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Titration of Ovalbumin
Final Project Report
Group W4
Gregory Moy
Rhonda Quain
Akshay Sateesh
Joyce Yang
May 7, 1999
Table of Contents
Abstract…………………………………………………………2
Introduction……………………………………………………..2
Background……………………………………………………..3
Data Analysis……………………………………………...4
Materials and Methods…………………………………………5
Initial Preparations………………………………………..5
Error Analysis…………………………………………….6
Results…………………………………………………………..8
Discussion……………………………………………………..10
Appendix………………………………………………………15
References……………………………………………………..18
1
Abstract
The main objectives of this experiment were (1) to compare the titration curves and the
numbers of ionizable residues in ovalbumin to those obtained from literature, (2) to determine
whether ovalbumin is reversible by comparing titration curves of pH 2 to 12,12 to 2, 7 to 2, and 7
to 12, and (3) to determine the optimum pH range of reversibility. Reversibility is expected if the
addition of an acid to the protein causes the titratable sites to be ionized, changing the pH
accordingly, and then the addition of a base to this same protein solution causes the reverse to
occur. By comparing different titration curves, it was deduced that any titration above or below
this pH range would permanently deform the protein, and would result in it being irreversible;
however, any titration within the pH range of 2 to 12.4 yielded the same titration curve. The
number of ionizable sites were calculated and compared to the theoretical ionizable sites
obtained from literature to determine the conformation of ovalbumin at a certain pH with respect
to a particular amino acid. It was found that such conformational changes cannot be predicted as
the number of ionizable sites differed depending on the start and end point of the titration.
Introduction
Individual amino acids have at least two sites that can be titrated: a carboxyl group and an
amino group. Seven amino acids have an extra acidic or basic residue that can be titrated as well.
In a protein (series of individual amino acids attached by peptide bonds), the carboxyl group and
the amino group on each amino acid are tied up in the covalent peptide bond and cannot be
neutralized. Only the two end- terminal groups and those in the different residues are still
titratable, so that the titration curve of the protein (eg: egg albumin) reflects its amino acid
composition in a manner related to the types of residues that can be neutralized (Litt,
Giandomenico).
By titrating ovalbumin and analyzing its titration curve, the number of ionizable sites can
be determined and related to the total number of ionizable sites (determined from structure in
literature) in the protein. Quaternary structure of proteins (eg: protein folding) can inhibit all the
ionizable sites to be available for acid-base titrations and so the difference in the numbers of
ionizable sites are results of quaternary protein structure (Chang, 327). This is one hypothesis
that was tested by conducting these titrations.
Another objective was to determine the reversibility of the protein. This can be achieved
by titrating the ovalbumin from a low pH to a high pH (2-12) by adding base. Reversibility is
expected if the addition of an acid to the protein causes the titratable sites to be ionized, changing
the pH accordingly, and then the addition of a base to this same protein solution causes the
reverse to occur. The protein should revert back to its original pH and composition. The protein
is reversible if the points on the titration curve match the points on the curve if it was titrated
2
down (from 12 to 2). From literature, ovalbumin is expected to be reversible between pH 2 and
pH 11 (Cannan, 250).
From the above hypotheses we hoped to learn of the specific amino acids that were
hidden from protein folding and their contribution to the reversibility of the protein.
Background
Ovalbumin is a glycoprotein with a molecular weight of 45,000 grams per chain. Each
macromolecule consists of four identical chains. The sequence of ovalbumin was obtained from
the Protein Data Bank of Brookhaven National Laboratory and the number of titratable sites for
each amino acids was determined (Appendix 2.1). Glutamic acid, aspartic acid, and histidine
have acidic R-groups because their pKa is below seven. Lysine, arginine cysteine, and tyrosine
have basic R-groups because their pKa values fall within the basic range. There was a total of
105 titratable residue per chain.
As mentioned before, in protein titration, only specific amino acids are available for acidbase reactions. Of these amino acids, some are acidic and some are basic. When base is added
to the protein the OH- ion of NaOH binds to a H+ ion from the titratable residue groups.
However, adding a small number of moles of OH- is not sufficient to ionize the entire protein.
