Animal Physiology Lab Report: Oxygen Saturation and Bohr Effect

This lab report investigates the effect of partial pressure of oxygen and pH on hemoglobin saturation in sheep blood, focusing on oxygen dissociation curves and the Bohr effect. The experiment involved measuring the percentage transmittance of sheep blood at varying partial pressures of oxygen and two different pH levels (6.8 and 7.4) to determine the oxygen affinity of hemoglobin. The results showed that as the partial pressure of oxygen increased, hemoglobin saturation also increased, and a lower pH resulted in a higher partial pressure of oxygen, indicating a lower oxygen affinity. This aligns with the Bohr effect, which describes the impact of pH on oxygen binding. The study also highlights the importance of these physiological mechanisms in ensuring efficient gaseous exchange within the body, particularly during increased metabolic demands. Although the P50 value could not be determined for a pH of 7.4, the overall findings support the initial hypothesis that increasing oxygen partial pressure and pH would affect hemoglobin saturation.

Running head: ANIMAL PHYSIOLOGY 1
Animal Physiology
Name
Institution

ANIMAL PHYSIOLOGY 2
Investigating the Effect of Partial Pressure of Oxygen on the Saturation of Hemoglobin and the
Bohr Effect
Introduction
Oxygen plays a very important role in cellular respiration. Oxygen penetrates tissues
through diffusion in smaller organisms. However, when these organisms have grown to a
particular size, gaseous exchange through diffusion becomes non-sustainable and they, therefore,
have to exchange gases in other ways. Animals have special organs that aid in gaseous exchange.
To maximize gaseous exchange animals have a circulatory system that transports blood all over
the body. Blood has red blood cells that have hemoglobin which carries oxygen around the body.
Hemoglobin can be defined as a protein molecule present in red blood cells that facilitate the
movement of oxygen from the lungs to body tissues (Jorge, Ribeiro, Santos & de Fátima Sonati,
2016). Hemoglobin also gives the blood a bright red color. A single molecule of hemoglobin can
bind to four oxygen molecules due to hemoglobin’s high oxygen affinity. The amount of oxygen
that can bind with hemoglobin is determined by the partial pressure of oxygen (PO2).
The binding of oxygen and hemoglobin in the red blood cells is responsible oxygen
dissociation curves’ sigmoid shape that is seen on most graphs. An oxygen dissociation curve
can be described as a curve that reveals the correlation between the saturation of oxygen in
hemoglobin and the partial pressure of oxygen (Bain, Bates & Laffan, 2016). This curve shows
hemoglobin’s oxygen affinity as the PO2 changes. An oxygen dissociation curve is determined
by the PO2 and the percentage of hemoglobin which is saturated with oxygen. If the curve shifts
to the right then it is an indication that the hemoglobin’s oxygen affinity has reduced. As a result,
hemoglobin releases oxygen to the tissues of the body. Conversely, if the curve moves towards
the left with a changing pH then it is an indication that hemoglobin’s affinity for oxygen is high.

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As a result, hemoglobin responds by taking up and retaining oxygen. The amount by which the
curve shifts are represented by P50O2. P50 is the partial pressure of oxygen at 50% saturation of
hemoglobin (Bain et al., 2016).
Low oxygen affinity can result from an increase in the concentration of carbon (IV) oxide
thus forcing the curve to shift to the right. This reduction in hemoglobin’s oxygen affinity is
described by Bohr’s effect which claims that a change in PO2 is caused by changes in blood's
pH. At normal temperature and pH, Bohr’s effect value is approximately 0.45 (Bain et al., 2016).
Bovo et al. (2015) performed a similar experiment on rattlesnakes and they found out that
an increase in hemoglobin saturation was affected by an alkaline tide after meal ingestion. This
experiment was performed to investigate the effect of partial saturation of oxygen and pH on
hemoglobin saturation in sheep blood. My prediction of the outcome is that an increase in PO2
will increase hemoglobin saturation. Additionally, a decrease in pH will increase hemoglobin
affinity thus shifting the curve to the right.
Methods
During this experiment, students used two different pH values to test the oxygen
dissociation of sheep blood. Haemolysate was used to collect data in the lab. We started the
experiment by setting a vacuum and ensuring that the manometer reading was 300mmHg. A
sidearm test tube was connected to the vacuum and the vacuum slowly opened until a manometer
reading of 300mmHg was attained. We held the tube in an upright position while slowly turning
the vacuum to prevent hemolysate from bubbling up in the tube.
In the second part of the experiment, we collected data for the oxygen dissociation
curves. We plugged in the spectrophotometer and turned it on to give it time to start up. We then

