THE HEMOGLOBIN PAGE
RBC's pick up O2 at the lungs and distribute that O2 to all cells of the body. The O2 carrying molecule in blood is hemoglobin (Hb). RBC's are essentially bags of heme dedicated to carrying O2. Hemoglobin is an RBC protein which can reversibly bind O2. About 25% by volume and 33% by weight of an RBC is made up of hemoglobin (Hb). Hemoglobin is the protein which imparts the red color to blood.
Hemoglobin (Hb) in g/100 mL of blood (% w/v)
14-20 g of hemoglobin/100 mL of blood in infants
13-18 g of hemoglobin/100 mL of blood in adult males
12-16 g of hemoglobin/100 mL of blood in adult females
Hemoglobin is a conjugated metallo-protein with 4 globular polypeptide subunits; 2 alpha and 2 beta globins. Each globin contains a porphyrin ring structure called heme (a porphyrin ring is made of 4 Nitrogen containing pyrole groups held together by methyl bridges). Each heme group covalently binds a central ferrus iron (Fe2+). It is the ferrus iron (Fe2+) in the middle of each of the heme rings which can reversibly bind O2. Of the 6 potential binding sites on each of the 4 Fe2+'s; 4 are bound to the porphyrin ring (the heme ring), 1 is bound to a histidine (the amino acid His) residue within the globin portion of the hemoglobin, and the remaining binding site is available to bind 1 molecule of O2, carbon monoxide (CO), or something else. Thus, each hemoglobin can carry 4 O2's. One RBC contains 250 million Hb molecules (ie. 1 RBC can carry as many as 1 billion molecules of O2).
Fetal Hb (humans & cattle) has gamma instead of beta globins. Alpha and gamma globins have a higher affinity for O2 than do beta subunits. Thus, fetal Hb has a higher affinity for O2 than does adult Hb. This higher affinity for O2 assists the fetal Hb in extracting O2 from maternal Hb. A pronounced response to the Bohr Effect allows fetal Hb to unload O2 at the tissues. The Bohr effect will be discussed in detail below; basically, the Bohr effect refers to a mechanism whereby acid in the tissues assists in O2 unloading from Hb, to promote O2 delivery to the tissues; the Bohr effect is very strong in the fetus. We will say more about fetal Hb below!
Greater than 98% of the O2 carried in the blood is bound to Hb; the rest is in dissolved form in the plasma; remember, gases can be dissolved in liquids, much the same way that a carbonated beverage contains a large amount of dissolved CO2. O2 "loading" onto Hb occurs in the lungs. As Hb picks up O2 in the lungs, it unloads CO2 and H+. When O2 binds to Hb, the Hb assumes a new conformation called "oxyhemoglobin," which has a bright red hue. O2 "unloading" occurs in the tissues to give "deoxyhemoglobin" (reduced Hb) which is dark red. As Hb unloads O2 at the tissues, it picks up CO2 and H+.
Approximately 20% of the CO2 carried in blood is bound to amino acids of the globin portions of hemoglobin. Thus, Hb carrying CO2 is called "carbaminohemoglobin." Carbaminohemoglobin has a blue tinge to it, such that venous blood is darker than arterial blood (NOTE: venous blood is NOT blue because of a lack of O2); arterial blood is red and venous blood is darker with a cyan blue tinge. Approximately 60% of the CO2 carried in the RBC is handled by the carbonic anhydrase enzymes of the red blood cell membranes (see bicarbonate buffering equation). The remaining 20% of the CO2 is carried as dissolved gas in plasma; the same way that CO2 gas is dissolved in carbonated beverages!
O2 and CO2 carrying functions of Hb
The Haldane Effect: The Haldane effect states that deoxygenated Hb has a greater affinity for CO2 than does oxyHb. Thus, O2 release at the tissues facilitates CO2 pickup while O2 pickup at the lungs facilitates CO2 release. Likewise, CO2 pickup at the tissues facilitates O2 release while CO2 release at the lungs promotes O2 pickup. In reality, the exchange of O2 and CO2 is occurring at the same time so O2 loading and unloading assist CO2 unloading and loading at the lungs and tissues, respectively. Similarly, CO2 loading and unloading assist O2 unloading and loading at the tissues and lungs, respectively!
The Bohr Effect: The Bohr effect states that deoxygenated Hb has a greater affinity for H+ than does oxyhemoglobin. Thus, O2 release at the tissues facilitates H+ pickup while O2 pickup at the lungs facilitates H+ release. Likewise, H+ pickup at the tissues facilitates O2 release while H+ release at the lungs promotes O2 pickup. In reality, the exchange of O2 and H+ is also occurring at the same time so O2 loading and unloading assist H+ unloading and loading at the lungs and tissues, respectively. Similarly, H+ loading and unloading assist O2 unloading and loading at the tissues and lungs, respectively!
