A Molecule of Hemoglobin Can Reversibly Combine with Four Molecules of Oxygen

Mammalian hemoglobin consists of four units, each containing one heme and its associated protein (globin). Globin is a polypeptide composed of 140 to 150 amino acids. Heme is a protoporphyrin consisting of four pyrroles with a ferrous iron at the center. Each ferrous iron can combine reversibly with a single molecule of oxygen. The complete hemoglobin molecule has four hemes each with its associated globin and thus can combine reversibly with up to four molecules of oxygen (Figure 48-2). The type and sequence of amino acids that compose globin are critical to oxygen binding. Without the presence of globin, oxygen would irreversibly oxidize the ferrous iron to ferric iron. The amino acids in globin cradle the heme and limit access of oxygen to the ferrous iron. This prevents oxidation and allows uptake and release of oxygen in response to local PO₂. The type and sequence of amino acids that compose globin define the different types of mammalian hemoglobin. Adult hemoglobin contains two alpha (α) and two beta (β) amino acid chains; fetal hemoglobin contains two α and two gamma (γ) chains. Closely related species, such as humans and anthropoid apes, have similar amino acid sequences on the side chains, whereas more divergent species have more differences in amino acid sequences.

Each hemoglobin molecule can reversibly bind up to four molecules of oxygen, one with each heme. The reversible combination of oxygen with hemoglobin is shown in the oxyhemoglobin dissociation curve (Figure 48-3). The binding of oxygen is a four-step process, and the oxygen affinity of a particular heme is influenced by the oxygenation of the others. This means that when the first heme unit is oxygenated, oxygen affinity of the second heme unit is increased, and so on. These heme-heme interactions are responsible for the sigmoid shape of the oxyhemoglobin dissociation curve.

The Binding of Oxygen and Hemoglobin Is Determined by Oxygen Tension

Figure 48-3 shows that the oxygen content of blood—that is, the amount of oxygen combined with hemoglobin—is determined by PO₂. At a PO₂ of more than approximately 70 mm Hg, the oxyhemoglobin dissociation curve is virtually flat, which indicates that further increases in PO₂ add little oxygen to hemoglobin. At this point, the hemoglobin is saturated with oxygen because each iron atom is associated with an oxygen molecule. The fact that hemoglobin becomes virtually saturated with oxygen at a PO₂ of more than 70 mm Hg has important clinical consequences. Many animals live at altitudes considerably above sea level, where the lower barometric pressure results in a low PIO₂ (inspired oxygen tension). Although these animals have a lower PaO₂ (arterial oxygen tension) than their sea-level–dwelling counterparts, they are still able to transport sufficient oxygen to their tissues because their hemoglobin is well saturated with oxygen. Clearly, at extreme altitudes, hemoglobin begins to desaturate.

One gram of saturated hemoglobin can hold 1.36 to 1.39 mL of oxygen; therefore, average mammalian blood with 10 to 15 g of hemoglobin per deciliter has an oxygen capacity of 13.6 to 21 mL of oxygen per deciliter (volume percentage [vol%]) when hemoglobin is saturated with oxygen. The oxygen capacity of the blood is the maximal amount of oxygen that can be carried in the blood at any given time. Anemia, a reduction in the number of circulating erythrocytes (red blood cells) with a consequent reduction in the amount of hemoglobin in the blood, decreases oxygen capacity. When the hemoglobin content of blood increases, oxygen capacity increases as well. The latter occurs during exercise; contraction of the spleen forces more erythrocytes into the circulation. More erythrocytes than normal in the blood is known as polycythemia, and these red blood cells increase the oxygen capacity of the blood.

When the PO₂ is less than 60 mm Hg, the oxyhemoglobin dissociation curve has a steep slope. This is in the range of tissue Po₂ at which oxygen is unloaded from the blood. Tissue PO₂ varies in accordance with the blood flow/metabolism ratio, but average tissue PO₂ is 40 mm Hg. Blood exposed to such a PO₂ loses 25% of its oxygen to the tissues. In rapidly metabolizing tissues in which tissue PO₂ is lower, more oxygen is unloaded from the blood. The oxygen remaining in combination with hemoglobin forms a reserve that can be tapped in emergencies.

Oxygen content is a term that describes the amount of oxygen in the blood, most bound to hemoglobin. When hemoglobin is saturated with oxygen, oxygen content and oxygen capacity are equal. When oxygen leaves the blood in the tissues, oxygen content decreases, but the oxygen capacity remains the same.

The Oxyhemoglobin Dissociation Curve Can Be Displayed with Percent Saturation of Hemoglobin as a Function of Oxygen Tension

Percent saturation of hemoglobin is the ratio of oxygen content to oxygen capacity. Hemoglobin is more than 95% saturated with oxygen when it leaves the lungs of an animal at sea level. Percent saturation of mixed venous blood averages 75%; venous oxygen tension () averages 40 mm Hg. Although all mammals have similarly shaped oxyhemoglobin dissociation curves, the position of the curve with regard to PO₂ varies (Figure 48-4). This can be described by measurement of P₅₀, the partial pressure at which hemoglobin is 50% saturated with oxygen. A higher P₅₀ is generally found in small mammals and allows unloading of oxygen at a high PO₂ to satisfy their higher metabolic demands.

The Affinity of Hemoglobin for Oxygen Varies with Blood Temperature, pH, Carbon Dioxide Tension, and the Intracellular Concentration of Certain Organic Phosphates

An increase in tissue metabolism produces heat, which elevates blood temperature and shifts the oxyhemoglobin dissociation curve to the right (increases P₅₀). Such a shift facilitates dissociation of oxygen from hemoglobin and releases oxygen to the tissues; hemoglobin is then said to have “less affinity” for oxygen. Conversely, excessive cooling of the blood, as occurs in hypothermia, shifts the dissociation curve to the left. Because of this increased affinity of hemoglobin for oxygen, tissue PO₂ must be lower than usual to release oxygen from hemoglobin.

Changes in carbon dioxide tension (PCO₂) and pH also affect the affinity of hemoglobin for oxygen. The shift in the oxyhemoglobin dissociation curve resulting from a change in PCO₂ is called the Bohr shift. This shift results in part from the combination of carbon dioxide with hemoglobin, but mostly from the production of hydrogen ions, which decrease the pH. A change in pH alters the oxygen binding by changing the structure of hemoglobin. As a result, a higher, more alkaline pH shifts the oxyhemoglobin dissociation curve to the left, and a lower, more acidic pH shifts the curve to the right (Figure 48-5). The Bohr effect is not constant among species; a given change in pH produces a greater shift in the dissociation curve for small mammals than for large mammals, supposedly ensuring the delivery of oxygen during high rates of metabolic activity, when carbon dioxide production is greatest.

A solution of mammalian hemoglobin generally has a higher affinity for oxygen than does whole blood until organic phosphates, such as 2,3-diphosphoglycerate (2,3-DPG) and adenosine triphosphate (ATP), are added to the solution. In erythrocytes, DPG has a molar content equivalent to that of hemoglobin, much higher than in other cells. This DPG regulates the combination of oxygen with hemoglobin. When concentrations of DPG are high, as occurs under the anaerobic conditions imposed by altitude or anemia, the oxyhemoglobin dissociation curve is shifted to the right (P₅₀ increases), and the unloading of oxygen is facilitated. In contrast, a reduction in DPG levels, as can occur in stored blood, shifts the dissociation curve to the left. Not all forms of hemoglobin bind DPG equally. Ruminant hemoglobin in general is unresponsive to DPG; elephant hemoglobin binds DPG weakly; and some forms of fetal hemoglobin do not bind DPG.