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~SSantana, M A and Villegas, L (1991) Plant Physiol 95, 335-336
~6Sherbet, G V (1978) 'The Biophysical Characterization of the Cell
Surface', Academic Press, London
JTRice, R H and Horst, J (1972) Virology 49, 602-610
tSRocha, J, Krmpf, J, Ferrand, N, Amorim, A and Ritter, H (1991)
Electrophoresis 12, 313-314
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33, 65-69
2°Andrzejewski, C, Jr, Young, P J, Cines, D B and Silberstein, L E
(1991) Transfusion 31,236-244
21Van Liedekerke, B M, Nelis, H J, Kint, J A, Vanneste, F W and De
Leenher, A P (1991) Pharm Sci 80, 11-16
22Bassett, P, Braconnier, F and Rosa J (1982) J Chromatogr 227, 267-
304
23Bassett, P, Beuzard, Y, Garel, M C and Rosa, J (1978) Blood 51,971-
980
24Celis, J E, Ratz, G P, Celis, A, Madsen, P, Gessen, B, Kwell, S,
Nielsen, H V, Ydel, H, Lauridsen, J B and Basse, B (1988) Leukemia
2, 561-601
A m i n o Acid Titrations Using a Spreadsheet Program
RONALD W BROSEMER
Department o f Biochemistry and Biophysics and the
Program in Basic Medical Sciences
Washington State University
Pullman, W A 99164-3250, USA
Introduction
Introductory biochemistry laboratory courses often include the
titration of an amino acid or peptide in order to illustrate the
principles of acid dissociation and the operation of a p H meter.
This is a valuable exercise, but I have concluded that there is an
application that is more valuable to students than the mechanics
of using a pH meter, namely the computer spreadsheet.
Techniques for using a p H meter can be readily acquired in any
laboratory setting, but introduction to spreadsheets requires, for
most students, a more formal approach. For this reason, I have
substituted the direct titration of an amino acid, that has been a
mainstay of the introductory laboratory course for decades, with
a titration based upon calculations performed by a spreadsheet
program. Upon introduction of this exercise, I had expected a
large percentage of these third- and fourth-year students to have
had experience with spreadsheets. The reality is that, despite
widespread acquaintance with word processing, almost no
students had used a spreadsheet; in fact, several did not know
what a spreadsheet was.
The algebraic basis for the spreadsheet titration is straight-
forward but confusing for the majority of these students, who are
for the most part in allied health professions. The mathematical
background must be carefully covered before undertaking the
mechanics of the spreadsheet. I have found the most efficient
approach to teaching the spreadsheet technique is discussion
while the students are at computer terminals working on the
titration exercise itself. Additional assignments allow students to
apply the methods they have learned to new problems without
instructor supervision.
This exercise results in a titration curve with sufficient points
to produce a smooth curve along the entire pH range from 0 to
14; charge vs p H is the actual plot employed. The example
shown below is for an amino acid that has three titratable
groups; amino acids or peptides with a different number of
titratable groups require modification of the spreadsheet. The
