Determination of the Buffering Capacity of Hemoglobin

Determination of the Buffering Capacity of Hemoglobin

Group W3

Sthita Das

Lauren Entrekin

Phillip Oh

Keith Somma

April 30, 1998

The purpose of this experiment was to investigate the physiological properties of hemoglobin which constitute its buffering capacity. To achieve this goal, multiple titrations were performed in order to find pK values and isoelectric points. Ovalbumin was also studied in a similar fashion to ensure the accuracy of the procedure. Using the knowledge of the amino acid content of both ovalbumin and hemoglobin, the amount of base needed to reach the pKa and the isoelectric point was determined for each buffer. These molar amounts were compared to determine which of the proteins was the superior buffer. Average pKa values were 5.43 and 3.93, while isoelectric points were 6.87 and 4.62 for hemoglobin and albumin, respectively. These values are consistent with literature values obtained from Lehninger3. It was then determined that 2.3256 mL and 5.4192 mL of NaOH needed to be added to hemoglobin and ovalbumin, respectively, to reach the pKa. According to experimental calculations, 3.3 mL and 1.47 mL of NaOH were used for hemoglobin and ovalbumin, respectively. The discrepancy in the albumin values is caused by experimental error: because the carboxyl groups were still mostly in their ionized form, much less NaOH was needed to reach pKa than expected theoretically. The hemoglobin error was caused by the ionization of the side chains of Histadine.

+Methods

Hemoglobin is a protein in the blood of many animals that transports oxygen to the tissues; it is found in the erythrocytes of vertebrates. When hemoglobin forms an unstable, reversible bond with oxygen, it is called oxyhemoglobin and is bright red; in the reduced state it is purplish-blue. As is evident from the three dimensional structure diagram on the title page, each hemoglobin molecule is made up of four iron-containing, oxygen-binding subunits (called hemes) chemically bonded to a large protein unit (globin), forming a tetrahedral structure. Heme, which accounts for only 4 percent of the weight of the molecule, contains all the iron and gives a red color to the molecule. Globin consists of two linked pairs of polypeptide chains.

Erythrocytes, more commonly known as red blood cells, are specialists in carrying oxygen (O2) from the lungs to the tissues of the body and for carrying carbon dioxide (CO2) in the opposite direction. Hemoglobin, which is responsible for the red color of blood, is the oxygen-carrying protein in erythrocytes. Carbonic anhydrase is the enzyme that, by catalyzing the conversion of carbon dioxide to another chemical species, allows the blood to take up carbon dioxide rapidly from the tissues and release it rapidly in the lungs. Hemoglobin uses atoms of iron for reversibly binding oxygen, whereas carbonic anhydrase uses atoms of zinc at its catalytic center.

Each heme subunit is 500 times larger than the molecule of oxygen that it carries. The reasons behind hemoglobin’s large tetragonal structure demonstrate the importance of the protein in the numerous reactions in the oxygen – carbon dioxide transport.

A determining factor in hemoglobin’s oxygen affinity is acidity, or the concentration of protons (hydrogen ions, or H+). When a subunit of hemoglobin binds to oxygen, the subunit not only changes its shape but also becomes a stronger acid and releases a proton. The oxygenation of one subunit of hemoglobin (HHb+) to form oxyhemoglobin (HbO2) can be expressed by the following equilibrium:

HHb+ + O2 « HbO2 + H+ (eq. 1)

The balance of this reaction can be shifted forward or in reverse by a change in the concentrations of either reactants or products. Raising the concentration of O2 favors the forward direction and the binding of O2, whereas raising the concentration of H+ (increasing the acidity) favors the reverse direction and the release of O2.

