Buffering Capacity of
Hemoglobin
BE 210
Spring 1998
Erin Butler
Elizabeth Hamme
Alexander Taich
Bryan Wells
Group R5
April 30, 1998
Abstract
The purpose of this experiment was to determine the buffering capacity of hemoglobin
and to compare its buffering capacity to that of ovalbumin and potassium phosphate.
Additionally, the total number of buffering sites for each of these proteins was determined and
compared. A Fisher Scientific Accumet Model 925 pH meter was used to measure the pH of
several solutions so that titration curves, the buffering capacity, and the number of buffering sites
could be obtained. Hemoglobin was found to have the greatest buffering capacity per mole of
7.17*107 1.31*107 over a pH range of 5.5 to 9, as compared to 2.6*107 + 5.54*106 per mole of
ovalbumin and 4.9*106 + 2.45*105 per mole of potassium phosphate. It was also determined
that hemoglobin had the greatest number of buffering sites per mole of 32.27 + 2.23, as
compared to 13.1 + 2.63 sites per mole for ovalbumin and 0.92 + 0.05 sites per mole for
potassium phosphate. There was found to be a direct relation between buffering capacity and the
number of buffering sites, and the values for the number of buffering sites corresponded very
closely to the theoretical values.
1
Background
Hemoglobin is a globular protein, abundant in red blood cells. It contains two identical
copies of the -globin and two identical copies of the -globin polypeptide chains. Each of these
chains contains a heme group, a ring-shaped structure with a single central iron atom1.
Hemoglobin possesses a certain capacity to act as a buffer in the blood of living
organisms. For example, in the human body, hemoglobin helps to maintain blood pH between
7.38 and 7.44(6). Since bovine hemoglobin will be used in this experiment it will be the only
type of hemoglobin referred to for the remainder of the paper. As a buffer, hemoglobin
counteracts any rise in blood pH by releasing H+ ions from a number of atomic sites throughout
the molecule. Similarly, a number of H+ ions are bound to, or are ‘taken up’ by the molecule,
acting to counteract a decrease in pH. In order to determine a theoretical value for the number of
buffering sites and the overall buffering capacity of bovine hemoglobin, a knowledge of the
amino acid composition of the molecule is required.
The hemoglobin protein contains all 20 amino acids in varying quantities, with the
exception of isoleucine. There are a total of 141 amino acids on each  chain and 145 on each 
chain. Considering that there are two of each of the  and  chains, this provides for a total of
572 amino acids and a molecular weight of 62,015.14 g/mol for the protein. The amino acid
composition for hemoglobin is listed in Table 1 below.
2
Name
Alanine
Arginine
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
Amino Acid Composition
Symbol
# per Alpha-Chain
Ala, A
20
Arg, R
3
Asn, N
3
Asp, D
8
Cys, C
0
Glu, E
5
Gln, Q
1
Gly, G
9
His, H
10
Ile, I
0
Leu, L
20
Lys, K
11
Met, M
1
Phe, F
7
Pro, P
6
Ser, S
13
Thr, T
8
Trp, W
1
Tyr, Y
3
Val, V
12
Total
141
# per Beta-Chain
16
4
7
9
1
8
3
11
6
0
17
13
3
10
4
5
6
2
2
18
145
Table 1: Amino acid composition of hemoglobin4.
The basic structure of an amino acid consists of an alpha carbon (central carbon – see
Figure 1) with four attached groups. Three of these groups are the same in all of the 20
biologically important amino acids: a hydrogen atom (H), a carboxyl group (COOH) which
behaves as an ionizable acid, and an amino group (NH2) which behaves as an ionizable base.
Due to the peptide bonds that link the individual amino acids in polypeptide chains, the carboxyl
and amino groups do not ionize and therefore do not contribute to the buffering capacity of the
molecule. It is therefore the fourth group, the R-group, which we anticipate is responsible for
any type of buffering that occurs.
NH2
R--C--COOH
H
Figure 1: Basic structure of an amino acid2.
