Effect of 2,3-bisphophoglycerate (2,3-BPG) on haemoglobin

Effect of 2,3-bisphophoglycerate (2,3-BPG) on haemoglobin

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When 2,3-bisphophoglycerate (2,3-BPG) binds to haemoglobin, a higher partial pressure of oxygen is needed to bring about 50% saturation of with oxygen.

What is the physiological significance of this and its molecular basis?

How would this affect the oxygen dissociation curve of haemoglobin and would it alter the Bohr effect?

Note regarding editing
In its original form this was a multiple choice question that had remained unanswered for a year, possibly because the detractors are very bad. Because the topic it covers is thought to be of general interest it has been reformulated as a standard question. For the record, the original question asked which of the following were correct: (1) 2,3-BPG in red blood cells causes the oxygen dissociation curve to shift to the left. (2) The binding of 2,3-BPG to haemoglobin lowers the affinity of the haemoglobin for oxygen. (3) Binding of 2,3-BPG to haemoglobin reduces the Bohr effect. (4) When 2,3-BPG is absent, oxy-haemoglobin is less likely to unload oxygen. The 'correct' answer was given as (2), but the poster thought that (4) was also correct.

The oxygen dissociation curve for haemoglobin (Hb) in the absence and presence of 2,3-BPG is shown in (i) below: It can seen that the curve has shifted to the right (MC-1 incorrect) and the oxygen affinity is obviously decreased, as it takes a higher concentration (pressure) of oxygen to achieve the same percentage saturation (MC-2 correct - and so is option MC-4 as far as I and @JM97 can see, agreeing with the poster. MCQs are an educational abomination!).

The physiological significance of this is considered in detail in my answer to another question, but, in short, it ensures that an adequate proportion of the (smaller) amount oxygen taken up at the lower pressure in the lungs is released in the tissues (3 minus 4, cf. 1ʹ minus 2).

To address the interaction between the effect of 2,3-BPG and the Bohr Effect one needs to consider the corresponding curves for the effect of hydrogen ions (falling pH), shown in (ii), above (see also my detailed answer to this question). It can be seen that the effect of hydrogen ions is similar to that of 2,3-BPG. Can these two have independent (additive) effects? It is known from molecular studies that they both interact with and stabilize deoxy-haemoglobin at different positions (see ii, below), and in one model of the allosteric mechanism they can be considered as shifting the equilibrium from the tense (deoxy-) to the relaxed (oxy-) state of haemoglobin: (i) Generalized diagram of allosteric equilibrium of tetrameric protein between relaxed state (where it can bind substrate/ligand) and tense state. Negative effectors favour the tense state. (ii) Illustration of this for haemoglobin, where H+ ions protonate His-146 of the β-subunits and 2,3-BPG forms ionic bridges between the two β-subunits.

So it would appear that Bohr effect can still augment the 2,3-BPG effect, but I would imagine that the extent of this augmentation would depend on the initial position of equilibrium, and I would not expect it to be additive - I may be wrong. (Is MC-3 correct or not? A school student can hardly be expected to know unless he had been fed the 'correct' answer. Perhaps he is intended to interpret the option as meaning “does the 2,3-BPG effect work in the opposite direction to the Bohr effect”. You may now understand why I loath MCQs.)


The diagrams are my own, based on various sources I have used over the years. To check whether what I have written is correct, the reader may wish to check any of the many accounts online, including the following which have references to original material:

Why does 2/3 DPG increase at altitude?

Accordingly, the main role of the 2,3-DPG change at high altitude (and also in acid-base disorders) is to maintain the oxygen dissociation curve of human blood at (or near) its original position. This conclusion seems to be valid for man resting at altitudes up to 7000 m.

One may also ask, what is the function of 2/3 DPG? &hellipthe blood), carbon dioxide, and 2,3-diphosphoglycerate (2,3-DPG a salt in red blood cells that plays a role in liberating oxygen from hemoglobin in the peripheral circulation). These substances do not bind to hemoglobin at the oxygen-binding sites.