program employed is Microsoft Excel version 2.1 for MS-DOS
(IBM) or version 2.2 for Macintosh. A comparable exercise that
B I O C H E M I C A L E D U C A T I O N 20(2) 1992
calculates the net charge on an amino acid for a single pH value
is described by Atkinson et al.l
The program described below requires entering the values for
pK and charges on the various ionic forms directly into the
formulae. A more versatile program allows input of pK values
(cells E16 to E18) and charges (cells B16 to B19) into
spreadsheet ceils with subsequent reference to these cells in the
formulae. I have found this latter approach a bit too complex for
the first-time spreadsheet user, since it introduces the concept of
relative vs absolute values. Those with some experience benefit
from this modification.
The description for producing an xy plot is included, since this
is usually not readily apparent from documentation accompany-
ing the Excel program.
Most operational details are included in this description. Some
instructors may prefer supplying fewer details in order to
encourage students to learn more by their own efforts and
mistakes. The spreadsheet data, spreadsheet formulae, titration
plot, and plot of mol fractions for glutamic acid are shown
below; each of these, except for the spreadsheet formulae, is
distributed with the experimental description. The students can
thus refer to these examples when programming the titration
curve for another amino acid.
Charge of an amino acid as a function of pH
The net charge on an amino acid or peptide is pH-dependent,
since they all contain two or more weakly ionizable acid groups.
Application of the Henderson-Hasselbalch equation allows
calculation of the net charge at any pH if the pK values for the
ionizable groups are known.
In an actual titration experiment, an amino acid or peptide is
titrated with known amounts of acid or base and the pH
monitored as a function of acid or base added. In order to
illustrate the usefulness of spreadsheet calculations, a model
titration is plotted via a series of calculations that relate net
charge with pH. Glutamic acid is used as example. Each student
will be assigned another amino acid together with the corre-
sponding pK values. You are to submit a spreadsheet and
titration curve analagous to those shown for glutamic acid.
Calculations
The single-letter abbreviation for glutamic acid, E, is used in the
formulae. You are responsible for the structural formulae for
each of the ionizable forms of glutamic acid and of the amino
acid assigned to you.
The three ionizable groups of glutamic acid are: ct-COOH
(pK1 = 2.1); 3,-COOH (pK2 = 4.3); ~t-NH3 + (pK3 = 10.0).
The Henderson-Hasselbalch equation for the acid H A is:
pH = pK + log [(A-)/(HA)]
or:
(A-)/(HA) = 10 ( p H - p K )
The abbreviations H3E +, H2E , H E - , and E = are used for each
of the four ionizable forms of glutamic acid.
Applying the above formula to each of the ionizable groups of
glutamic acid:
(H2E)/(Hs E+) = 10(pH-2'I) (1)
(HE-)/(H2E) = 10(pH-4"3) (2)
(E~)/(HE -) = 10(pH-IO'O) (3)
The relative amount of each ionizable form can be expressed as a
ratio of the concentration of that form to the total concentration.
112