Thus, only the H+ ions from the amino acids with the strongest OH- affinity will be ionized. This
affinity for OH- ions is related to the pKa of the titratable residue. A lower pKa represents a
stronger acid and the ability of the acid to dissociate increases. Since it is more likely to
dissociate it is more likely to bind to the OH- ion from the NaOH. Hence at any given pH, there
are several amino acids that are partially ionized. To calculate the moles of H+ ions from each
amino acid ionized by the OH- added the following equation was used:
[ A ]
( x)
(1)
pH  pKa  log(
)  pKa  log
[ HA]
( HAo  x )
where x is the moles of OH- ionizing a certain amino acid and HAo is the corrected initial moles
of that amino acid. (Swanson, 6417)
To calculate the corrected initial moles of a certain amino acid at a given pH the
following equation was used:
10 pH  pKa
% ionized  pH  pKa
(2)
10
1
Our second objective is to study the reversibility of ovalbumin. Reversibility is defined
as the ability of a protein to maintain its structure when titrated from a low pH to a high pH and
vice versa. Thus, the number of H+ ions ionized at a certain pH should be the same whether the
protein is titrated from pH 2-12 or pH 12-2 (Tanford, 254).
3
Background-Data Analysis
As mentioned in the background, at any given pH there exists more than one amino acid
being ionized. Therefore when adding base/acid, a fraction of the amount added reacts with each
of the amino acids that are present in solution. To determine how much of the base/acid reacts
with the individual amino acids, the Henderson-Hasselbach equation was used (Equation 1). By
using the composition of amino acids in ovalbumin, an initial number of moles of the respective
amino acids were calculated. For example, at a pH of 2.9, the only two amino acids that are able
to react with the base/acid added are aspartic acid and glutamic acid. If base was added, all the
other amino acids have been untouched (less than 1%) and if acid was added all the other amino
acids have been completely ionized (or more than 99%). Below is an example calculation if base
was added to the solution at a pH of 2.9:
x
pH  log(
)  3.86
5.10 X 10 7  x
2 X 10 4  x
pH  log(
)  4.25
3.03 X 10 7  x
When the two equations are solved simultaneously, the amount of base that reacts with the
aspartic acid (x) and the new pH can be determined. Using this method of analysis, a titration
curve can be constructed for ovalbumin (see Appendix 2.3). This curve represents the protein
when all potential titration sites are available and when the protein is reversible at any range of
pH. When the experimental curves are plotted (pH 2-12 and pH 12-2), the number of ionizable
sites can be determined by analyzing the curve using the same technique to construct the
theoretical curve. To calculate the number of titratable residues, the number of moles used to
neutralize a given species of amino acid is taken from the titration curve. This value is used to
obtain the percent composition of the amino acid in question in the ovalbumin chain by the
equation:
%composition A.A.in ovalbumin = moles of titrant to neutralize
total moles of ovalbumin
(3)
where the total moles of ovalbumin is 2.34E-05 mol (see Appendix 1.1).
The following equation solves for the number of titratable sites of the particular amino
acid:
experimental # of ionizable sites = (% comp)(MW ovalbumin)
(4)
(MW of AA)(4)
A reversibility test must be done to determine whether the protein is reversible at a given
pH range. To do this, a series of solutions is prepared, containing sufficient acid to bring each
solution to pH 2. Different amounts of base are then added to each solution, so that final pH
values of say, pH 3, pH 4, pH 5, etc. are reached. Calculations for the number of hydrogen ions
bound or dissociated are made for the final pH values. If the resulting values fall precisely on the
predicted titration curve obtained, then the acid portion of the titration curve is reversible. From
4
this, the optimum pH range of reversibility can be determined for ovalbumin (approximately
between pH 2 and pH 12).
Methods and Materials
1.
2.
3.
4.
5.
6.
7.