3
In determining the buffering capacity and number of buffering sites per molecule, only
the ionizable groups, those amino acids that contain R-groups with additional acid or base
groups, will be considered. These amino acids include arginine, aspartic acid, cysteine, glutamic
acid, histidine, lysine, and tyrosine. Aspartic and glutamic acid both have carboxyl groups as
part of their R-groups with pKa values of 3.86 and 4.25 respectively. Histidine, even though it
possesses a secondary amino group within its R-group (i.e. NH+), will be treated as an acid since
the pKa value for this R-group, which is 6.00, lies within the acidic range. Arginine, cysteine,
lysine, and tyrosine all have literature R-group pKa values of 12.48, 8.50, 10.53 and 10.07
respectively, which fall within the basic range and will therefore be treated as the base groups2.
The breakdown of the total number of potential buffering sites on the hemoglobin molecule is
shown in Table 2 below.
Number of Potential Buffering Sites per Bovine Hemoglobin Molecule
(from literature)
26 Glutamic acid (Acid)
34 Aspartic acid (Acid)
48 Lysine (Base)
14 Arginine (Base)
32 Histidine (Acid)
2 Cysteine (Base)
10 Tyrosine (Base)
Table 2: Numerical breakdown of potential hemoglobin buffering sites4.
The buffering capacity of hemoglobin is its ability to maintain the pH of a solution within
a certain range. A protein with a large buffering capacity acts as a stronger buffer in comparison
to a protein with a small buffering capacity. The difference in buffering capacity is due to the
number of buffering sites; those proteins with a large buffering capacity have a larger number of
buffering sites, and the reverse is also true. It requires an increased amount of titrant to observe
4
the same pH change in a buffering solution as may be observed in a non-buffering solution. By
this argument we define the buffering capacity of a substance as the following:
Buffering Capacity = NExperimental /NTheoretical
(Equation 1)
where NExperimental is the experimental moles of titrant added to buffer solution over pH change
and NTheoretical is the theoretical change in moles of H+ over same pH change.
The buffering capacity is based on the fact that there is more titrant being added to the
buffered solution then would normally be required for the observed pH change (i.e. change in H+
moles). The number of buffering sites is therefore thought of as all such sites responsible for this
increase in titrant per molecule of buffer (see Equation 2). It should be noted that the terms in the
numerator are determined over the same pH range.
# Buffering Sites = (NExperimental – NTheoretical)/ NBuffer
(Equation 2)
where NBuffer is the number of moles of buffer in solution.
Another large molecule with a noticeable buffering capacity is ovalbumin, which
composes over fifty percent of the proteins in egg whites. Ovalbumin is a glycoprotein
consisting of 385 amino acids, with a molecular weight of 45,000 g/mol5. This protease inhibitor
consists of a polypeptide with up to two phosphate groups per mole. The number of buffering
sites in ovalbumin will be compared to those present in hemoglobin in order to verify the
methods used in the above equations, since there is a comparable literature value for number of
sites available. From its amino acid sequence and the same process as discussed above for
hemoglobin, the breakdown of potential buffering sites for the ovalbumin molecule is displayed
in Table 3 below.
5
Number of Potential Buffering Sites per Ovalbumin Molecule
(from literature)
33 Glutamic acid (Acid)
14 Aspartic acid (Acid)
20 Lysine (Base)
15 Arginine (Base)
7 Histidine (Acid)
6 Cystein (Base)
10 Tyrosine (Base)
Table 3: The potential number of buffering sites per molecule of ovalbumin4.
Additionally, both the buffering capacities of bovine hemoglobin and ovalbumin can be
compared to that of potassium phosphate (KH2PO4) for additional comparison.
It must be emphasized that the number of buffering sites presented in both Tables 2 and 3
consist of the maximum number of sites that could possibly participate in the buffering process.