Since (2) Enter 0 (= zero, not the letter) into B1.

(3) Enter into cell C1 the formula that adds 0.25 to the adjacent
(total concn of glutamic acid) = (HaE +) + (H2E) + (HE-) + (E ~) cell on the left. Do not forget to start the formula with =. Copy
C1 into D1 to BF1. If the value in BF1 is not 14, there is an error.
then Remove the copy function by simultaneously pressing shift +
control [IBM] or shift + command [Macintosh] while typing a
1 = [H3E+] + [H2E] + [HE-] + [E =] (4) period.
(4) Enter eqn 1 into B5, eqn 2 into B6, and eqn 3 into B7. Use a
where: [H3E+] = (H3E+)/(total concn of glutamic acid), etc for cell reference for the pH value and enter the numerical value for
each of the ionizable species, (These are mole fractions.) the pK. The parentheses in the formula must be those found as
From the mole fraction of each species, one can calculate the upper case on the 9 and 0 keys; other parentheses on the
net charge. keyboard cannot be entered into formulae. Exponents are
entered as ^ (upper case on the 6 key).
Net charge on glutamic acid = (5) Enter eqn 9 into B4. Note that all three exponential factors
(+1 x [H3E+]) + {0 × [H2E]} + {-1 x [HE-]} + {-2 x [E=]) (5) in eqn 9 are already entered into cells B5 to B7, so you need only
refer to the cells rather than type out the whole equation.
In order to perform the final calculation, it is necessary to (6) Enter into B10 a formula that inverts the value in B4.
express all factors in terms of pH and pKs; ie, all factors must be (7) Enter eqn 10 into B l l . Note that both factors in the equation
expressed in terms of ratios of ionic species. have been calculated, so you need only refer to the cells rather
than type out the whole equation.
Dividing eqn (4) by [HaE ÷] (8) Enter eqn 11 into B12 and eqn 12 into B13. Time saving
hint: you can perform this operation by copying B l l into B12
1 [H2E] [HE-] [E =] and B13; you need not type any formulae directly into B12 or
[H3E+] - 1 + [HaE+] + [H3E+] + [H3E+] (6) B13. Why does this work in this case?
(9) Enter eqn 5 into B2. Be sure to use the correct charges for
each ionic form: they may not be the same as for glutamic acid.
By judicious use of ratios that equal 1 For negative charges, you may need to use parentheses to
separate the different algebraic operations (it will depend upon
[HE-] [HE-] [H2E] how you set up the formula).
= x (7)
[H3E ÷] [nEE] [H3E ÷] (10) Save! Actually, you should have already saved several
times. When you first lose a long document because you failed to
[E =] = [E-] [HE-] x ~H2E] (8) save (this happens to everyone once and usually only once), you
[HaE +1 [HE-] x [H2E] [HBE +] will never again forget to save at regular short intervals.
(11) Split the screen so that column BF is visible on the right and
By referring to eqns 1-3 and eqns 6-8, each mol fraction can be the first columns are visible on the left; use the small vertical
calculated in terms of pH and pKs. black bar in the lower left-hand comer of the screen next to the
arrow to split the screen.
1/[H3 E+] = 1 + {10(pH-2"I)) + {10(pH-2"I) X 10(pH-4'3)} (12) Select cells B2 to B13. Copy. Select cells C2 to BF13. Paste.
q- {10 (pH-2"I) X 10(pH-4'3) X 10(pH-10"0)) (9) (See what a time-saver the split screen is?) Remove the copy
function by simultaneously pressing shift + control [IBM] or
[H2E] = 10(pH-2'I) X [H3E+] (10) shift + command [Macintosh] keys while typing a period.
(13) Save.
[HE-] = 10(pH-4"3) x [H2E ] (11) (14) Select the printer and page setup. Print the first page only.
Use the page preview before printing; this often saves time since
[E =] = 10(pH-10"0) X [HE-] (12) changes can be made before printing.
(15) Change the display so it shows the formulae (Options:
Since the net charge on glutamic acid Display: Formulas). Adjust the width of the columns so that the
complete formulae are visible. Print the first three columns.
= {+1 x [H3E+]} + {0 x [H2E]} + { - 1 x [HE-]} Revert to display of values.
+ { - 2 x IE=I} (5) (16) Make an x y plot of the pH vs Total Charge data. Select
'rows' as explained in the accompanying directions.
it is possible to calculate the net charge as a function of pH and (17) Submit three printouts: the first page of the spreadsheet
pKs. with the data; the first three spreadsheet columns with the
formulae; the x y plot.
Assignment (18) By referring to your plot, what is characteristic about the
A copy of an Excel spreadsheet and the resulting graph are pH values when the charge on the amino acid is half way
shown for the titration of glutamic acid. Using this spreadsheet between two integers (eg, +1.5, +0.5, -0.5, -1.5)? What is
as model, write your own spreadsheet and plot the resulting characteristic about the pH values when the charge is an integral
charge vs pH relationship for an amino acid assigned to you. Use value (eg, +2, +1, 0, - 1 , - 2 ) ?
pH values in increments of 0.25 in the range from 0 to + 14. The
pK values for all ionizable groups are shown in an accompanying Supplementary Assignment
table [distributed to students as an addendum]. Note that you For additional credit, plot the mol fraction of each of the four
must assign the correct values of charge for each of the ionizable ionic species of your amino acid (on the y-axis) as a function of
species for your amino acid; the values for glutamic acid do not pH (on the x-axis). Generate one figure containing all four plots.
necessarily apply to other compounds. The data have already been calculated on your spreadsheet.
You must arrange the pH and mol fraction data such that they
Notes on Using the Spreadsheet can be transferred to an xy plot. There are several ways to do
(1) Type text into cells A1, A2, A4 to A7, A10 to A13, A15 to this. One of the simplest is to insert a row above each of the four
A19, B9, B15 to B19, D16 to D18, E16 to E18. mole fraction rows. Do this by selecting the entire row above

