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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