All of our experimental values for buffering capacity and number of buffering sites are
determined over a specific pH range (e.g. pH = 5.5 to 9), where not all of the sites listed in
Tables 2 and 3 will buffer. For example, at a pH of 5.5 glutamic and aspartic acid will already
be mostly ionized based on their pKa values of 4.25 and 3.85, respectively. Similarly, arginine
hardly even begins to release an H+ ion from the NH3+ group in its R-group at a pH of 9. The
theoretical number of total buffering sites must therefore be corrected for the corresponding pH
range by multiplying each number of specific buffering sites by the change in % ionization of
the particular amino acid. The % ionization of a substance at a given pH is defined by the
following equation3:
% Ionization = 10^(pH – pKa)
(10^(pH-pKa) + 1)
(Equation 3)
where pH is the experimental pH recorded and pKa is the literature value for each respective Rgroup of the amino acid. For example, consider the titration of histidine from a pH of 5.5 to 9.
6
At a pH of 5.5, using equation 3 and the fact that the R-group pKa for histidine is 6.00, the
histidine R-group is already  24% reacted (i.e. 24% of all histidines on the given molecule have
released their H+ ions from the NH+ group on the R-group). Repeating the above process, at a
pH of 9, histidine is  100% reacted. Therefore, over the pH range of 5.5 to 9 approximately
76% of all available histidines (i.e. 24.3 of 32 total histidines in bovine hemoglobin) react and
therefore contribute to the buffering capacity. The theoretical number of buffering sites for
either hemoglobin or ovalbumin over a specific pH range is determined by summing the products
of (%ionization * max number of buffering sites) for each particular amino acid. Specifically,
the formula is as shown below in Equation 4, where %I is the change in percent ionization over
the specified pH range and #AA is the theoretical total of amino acids with the relevant R-groups
(i.e. max number of potential buffering sites) obtained from the amino acid sequencing of the
protein.
Theoretical # of buffering sites = (%I * #AA)
(Equation 4)
7
Materials
1. Fisher Scientific Accumet Model 925 pH meter with combination glass-silver/silver chloride
electrode and swing arm electrode holder
2. pH buffer standards (4, 7, 10)
3. Thermometer
4. Magnetic stirrer and stirring bar
5. P-1000 Micropipette and tips
6. 1 M concentrations of HCl and NaOH solutions
7. Assorted glassware
8. Freeze-dried bovine H2500 Hemoglobin (in powder form)
9. Ovalbumin (in powder form)
10. Potassium phosphate, monobasic (in powder form)
Procedure
The Fisher Scientific pH meter was calibrated using buffer standards of pH 4, 7, and 10,
following the procedure as listed in the pH meter manual.
Deionized water was titrated in order to serve as the standard for a non-buffer. One gram
of NaCl was added to the water and the pH was lowered to 2.0 with the addition of 1 M HCl.
The solution was then titrated up to a pH of 11 with the addition of 13.5 ml of 0.1 M NaOH.
During the titration upward, the pH was recorded at each small volume of base added and the pH
was plotted against the volume of NaOH added in order to form a titration curve.
Five trials were performed of the hemoglobin titration. The specifications for each trial
can best be seen by looking at Table 4. In each trial, NaCl was added to the solution. The salt
8
was used to introduce ions into the solution in order to stabilize the readings on the pH meter.
From the initial pH, which was approximately 7 for each trial, the pH was lowered with 1 M HCl
to a low pH and then raised to a high pH with some volume of a known concentration of NaOH.
The concentration of NaOH used was 100 times more concentrated than the hemoglobin solution
in the last three trials, but of varying concentrations in the first two trials. During the titration
upward, the pH readings were recorded as increments of 0.75 ml of the NaOH solution was
added. The same procedure was followed while titrating back down to a low pH. The acid and
base were introduced into the hemoglobin solution via the P-1000 Micropipette.