BIOCHEMICAL EDUCATION 20(2) 1992

 113

A B C i
D E F G H
1 pH= 0 0.25 0.5 0.75 I 1.25 1.5 1.75
2 Net Charge,= 0.992118529 0.986069 0.975489 0.957218 0.926341 0.876022 0.798668 0.68976(
3
4 1/[H3Glu]= 1.00794368 1.014127 1.025123 1.044681 1.079473 1114138 1.25'1587 1.447943
5 [H2GIuI/[H3GluI= 0.007943282 0.014125 0.025119 0.044668 0.079433 0.141254 0.251189 0.446684
6 [HGIu]/[H2GIu]= 5.01187E-05 8.91E-05 0.000158: 0.000282 0.000501i 0.000891 0.001585 0.00281~
7 [Glu]/[HGlu]= IE-10 1.78E-10 3.16E-10 5.62E-10 IE-09 1.78E-09 3.16E-09 5.62E-0~
8
9 Mol Fract Mol Fract Mol Fract Mol Fract Moi Fract Mol Fract Mol Fract Mol Fract
I0 H3GIu 0.992118924 0.98607 0.975493 0.95723 0.926378 0.876133 0.798986 0.690635
11 H2GIu 0.007880681 0.013929 0.024503 0.042758 0.073585 0.123757 0.200696 0.308495
12 HGlu 3.9497E-07 [i 1.24E-06 3.88E-06 1.21E-05 3.69E-05 0.00011 0.000318J 0.000869
13 Glu 3.9497E-17 2.21E-16 1.23E-15 6.78E-15 3.69E- 14 1.96E-13 1.01E-12 4.89E-12
14
15 Ionic Form Charge/Molec
16 H3GIu I pKl= 2.1
17 H2Glu 0 pK2-- 4.3
18 HGlu -1 pK3= l0
19 Glu -2

Figure 1 Charge vs pH spreadsheet for glutamic acid: data

A B c
1 pH= 0 =B1+0.25
2 Net Charge,= =l*Bl0+0*Bl l+(-l*Bl2)+(-2*Bl3) =l*Cl0+0*Cl I+(-I*C12)+(-2"C13)
3
4 ll[H3Olu]= ~l+B5+(B6*I35)+(B7*B6*B5) !=1+C5+(C6"C5
+~)~C_7*C6"C5)_.
5 [H2GIu]/[H3GIa]= :=10~al-2.1) =10~Cl-2.1)
6 [HGlul/[H2Glul= !=10~B1-4.3) I=10A(Cl-4.3)
7 [Glu]/[HGlu]= =I0~BI-10) ...... ~IOA(CI-IO)
8
9 Mol Fract Mol Fract
10 H3Glu =1/134 =1/t24
11 H2GIu =B5*B 10 =C5"C10
12 HGlu "-B6*B11 =C6"C11
13 GIu =B7*BI2 =C7"C12
14
15 Ionic Form Charge/Molec
16 H3Glu 1
17 H2Glu 0
18 HGIa [-1
19 Glu -2

Figure 2 Charge vs pH spreadsheet for glutamic acid: formulae

which you wish to add a new row; choose: Edit: Insert. Into each
of the four vacant new rows, paste the pH data from row 1. The
four adjacent sets of pH and mole fraction data can be plotted on
a single chart. Once you have opened a new chart, this same
chart must be used for adding subsequent data sets. It is easiest
to activate this chart by using the window menu.
Directions for Making an xy Plot with Excel
These directions apply to data that are in rows {or columns}. On
the Excel spreadsheet, select two adjacent rows {or columns} of
data to be plotted. Select only the data points themselves; do not
include titles. Copy the selection: File: New: Chart: OK: Edit:
Paste Special: Rows {or Columns}: Categories in First Row {or
Column}: do not select Series Names in First Column: OK.
Unless you have already added data to this chart, you can ignore
Replace Existing Categories; if you already have data plotted on
the chart and wish to add new data, do not select Replace
Existing Categories. Choose: Gallery: Scatter: 3 (or any other
choice): OK.
Axes labels and a title can be added as follows. If values on
either or both axes must be moved, select the appropriate axis by
placing the tip of the cursor on the axis and clicking. Two hollow
circles or squares appear at each end of the axis. If the y axis is
selected: Format: Scale: Category axis crosses at: enter the

BIOCHEMICAL E D U C A T I O N 20(2) 1992

114

1 F-.qmmm I Determination of Accessible Bed Volume of Gels by

N o.5 I
Short Column Filtration
e mm
O P MALHOTRA, l PRAKASH PRABHAKAR 1 and
t 0 mmmm
ARVIND M KAYASTHA 2
C -0.s - -

h IIIIii Department of Chemistry 1

a *1 IIIIllm
r"
mm mmmm! and
g i
School of Biotechnology 2
-~.5 I
e Banaras Hindu University
mill n i n .I. . . . . . . .
-2 J Varanasi 221 005, India
0 2 4 6 8 10 12 14