Similarly, it is asked, why does Bpg increase at high altitudes?

2,3 Bisphosphoglycerate (2,3-BPG) stabilizes the T- (taut oxygen unbound) form of haemoglobin thereby reducing its affinity to bind to oxygen. 2,3-BPG is found to be elevated in people living at high altitudes. However, haemoglobin content also increases so that higher amounts of oxygen can be captured.

How does altitude affect your body?

Within the first few hours of altitude exposure, water loss also increases, which can result in dehydration. Altitude can also increase your metabolism while suppressing your appetite, meaning you'll have to eat more than you feel like to maintain a neutral energy balance.

Where does Bpg bind to hemoglobin?

See full answer to your question here. Likewise, what does Bpg bind to?

Glycolysis and Gluconeogenesis 2,3-BPG binds to the beta subunit of the T (taut) state of hemoglobin, deoxyhemoglobin, the less active form. The greater affinity of 2,3-BPG for hemoglobin compared to oxyhemoglobin allows oxygenated hemoglobin to release its oxygen to needy tissue, such as the lungs.

Beside above, how many Bpg molecules bind to hemoglobin? 2,3-BPG binds to hemoglobin in the center of the tetramer to stabilize the T state (E.g. in muscle tissues).

Regarding this, where does co2 bind to hemoglobin?

Second, carbon dioxide can bind to plasma proteins or can enter red blood cells and bind to hemoglobin. This form transports about 10 percent of the carbon dioxide. When carbon dioxide binds to hemoglobin, a molecule called carbaminohemoglobin is formed. Binding of carbon dioxide to hemoglobin is reversible.

How does 2/3 bpg affect hemoglobin?

The accumulation of 2,3-BPG decreases the affinity of hemoglobin for oxygen. In tissues with high energetic demands, oxygen is rapidly consumed, which increases the concentration of H + and carbon dioxide. Through the Bohr effect, hemoglobin is induced to release more oxygen to supply cells that need it.

Erythrocyte adaptive metabolic reprogramming under physiological and pathological hypoxia

Purpose of review: The erythrocyte is the most abundant cell type in our body, acting as both a carrier/deliverer and sensor of oxygen (O2). Erythrocyte O2 delivery capacity is finely regulated by sophisticated metabolic control. In recent years, unbiased and robust human metabolomics screening and mouse genetic studies have advanced erythroid research revealing the differential role of erythrocyte hypoxic metabolic reprogramming in normal individuals at high altitudes and patients facing hypoxia, such as sickle cell disease (SCD) and chronic kidney disease (CKD). Here we summarize recent progress and highlight potential therapeutic possibilities.

Recent findings: Initial studies showed that elevated soluble CD73 (sCD73, converts AMP to adenosine) results in increased circulating adenosine that activates the A2B adenosine receptor (ADORA2B). Signaling through this axis is co-operatively strengthened by erythrocyte-specific synthesis of sphingosine-1-phosphate (S1P). Ultimately, these mechanisms promote the generation of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific allosteric modulator that decreases haemoglobin--O2-binding affinity, and thus, induces deoxygenated sickle Hb (deoxyHbS), deoxyHbS polymerization, sickling, chronic inflammation and tissue damage in SCD. Similar to SCD, plasma adenosine and erythrocyte S1P are elevated in humans ascending to high altitude. At high altitude, these two metabolites are beneficial to induce erythrocyte metabolic reprogramming and the synthesis of 2,3-BPG, and thus, increase O2 delivery to counteract hypoxic tissue damage. Follow-up studies showed that erythrocyte equilibrative nucleoside transporter 1 (eENT1) is a key purinergic cellular component controlling plasma adenosine in humans at high altitude and mice under hypoxia and underlies the quicker and higher elevation of plasma adenosine upon re-ascent because of prior hypoxia-induced degradation of eENT1. More recent studies demonstrated the beneficial role of erythrocyte ADORA2B-mediated 2,3-BPG production in CKD.