Fisher Scientific pH meter
Burettes
Micropipettes
1M NaOH and HCl
Sigma Egg/Chicken Albumin, Grade II (A-5253)
Analytical Balance
Volumetric Flasks
Initial Preparations
The first step in the experiment was the determination of the solubility of ovalbumin. By
taking a five-gram sample of ovalbumin and submersing it in 100mL of deionized water,
microfuging, and dessicating for a week, the solubility was obtained. After the dessication
process, the final sample was weighed to be 4.25 grams of ovalbumin. Therefore, the solubility
of ovalbumin was determined to be 4.25g ovalbumin per 100mL deionized water, or 0.0425g/ml
ovalbumin. This solubility was used throughout our experimentation because our different
samples were microfuged each time and then the liquid solution was used in our titrations
because it was already known that only 0.0425 g/mL would be soluble.
The next step was the preparation of the titrants. The titrants used in this experiment were
1M NaOH and 1M HCl. The molarity of NaOH and HCl that was initially used was 5X10-4 M to
compensate for the small amount of ovalbumin, 5g, being used; however, after the first week of
experimentation, it was instantly noticed that the pH barely changed after a significant amount of
titrant was added. This molarity was determined using the number of amino acid residues and
their percent compositions in ovalbumin, the molecular weights of these amino acids, and the
number of moles of each found. It was then determined that there were a total of 5.17X10-6 moles
of ovalbumin in a 5 gram sample. After trying to titrate with this very dilute solution, it was
decided that a stronger solution was definitely needed in order for the pH to be changed from 2 to
12 and 12 to 2. After titrating with the 1M NaOH and 1M HCl solutions for a few trials, it was
determined that these molar titrants were much more adequate than the very dilute solution.
After deciding to use 1M titrants, the pH meter was standardized using the Fisher Scientific
Manual. This standardization was performed every week. Since one of the main goals of this
experiment was to determine the reversibility of ovalbumin, several titrations of the ovalbumin
using the 1M NaOH and HCl were performed. The titrations that were performed consisted of
pH 2 to 12, 12 to 2, 7 to 2, and 7 to 12. For example, the titrations performed for a pH of 12 to 2
used HCl as the titrant, and then to go back from the pH of 2 to 12, NaOH was used to turn the
5
solution from acidic to basic. The acid or base was added to the ovalbumin solution in
increments of 0.2 ml.
In preparation for this experiment, a sample of ovalbumin was taken and mixed with water,
and the pH was taken. An approximate pH of 7.325 was read from the pH meter. As mentioned
above, it was noticed during this trial that not all the ovalbumin had dissolved in the deionized
water, therefore, the ovalbumin was microfuged and constantly stirred so that the pH of a wellmixed was read after the titrant was added. This solubility factor was also taken into account
when the %ionized and number of titratable sites calculations were completed.
As an afterthought to our experiment, it was suggested that we try a titration using saline
solution to determine the ionic strength of the ovalbumin. This titration was attempted the last
week of experimentation, and the above methods for the titrations using ovalbumin submersed in
deionized water was followed for the ovalbumin submersed in saline solution.
Using equation 2, the percent hydrogen ions that were ionized was calculated at different pHs
to determine the amino acids that were able to undergo acid/base titrations in the solution at a
given pH (see Appendix 2.2).
Error Analysis
Not all residues will show up in the titration, and this must be accounted for in some way.
The greatest possibility is that the amino acid is hidden within the chain’s structure, or
conformation. If, for example, one aspartic acid site is missing from the titration, it needs to be
concluded whether the residue is not showing up because of the conformation or because of the
experimental error.
The methods in the experiment propagate inherent uncertainty to the experimental
number of titratable residues for each amino acid. Table 1 shows the uncertainties of various
components of the experiment.
Source
pH meter
Burette
Concentration of titrant
TOTAL
Table 1: Sources of uncertainty in procedure.
Uncertainty
±0.001 pH units
±0.05 mL
±0.005 M
2.22%
To effectively account for these uncertainties in the discussion, the maximum percent
uncertainty is calculated by the following formula:
Maximum % uncertainty = %unc pH meter + % unc burette = 2.22%
The following equation (4) is used to calculate the maximum uncertainty in the number of
sites:
6
Max unc. in # of sites = (% unc.)(% comp)(MW ovalbumin)
(MW of AA)(4)
(see Appendix 1.2)
Table 3 lists the maximum uncertainty in the number of sites for each particular amino acid.
