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Hemoglobin lab.pptx
- 1. Lab 2: Hemoglobin-Oxygen Binding
- 2. Hemoglobin protein is made of 4 polypeptide chains Each of these 4 chains contains a heme group • The heme group contains Fe2+ that can bind O2
- 3. Hemoglobin and HbO2 • Hemoglobin exists in the blood as the oxygenated form (HbO2) and the deoxygenated form (Hb) • Differences in partial pressures of oxygen (PaO2) between the systemic capillaries and peripheral tissue allows O2 to diffuse into the cells • The % of HbO2 and PaO2 are compared via oxygen-hemoglobin dissociation curve ⇌
- 4. Oxygen-Hemoglobin Dissociation Curve (Pages 1-3) P50: the pressure at which half of the hemoglobin is bound to oxygen Cooperative binding: the (un)binding of one molecule promotes the (un)binding of the next molecule
- 5. Absorbance How a spectrophotometer works: We use absorbance of visible light to study the Hb-O2 relationship.
- 6. As HbO2 is deoxygenated to Hb, different concentration leads to different absorption at 660nm of wavelength Molar absorptivity How does the deoxygenation of Hb change its absorption?
- 7. Be sure to set the spectrophotometer to Absorbance mode Zero the spectrophotometer with a blank tube between samples and different experimental conditions Reminders for operation of spectrophotometer
- 8. Hb-O2 dissociation curve Deoxygenate the oxygen from PaO2 760 mmHg in 140mmHg intervals Measure absorbance of Hb-O2 after every decrement until PaO2 reaches 0 Spectrophotometer
- 9. Using the absorbance data, calculate the % O2 saturation of hemoglobin at each step For example, a 280 mm Hg decrease in pressure: (760–280) = 480 mmHg Calculate the PaO2: (0.21 x 480) = 101 mmHg Construct an oxygen-hemoglobin dissociation curve for the sample (control and treatment) % Saturation = (A–B)/(A–C) x 100% Hb-O2 dissociation curve C = Absorbance before deoxygenation (before removing oxygen) B = Absorbance after each deoxygenation step (in between) A = Absorbance after complete deoxygenation (after removing oxygen)
- 10. Right Shift = Release of Oxygen lower affinity Left Shift = Loading (binding) of Oxygen higher affinity What shift did you see for pH and cold treatments?
- 11. Factors Affecting the Oxygen-Hemoglobin Dissociation Curve CADET faces right (↑ in CO2, acidity, DPG, Exercise, Temperature)
- 12. P50 is reached when _____ A) the partial pressure of O2 in blood is 50 mmHg B) hemoglobin is 50% saturated with oxygen
- 13. If the oxygen-hemoglobin dissociation curve moves to left, the affinity between O2 and Hemoglobin is ____ A. Lower B. Higher
- 14. pCO2, acidity and temperature are higher in capillary circulation than in arterial blood. In this circumstances, Hb has lower affinity to oxygen. That enables: A. Hb to release oxygen B. Hb to hold on to oxygen
- 15. O2 binding affinity of myoglobin vs hemoglobin: which one has a higher oxygen- binding affinity?
- 16. O2 affinity fetal vs adult hemoglobin: which one has a higher oxygen-binding affinity?
Editor's Notes
- On the surface of red blood cells, there are millions of hemoglobin molecules. The hemoglobin protein is made up of four polypeptide subunits, each of which contains a heme group. This a ring-like structure that contains iron. The iron in the heme group binds to molecular oxygen, allowing the red blood cells to transport oxygen to other tissue. The binding of oxygen is reversible.
- Hemoglobin binds oxygen to form oxyhemoglobin (HbO2), the main oxygen carrier in human body. Difference in the partial pressure of oxygen between systemic capillaries and peripheral tissue allows oxygen to diffuse into cells. On the oxygen-hemoglobin dissociation curve, the % of HbO2 and PaO2 are plotted. The tissues sit at a lower resting oxygen concentration making the affinity for oxygen lower allowing oxygen to unload. In the lungs, hemoglobin is saturated and has a high affinity for oxygen allowing oxygen to load.
- The sigmoidal curve shape is due to Cooperative binding of the four hemoglobin subunits – as one subunit binds O2, it increases the O2 affinity for remaining subunits. oxygen affinity of 3-oxyhemoglobin is ~300x that of deoxyhemoglobin. This allows hemoglobin to be far more dynamic in binding O2, and to rapidly transition from a low oxygen-bound state to an oxygen saturated state. It also allows hemoglobin to off-load O2 rapidly, as affinity for O2 decreases. P50 is the pressure at which half of the hemoglobin is bound to oxygen
- The sequence of events of a spectrophotometer are as follows: The light source sends light into a monochromator and is diffracted into a spectrum that is split into two beams. These two beams scan through the sample that you are measuring. Some fractions of light are transmitted through the sample and some are reflected off of it. The transmitted light and reflected light are measured by the photo detector device which compares the relative intensities The spectrophotometer then reads out an absorbance value.
