Oxygen Dissociation Curve and Bohr Effect Using Haemolysate

Oxygen diffusion is essential for the survival of living animals. Differences in the surface area and relative thickness of respiratory surfaces among vertebrates have been shown to influence the rates of oxygen diffusion and the levels of aerobic activity (Gillooly et al., 2016). Different animals have developed specialized organs in which gas exchange can take place, such as the gills in fish and the lungs in humans. These specialized organs facilitate the transportation of oxygen molecules from the lungs to body tissues (Resource, n.d.). This transport system depends on the red blood cells or erythrocyte, which contains haemoglobin molecules.

Haemoglobins (Hb) plays an important role in the facilitation of diffusion and transportation of oxygen (Hsia, n.d.). In the blood, haemoglobin carry out the binding of oxygen molecules to the red blood cell (Resource, n.d.). One hemoglobin molecule has four heme, the iron-containing and oxygen-binding portion of the hemoglobin, and thus meaning that each haemoglobin molecule is capable of binding to a maximum of 4 oxygen molecules (Resource, n.d.). Since hemoglobin have a high affinity for oxygen, oxygen molecules are bound to hemoglobin as it diffuses into the red blood cells (Resource, n.d.). When oxygen is bound to hemoglobin, the hemoglobin molecule is now oxygenated, and it is now called oxyhemoglobin which possesses a bright red-colour (Resource, n.d.). On the other hand, deoxygenated hemoglobin possesses a darker, purplish- red colour. This difference in colours can be detected using a spectrophotometer which measures the reflection and transmission of light in solutions.

Factors such as temperature, pH, carbon dioxide, carbon monoxide, and 2,3-BPG can affect the affinity of hemoglobin to oxygen. When the concentration of compounds such as carbon dioxide increases, this can cause a decrease in the blood pH and as a result, a decrease in the amount of oxygen molecules bounded to hemoglobin (Riggs, n.d.). This is referred to as the Bohr effect. This effect causes the sigmoid-shaped display of the oxygen dissociation curve (Hsia, n.d.).

In this experiment, the % transmittance of sheep blood (haemolysate) during the process of gradual deoxygenated is measured using the spectrophotometer. Oxygen dissociation curves, where the % saturation is plotted against the partial pressure of oxygen (PO2), are created to show the relationship between the effect of the changing PO2 on % oxygen saturation of hemoglobin and the effect of pH on the affinity of hemoglobin to oxygen molecules (Hsia, n.d.). P50 on the curves is defined as the oxygen tension during which the hemoglobin-oxygen binding sites are 50% saturated (Hsia, n.d.).

As a result of the Bohr effect, it is hypothesized that as the pressure of the vacuum increases, the blood or haemolysate will become more deoxygenated, meaning that there is now a reduced affinity to oxygen, and thus causing a right shift in the oxygen dissociation curve. Furthermore, it is predicted that as the pressure of the vacuum increases, the % transmittance of the blood would decrease as a result. The Hb Saturation (%) is predicted to increase as the partial pressure of oxygen (mmHg) is increasing.

Methods

This experiment involves four parts and the experiment is performed as described in the BIO202 lab manual. As stated in the lab manual, half of the class will perform the experiment using the haemolysate buffered at pH 7.4 and the other half will test the haemolysate buffered at pH 6.8 in groups of 4 (pH 6.8 was collected as a group with: Sarah Boganee, Divya Sharma, and Sze-nga Cecilia Yeung; data for pH 7.4 was collected from Manaal Ali and Fahad Ahmed).

Before starting the experiment, the procedures to using the vacuum properly should be practiced. While using the vacuum, it should be noted that if the vacuum valve is opened too quickly, an overshoot of the target reading can occur. When this happens, the vacuum should be turned off and the stopper should be removed from the side arm test tube to reduce the vacuum pressure. If the vacuum is opened too quickly, this can also cause haemolysate to bubble up in the side arm tube. To prevent this from occurring, the vacuum should be increased slowly, and the side arm tube should be placed in an upright position, until the haemolysate becomes deoxygenated or darker in colour, before tilting the tube.

Part 2 of the experiment is to collect data for creating the oxygen dissociation curves. To prepare, the spectrophotometer should be plugged-in and turned on during the preparation of the other materials. The spectrophotometer is set to measure the & transmittance (%T) and the wavelength is set to 625nm. Following, label 9 clean test tubes for the reference blank and each vacuum pressure level that will be tested: buffer (blank), 0 mmHg, 300 mmHg, 400 mmHg, 500 mmHg, 550 mmHg, 600 mmHg, 650 mmHg, and 700 mmHg. To avoid any mistakes, any condensation and/or fingerprints sitting on the outside of the sample tubes should be wiped with paper towel before placing into the spectrophotometer. Furthermore, using 2.5 ml of the buffer, appropriate for the corresponding pH, blank the spectrophotometer. This should only be done once. Then, transfer 2.5 ml of the haemolysate into the 0 mmHg tube and allow it to go to room temperature before taking the % transmittance reading. Additionally, transfer 2.5 ml of fresh haemolysate to the side arm test tube. Note that before starting the vacuum, turn on the manometer and ensure that the stopper has been placed in the top of the test tube. Following, set the vacuum to 300 mmHg and maintain it for 5 minutes while another student shakes the sample tube. Shaking the tube will expose the maximum amount of blood surface area to the vacuum. After having exposed the sample to the vacuum for 5 minutes, turn off the vacuum, remove the stopper, and using a pipette, transfer the sample to the appropriately labelled test tube and measure the % transmittance from the spectrophotometer. This step should be done carefully to avoid creating any air bubbles while pipetting and quickly because the blood will begin to re-oxygenate once the stopper has been removed. These steps of placing the haemolysate into the vacuum and recording the % transmittance is then repeated for each vacuum setting (400, 500, 550, 600, 650, 700) by using a fresh haemolysate sample each time.