Amino Acid
Maximum uncertainty in # of sites
Aspartic acid
±0.31
Glutamic acid
±0.72
Histidine
±0.16
Cysteine
±0.68
Tyrosine
±0.20
Lysine
±0.08
Arginine
±0.40
Table 3: Maximum uncertainty in the number of sites for each amino acid, using 2.22%
maximum uncertainty. (See Appendix 1).
These uncertainties differ from one amino acid to the next because of the different
molecular weight and percent composition of each respective amino acid. Due to the relatively
small uncertainties of some amino acids (such as histidine, tyrosine and lysine), the conclusions
drawn from the experiment for those amino acids can be discussed with good confidence.
Because of the larger maximum uncertainties for the others, for example glutamic acid and
cysteine, it would be difficult to definitively determine the number of hidden sites.
Results
7
The following is a compilation of the titration curves from the different weeks of
experimentation. All trials of the same titration are superimposed onto one graph.
14
12
10
pH
8
6
Week 2
4
Week 1
2
0
0
0.005
0.01
0.015
0.02
0.025
moles OH- added
Figure 1.
A graph of the titration curves from pH 7 to 12 from trial weeks 1 and 2.
8
7
Week 3
6
Week 1
pH
5
4
3
2
1
0
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
mol added
Figure 2.
A graph of the titration curves from pH 7.5 to 2 from trial weeks 1 and 3.
8
Figure 3.
A graph of the titration curves from pH 2 to 12 and 12 to 2 from trial weeks 1 and 2.
Below are the results obtained to achieve the first objective of the experiment. The
calculations leading up to these numbers are presented in the background. The experimental %
composition of each amino acid was calculated using equation (3). This % was found using
simultaneous Henderson Hasselbach equations (Equation 1). The equations were solved using
excel and the total moles of amino acid ionized was found. See Appendix (2.1). Finally, using
equation (4) the number of titrated sites for each amino acid was found.
Literature Values
Week 1 Sol 1 2 to 12
12 to 2
Sol 2
7 to 2
12 to 2
Week 2 Sol 1 7 to 12
12 to 2
Sol 2
2 to 12.5
12.5 to 2
Week 3 Sol 1 7 to 2
Uncertainty
Asp
14
13.7
13.54
12.55
13.67
--13.42
13.73
13.45
12.7
.31
Glu
33
32.4
30.1
31
30.1
--32.2
29.12
31.4
30.8
.72
His
7
6.9
6.7
6.7
6.5
0.3
6.7
6.33
4.3
6.7
.16
Cys
6
5.49
5.3
--5.2
5.62
5.89
5.46
4.9
--.68
Tyr
10
8.14
9
--8.89
9.52
9.2
8.9
7
--.20
Lys
20
17.66
18.2
--19.6
19.65
17.5
18.2
15.4
--.08
Arg
15
6.3
6.8
--6.9
6.4
7.76
7.42
7.46
--.40
Table 4. A summary of the number of titratable sites calculated for each amino acid from the respective trials,
compared to the literature values.
9
Literature Values
Week 1 Sol 1 2 to 12
12 to 2
Sol 2
7 to 2
12 to 2
Week 2 Sol 1 7 to 12
12 to 2
Sol 2
2 to 12.5
12.5 to 2
Week 3 Sol 1 7 to 2
Asp
14
0
0 or 1
1 or 2
0 or 1
--0 or 1
0
0 or 1
1 or 2
Glu
33
0 or 1
2, 3 or 4
1, 2 or 3
2, 3 or 4
--0 or 1
3 or 4
1 or 2
2 or 3
His
7
0
0
0
0 or 1
--0
1
3
0
Cys
6
0 or 1
0 or 1
--0 or 1
0 or 1
0
0 or 1
1 or 2
---
Tyr
10
2
1
--1
0 or 1
1
1
3
---
Lys
20
2
2
--0
0
3
2
4
---
Arg
15
8 or 9
8 or 9
--8 or 9
8 or 9
7 or 8
7 or 8
7 or 8
---
Table 5. Experimental number of hidden sites determined for each amino acid from each respective trial.