Thus, CO2 is transported by CO2 binding to Hb (ie. 20% of CO2 is transported in the form of carbaminohemoglobin), via the carbonic anhydrase method (60%) and as CO2 dissolved in plasma (20%). Carbonic anhydrase is an enzyme that promotes conversion of CO2 + H2O to H2CO3 (carbonic acid). Most of the CO2 removed from the body, then, is carried to the lung in the form of carbonic acid. When the blood reaches the lungs, the CO2 transported in plasma is blown off in the expiratory gas, the CO2 transported to the lungs bound to hemoglobin is released and blown off in the expiratory gas, and the CO2 transported to the lungs in the form of carbonic acid is converted back to CO2 and blown off in the expiratory gas. See a picture developing here... no matter how the CO2 is transported to the lungs, it is all blown off in the expiratory gas.
Figure: shifts in the O2 loading/unloading curve: The arrows in the figure indicate the direction in which each specified effect will shift the curve. As you interpret this figure, remember that a left shift represents an increase in affinity of Hb for O2. A left shift means that Hb will be saturated (loaded) with O2 in the presence of lower O2 concentrations. A right shift represents a decrease in affinity of Hb for O2, meaning that O2 is more easily released (unloaded) from the Hb.
H+ and CO2 binding to Hb, in the tissues, shifts the O2 loading curve to the right, meaning that the amount of O2 required to maintain O2 saturation is increased (ie. the affinity for O2 is decreased). This effect (this right shift caused by CO2 and H+ binding) promotes O2 unloading from hemoglobin at the tissues; this obviously promotes O2 delivery to the tissues!
In the lungs, the O2 loading curve is shifted back to the left as H+ and CO2 are released (ie. the affinity of Hb for O2 is increased and a decreased concentration of O2 is required to saturate Hb with O2). This effect at the lung, increases the affinity of Hb for O2 and promotes O2 loading onto Hb!
These shifts in the O2 loading/unloading curve are particularly important to O2 unloading at the tissues. Without this shift, the O2 concentration in the tissues would have to be considerably decreased for O2 to be unloaded from Hb (ie. the tissue would have to become severely hypoxic to achieve O2 release, which is not the case). O2 can be effectively released at the tissues because of H+ and CO2 pickup (the Bohr and Haldane effect), with very little decrease in tissue O2 concentration.
Note: the right shift in the O2 loading curve at the tissues is partly due to H+ pickup (80% due to the Bohr effect) and partly due to CO2 pickup (20% due to the Haldane effect). Remember, the Bohr effect is even more pronounced in the fetus!
The Bohr and Haldane effects are allosteric effects caused by binding of different ligands at different spots on Hb. "Allosteric" effects are associated with "shape changes" in molecules. The Bohr and Haldane effects change the shape of the hemoglobin molecule, resulting in an increase in the affinity of the Hb for O2 at the lung and a decrease in the affinity of Hb for O2 at the tissue. The shape changes in Hb, therefore, promote O2 uptake at the lung and O2 release at the tissues. Together, the reactions are referred to as HALI (heterotropic allosteric ligand interaction = the sum effect of the Bohr-Haldane Effects).
If Bohr and Haldane had worked together, they would have adequately described this overall sequence of events. Thus, the 3 components (H+, O2, CO2) facilitate the movement of one another onto and off of the Hb molecule.
2,3-DPG is a metabolite found in the anaerobic glycolysis pathway. Remember, RBC's must produce ATP through anaerobic glycolysis as RBC's have no mitochondria. One molecule of DPG interacts with one molecule of Hb. 2,3-DPG binds to partially deoxygenated Hb. By partially deoxygenated Hb, we mean Hb which has lost the O2 from the beta chains but still has O2 on the alpha chains. At the tissues, the beta chains lose their O2 very easily, but the alpha chains continue to hold on to their O2 very tightly. DPG slides into the cavity left between the two beta chains after they release their O2; DPG is heavily negatively charged and binds nicely to the positive charges provided by the amino acids of the beta globin chains. To put it simply, after the beta chains release their O2, which comes off fairly easily at the tissues, a molecule of DPG slides between the 2 deoxygenated beta chains.
DPG binding between the deoxygenated beta chains changes the shape of Hb (an "allosteric" effect) and this shape change promotes the release of the O2's from the 2 alpha chains at the tissue. When O2 loads back onto the beta-globins at the lungs, the 2,3-DPG is physically squeezed out from between the beta chains.