Acid affects the binding of oxygen to hemoglobin. One source of acidification is lactic acid, a metabolic product made by muscle cells in extracting energy from glycogen. The other is carbon dioxide, which is hydrated (combined with a molecule of water [H2O]), under the catalytic influence of carbonic anhydrase to make the bicarbonate ion (HCO3-), accompanied by the release of a proton. This reaction can be expressed by the following equilibrium:

CO2 + H2O « HCO3- + H+ (eq. 2)

As the erythrocytes pick up carbon dioxide from the tissues, the hydration of CO2 via carbonic anhydrase generates acid (H+). The increase in H+, in turn, drives the reaction in eq. 1 in reverse, thus favoring the release of O2. Once the erythrocytes reach the lungs, their release of CO2 via the reverse of reaction in eq. 2 diminishes H+ and so drives reaction in eq. 1 forward, favoring the uptake of O2.

There is another important aspect to the effect of acidity on the oxygenation of hemoglobin via reaction in eq. 1: one having to do with buffering, or minimizing changes in the acidity of the blood. As shown by reaction in eq. 2, carbon dioxide entering the blood from the tissues is hydrated by carbonic anhydrase in the erythrocytes with the release of protons. The protons could seriously acidify the blood traversing the tissues were it not for the fact that these protons are at the same time being taken up by oxyhemoglobin as it releases oxygen - the reverse of reaction in eq. 1. Conversely, in the lungs the loss of carbon dioxide from the blood would seriously deplete H+ but for the fact that the hemoglobin present is releasing protons as it binds to oxygen - reaction in eq. 2. Loss of carbon dioxide thus helps drive the oxygenation of hemoglobin in the lungs, while gain of carbon dioxide drives the release of oxygen from oxyhemoglobin in the tissues. The involvement of protons in both the reaction in eq. 1 and the reaction in eq. 2 provides the basis for this balance while simultaneously allowing the transport of large amounts of potentially dangerous acid without significant changes in the acidity of the blood.

Apparatus and Materials

1.  Fisher Scientific Accumet Model 625 pH meter with combination glass-silver/silver chloride electrode and swing arm electrode holder

2.  Thermometer

3.  1000 mL and 200 mL micropipets

4.  Magnetic stirrer and stirring bar

5.  Assorted glassware and plasticware

6.  Phenolphthalein and methyl red indicator solutions

7.  Mettler balance

8.  100 mL volumetric flasks

9.  Bovine hemoglobin obtained from Sigma Corp, #H2500

10.  Chicken egg albumin obtained from Sigma Corp, #A5253

11.  1M solutions of HCl and NaOH to be diluted to 0.1M for titration

Procedure

Each week prior to the commencement of the actual experiments, the pH meter was standardized following the standardization procedure found in the BE 210 Lab Manual2. Using standard titration procedures, both met-hemoglobin and egg albumin were titrated to determine their respective pK values and isoelectric points.

Week 1:

The first concentration of hemoglobin used was 0.008g/dL. This very dilute concentration caused the pH meter to be unsuccessful in giving a consistent reading. Therefore, the concentration of hemoglobin was increased to 0.08g/dL. This concentration did not fare better: it led to further inconsistency in the pH meter’s output. Finally the concentration was raised to 1g/dL, and NaCl salt was added to the hemoglobin. Salt did not affect the pH, but when dissolved in the hemoglobin solution, the Na+ and Cl- ions allowed electricity to be conducted through the solution. Therefore, the current could flow and the deionized water acted as an insulator. Before the titration took place, 0.1M HCL was added to bring down the pH of hemoglobin to below the pKa value. Titration was initially performed by adding 0.1 mL of NaOH at a time and then increasing the volume of titrant to 0.2mL at a time. Another titration of 0.1g/dL hemoglobin was conducted, but this again led to an inconsistent reading because the hemoglobin concentration was too low. The data that was obtained for the 1g/dL hemoglobin was then plotted on a pH vs. titrant volume graph and both the pK values and the isoelectric point were determined. pK values were determined by fitting a 3rd degree polynomial trendline to the regions of the graph showing points of inflection. By setting the second derivative of the trendline equation equal to zero, the pK value (represented by the pH at the inflection point) was determined in each case. The first and second pK value for each trial were averaged to find the isoelectric point.

