Buffering Capacity of Hemoglobin

BE 210Spring 1998

Erin ButlerElizabeth HammeAlexander Taich

Bryan WellsGroup 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.

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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.

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Amino Acid Composition

3

Name Symbol # per Alpha-Chain # per Beta-ChainAlanine Ala, A 20 16Arginine Arg, R 3 4

Asparagine Asn, N 3 7Aspartic acid Asp, D 8 9

Cysteine Cys, C 0 1Glutamic acid Glu, E 5 8

Glutamine Gln, Q 1 3Glycine Gly, G 9 11

Histidine His, H 10 6Isoleucine Ile, I 0 0Leucine Leu, L 20 17Lysine Lys, K 11 13

Methionine Met, M 1 3Phenylalanine Phe, F 7 10

Proline Pro, P 6 4Serine Ser, S 13 5

Threonine Thr, T 8 6Tryptophan Trp, W 1 2

Tyrosine Tyr, Y 3 2Valine Val, V 12 18

Total 141 145

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.

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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

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:

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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.

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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) (Equation 3)(10^(pH-pKa) + 1)

where pH is the experimental pH recorded and pKa is the literature value for each respective R-

group of the amino acid. For example, consider the titration of histidine from a pH of 5.5 to 9.

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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)

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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 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

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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 Trial 5[Hb] 1.61*10-4 M 3.21*10^-4 M 1.69*10^-4 M 1.69*10^-4 M 1.69*10^-4 M

Initial Volume of Hb Solution

100 ml 100 ml 100 ml 100 ml 149 ml

Salt added Approx. 4 g Approx. 4 g 1.03 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 4.83*10^-5

pH range 5.53-9.61 5.31-9.05 3.70-10.09 4.32-9.10 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.

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

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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.

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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.

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Hemoglobin Trials Exp. Buff Sites* Buff Cap. per mole of bovine hemoglobin*

Trial #1 35.81 8.40E+07Trial #2 30.64 6.23E+07Trial #3 34.60 7.55E+07Trial #4 31.52 8.59E+07Trial #5 28.78 5.07E+07

Theoretical # buffering sites 30.10 N/AAverage experimental value 32.27 7.17E+07

Standard deviation 2.89 1.50E+0795% 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.

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The buffering data for both

ovalbumin titrations are listed in

Table 6. Ovalbumin Exp. Number of Buffering

Sites per Molecule*Buffering Capacity per

mole of Ovalbumin*Trial 1 14.4 2.9E+07Trial 2 11.7 2.3E+07

Theoretical # of buff. Sites 13.3 N/AAverage exp. # of buff. Sites 13.1 2.6E+07

Standard Deviation 1.9 4.0E+0695% 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.

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The buffer data for this trial is

displayed below in Table 7.Potassium Phosphate KH2PO4

Buffering Capacity per mole 4.9E+06Exp. # of Buffering Sites 0.92Theo. # 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.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.

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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.

Buffering Substance Experimental Number of Average BufferingBuff. Sites per Molecule Capacity per Mole

Hemoglobin 32.27 8.23E+07Ovalbumin 13.10 2.63E+07

Potassium Phosphate 0.92 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.

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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.

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 PrecisionMicro Pipette 0.5%

Graduated Cylinder 0.5mlpH Meter 0.001

Electronic Scale 0.001gTable 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

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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.

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.

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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

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

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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

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.

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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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