The experimental number of hidden sites is indeterminate for some due to uncertainty in protocol (See Table 3).
Titration
2 to 12
12 to 2
7 to 2
7 to 12
Asp (14)
13.72
0.02
13.52
0.11
12.63
0.11
---
Glu (33)
29.61
0.69
31.53
1.04
30.9
0.14
---
His (7)
6.615
0.40
6.58
0.15
6.65
0.07
0.3
Cys (6)
4.98
0.73
5.53
0.33
---
Tyr (10)
8.52
0.54
9.18
0.32
---
Lys (20)
17.93
0.38
18.50
0.88
---
Arg (15)
6.86
0.63
7.15
0.42
---
5.62
9.52
19.65
6.4
Table 6. Average of total titrated sites of each amino acid for a particular start and end pH.
Discussion
Ionizable Sites
Table 4 and Table 5 illustrate the summary of results obtained to achieve the first
objective of this experiment. Evidently, the number of titratable sites for each amino acid, differ
depending on the start and end points of the titration.
Aspartic Acid
When titrating from pH 2-12, it was found that all the titratable sites for aspartic acid
were available (13.7  0.31 sites). In contrast, when titrating from pH 7-2, it was found that only
12.7  0.31 sites were available, indicating that at least one titratable site was hidden in the
10
conformation of the protein. Aspartic acid completely ionizes at a range of pH 2-6 and therefore
the start and end points of the titration should not be a factor in the difference of number of
titratable sites. In the experiment itself all the Aspartic acid was ionized within a pH range of
2.10 – 4.49 for the titration of 2-12 and a pH range of 2.93 – 5.72 for the titration from pH 7-2.
The difference in the pH ranges and the titratable sites can be attributed to the fact that for the
titration of pH 2-12, acid was added to the protein causing a conformational change before any
titration began. Whereas when the titration began at a pH of 7, the protein was at its natural state
before titration began. From the titrations of pH 2-12 and 12-2, the results showed the same
number titratable sites.
Glutamic Acid
Results for glutamic acid show that the number of experimental ionizable sites did not
correspond, for the most part, to the theoretical number of sites (33). For the titrations from pH
2-12 and 12-2, it cannot be determined whether all the sites were ionized or one site was missing.
These inconclusive results are due to the uncertainty in the protocol of the experiment. For the
majority of the other trials, up to 4 sites were missing or hidden during the titrations. Once again
due to the acid or base added, the hidden sites can be attributed to the quaternary structure of
ovalbumin. With a high molecular weight, glutamic acid has more surface area than the other
amino acids, which could explain its ability to be hidden or covered up easily by other molecules
in the protein.
Histidine
With the exception of the titrations done from pH 2-12.5 and 12.5-2, all the sites of
histidine were ionized which shows that they were available for acid-base titrations. For the pH
of 7-12, the number of ionizable sites was found to be 0.3  0.16 sites. This is very different
from the theoretical value of 7 sites. However at a pH of 7, most of histidine is ionized, so that
only 0.3 sites are left beyond a pH of 7. Moreover, from a pH of 7-2, the number of titratable
sites was found to be 6.7  0.16 sites. When superimposing the two titration curves, all of the
sites are accounted for by titration and so none are missing or hidden.
Cysteine, Tyrosine, Lysine
For these amino acids, the same analysis was done as discussed above. The number of
possible missing sites for Cysteine ranged from 0-2. Cysteine was mostly inconclusive due to the
relatively large uncertainty (0.68 sites). The number of sites missing for tyrosine ranged from 0
to 3. The number of sites missing for lysine ranged from 0 to 4. The difference in ionized sites
may be due to conformational changes of ovalbumin that occurred with the addition of large
amounts of acid and base to the protein to bring the pH to 2 or 12, or the nature of the protein
itself at a certain pH change. Once again for the titration from pH 2-12.5 and 12.5-2, the results
disagreed with the theoretical value due to the permanent deformation of the protein itself.