Summary: Taken together, these findings revealed the differential role of erythrocyte hypoxic metabolic reprogramming in normal humans at high altitude and patients with CKD vs. SCD patients and immediately suggest differential and precision therapies to counteract hypoxia among these groups.

The effect of phosphate loading on erythrocyte 2,3-bisphosphoglycerate levels

Background: Phosphate supplementation has been used in an effort to enhance athletic performance by increasing erythrocyte 2,3-bisphosphoglycerate levels ([2,3-BPG]) and hence improve oxygen offloading from haemoglobin. Claimed effects of phosphate loading upon both exercise performance and erythrocyte [2,3-BPG] are inconsistent, and the basis of any change in [2,3-BPG] is unknown.

Methods: We analysed plasma inorganic phosphate concentration ([P(i)]) and erythrocyte [P(i)] and [2,3-BPG] in venous blood samples from 12 healthy subjects. We re-examined a subset of five of these subjects after 7 days of phosphate loading.

Results: There were significant positive correlations between plasma [P(i)] and erythrocyte [P(i)] (r(2)=0.51, p=0.009) and between erythrocyte [P(i)] and [2,3-BPG] (r(2)=0.68, p<0.001). Following phosphate loading, there was a 30% increase in plasma [P(i)] (1.02+/-0.22 to 1.29+/-0.15 mmol/l (mean+/-S.D.), p=0.03) and a 25% increase in erythrocyte [2,3-BPG] (6.77+/-1.12 to 9.11+/-1.87 mmol/l cells, p=0.03). There is no relation between [2,3-BPG] and plasma [P(i)].

Conclusions: Phosphate loading increases both plasma and erythrocyte phosphate pools and the rise in [2,3-BPG] is probably a consequence of the rise in cell [P(i)].

Oxygenation of hemoglobin in the presence of 2,3-diphosphoglycerate. Effect of temperature, pH, ionic strength, and hemoglobin concentration

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Function, Metabolism, and Regulation of Organic Phosphates in Erythrocytes

2,3-DPG, ATP, inositol hexaphosphate (IHP), and other organic phosphates bind to hemoglobin and decrease its affinity for oxygen. IHP, the principal organic phosphate in avian erythrocytes, is the most negatively charged of these compounds and binds the most tightly however, it is not found in human red cells.

2,3-DPG levels in the erythrocytes help regulate hemoglobin oxygenation ( Figure 28-7 ). Unloading of oxygen at the PO2 in tissue capillaries is increased by 2,3-DPG, and small changes in its concentration can have significant effects on oxygen release. At the pH prevailing in the erythrocyte, the net charge on the 2,3-DPG molecule is −5. The binding site between the two β chains of hemoglobin contains eight positively charged amino acid residue side chains contributed by the Va11, His2, Lys82, and His143 of each chain ( Figure 28-8 ). These residues are highly conserved in mammalian hemoglobins, indicating their importance for normal hemoglobin function. In placental mammals, a fetus receives its oxygen by diffusion from the maternal circulation, across the placenta, into the fetal circulation. To ensure that the oxygen flow is adequate, the pressure gradient from mother to fetus is increased by increasing the affinity of fetal hemoglobin for oxygen. This decreases the partial pressure of oxygen in the fetal circulation, thereby increasing the rate of transplacental diffusion.

FIGURE 28-7 . Effect of 2,3-bisphosphoglycerate (2,3-DPG) on the oxygen saturation curve of hemoglobin. Note that 2,3-DPG decreases the affinity of hemoglobin for oxygen.

FIGURE 28-8 . Schematic representation of the side chains of the β subunits of human hemoglobin that participate in binding to 2,3-DPG. The binding cavity is lined with eight positive charges (four from each β subunit) that react with five negative charges on 2,3-DPG. Fetal hemoglobin binds 2,3-DPG much less tightly than does maternal hemoglobin because its γ chains (the counterpart of β-chains) contain Ser at γl43 in place of His at βl43.

