Hemoglobin

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 O_2. 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 structure

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!

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.

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-Diphosphoglycerate (2,3-DPG)

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.

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

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.

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.