- If we looked at the absorption of light at 800 nm, we would not be able to tell a difference between oxygenated and deoxygenated hemoglobin.
- In this part of the experiment, students will use the vacuum gauge provided to gradually remove oxygen from the flask with a side arm cuvette. This multi-step deoxygenation process will create various partial pressure of oxygen and allow hemoglobin and oxygen to disassociate gradually. Be aware that the readings of the vacuum gauge is compared to the atmospheric pressure. Make sure you understand the differences between mm Hg vacuum and mm Hg pressure. Zero reading from the gauge means approximately 760 mm Hg.
- Remind students to keep flask closed when deoxygenating. mm Hg is a unit of pressure measurement, specifically for measuring pressure in liquids and gases. It stands for millimeters of Mercury and refers to the height of a column of mercury (Hg) in a manometer. For example, a 280 mm Hg decrease in pressure: (760–280) = 480 mmHg Calculate the PaO2 at that pressure by multiplying it by the fraction of air that is oxygen (21%) (0.21 x 480) = 101 mmHg.
- BPG/DPG – Increase shifts curve to the right, decrease to the left. Acts as a heteroallosteric inhibitor by binding preferentially to deoxyhemoglobin, lowering hemoglobin’s affinity for oxygen and shifting the curve right. It interacts with deoxygenated hemoglobin beta subunits and so it decreases the affinity for oxygen and allosterically promotes the release of the remaining oxygen molecules bound to the hemoglobin; therefore it enhances the ability of RBCs to release oxygen near tissues that need it most Temperature – Increasing temperature weakens the O2 – Hb bond, decreasing the concentration of oxyhemoglobin. Acidity – H+ and O2 compete for the heme group, so at lower pH (higher H+ conc.) there is less oxyhemoglobin, shifting the curve right. An increase in CO2 will also cause an increase in acidity (CO2 + H2O <-> HCO3- + H+). O2 affinity is inversely related to both acidity (H+ conc) and CO2, referred to as the Bohr effect. Exercise causes an increase in acidity, temperature and metabolic intermediates and a decrease in oxygen in your muscle tissues. This causes an increased dissociation of oxygen from your blood flowing through your muscles, supplying them with much needed oxygen. Your body increases blood flow to your muscles to supply them with more oxygen. Warming up before exercise can help prime your muscles to receive ample oxygen by kick starting metabolism and increasing temperature.
- B
- B-Review page 6
- A-review page 7
- Like hemoglobin, myoglobin is a cytoplasmic protein that binds oxygen on a heme group. It harbors only one globulin group, whereas hemoglobin has four. Although its heme group is identical to those in Hb, Mb has a higher affinity for oxygen than does hemoglobin. This difference is related to its different role: whereas hemoglobin transports oxygen, myoglobin's function is to store oxygen. The steep (hyperbolic dissociation curve) indicates that O2 affinity is higher for myoglobin than for hemoglobin. This makes myoglobin the oxygen storage molecule, while hemoglobin is the oxygen transport molecule. The cooperative binding of O2 to hemoglobin allows it to dynamically load and offload O2 as it travels around the body, while the higher O2 affinity of myoglobin makes it a good O2 storage molecule to facilitate a build up of O2 in the muscles that can be released when needed.
- 1 - Higher affinity than adult hemoglobin (also a lower affinity for DPG/BPG). 2 - Fetal hemoglobin needs to extract oxygen from the maternal blood. During pregnancy, the mother will also increase DPG/BPG concentration, shifting the adult curve to the right, making it easier for fetal hemoglobin to extract O2. Fetal hemoglobin (HbF) is the dominant form of hemoglobin present in the fetus during gestation. HbF is produced by erythroid precursor cells from 10 to 12 weeks of pregnancy through the first six months of postnatal life. HbF contains two alpha and two gamma subunits, while the major form of adult hemoglobin, hemoglobin A (HbA), contains two alpha and two beta subunits. The genes that express gamma chain proteins are present in the beta chain locus on chromosome 11. The gamma subunit differs from its adult counterpart in that it contains either an alanine or a glycine at position 136, both of which are neutral, nonpolar amino acids. This difference introduces conformational changes to the protein that gives rise to several physiological differences in oxygen delivery that are important in the fetal circulation. Fetal hemoglobin has a vital role in the transport of oxygen from maternal to fetal circulation. Oxygen transfer from the maternal circulation to the fetal circulation is made possible by HbF having a high oxygen affinity but decreased affinity to 2,3-bisphosphoglycerate relative to HbA. The HbF oxygen dissociation curve is left-shifted in comparison to HbA. The partial pressure at which HbF is half saturated with oxygen (P50) is 19 mm Hg, compared to 27 mm Hg for HbA. This value indicates that HbF has a high affinity for oxygen, giving HbF the ability to bind oxygen more readily from the maternal circulation.