Week 2:

Two trials of hemoglobin and two trials of egg albumin (both with concentrations of 1g/dL) were performed. As in the previous week NaCl salt was added to both the samples, in order to get accurate pH readings. Initially 0.1M HCl was added to bring down the pH of both the 1g/dL concentration of hemoglobin and egg albumin. The hemoglobin was titrated first by adding 0.2mL of NaOH as the titrant. The egg albumin trials were performed by titrating in a similar fashion using 0.2mL of NaOH. The data for theses two separate trials were then plotted on a pH vs. titrant volume curve to determine the pK values and the isoelectric point using the same regression procedure as was used in week 1.

Week 3:

For the final trail, a 1g/dL sample of egg albumin was titrated. Again, NaCl salt was added to the sample before titration as well as 0.1M HCl until the pH dropped below the first pK value. A single trial was then performed by adding 0.2mL of NaOH to the sample. Finally these data was plotted on a graph and both the pK values and the isoelectric point were determined in the same manner as in previous trials. pK values and isoelectric points for both hemoglobin and egg albumin were averaged for the three trials.

Titration curves showing the relationship between volume of titrant added and pH of the mixture were used in this experiment to find the pK values. Three trials were performed for both hemoglobin and albumin, resulting in six pH-volume graphs. These graphs are shown in Figures 2-7. In each graph, two points of inflection can be observed. By fitting a trendline to the section of the curve surrounding the inflection point, an equation can be obtained. This task was performed for each inflection point in each trial. Second derivatives were taken of the equations, and by setting these equations equal to zero, inflection points were obtained. These inflection points represent the pK values, or the pH values at the equilibrium constants.

For the first hemoglobin trial, multiple concentrations were used in order to find a correct titration curve. One of the early attempts, using 0.8g of hemoglobin per 100 mL of water is plotted in Figure 1. This curve shows no visible inflection points, so the concentration of Hb was modified. Concentrations of 1g of hemoglobin per 100 mL of water produced titration curves with noticeable inflection points.

After settling on the correct amount of Hb to use, the three trials were performed. Trendlines were plotted on the graphs as described in the procedure. An example graph of the section surrounding an inflection point fitted with a trendline is shown in Figure 8. Findings from the hemoglobin trendlines are summarized below in Table 1:

Table 1: Summary of pK and isoelectric data for hemoglobin titrations

Literature values for the isoelectric point of hemoglobin give a pH of 6.8.3 The value obtained experimentally differs by this value by 0.07.

Results for the egg albumin trials are summarized in Table 2.

Table 2: Summary of pK and isoelectric data for albumin titrations

Values obtained from the same literature source cite the isoelectric point for egg albumin as 4.6.3 The experimental value differs by 0.02. The author of the source, however, only presents his data to two significant figures. Restricting the experimental value to two significant figures yields the same value as that obtained from literature.

In both Table 1 and Table 2, R2 values are displayed. In every case, these values are within less than 0.01 of the ideal value, 1. This shows excellent correlation between the plotted trendlines and the actual data points. Due to this near equivalence, it is possible to say that the trendline error is minimal.

Figure 1: Titration graph for initial Hb trial. No visible inflection points show that there was an error in the concentrations. Another error in this trial occurred because the initial pH was not brought down to below the pKa value. Modifying concentrations and lowering the pH to below the first pK value yielded curves with inflection points, as shown in Figures 2-7.

Figure 2: Titration graph for the first hemoglobin trial. Inflection points representing pK values are indicated by *, while the isoelectric point is indicated by a PI.

Figure 3: Titration graph for trial 2 of hemoglobin. pK values and isoelectric points are in the same range as in Figure 2.

Figure 4: Titration graph for trial 3 of hemoglobin.
Figure 5. Titration graph for trial 1 albumin. Inflection points representing pK values are indicated by *, while the isoelectric point is indicated by a PI.

Figure 6. Titration graph for trial 2 albumin.

Figure 7. Titration graph for trial 3 albumin.

Figure 8. Trendline fitted to second inflection point of first hemoglobin trial. The equation of the trendline and its R2 value are displayed in the lower right corner. The pH at the inflection point represents the pKb value for this trial.