Arginine
11
The data collected for arginine cannot be used to conclude how many titratable sites were
available because the pKa of Arginine is 12.48 and the maximum titration curve went up to 12.5
in which the protein began to denature and a foul odor resulted. Thus the titratable sites
calculated for Arginine only account for the sites ionized up to pH 12. However, the same
variation in the number of sites was found for the titration from pH 2-12.5 and vice-versa.
Table 6 gives a more general summary of the results obtained to achieve the first
objective of this experiment. For each type of pH titration an average of the number of titrated
sites of each amino acid is given. However, not enough trials have been done for each type to be
able to use these numbers with enough confidence. Moreover, one of the solutions was titrated
up to 12.5 to find reversibility instead of just pH 12, thus the protein was denatured and the
results were skewed. This is the reason that the discussion focus on the results of each individual
amino acid with respect to a particular titration.
Determining the number of sites hidden due to conformational changes is useful as a hint
to determine in what titrations the protein changes in behavior. For example, the number of
titratable sites varied greatly in the titration from pH 2-12.5 and vice-versa, from the theoretical
values and from the other experimental values. This might indicate that the protein cannot be
subjected to an acid reaction followed by a base reaction to return to the same pH and
conformation. When acid is added to ovalbumin, the pH changes respectively. When the same
amount of base is added, the protein should revert back to the same pH and conformation. This
concept is called reversibility. With this specific definition of reversibility, one cannot use the
number of titratable sites to determine the optimum pH range of reversibility. Instead, the
number of moles of acid or base reacted with the protein needs to be used.
Reversibility
Figure 3 illustrates the graph of the titration curves from pH 12 to 2, 2 to 12, 2 to 12.5 and
12.5 to 2. This graph depicts that ovalbumin is not reversible from a pH range of 2 to 12.5. This
is emphasized by the fact that when the protein is titrated from 2 to 12, 12 to 2, and then back up
to 12.5, the same titration curve occurs; however, when the protein is then titrated back down to
2, a different titration curve results. Using the definition of reversibility, the number of moles of
base or acid added for any one set of titration curves needed to be analyzed. If the number of
moles of acid and base added at a given pH should be the same. Hence, the test of significance
was used to determine whether the amount added and the titration curves were significantly
different.
12
Titration
2-12
12-2
2-12
2-12.5
2-12.5
12.5-2
T-Statistical
4.33
T - Critical
1.25
5.23
2.66
2.15
3.54
Table 6 – Summary of significance tests for the titration curves (see Figure 3).
If the t-Critical is less than the t-Statistical, using regression analysis on Excel 97, the sets
of data are not significantly different. Clearly from the above table, the curve for the titration
from 12.5-2 is significantly different and therefore redeeming the protein irreversible from that
pH range. The titration curve from pH 12.5 to 2 also shows that this protein cannot be brought to
such high pH’s without its conformation changing permanently. It is thereby concluded that
ovalbumin has an optimum pH range from 2 to 12.4, but once this range is exceeded, the protein
is irreversible and begins to denature. Furthermore, during this titration curve, a white precipitate
emerges as the protein becomes more and more acidic. This precipitate is a new addition to the
sulfuric-like smells that emanated during most of the titrations. Due to these observations and
trouble-shooting trials of titrating water and saline solutions, it was determined that ovalbumin’s
behavior during titrations was not predictable.
Amendments for future reference
Another significant finding that occurred early on in our experimentation was the fact that
we needed to add 1M NaOH and 1M HCl to titrate our protein. We originally calculated that we
would only need to add 5X10-4 moles, but the addition of such a diluted titrant resulted in
insufficient pH changes. As a result, we increased the molarity of titrant to 1M thus increasing
number of moles of acid/base added. While our calculations for the number of titrated sites of
each amino acid were comparable to literature values, it is still to be determined as to where all
the excess NaOH and HCl went.
A possible explanation is that the acid or base might be reacting with the solvent rather
than the ovalbumin. When performing a titration of deionized water, however, the water seemed
to react with all of the base or acid added. According to the titration, the water is not the cause
for adding excess base/acid. Another possible solution is to dissolve the ovalbumin in a saline
solution. This would neutralize any isoionic charges on the side-chains of the protein. The NaCl
did not affect the pH readings and it did not change the titration from pH 7-2. This result
indicated that using saline solution could not correct for the excess base/acid added either.