Different species increase transplacental diffusion in different ways. In humans and other primates, adult hemoglobin contains two α and two β chains, while fetal hemoglobin has two α and two γ chains. Although the amino acid sequences of β and γ chains are similar, they differ in position 143, which is part of the 2,3-DPG binding site. In the γ chain, Hisl43 is replaced by Ser, thus reducing the charge in the 2,3-DPG binding site from +8 to +6. Thus, 2,3-DPG binds less tightly to fetal hemoglobin, the oxygen affinity of which is thereby increased relative to that of HbA at the same concentration of 2,3-DPG. Thus, erythrocytes in fetal primates, despite having 2,3-DPG concentrations equal to those in adult erythrocytes, have higher oxygen affinity than maternal red blood cells, allowing for maternal to fetal oxygen transport.

In other mammals, including horse, dog, pig, and guinea pig, fetal hemoglobin is not structurally different from adult hemoglobin, and transplacental diffusion is facilitated by a reduced concentration of 2,3-DPG in fetal erythrocytes. In ruminants, hemoglobin does not bind 2,3-DPG because the β chains are too far apart. However, fetal hemoglobin in ruminants has a higher affinity for oxygen than does adult hemoglobin because of other structural differences. These three different solutions to the problem of the need for transfer of oxygen to the fetus are an example of convergent evolution.

In humans, 2,3-DPG is the most abundant phosphate compound in the red cell. Its concentration is 5 mmol/L, approximately the same as that of hemoglobin tetramer. The concentration of ATP is also high, 1.3 mmol/L. Although ATP has roughly the same affinity for hemoglobin as does 2,3-DPG, it has little effect on oxygen affinity because it is mostly present as ATP-Mg 2+ , which binds weakly to hemoglobin. 2,3-DPG is formed by rearrangement of 1,3-bisphosphoglycerate, an intermediate in glycolysis ( Chapter 13 ). The rearrangement, catalyzed by bisphosphoglycerate mutase, requires 3-phosphoglycerate as cofactor and is allosterically stimulated by 2-phosphoglycerate ( Figure 28-9 ). Inorganic phosphate appears to be a negative allosteric modifier. 2,3-DPG is also a cofactor for the phosphoglycerate mutase of glycolysis. Bisphosphoglycerate phosphatase converts 2,3-DPG to 3-phosphoglycerate. Identical electrophoretic and chromatographic patterns and copurification of the two activities suggest that the catalytic sites for bisphosphoglycerate mutase and phosphatase may reside in the same protein. This hypothesis is supported by the report of an individual with an extremely low intraerythrocytic concentration of 2,3-DPG whose red blood cells lacked both bisphosphoglycerate mutase and phosphatase activities. Trace amounts of 2,3-DPG were present to act as cofactor for phosphoglycerate mutase and permit glycolysis to proceed. The 2,3-DPG deficiency diminished oxygen delivery to the tissues and produced a mild erythrocytosis. There was no hemolysis, and the disorder was clinically silent. Patients who have pyruvate kinase deficiency have above-normal levels of 2,3-DPG, whereas those who have hexokinase deficiency have below-normal levels. Appropriate erythropoietic responses are seen in both types (see below). The concentration of 2,3-DPG in the red cell can be altered by 15-25% in less than 12 hours. The most probable mechanisms involved are summarized below.

FIGURE 28-9 . Formation of 2,3-bisphosphoglycerate (2,3-DPG) in erythrocytes. Formation of 2,3-DPG occurs as a shunt from the main pathway of glycolysis, and free energy is used that otherwise would have been employed in the formation of ATP. ⊕ Positive allosteric modifier ⊖ negative allosteric modifier Pi, inorganic phosphate.

The binding of 2,3-DPG to deoxyhemoglobin decreases the amount of free 2,3-DPG available for participation in other reactions and causes increased 2,3-DPG synthesis at the expense of 1,3-bisphosphoglycerate. A decrease in oxygen saturation of hemoglobin may act in the same way.

