Because these respiratory pigments have a high affinity for oxygen and/or carbon dioxide, greater amounts of these gases may be transported than would normally occur if the gases were simply dissolved in water. However, the affinity of the respiratory pigment for O₂ or CO₂ is dependent, along with other factors, upon pH, temperature, and concentration of the gases. Prior to coming to the laboratory, review the dynamics of the relationships between pigments (e.g., hemocyanin and hemoglobin) and gases (Hickman et al., 2004; pages 672-674).

Knowns: 

1. The affinity of hemoglobin for oxygen changes with pressure.

2. Oxygen concentration in liquids changes with pressure.

Question: What is the basis for the relationship of hemoglobin and pressure? 

Hypothesis: Association of hemoglobin and oxygen will decrease as partial pressure of oxygen decreases.

Task: Using the vacuum manometer provided, determine the oxygen dissociation curve for hemoglobin, a respiratory pigment found in erythrocytes of vertebrates and other animals, by creating various partial pressures of oxygen. 

[The following was accomplished by the lab technician]: The hemoglobin to be used was first freed from the erythrocytes from a blood sample of a mammal and the blood centrifuged until a clear amber supernatant was obtained. A spectrophotometer (Spectronic 20) was set at 606 nanometers and the supernatant containing the hemoglobin with saline was diluted to achieve a 50-70% transmission. The hemoglobin was oxygenated by gently agitating the supernatant in a flask that was open to the atmosphere.

PROCEDURE: First, practice with your partners, achieving known reduced pressures in an empty side-arm flask as follows (Be cautious because the apparatus is delicate):

1. Turn the valve to open the flask to the vacuum pump.

2. Turn on the vacuum pump and increase suction (vacuum) by gradually turning the regulator on the intake side of the pump while a partner watches the movement of the gauge. Turn the valve on the stopper to close off the flask when you reach the desired vacuum level.

When you have become proficient at controlling pressure changes, open the regulator to allow pressure to reduce to ambient pressure, remove the stopper and add the supernatant containing just enough hemoglobin to the flask to fill the side arm to a level appropriate to take readings using the spectrophotometer and proceed with the following steps: 

3. Standardize the Spectrophotometer with blank of DIHOH.

4. At ambient pressures (e.g.760 mm Hg), gently agitate the hemoglobin solution briefly, then tip solution into side arm. Clean the outside of the side-arm if necessary. Place the side-arm filled with the supernatant into the spectrophotometer that has been set to 100% transmission (T) with saline and determine the % transmission. Record the % transmission in the table below. 

5. Next, proceed to reduce pressure by 140 mm Hg (i.e. to 620 mm Hg) by suction aspiration. When this vacuum is reached, use the valve to close the connection of the flask to the system as practiced before.

6. Gently agitate the supernatant and hemoglobin in the flask in order to increase the exposure of the hemoglobin to the reduced pressure and facilitate equilibrium. Do this for at least 1 min. Determine % transmission of supernatant. 

7. Repeat this series of steps to achieve pressures in the flask of 480, 340, 200, and 60 mm Hg.

8. Reduce vacuum to ambient pressure. Add 0.3 g of sodium hydrosulfite (Na₂S₃O₄ . 2H₂O) to the flask containing the hemoglobin, gently agitate as before and determine % transmission. Sodium hydrosulfite will completely deplete the sample of oxygen and simulate a vacuum unachievable with the apparatus, i.e. a vacuum of 760 mm Hg. 

[Make sure you understand the difference between mm Hg vacuum and mm Hg pressure.]

Calculations: Convert all total pressure readings to partial pressure of oxygen and enter into the table below. Remember that you must convert vacuum levels to absolute pressures first by subtracting them from 760 mm Hg. For example, a 280 mm Hg decrease in pressure (760-280) = 480 mm Hg pressure; the partial pressure for oxygen at that pressure (.21 x 480) = 101 mm Hg.

Assume that the %T of hemoglobin at standard pressure corresponds to 100% saturation and that the %T after addition of sodium hydrosulfite corresponds to 0% saturation. Plot %T on the Y axis against % saturation on the X axis, and fit a straight line to these points. (Clicking on each below opens a new window with the graph. You would be really clever if you printed each graph out and brought them to lab. )

On a different chart, illustrated below, plot % saturation (Y axis) against partial pressure as determined from the first graph to produce an oxygen dissociation curve for hemoglobin. 

Plot % saturation at each partial pressure against partial pressures of oxygen to produce an oxygen dissociation curve for hemoglobin. Compare your graph to a standard oxygen dissociation curve such as the one illustrated in your text book.

Questions:

Do your results support your hypothesis?
 
 
 
 

Define dependent and independent variables. In this procedure, which are the dependent and which are the independent variables?
 
 
 

Describe the effect a change in altitude would have on your capacity to use oxygen. Can you envision a maximum altitude at which respiratory efficiency would be zero?
 
 
 
 

What does this curve indicate regarding hemoglobin's affinity for oxygen at the level of the lungs versus at the level of the tissues? How does this difference relate to the advantage of a respiratory pigment for gas transport? 

Go back to the syllabus.