Introduction
Cross-linked dextran and polyacrylamide gels have been used
Figure 3 Titration of glutamic acid widely in the fractionation of proteins) Since cross-linking is a
random process, each gel has a distribution of pore sizes. Smaller
pores predominate in highly cross-linked gels and there is a
1
UUmll •,$• ~**+++ ÷,, $ e $ o o o °' ,~
larger proportion of large pores in gels of lesser degree of cross-
M
k, • ~0 D •
l *
• o
IL linking. When such gels are suspended in buffer and packed in a
0
I o.a --- -¢ O O • • o
column, some of the column buffer is located inside the gel
I [] particles (V1) and some between the gel particles or in the void
HE- * o
F o.s i itJ
volume (V0). Whereas the buffer in the void volume is equally
6-
r I..1~+ D accessible to all proteins (and other solutes), the accessibility of
a the buffer held in the gel particles to a protein depends on the
C 0.4 l b _ LJ
Di • size of the latter. Very large proteins, which cannot penetrate
t • [ 0 --I-~E ° ° *• I any gel pore, are said to be 'completely excluded'. These can
i 0,2
0 00 ••
equilibrate with the buffer in the void volume only. At the other
D • • O
n 00 [ .ll [] extreme are very small proteins, which can pass freely through
o
all pores and consequently equilibrate with buffer held in the gel
2 4 6 8 10 12 I
particle and that present in the void volume (ie V1 - Vo).
Proteins of intermediate sizes may penetrate only some of the
pores and can, therefore, equilibrate with buffer of the void
Figure 4 Mole fraction of ionic species of glutamic acid vs pH volume plus a fraction of that in the gel particles (V0 + KdVO.
This explains the differential retardation of proteins of different
sizes when these are passed through gel columns and forms the
lowest value on the y axis: OK. If the x axis is selected: Format: basis of molecular weight determination by gel filtration. 2'3
Scale: Value axis crosses at: enter the lowest value on the x axis: The fraction of bed volume accessible to a given protein (Kd)
OK. Choose: Chart: Attach Text: Title: OK: type the title [more may be computed from a series of determinations of elution
than one line can be entered in the title by using control + enter volumes of proteins of different sizes. In the present communi-
(IBM) or command + return (Macintosh)]: Enter: Chart: cation a method is described for rapid and direct determination
Attach Text: select either Value axis (= y axis) or Category axis of Ku, which may also be used for molecular weight deter-
(= x axis): OK: enter the axis legend (more than one line can be mination. It involves determination of dilution undergone by a
entered as described above): Enter. Repeat for the other axis. protein when its solution is applied on a short, tightly packed gel
If more sets of data are to be entered, repeat the above column.
directions, except open the existing chart file instead of opening When a protein solution is applied to a short, tightly packed
a new file. gel column, dilution undergone by the protein is related to the
For printing select: File: Page setup: select your choices: OK. volume of accessible buffer.
Turn on the printer. Choose: Print: select Best or Faster (if
available): Page Preview (if desired): OK. Vol solution applied + Vol accessible buffer
Dilution (D) = Vol solution applied
Reference
tAtkinson, D E, Clarke, S G, Rees, D C and Barkley, D S (1989) Rearranging,
Dynamic Models in Biochemistry, pp. 43-48, N Simonson & Co,
Marina del Rey Vol accessible buffer = Vol solution applied x (D - 1) (1)

Recall that volume of accessible buffer = Vo + KdV1 (2)

The void volume (V0) may be found by determining the dilution

undergone by a protein which is completely excluded (ie where
Kd = zero). It is not always necessary to determine V1
experimentally. It may be obtained by subtracting V0 from the
total bed volume, because the volume occupied by dry gel
particles is very small and may be neglected. However, for very
precise measurements specially when extensively cross-linked
gels are used, it can be determined experimentally by finding out
dilution undergone by a protein whose molecular weight is below
the fractionation range of the gel. For such proteins Kd is unity
and volume of accessible buffer is equal to V0 + V1. Alterna-

BIOCHEMICAL EDUCATION 20(2) 1992