The intraerythrocytic pH affects 2,3-DPG concentration. A decrease in pH increases the amount of bound 2,3-DPG by increasing the concentration of deoxyhemoglobin, which acts as described in the preceding paragraph. An increase in pH stimulates glycolysis, which tends to increase the concentration of all glycolytic intermediates, including 2,3-DPG. A decrease in pH within the physiological range also decreases the activity of bisphosphoglycerate mutase and increases the activity of bisphosphoglycerate phosphatase.

As erythrocytes age in vivo, their oxygen affinity increases. The concentration of 2,3-DPG in young red cells is higher than that in old red cells. This may reflect a general change in activity of bisphosphoglycerate mutase and phosphatase. Since erythrocytes are unable to synthesize proteins, inactivated enzymes cannot be replaced.

There may also be genetic control over 2,3-DPG levels. The ATP concentration in erythrocytes is under hereditary control, and in hooded rats, levels of ATP and 2,3-DPG appear to be genetically influenced. However, this finding may be of no importance in producing rapid, short-term, adaptive changes.

These processes are final-step controls that directly influence 2,3-DPG concentrations. The primary stimuli that trigger these final steps include the following:

Decreased delivery of O2 to tissues as a result of anemia, altitude, cardiac insufficiency, or pulmonary disease.

Thyroxine (which may directly stimulate bisphosphoglycerate mutase), androgens (which act partly by increasing erythropoiesis), and other hormones.

Polycythemia, which decreases the intraerythrocytic concentration of 2,3-DPG.

Whether the shift in the O2-dissociation curve that accompanies changes in the concentration of 2,3-DPG is beneficial depends largely on the oxygen saturation of arterial blood. The 2,3-DPG concentration can vary widely among patients with the same disease. For example, in severe pulmonary disease, the increase in 2,3-DPG concentration ranges from 0% to 100% in leukemia with depressed production of erythrocytes, elevations of 20–150% occur in iron deficiency, increases range from 40% to 75%.

2,3-DPG and, to a lesser extent, ATP concentrations decrease rapidly in blood that is stored even for a few days in the acid-citrate-dextrose (ACD) medium used by many blood banks. As a result, oxygen affinity is increased and the ability of blood that is transfused to supply oxygen to the tissues is decreased. Volunteers who received such blood had an increase in oxygen affinity that did not return to normal for 6–24 hours. The therapeutic significance of these changes is not clear. The greatest effects should occur in patients who receive numerous transfusions over a period of about 6 hours, so that a significant fraction of their circulating erythrocytes has increased oxygen affinity.

Traditionally, red cell survival has been the main criterion of the quality of stored blood. However, cell survival does not necessarily correspond to maintenance of adequate organic phosphate levels. Studies on the composition of the storage medium needed to prevent this metabolic loss of organic phosphates show the following:

Citrate-phosphate-dextrose (CPD) medium is better than ACD for maintaining organic phosphate levels and for preventing a reduction in P50, probably because of the higher pH of CPD. In 1971, 90% of blood banks in the United States used ACD and only 10% used CPD by 1975, the reverse was true. The shelf life of cells is the same for both media (21 days), but cells stored in CPD function better physiologically when transfused.

Supplementation with inosine generates a supply of ribose 1-phosphate and provides a potential glycolyti substrate that can be metabolized to 2,3-DPG. Pyruvate and fructose, which help maintain a supply of oxidized NAD + , potentiate the effect of inosine. This modification must be balanced against the possibility of hyperuricemia ( Chapter 27 ) caused by transfusion of large amounts of inosine-containing blood.

Supplementation with dihydroxyacetone phosphate could provide a glycolytic substrate without the risk of hyperuricemia.