Titrations of Hemoglobin
Trial 1
Trial 2
Trial 3
Trial 4
[Hb]
1.61*10-4 M 3.21*10^-4 M 1.69*10^-4 M 1.69*10^-4 M
Initial Volume
100 ml
100 ml
100 ml
100 ml
of Hb Solution
Salt added
Approx. 4 g Approx. 4 g
1.03 g
1.03 g
[NaOH]/[HCl]
0.05 M
0.1 M
1.64*10^-2 M 1.64*10^-2 M
pH range
5.53-9.61
5.31-9.05
3.70-10.09
4.32-9.10
Trial 5
1.69*10^-4 M
149 ml
1.03 g
4.83*10^-5
8.99-4.29
Table 4. Table of specifications for hemoglobin titration.
Two titrations of albumin were also performed. In the first trial, 1.57 g of albumin was
added to 100 ml of H2O, and 1.01 g of NaCl was added to the solution in order to stabilize the
pH meter. The pH was lowered from approximately 7 to a pH of 2.643 with 1 M HCl, and it was
raised to a pH of 10.944 with 3.49x10-2 M NaOH. The basic solution was 100 times more
concentrated than the albumin solution. The pH was recorded as the base was added in
increments of 0.75 ml; it required 99.5 ml of base to raise the pH to 10.944. For the second trial,
1.576 g of albumin was added to 100 ml of H2O. An amount of 1.01 g NaCl was added to the
solution and the pH was lowered to 2.775 to begin the titration. The same concentration of
NaOH was used as in the first trial to raise the pH to 10.994 and the pH and volumes were again
recorded. For the second trial, 103 ml of base were required to bring the pH up to this level.
9
In addition to the hemoglobin and albumin titrations, a titration was performed on
potassium phosphate. Fifty ml of a 0.1259 M concentration of KPO4 was lowered with 1 M HCl
from a pH of approximately 7 to a pH of 2.371. With the addition of 47 ml of 0.252 M NaOH,
the pH was raised to 11.469.
For each trial, a titration curve was plotted comparing the volume of NaOH added to the
pH. Both the buffering capacity, as well as the total number of buffering sites were determined
from Equations 1-4.
10
Results
A titration curve of pH versus volume of titrant added was plotted for each titration trial.
An exemplary curve can be seen in Figure 2 and the titration curves for the remaining
hemoglobin and ovalbumin trials can be found in Appendix B.
Figure 2: Trial #3 hemoglobin titration curve.
The specific buffering capacities and the number of buffering sites per molecule of
hemoglobin for each trial can be found in Table 5 below. An exemplary format of the
calculations used to find the buffering capacity and the number of buffering sites for each of the
trials are outlined in Table A1 in Appendix A. The theoretical number of buffering sites was
found using Equations 3 and 4, as discussed in the background.
11
Hemoglobin Trials
Trial #1
Trial #2
Trial #3
Trial #4
Trial #5
Theoretical # buffering sites
Average experimental value
Exp. Buff Sites* Buff Cap. per mole of
bovine hemoglobin*
35.81
8.40E+07
30.64
6.23E+07
34.60
7.55E+07
31.52
8.59E+07
28.78
5.07E+07
30.10
N/A
7.17E+07
32.27
Standard deviation
2.89
1.50E+07
95% Confidence Limit
2.53
1.31E+07
Absolute Error
2.17
N/A
*Table values have been determined over a specific pH range of 5.5 to 9.
Table 5: Calculated results of individual hemoglobin titrations.
The titration curve of the best ovalbumin trial is displayed in Figure 3 below; the titration
curve obtained for the second ovalbumin trial can be found in Figure B5 in Appendix B.
Figure 3: Notice that the titration curve for ovalbumin trial #1 is much more pronounced than
that of hemoglobin trial #3. This is demonstrative of a lower buffering capacity.
12
The buffering data for both ovalbumin titrations are listed in Table 6.
Ovalbumin
Exp. Number of Buffering
Buffering Capacity per
Sites per Molecule*
mole of Ovalbumin*
Trial 1
14.4
2.9E+07
Trial 2
11.7
2.3E+07
Theoretical # of buff. Sites
13.3
N/A
Average exp. # of buff. Sites
2.6E+07
13.1
Standard Deviation
1.9
4.0E+06
95% Confidence Limit
2.63
5.54E+06
Absolute Error
0.2
N/A
*Table values are calculated for a specific pH range of 5.5 to 9.