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set the spectrophotometer to measure percentage transmittance (%T) and after which we set a
wavelength of 625 nm. We then labeled 9 test tubes as follows: reference blank, 0mmHg,
300mmHg, 400mmHg, 500mmHg, 550mmHg, 600mmHg, 650mmHg, and 700mmHg. Each
group of students blanked the spectrophotometer using 2.5 ml of the appropriate buffer
depending on the choice of pH (6.8 or 7.4). We then transferred 2.5 ml of hemolysate to the 0
mmHg test tube and allowed the sample to settle to room temperature. The outer part of the test
tube was wiped clean to get rid of any fingerprints or condensations. We placed the test tube in a
spectrophotometer and noted the % transmittance value. We then transferred 2.5 ml of fresh
hemolysate into the sidearm test tube and zeroed the manometer. We set the vacuum to 300
mmHg and waited for 5 minutes while shaking and tilting the sample tube to expose the
maximum surface area of the blood sample to the vacuum. The vacuum was turned off after the 5
minutes and a pipette used to transfer the whole sample into an appropriately labeled
spectrophotometer tube. We recorded the % transmittance reading from the spectrophotometer in
a table. We repeated the above procedure for the other vacuum settings; 300mmHg, 400mmHg,
500mmHg, 550mmHg, 600mmHg, 650mmHg, and 700mmHg.
My partner and I collected data for a pH of 7.4. After recording all the readings in a table,
we converted the manometer readings from the experiment to PO2 and % transmittance to %
oxygen saturation. We then plotted % saturation against partial pressure of oxygen on an oxygen
dissociation curve. We exchanged our data with Fellicia Henamona and Brahmleen Kaur.

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Results (Figure)
Figure 1: A graph of Oxygen Dissociation Curve for both pH values of 6.8 and 7.4. % Hb saturation was
plotted against partial pressure of oxygen after testing % transmittance of sheep blood in relation to
manometer reading. A vacuum was used to analyze the blood and the % Hb saturation was calculated
from % transmittance. P50 value was taken for the pH of 6.8. The P50 value reads 22 mmHg.
Results (Text)
From figure 1 above, we can observe that % hemoglobin saturation steadily rises with an
increase in PO2. At around 50 mmHg partial pressure of oxygen, the % hemoglobin saturation
for both 6.8 and 7.4 pH increases at a slower rate and flattens out becoming almost a constant.
The above figure also indicates that the value of % hemoglobin saturation does not go lower than
50 for a pH of 7.4. Therefore, we could not extrapolate the partial pressure of oxygen at P50 for a
pH of 7.4. However, for a pH of 6.8, the PO2 at P50 can be seen to be approximately 22 mmHg.
P50

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Based on figure 1 above, we can also see that the oxygen dissociation curves are shifted
according to the pH. The curve that represents a pH of 7.4 is shifted the left and it has higher
values of % hemoglobin saturation as compared to the curve representing a pH of 6.8.
Discussion
This experiment was performed to investigate the effect of the partial pressure of oxygen
and changing pH on percentage hemoglobin saturation. From the graph in figure 1 above, it is
visible that a low pH results in a high partial pressure of oxygen. This implies that at a low pH
hemoglobin’s affinity for oxygen is low. This low oxygen affinity can be caused by an increase
of carbon (IV) oxide in blood causing acidosis. As a result, hemoglobin releases oxygen to body
tissues to counter the rising levels of carbon (IV) oxide (Collins, Rudenski, Gibson, Howard &
O’Driscoll, 2015). A higher pH, on the other hand, leads to a low PO2. During this time,
hemoglobin has a high affinity for oxygen thus taking up oxygen from the lungs. The oxygen is
retained in hemoglobin until when tissues need it when the pH of blood drops. This relationship
between a change in pH and partial pressure of oxygen is in agreement with Bohr’s effect
(Collins et al., 2015). The concept of gaseous exchange and Bohr’s effect are important in the
human body because they ensure that the tissues of the body receive oxygen when they need it
thus facilitating cellular respiration and other important biological processes.
The graph in figure 1 also shows that the % hemoglobin saturation increases as oxygen’s
partial pressure increases. The curves then flatten out as the partial pressure of oxygen increases.
It is important to note that one molecule of hemoglobin can bind with four molecules of oxygen.
Hemoglobin has a property that increases its oxygen affinity once it has combined with a single
oxygen molecule thus facilitating the binding with the remaining oxygen molecules. Therefore,
at low oxygenation levels, the slope of the oxygen dissociation curve increases (Collins et al.,