After all the data has been collected, part 3 of the lab is to convert the manometer readings to the partial pressure of oxygen using the formula given in the lab manual: Partial Pressure O2 (mmHg) = 0.21 (D-W-M). This is done because the % saturation is plotted against the partial pressure of oxygen in an oxygen dissociation curve. The temperature and barometric pressure of the lab room should also be recorded from the barometer provided in the room. Using this temperature, the water vapour pressure, or W, can be determined from Table 2 in the lab manual. Refer to the appendix for the calculation steps.

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Finally, part 4 of the experiment is to convert % transmittance to % oxygen saturation of Haemoglobin. Refer to the appendix for the calculation steps. To create the oxygen dissociation curve from the data collected, standard curves involving the % oxygen saturation of haemoglobin to the % transmittance for each pH tested should be plotted first. The data for this standard curve is provided in the lab. Last but not least, clean up by removing all the labels from the test tubes used, place the used test tubes and stoppers in the containers by the sink, dispose of the Haemolyssate down the sink, turn-off and un-plug the spectrophotometer, turn off the manometers, return any used materials to their original positions, wipe the bench and wash your hands before leaving the lab.

Results (Figure):

P50 for pH 7.4 P50 for pH 6.8 (where Hb saturation is 50%)

Figure 1 Oxygen Dissociation Curves plotting partial pressure of oxygen (mmHg) vs. % saturation of Hb. Substance used: sheep blood. Data is obtained from part 2 of the experiment with the use of the vacuum and the measurements from the spectrophotometer.

Results

For the % transmittance, both pH 6.8 and 7.4 share similar values during each manometer readings. Additionally, from the data collected, as the pressure of the vacuum increases with each reading (0, 300, 400, 500, etc.), the % transmittance of both pH 6.8 and pH 7.4 decreases. The partial pressure of oxygen (PO2) in mmHg also decreases as the pressure of the vacuum increases. Furthermore, for the Hb saturation (%) of both pH 6.8 and 7.4, the values decrease as the PO2 decreases. The P50 of the trendline of pH 6.8 has the value of ~88 mmHg, and the P50 of the trendline of pH 7.4 has the value of ~93.04.

Discussion

The partial pressure of oxygen plays an important role in the determination of the degree of oxygen molecules binding to hemoglobin and oxygen dissociation from hemoglobin (Resource, n.d.). The affinity of oxygen molecules to hemoglobin increases as more oxygen molecules are bound and as a result, the oxygen dissociation curve shows that as the partial pressure of oxygen increases, the % saturation of Hb also increases (Resource, n.d.). This trend can be seen in Figure 1 and thus it can be interpreted that the results of the experiment support the hypothesis/predictions in which it was hypothesized that a right shift in the oxygen dissociation curve would occur, and predicted that as the pressure of the vacuum increases, the % transmittance of the blood would decrease as a result and that the Hb saturation (%) would increase as the partial pressure of oxygen (mmHg) increases.

Considering the structure of the hemoglobin and the fact that it is composed of 4 heme units in which oxygen binds in sequences (one after the other), a conformational change can occur when the first oxygen molecule binds. This conformational change allows the second oxygen molecule to bind more effectively (Resource, n.d.). When all 4 heme units are bound to oxygen, the hemoglobin molecule is said to be saturated. The more the oxygen molecules that are bound to hemoglobin, the more saturated it comes and therefore, an increase in the partial pressure of oxygen would cause an increase in the oxygen saturation of hemoglobin.

The pH also plays an important role in the overall shape of the oxygen dissociation curve. Referring back to the Bohr effect, the Bohr effect shows the relationship between pH and the affinity of hemoglobin to oxygen (Resource, n.d.). This means that the lower and the more acidic the pH, the higher the oxygen dissociation from hemoglobin (Resource, n.d.). When oxygen dissociation from hemoglobin is high, this will result in a right shift in the oxygen dissociation curve due to the low pH and the loss of affinity of oxygen molecules to hemoglobin. The opposite is also true in which a higher, more basic pH will result in a left shift of the oxygen dissociation curve.

Additionally, the oxygen dissociation curve also plays a role in the automatic control mechanisms which regulate the amount of oxygen that is delivered to the tissues throughout the body (Resource, n.d.). This is essential in oxygen reserves during immediate situations where body tissues require more oxygen. In highly active tissues such as muscles, oxygen dissociation from hemoglobin is higher and thus more oxygen molecules enter the tissues (Resource, n.d.). On the other hand, in tissues with lower metabolic rates, less oxygen is used and thus the partial pressure of oxygen within these tissues are relatively high and as a result, there’s less oxygen molecules dissociate from hemoglobin (Resource, n.d.). The body can therefore benefit from the Bohr effect in which the Bohr effect allows for more efficient transportation of oxygen throughout the blood and this is useful in situations such as an increase amount of oxygen being available in highly active muscles such as exercising skeletal muscles.

Finally, although the results provide support of the original hypothesis and predictions, there are a few improvements that could be made to the overall experiment. One improvement that could be made would be to properly shake the test tube during data collection to ensure no bubbles were created in the sample. Another improvement that could be made would be to conduct the experiment using a wider range of pH to account for the differences and influences that the pH could bring onto the oxygen dissociation curves.