Thus, DPG binding shifts the O2 loading/unloading curve further to the right, even further to the right than H+ or CO2 (ie. a fairly extreme shift) to help unload O2 at the tissues. Old RBC's don't produce DPG as effectively as young RBC's and, therefore, do not release O2 as readily at the tissues as do young RBC's.
In an anemia, DPG production by RBC's is increased to cause a further right shift in the O2 loading/unloading curve so that O2 unloading at the tissues is even more efficient (ie. the body tries to compensate for the loss of O2 carrying capacity by increasing the efficiency of O2 unloading from RBC's at the tissues). DPG production is also increased in persons living at high altitudes, and is one of the benefits obtained by athletes living at high altitudes; the increase in DPG makes O2 delivery to their tissues considerably more efficient! Remember, the basic physiological rationale for the "live high, train low," paradigm is...
living at high altitudes increases DPG production, mitochondrial concentrations, EPO production and hematocrit
training at low altitudes allows one to perform at peak levels of activity for longer periods of time, increasing the benefit of each exercise session
Hb without DPG only releases O2 with 50% efficiency (ie. without DPG, O2 is not unloaded from the alpha chains). Functionally speaking, the effective O2 carrying capacity of Hb without DPG is more similar to that of myoglobin than of normal Hb.
DPG in Refrigerated or Frozen Stored Blood:
Blood in cold storage contains glucose as a potential substrate for ATP production, but the glycolytic enzymes do not work well in the cold. Remember, DPG is produced as a byproduct of glycolysis. Thus, DPG levels decrease in refrigerated (stored) blood. Thus, if refrigerated whole blood is stored for too long, it becomes less efficient at O2 unloading, because it contains less DPG!
DPG decreases so much by the time the blood is 10 days old that the Hb affinity for O2 is actually increased such that less O2 is released from Hb which has been refrigerated for 10 days. For this reason, refrigerated blood is not normally stored beyond 21 days; if refrigerated blood older than 21 days is transfused, it picks up O2 at the lung but will not release the O2 at the tissues. Transfusion of blood older than 21 days actually impairs O2 transfer to the tissues. When older refrigerated blood is returned to the body, the levels of DPG present in the blood will gradually increase, but the initial decline in O2 carrying capacity following transfusion should be considered potentially dangerous. Blood which is to be used for planned autologous transfusions (planned surgeries, for example), should be frozen if it will not be used within a few weeks... and must be frozen if it is to be stored for 42 days or more. While improving cell freezing technologies (development of less toxic cryoprotectants) allows cells to be frozen indefinitely, frozen blood is generally used within 1 year, but is readily frozen for 10 years or more.
Remember, DPG levels are increased in people living at high elevations, such that the efficiency of O2 unloading from their Hb at the tissues is increased.
Fetal hemoglobin is very similar in structure to that of "adult" hemoglobin with a minor exceptions. Fetal Hb does not have beta subunits. Fetal Hb has alpha subunits, which are identical to the alpha subunits of adult Hb, and gamma subunits. Beta subunits in "adult" Hb are almost identical to the gamma subunits of fetal Hb, with only slight differences between the amino acid sequences of gamma and beta subunits. Even though the differences in the amino acid sequence of the gamma and beta subunits are minor, there are distinct functional differences between fetal gamma and adult beta subunits.
1. Alpha subunits have a higher affinity for O2 than either beta or gamma subunits. However, gamma subunits (fetal) have a higher affinity for O2 than do beta subunits. This helps the fetal circulation to obtain O2 from the beta subunits of maternal Hb. Thus, the oxygen loading/unloading curve for fetal Hb is shifted far to the left of the curve for adult Hb (fetal Hb will load at lower O2 concentrations).
2. Fetal Hb does not respond to DPG as fetal Hb has no beta globins for DPG binding.
3. Fetal Hb responds dramatically to the Bohr effect. When fetal Hb picks up H+ at the fetal tissues, the O2 saturation curve shifts strongly to the right to readily release O2 to the tissues. This exaggerated response of fetal Hb to H+ pickup (to the Bohr effect) more than compensates for the lack of DPG in fetal Hb.
changes in hemoglobin at birth
1. Hb quickly becomes less responsive to the Bohr effect. However, now that the neonate is "out of the womb," increased physical activity results in greater H+ production in the tissues; the resultant greater H+ concentration in the tissues helps unload O2 from Hb at the tissues.
2. A gradual increase in DPG production increases the efficiency of O2 unloading from Hb at the tissues, particularly as the neonate is aging and its fetal Hb is being replaced with "adult" Hb. The term "adult" Hb simply refers to Hb produced following birth, that is, Hb with alpha and beta subunits.
3) A gradual reduction in the affinity of Hb for O2 as gamma globin decreases and beta globin increases will also assist in O2 unloading from Hb at the tissues.
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Revised: January 05, 2009