technical
complexity and time limitations. As a result, undergraduate
experience with important electrophoretic applications such as
determination of isoelectric point is severely limited.
In order to alleviate this problem, an exercise has been
developed that employs agarose gel electrophoresis to separate
three molecules that migrate differentially due to the influence
of pH and molecular size. The colors of these substances enable
direct identification of resultant bands thus eliminating the
lengthy time periods needed for staining and destaining. Agar-
ose gels are easily prepared, inexpensive, and much less toxic
than polyacrylamide. The large pore size of the gel enables rapid
movement of most molecules and allows the completion of the
electroi~horetic exercise within one three-hour laboratory
period. ° Varying the pH conditions enables students to observe
directly the inflence of charge on electrophoretic mobility and to
estimate the isoelectric point of each of the molecules employed.
Aim of the Practical
The students are given a mixture containing three colored
molecules (blue dextran, cytochrome c and DNP-lysine) and
asked to separate them under different pH conditions using
agarose gel electrophoresis. The properties of these substances
are outlined in Table 1. Each of the colored bands is identified
and the electrophoretic results at pH 3, 7, and 11 are compared.
Students are asked to determine the net charge of each type of
molecule at each pH and to estimate the range of the isoelectric
point of each substance.
Table 1 Characteristics of biological molecules
Charge
Name Color M, pH 3 pH 7 pH 11 pl
Blue dextran 4"6 Blue 2 000 000 + 0 - ~7.0 a
Cytochrome c4"6 Red 12 400 + + - 10.70
DNP-lysine7 Yellow 348 + - - <5.56
aThe charge of blue dextran's chromophore is influenced by pH;
however, the high Mr prevents its migration into the gel
BIOCHEMICAL E D U C A T I O N 20(1) 1992
Materials
Colored molecules A stock solution containing blue dextran
(25 mg/ml), cytochrome c (15 mg/ml) and DNP[N,-2,4-dinitro-
phenyl]-L-lysine (12 mg/ml) was prepared in 0.02 M phosphate
buffer, pH 7.0 and stored at 4°C. These substances were
purchased from Sigma Chemical Co (St Louis, MO, USA).
Electrophoretic buffers Buffers at pH 3, 7, and 11 were
prepared by adding an appropriate pHydrion capsule (Sigma
Chemical Co) to distilled water according to the manufacturer's
directions. Alternatively, buffers may be prepared as follows.
For pH 3.0, add 0.05 M trisodium citrate to 91 ml 0.05 M
citric acid for a final volume of 100 ml. 1
For pH 7.0, add 30 ml of 0.2 M sodium hydroxide to 50 ml
0.2 M sodium dihydrogen phosphate and dilute to 100 ml final
volume with distilled water.1
For pH 11, add 0.1 M sodium carbonate to 5.5 ml 0.1 M
sodium dicarbonate for a final volume of 100 ml. 1
Agarose gels Gels (1.0% w/v) were prepared in the appropriate
pH buffer immediately before the laboratory as described in
Maniatis et al. 5 An 8 tooth comb (0.8 mm thickness) was used to
form sample wells. After the gels had solidified, buffer at the
appropriate pH was added to cover the gel and the well comb
removed. Gels submerged in this manner may be stored at 4°C
overnight and warmed to room temperature before use.
Equipment A Baby Gel Electrophoresis unit or Horizon 58 Gel
Electrophoresis apparatus (BRL Life Technologies, Inc,
Bethesda, MD, USA) was used to carry out the electrophoresis.
Gel volumes of 12 ml and 25 ml were employed for each of the
gel units, respectively.
Experimental Procedure
The experiment is designed for pairs of students. For a class of 12
students (6 pairs) one gel should be prepared with each of the pH
buffers to be investigated. Power supplies can cause shocks and
electrocution. Therefore, students should be warned not to
touch any part of the apparatus while it is running. The 2,4-
dinitrophenyl moeity of DNP lysine is a potential allergen and
should be handled with care.
Electrophoresis Avoiding the two outside lanes, load one lane
on each of the agarose gels (pH 3, 7, 11) by placing 10 Ixl of the
colored molecule stock solution carefully into the well.
Once the gel is fully loaded, place the top on the electro-
phoresis apparatus and connect the leads to the power supply.
Electrophorese samples at 50 volts for 1 hour. Turn off the
power supply, disconnect the leads, and remove the gel tray (or
glass plate) containing the gel. Gently transfer the gel from the
gel tray to a white sheet of paper using a spatula. Observe the
location of bands on the gel at each pH. Identify the bands
corresponding to each of the colored molecules (blue dextran =
blue; cytochrome c = red; DNP-lysine = yellow). Compare
the extent of migration of each type of molecule at various pH
values.
Discussion Questions
(1) Briefly describe the migration of each molecule at pH 3, 7,
and 11.
(2) What net charge (+, - , 0) does DNP-lysine possess at pH 3?
pH 7? pH 11?
(3) What net charge (+, - , 0) does cytochrome c possess at pH
3? pH 7? pH 11?
(4) What can you conclude about the charge (+, - , 0) of blue
dextran at each pH?
(5) Define, in your own words, the term isoelectric point.
(6) Do the results of this experiment enable you to estimate the
isoelectric point of any of the colored molecules employed?
42
(7) How would you change the experiment to improve the
accuracy of isoelectric point determination?
