The binding site for 2,3-BPG is located in the center of the deoxyhemoglobin tetramer, formed by the two β-globin chains. Within this pocket, there are several positively charged amino acids that form ionic bonds with the negatively charged phosphate groups of 2,3-BPG. It should be noted that if one of these amino acids is replaced, it can result in hemoglobin variants with abnormally high oxygen affinity, which can be compensated for by an increase in red blood cell production (erythrocytosis). When hemoglobin is oxygenated, the pocket narrows and causes the release of 2,3-BPG.

The presence of 2,3-BPG significantly reduces the oxygen affinity of hemoglobin, resulting in a high oxygen-dissociation curve shift to the right. This allows hemoglobin to efficiently release oxygen at the partial pressures found in the tissues. In conditions of chronic hypoxia, such as chronic obstructive pulmonary disease (COPD) or at high altitudes, the concentration of 2,3-BPG in red blood cells increases as a response. This elevation of 2,3-BPG levels also occurs in chronic anemia, where there is a decreased availability of red blood cells to supply oxygen to the body. Higher levels of 2,3-BPG lower the oxygen affinity of hemoglobin, enabling greater unloading of oxygen in the capillaries of tissues.

When blood is stored using current media, there is a gradual depletion of 2,3-BPG, resulting in stored blood displaying abnormally high oxygen affinity and improper unloading of oxygen in the tissues. Transfused red blood cells are able to restore their depleted supplies of 2,3-BPG within 6-24 hours. However, transfusing severely ill patients with large quantities of 2,3-BPG-depleted blood may compromise their condition. To address this, stored blood is treated with a "rejuvenation" solution that rapidly restores 2,3-BPG levels. It should be noted that rejuvenation also restores ATP lost during storage.

In terms of CO2 binding, most of the CO2 produced during metabolism is transported as bicarbonate ion. However, a portion of CO2 is carried as carbamate, which is bound to the terminal amino groups of hemoglobin. This binding of CO2 stabilizes the deoxy form of hemoglobin, resulting in a decrease in its oxygen affinity and a right shift in the oxygen-dissociation curve. In the lungs, CO2 dissociates from hemoglobin and is released through exhalation.
2,3-BPG binding site: One molecule of 2,3-BPG binds to a pocket, formed by the two β-globin chains, in the center of the deoxyhemoglobin tetramer (Fig. 3.11). This pocket contains several positively charged amino acids that form ionic bonds with the negatively charged phosphate groups of 2,3-BPG. [Note: Replacement of one of these amino acids can result in hemoglobin variants with abnormally high oxygen affinity that may be compensated for by increased RBC production (erythrocytosis).] Oxygenation of hemoglobin narrows the pocket and causes 2,3-BPG to be released.
c.
Oxygen-dissociation curve shift: Hemoglobin from which 2,3-BPG has been removed has high oxygen affinity. However, as seen in the RBC, the presence of 2,3-BPG significantly reduces the oxygen affinity of hemoglobin, shifting the oxygen-dissociation curve to the right (Fig. 3.12). This reduced affinity enables hemoglobin to release O2 efficiently at the partial pressures found in the tissues.
d. 2,3-BPG levels in chronic hypoxia or anemia: The concentration of 2,3-BPG in the RBC increases in response to chronic hypoxia, such as that observed in chronic obstructive pulmonary disease (COPD) like emphysema, or at high altitudes, where circulating hemoglobin may have difficulty receiving sufficient O2. Intracellular levels of 2,3-BPG are also elevated in chronic anemia, in which fewer than normal RBC are available to supply the body’s oxygen needs. Elevated 2,3-BPG levels lower the oxygen affinity of hemoglobin, permitting greater unloading of O2 in the capillaries of tissues (see Fig. 3.12).
e. 2,3-BPG in transfused blood: 2,3-BPG is essential for the normal oxygen transport function of hemoglobin. However, storing blood in the currently available media results in the gradual depletion of 2,3BPG. Consequently, stored blood displays an abnormally high oxygen affinity and fails to unload its bound O2 properly in the tissues. Thus, hemoglobin deficient in 2,3-BPG acts as an oxygen “trap” rather than as an oxygen delivery system. Transfused RBC are able to restore their depleted supplies of 2,3-BPG in 6–24 hours. However, severely ill patients may be compromised if transfused with large quantities of such 2,3-BPG–depleted blood. Stored blood, therefore, is treated with a “rejuvenation” solution that rapidly restores 2,3-BPG. [Note: Rejuvenation also restores ATP lost during storage.] 4. CO2 binding: Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion (see Fig. 1.12 on p. 9). However, some CO2 is carried as carbamate bound to the terminal amino groups of hemoglobin (forming carbaminohemoglobin as shown in Fig. 3.8), which can be represented schematically as follows:
The binding of CO2 stabilizes the T, or deoxy, form of hemoglobin, resulting in a decrease in its oxygen affinity (see p. 28) and a right shift in the oxygen-dissociation curve. In the lungs, CO2 dissociates from the hemoglobin and is released in the breath.