Table 6. Ovalbumin buffering data.
The titration curve for the lone potassium phosphate titration trial is shown in Figure 4.
Figure 4: Titration curve for potassium phosphate titration trial.
The buffer data for this trial is displayed below in Table 7.
Potassium Phosphate KH2PO4
Buffering Capacity per mole
4.9E+06
Exp. # of Buffering Sites
0.92
Theo. # of Buffering Sites
0.97
% Difference
5.0%
*Table values are calculated for pH range of 5.5 to 9.
Table 7: Trial buffer data for potassium phosphate.
13
Discussion
The titration curve of hemoglobin, as shown in Figure 2, is noticeably different than the
graph shown in Figure 5, below, which is the curve for the titration of deionized water. Notice
the large jump in pH from 3 to 9, whereas the hemoglobin curve increases very gradually. This
gradual behavior is characteristic of a good buffer. By looking at the titration curves, it should
be apparent then that hemoglobin is a better buffer than ovalbumin, which is a better buffer than
potassium phosphate.
Figure 5: Titration curve for deionized water; this serves as a standard non-buffer.
In addition to the titration curves, the numerical results also verify the above stated
observation (see Table 8, below). A greater number of buffering sites is indicative of a more
effective buffer.
14
Buffering Substance
Hemoglobin
Ovalbumin
Potassium Phosphate
Experimental Number of
Buff. Sites per Molecule
32.27
13.10
0.92
Average Buffering
Capacity per Mole
8.23E+07
2.63E+07
4.88E+06
Table 8: Comparison of buffering sites and buffering capacity per mole of each buffering
substance.
As stated in the background, the number of buffering sites is directly related to the
buffering capacity of each substance. The difference in buffering capacity is due to the number
of buffering sites; those proteins with a large buffering capacity also have a larger number of
buffering sites, and the reverse is true as well. The linear relationship between number of
buffering sites and buffering capacity is shown in Figure 6 below.
Figure 6: Display of the direct linear relationship between buffering capacity and number of
sites.
Another trend that was observed was the average number of buffering sites was higher
than the theoretical values for each of the titrations that were performed. This difference can
possibly be explained by the formation of bicarbonate, another buffer, but this will be discussed
later in the error analysis.
15
It should also be noted that for each trial, the number of buffering sites and buffering
capacities was computed over a pH range of 5.5 to 9. The reason for this was that it was the
largest range covered for each of our trials. This range had to be consistent in order for a valid
comparison of values to be made between the three buffers, since the number of sites and
capacity is dependent on the pH.
Sources of error in this experiment, as in any, can be divided into systematic error that is
inherent in methods and materials and accidental or human error. Apparatus precision
limitations are presented in Table 9.
Apparatus
Precision
Micro Pipette
0.5%
Graduated Cylinder
0.5ml
pH Meter
0.001
Electronic Scale
0.001g
Table 9: Apparatus precision limitations.
The error in concentration of the NaOH and HCl solutions, which originates in error in
the micro pipette and the graduated cylinder for an average trial (0.035M for albumin titrations
and 0.016M for hemoglobin), is about 1%. Since the imprecision of the graduated cylinder
contributes the most to that value, a more precise way of measuring relatively large quantities
would eliminate much of this error. The error in concentration translates into about 1%
uncertainty in the final calculations of both buffering capacity per mole as well as the number of
buffering sites. The imprecision in the micro pipette translates into 0.5% uncertainty in the
calculations of the buffering capacity per mole and the number of buffering sites; the imprecision
in pH meter translates into 0.2% uncertainty in the final results. The error in the electronic
balance that was used to determine the mass of hemoglobin had a negligible effect on the final
results. Finally, the error in the graduated cylinder results in an imprecise determination of total
volume of hemoglobin solution used and translates into 0.3% uncertainty in the final results.