Discussion of Results
During this laboratory exercise, students observe the migration
of each of the three distinctly colored biological molecules under
different pH conditions. Typical results of the exercise are shown
diagramatically in Figure 1. While blue dextran remains in the
sample wells, it does shift toward the negative pole at pH 3 and
toward the positive pole at pH 11.
pH 3 pH 7 pH 11
POSITIVE
sample _ I
well ~
NEGATIVE
Figure 1 Migration patterns for blue dextran (I), cytochrome C
( ~ ) and DNP-lysine (m9 following agarose gel electrophoresis at
pH 3, 7, and 11
DNP-lysine has two functional groups whose dissociation
constants are influenced by pH. The ct-carbox~(l group has a pK
of 2.18. 7 The pK of the a-amine group is 8.95/Since it is linked
to the DNP moeity, the R-functional group or the ~-N is not
affected by pH. At pH 3, the et-carboxyl group has dissociated
while the a-amino group remains protonated. The net charge on
this molecule is positive and it therefore migrates to the negative
pole. At pH 7 and pH 11, the isoelectric point (5.56) for DNP-
lysine has been exceeded. The molecule exhibits a net negative
charge and therefore migrates to the positive pole.
Buffers at pH 3 and pH 7 are lower than the isoelectric point
(pH 10.7) for cytochrome c. Under these conditions, the
molecule has a net positive charge and migrates to the negative
pole. At pH 11, cytochrome c has a negative charge and migrates
to the positive pole.
Blue dextran does not migrate into the gel at any pH. This
reflects the large size of this molecule. At pH 7.0, this molecule
appears to carry no charge and it remains equally distributed
within the sample well. The chromophore of blue dextran
acquires a positive charge at pH 3 and a negative charge at pH
11. As described above, this causes the molecule to become
concentrated at the gel interface closest to the oppositely
charged pole.
Students participating in this exercise quickly realize that the
properties of specific functional groups and the response of each
group to variations in pH can dramatically affect the net charge
of biological molecules. The effects of pH on the pK and net
charge of the molecules can be easily seen by visualizing the
migratory patterns following electrophoresis. Incremental
changes in the pH can be used by the students to ascertain the
approximate pI for each of the molecules.
Acknowledgement
The authors want to thank Dr John W Dooley for the figure preparation
and Mrs Jennifer M Fisher for the manuscript preparation.
References
I Plummer, D T (1987) An Introduction to Practical Biochemistry,
McGraw-Hill Book Co, New York, NY, USA
2Campbell, Mary K (1991) Biochemistry, Saunders College Publishing,
Philadelphia, PA, USA
3Robyt, J F and White, B J (1987) Biochemical Techniques, Theory and
Practice, Brooks/Cole Publishing Co, Monterey, CA, USA
BIOCHEMICAL EDUCATION 20(1) 1992
4Boyer, R F (1986) Modern Experimental Biochemistry, Addison-
Wesley Publishing Co, Reading, MA, USA
-5Maniatis, T, Fritsch, E F, and Sambrook, J (1982) Molecular Cloning.
A Laboratory Manual, Cold Spring Harbor Laboratory, New York,
NY, USA
6Clark, John M, Switzer, R L (1977) Experimental Biochemistry, W H
Freeman & Co, San Francisco, CA, USA
7Bohinski, R C (1987) Modern Concepts in Biochemistry, Allyn &
Bacon, Inc, Boston, MA, USA
Spectrophotometric Determination Of Enzyme
Activity: Alcohol Dehydrogenase (ADH)
J R L WALKER
Department of Plant and Microbial Sciences
University of Canterbury
Christchurch, New Zealand
Introduction
The dehydrogenases are an important group of enzymes, which
may be assayed rapidly by UV spectrophotometry. These
enzymes use either N A D ÷ or N A D P ÷ as their coenzyme which.is
reduced during the dehydrogenation. A typical example is
alcohol dehydrogenase (ADH) which catalyses the reaction
Ethanol + N A D ÷ ~ Acetaldehyde + N A D H + H +
Reduced N A D ÷ (NADH) exhibits strong UV absorption at
340 nm whilst the oxidised form has virtually no absorption at
this wavelength. Therefore if one starts with a mixture of
ethanol, NAD + and enzyme in buffer, the reaction proceeds
until equilibrium is established. The reaction may be followed by
measuring the increase in absorbance of the solution at 340 nm
as N A D H is formed.
This series of experiments makes use of the change of
absorbance at 340 nm to monitor (a) the rate of reduction of
N A D ÷ by ethanol, and (b) the concentration of N A D H when
the reaction has reached equilibrium. From this, the equilibrium
constant for the reaction may be determined. The reversibility of
the reaction may also be demonstrated by showing the effect of
altering the concentration of products on the equilibrium
concentration of NADH.
Materials and Methods
Reagents Yeast A D H (from Sigma Chemical Co) made up as a
0.1% solution in water, 5 mM NAD + (also from Sigma), 0.05 M
phosphate buffer, pH 8.0, ethanol and other alcohols.
The following basic protocol may be used for all experiments.
Rate of reaction
The stock yeast A D H solution is diluted (try 1:10 for this part,
but if the rate is still too high to measure conveniently, the stock
enzyme should he diluted further). Set up two spectrophoto-
meter cuvettes as follows:
Reaction mixture Reaction Blank
0.05 M phosphate buffer (pH 8.0) 500 ixl 500 pJ
N A D + (5 mM) 200 pL1 200 I~1
Enzyme (diluted) 200/~1 200 p.I
Water 2.1 ml 1.9 ml
Total vol 3.0 ml 2.8 ml
Mix thoroughly. Zero the spectrophotometer at 340 nm using
the 'blank' cuvette and then place the 'reaction' cuvette in the