Hence, both water and saline did not account for the excess base or acid.
Since ovalbumin is categorized as a glycoprotein, there is a possibility that the sugar
molecules on the side-chains are still attached to the Grade II Sigma Egg Albumin protein. This
would change the molecular weight and the nature of the reactions that can occur during the
titrations. However, with these extra sugar molecules, the molecular weight would increase and
would result in a decrease in the number of moles of each of the amino acids. In order to titrate
the four chains of ovalbumin and nothing else, the protein needs to be purified. Possible
methods of purification were not discussed however, permanently deforming the protein with the
13
use of DTT is a possible solution. DTT will break the sulfide bonds of the quaternary structure
of the protein and permanently unfold the protein, hence sheltering it from any protein folding
during the titrations. This method is strongly suggested to see the affects of the titration without
the disulfide bonds.
A qualitative analysis of a small amount of ovalbumin solution was conducted to show
the unpredictability of ovalbumin. One portion of acid was added to one portion of ovalbumin
solution and universal indicator was used to determine its approximate pH (light red). When one
portion of base was added the solution returned to the original color (dark green). Next a doubleportion of the ovalbumin solution was used and the same portions of the acid and base were
added. The color in the end did not match the original color to start with (light green, not dark
green). Theoretically, the solutions should be at the same pH in the end because the amount of
acid added is completely ionized by the equal amount of base added. This shows that when
different portions of acid or base are added, ovalbumin reacts and behaves very differently.
Hence, using DTT, saline solutions and making sure the ovalbumin is stored between 0
and 5 degrees Celcius, may aid in determining the reason for the excess acid/base need to react
with the protein.
14
Appendix
1. Calculations
(1)
Example calculation for percent composition of aspartic acid
experimental % composition A.A.in ovalbumin = (4.19e-7 mol / 2.34e-5 mol) = 0.0180 %
experimental # of ionizable sites
(2)
= (percent comp)(MW ovalbumin)
(MW of AA)(4)
= (0.0180)(180000)
(59)(4)
= 13.7 sites
Example calculation for the maximum uncertainty in the number of titratable sites
Maximum unc. in # of sites= (% unc.)(% comp)(MW ovalbumin)
(MW of AA)(4)
= (±2.22%)(1.836%)(180000)
(59)(4)
= ±0.31 sites
2. Tables and Graphs
(1) Moles of amino acid in 4.25g Ovalbumin
Residue
Mol. Weight
Relevant pKa
Percent
Composition
# moles in
4.25g
ovalbumin
14 Aspartic Acid
59
3.86
1.836
4.34E-07
33 Glutamic Acid
73
4.25
5.353
1.27E-06
7 Histidine
70
6
1.089
2.63E-07
6 Cysteine
47
8.5
3.244
7.85E-07
10 Tyrosine
107
10.07
1.967
4.76E-07
20 Lysine
73
10.53
0.6267
1.52E-07
15 Arginine
59
12.48
2.378
5.75E-07
Total # of moles
5.17E-06
15
(2) % ionized at each pH
Proline
Aspartic Acid
Glutamic Acid
Histidine
Cysteine
Tyrosine
Lysine
Arginine
pH 2
50.58
1.3620
-
pH 3
91.10
12.13
5.32
-
pH 4
57.99
35.99
-
pH 5
93.24
84.90
9.09
-
pH 6
98.25
50.00
-
Proline
Aspartic Acid
Glutamic Acid
Histidine
Cysteine
Tyrosine
Lysine
Arginine
pH 7
90.91
3.07
-
pH 8
24.03
-
pH 9
75.97
7.84
2.87
-
pH 10
96.93
45.98
22.79
-
pH 11
89.49
74.69
3.21
pH 12
98.84
96.72
24.88
* Amino acids which are less than 1% ionized are taken as negligible and ones which are 99% or
more ionized are considered completely ionized and therefore not used in calculations.