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2015). As hemoglobin approaches full oxygen saturation, the curves flatten out. It is thus evident
that at a low PO2, hemoglobin is less saturated thus have a higher affinity for oxygen. As a
result, hemoglobin takes up oxygen from the lungs and becomes saturated. Once hemoglobin
becomes saturated with oxygen as oxygen’s partial pressure increases, affinity to oxygen reduces
and hemoglobin releases oxygen to body tissues (Collins et al., 2015).
The obtained data from the experiment did not produce curves that could reveal partial
pressure of oxygen at P50 for both pH values of 6.8 and 7.4 and thus P50 was incomparable for
the two curves. The unavailability of P50 for a pH of 7.4 could be due to errors that occurred
during the experiment. However, based on Bohr’s effect, P50 for pH of 7.4 could have been
lower than P50 for a pH of 6.8.
This study is important because it reveals the processes that take place inside the body to
ensure sufficient gaseous exchange between tissues. As the pH decreases, hemoglobin’s
saturation increases thus reducing % hemoglobin saturation. Therefore, oxygen is released into
tissues. This phenomenon is important during physical exercises because more oxygen can be
supplied to the muscles. The study reveals that the hypothesis stated before is correct that
increasing partial pressure of oxygen leads to an increase in the % hemoglobin saturation.
Additionally, an increase in pH lowers hemoglobin’s oxygen affinity.

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References
Bain, B. J., Bates, I., & Laffan, M. A. (2016). Dacie and Lewis Practical Haematology E-Book.
Elsevier Health Sciences.
Bovo, R. P., Fuga, A., Micheli-Campbell, M. A., Carvalho, J. E., & Andrade, D. V. (2015).
Blood oxygen affinity increases during digestion in the South American rattlesnake,
Crotalus durissus terrificus. Comparative Biochemistry and Physiology Part A:
Molecular & Integrative Physiology, 186, 75-82.
Collins, J. A., Rudenski, A., Gibson, J., Howard, L., & O’Driscoll, R. (2015). Relating oxygen
partial pressure, saturation and content: the haemoglobin–oxygen dissociation
curve. Breathe, 11(3), 194-201.
Collins, J., Rudenski, A., Gibson, J., Howard, L., & O’Driscoll, R. (2015). Relating oxygen
partial pressure, saturation and content: the haemoglobin–oxygen dissociation
curve. Breathe, 11(3), 194-201. doi: 10.1183/20734735.001415
Jorge, S. E., Ribeiro, D. M., Santos, M. N., & de Fátima Sonati, M. (2016). Hemoglobin:
Structure, synthesis and oxygen transport. In Sickle cell anemia (pp. 1-22). Springer,
Cham.

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Appendix
Figure 2: A graph of the standard curve for pH values of 6.8 and 7.4. Transmittance % was plotted
against % hemoglobin saturation using the standard values provided in class.
Sample Calculations
Partial Pressure of Oxygen
Partial pressure of oxygen (PO2) (mmHg)=0.21( D – W – M )
Where;
D is the barometric pressure measured in mmHg
W is the water vapor measured in mmHg

ANIMAL PHYSIOLOGY 10
M is the vacuum pressure measured in mmHg. This vacuum pressure is the reading taken from
the manometer.
0.21 is a constant.
PO2 ¿ 0.21 ¿
¿ 91.329 mmHg
% Hemoglobin Saturation
Using one of the regression line equations ( pH=6.8);
y=0.574 x+5.7
Where;
y=59.6
Therefore,
59.6=0.574 x+5.7
59.6−5.7=0.574 x
0.574 x=53.9
x=93.9024 %