16
Thus the total instrumentation error can be estimated to be 2%. It can be concluded, then, that
the apparatus is sufficiently precise for this particular experiment and with the exception of the
graduated cylinder, which contributes about 1.3% uncertainty to final results; no adjustments are
necessary in order to make this experiment more precise.
Since no theoretical or literature experimental values for buffering capacity of titrated
proteins are available, the error analysis will focus on the number of buffering sites per molecule.
This analysis will be indicative of the overall quality of the experiment since values for both
buffering capacity and number of buffering sites are derived from the same experimental data.
The 95% confidence limit for the average number of ionization sites in a hemoglobin
molecule was calculated to be equal to 2.53 sites. This value is indicative of good accuracy and
precision since the absolute error (difference between experimental value and the theoretical
value) of 2.17 sites is within the 95% confidence limits. The estimated instrumentation error can
account, in the worst case scenario, for 2% of that error or 0.64 sites. Formation of bicarbonate
through dissolution of CO2 in the hemoglobin solution and simple human error may account for
the rest of the difference between theoretical and experimental values.
Absolute error calculations were based on the theoretical values derived from the bovine
hemoglobin amino acid sequence. They are not literature values.
We are operating under the assumption that the freeze-drying process does not change the
actual amino acid sequence of the protein but does completely oxidize the heme group of the
protein so that the effects of dissolved O2 and CO2 binding to the protein can be dismissed. We
can conclude, then, that the buffer reactions are limited to the acid-base R-groups of the amino
acids throughout the polypeptide sequence and that the freeze-drying process does not affect the
17
buffering capacity of the protein and, therefore, our comparison of experimental values to the
theoretical values is valid.
Effects of CO2 in air can possibly explain the fact that the experimental values for the
number of ionization sites in a hemoglobin molecule were consistently higher than theoretical
values. The CO2 in the air could have reacted with the solution, creating bicarbonate, which
would have also acted as a buffer in the solution. With the addition of this other buffer, the
hemoglobin would have appeared to have an inaccurately larger buffering capacity. Since that is
the case, it can be hypothesized that eliminating CO2 from the experimental environment would
improve accuracy.
Since only two trials of ovalbumin titration were performed, very little can be said about
the precision other than the absolute error in number of ionizing sites per ovalbumin molecule
does fall within the 95% confidence limit. The results are quite accurate, with the experimental
values differing from theoretical values by only 1.5%. The above discussion on validity of the
theoretical values for the number of hemoglobin buffering sites applies to ovalbumin in the same
way. Similar to the hemoglobin trials, the number of ionizing sites was greater than theoretical,
which supports the above hypothesis of the effects of the atmospheric CO2 on the determination
of buffering capacity. Instrumentation uncertainties in phosphate and ovalbumin trials were
identical to hemoglobin, and thus all of the above systematic error discussion applies to these
trials.
The titration of the hemoglobin, ovalbumin, and potassium phosphate could have been
simplified and made more precise and accurate with the use of an automated titration device.
The human error involved in these titrations and in recording the pH was unavoidable and would
have been lessened with instrumentation similar to the Titron, which was developed by Group
18
R7. In addition, if this experiment were to be repeated, the range of pH recorded should be
larger (i.e. 1-14). By expanding this range, the theoretical number of buffering sites could be
counted directly from the amino acid sequence without the percent-ionized correction as
discussed in the background.
Based on the precision within our trials and the statistically verified closeness of the
results with the theoretical values for number of buffering sites, we conclude that the
experimental set-up was adequate in determining the buffering capacity of large proteins.
19
References
1. Alberts, et al. Essential Cell Biology. Garland Publishing: New York, 1998.
2. Bioengineering 210 Lab Manual: Spring 1998.
3. Devlin, Thomas M. Textbook of Biochemistry. Wiley-Liss, Inc.: New York, 1992.
4. http://www.unl.edu/stc-95/ResTools/BioTools/biotools11.html
5. Ovalbumin Product Description Manual
6. Webster, John G., Editor. Medical Instrumentation and Design, 2nd Edition. Houghton
Mifflin Company: Boston, 1992.
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