(3) Sample excel sheet for Aspartic Acid and Glutamic acid for week 2 solution 2. Used to
16
calculate number of titrated sites of each amino acid.
pH
Initial
OH- to Asp Final Asp Initial Glut OH- to Glu Final Glu
Asp
2.102 4.22E-07 6.20513E-08 3.60E-07
2.172 3.60E-07 5.6166E-08 3.04E-07
2.263 3.04E-07 5.11574E-08 2.53E-07
2.353 2.53E-07 4.58221E-08 2.07E-07
2.442 2.07E-07 4.03215E-08 1.66E-07
2.56 1.66E-07 3.56545E-08 1.31E-07
2.672 1.31E-07 3.05634E-08 1.00E-07 Initial Glut OH- to Glu Final Glu
2.8 1.00E-07 2.57988E-08 7.45E-08 1.20E-06 2.28002E-07 9.72E-07
2.879 7.45E-08 2.03061E-08 5.42E-08 9.72E-07 1.96789E-07 7.75E-07
3.015 5.42E-08 1.62738E-08 3.79E-08 7.75E-07 1.7466E-07 6.01E-07
3.108 3.79E-08 1.21378E-08 2.57E-08 6.01E-07 1.45305E-07 4.55E-07
3.204 2.57E-08 8.79624E-09 1.70E-08 4.55E-07 1.18361E-07 3.37E-07
3.34 1.70E-08 6.3202E-09 1.06E-08 3.37E-07 9.66854E-08 2.40E-07
3.428 1.06E-08 4.18478E-09 6.45E-09 2.40E-07 7.33418E-08 1.67E-07
3.51 6.45E-09 2.66465E-09 3.78E-09 1.67E-07 5.38952E-08 1.13E-07
3.611 3.78E-09 1.65648E-09 2.12E-09 1.13E-07 3.90249E-08 7.39E-08
3.697 2.12E-09 9.76023E-10 1.15E-09 7.39E-08 2.69991E-08 4.69E-08
3.79 1.15E-09 5.54311E-10 5.95E-10 4.69E-08 1.81639E-08 2.88E-08
3.901 5.95E-10 3.03345E-10 2.91E-10 2.88E-08 1.19012E-08 1.69E-08
3.996 2.91E-10 1.55464E-10 1.36E-10 1.69E-08 7.37024E-09 9.50E-09
4.088 1.36E-10 7.5549E-11 6.01E-11 9.50E-09 4.36678E-09 5.13E-09
4.17 6.01E-11 3.46975E-11 2.54E-11 5.13E-09 2.46472E-09 2.67E-09
4.284 2.54E-11 1.53823E-11 1.01E-11 2.67E-09 1.35769E-09 1.31E-09
4.412 1.01E-11 6.38822E-12 3.68E-12 1.31E-09 7.09186E-10 6.03E-10
4.21996E-07
6.03E-10 3.38615E-10 2.65E-10
4.497
2.65E-10 1.56764E-10 1.08E-10
4.625
13.73 sites
1.08E-10 6.69131E-11 4.08E-11
4.744
4.08E-11 2.6733E-11 1.41E-11
4.89
1.41E-11 9.73499E-12 4.36E-12
5.053
4.36E-12 3.16406E-12 1.20E-12
5.222
1.20E-12 9.14816E-13 2.82E-13
5.426
2.82E-13 2.26041E-13 5.62E-14
5.642
5.958
29.12 sites
17
References
Al Giandomenico, Dr. M. Litt. BE 210 Bioengineering Laboratory II, Laboratory Manual.
University of Pennsylvania, Spring 1998.
Tatsumi. Biochemistry. Vol 37 (35) pgs 12351-9; September 1998.
C. Tanford in T. Shedlovsky, ed. “Electrochemistry in Biology and Medicine.” John Wiley and
Sons Inc. New York, NY; 1955.
C. Tanford, S.A. Swanson, and W.S. Shore. “Hydrogen Ion Equilibria of Bovine Serum
Ovalbumin.” American Chemical Society. New York, NY. Vol. 77; 1955.
Keith Cannan, A. Kibrick, and A.H. Palmer. “ The Amphoteric Properties of Egg Albumin.”
Annals of the New York Academy of Sciences. New York University. Vol. 41 (243);
1941.
www.artsci.wustl.edu/~jhan
18