Hemoglobin (Hb) is the primary vehicle for transporting oxygen in the blood. Each hemoglobin molecule has the capacity to carry four oxygen molecules. These molecules of oxygen bind to the iron of the heme prosthetic group. [1]

When hemoglobin has no bound oxygen, nor bound carbon dioxide, it has the unbound conformation (shape). The binding of the first oxygen molecule induces change in the shape of the hemoglobin that increases its ability to bind to the other three oxygen molecules.

In the presence of dissolved carbon dioxide, the pH of the blood changes this causes another change in the shape of hemoglobin, which increases its ability to bind carbon dioxide and decreases its ability to bind oxygen. With the loss of the first oxygen molecule, and the binding of the first carbon dioxide molecule, yet another change in shape occurs, which further decreases the ability to bind oxygen, and increases the ability to bind carbon dioxide. The oxygen bound to the hemoglobin is released into the blood's plasma and absorbed into the tissues, and the carbon dioxide in the tissues is bound to the hemoglobin.

In the lungs the reverse of this process takes place. With the loss of the first carbon dioxide molecule the shape again changes and makes it easier to release the other three carbon dioxides.

Oxygen is also carried dissolved in the blood's plasma, but to a much lesser degree. Hemoglobin is contained in red blood cells. Hemoglobin releases the bound oxygen when carbonic acid is present, as it is in the tissues. In the capillaries, where carbon dioxide is produced, oxygen bound to the hemoglobin is released into the blood's plasma and absorbed into the tissues.

How much of that capacity is filled by oxygen at any time is called the oxygen saturation. Expressed as a percentage, the oxygen saturation is the ratio of the amount of oxygen bound to the hemoglobin, to the oxygen-carrying capacity of the hemoglobin. The oxygen-carrying capacity of hemoglobin is determined by the type of hemoglobin present in the blood. The amount of oxygen bound to the hemoglobin at any time is related, in large part, to the partial pressure of oxygen to which the hemoglobin is exposed. In the lungs, at the alveolar–capillary interface, the partial pressure of oxygen is typically high, and therefore the oxygen binds readily to hemoglobin that is present. As the blood circulates to other body tissue in which the partial pressure of oxygen is less, the hemoglobin releases the oxygen into the tissue because the hemoglobin cannot maintain its full bound capacity of oxygen in the presence of lower oxygen partial pressures.

The curve is usually best described by a sigmoid plot, using a formula of the kind:

A hemoglobin molecule can bind up to four oxygen molecules in a reversible method.

The shape of the curve results from the interaction of bound oxygen molecules with incoming molecules. The binding of the first molecule is difficult. However, this facilitates the binding of the second, third and fourth, this is due to the induced conformational change in the structure of the hemoglobin molecule induced by the binding of an oxygen molecule.

In its most simple form, the oxyhemoglobin dissociation curve describes the relation between the partial pressure of oxygen (x axis) and the oxygen saturation (y axis). Hemoglobin's affinity for oxygen increases as successive molecules of oxygen bind. More molecules bind as the oxygen partial pressure increases until the maximum amount that can be bound is reached. As this limit is approached, very little additional binding occurs and the curve levels out as the hemoglobin becomes saturated with oxygen. Hence the curve has a sigmoidal or S-shape. At pressures above about 60 mmHg, the standard dissociation curve is relatively flat, which means that the oxygen content of the blood does not change significantly even with large increases in the oxygen partial pressure. To get more oxygen to the tissue would require blood transfusions to increase the hemoglobin count (and hence the oxygen-carrying capacity), or supplemental oxygen that would increase the oxygen dissolved in plasma. Although binding of oxygen to hemoglobin continues to some extent for pressures about 50 mmHg, as oxygen partial pressures decrease in this steep area of the curve, the oxygen is unloaded to peripheral tissue readily as the hemoglobin's affinity diminishes. The partial pressure of oxygen in the blood at which the hemoglobin is 50% saturated, typically about 26.6 mmHg (3.5 kPa) for a healthy person, is known as the P50. The P50 is a conventional measure of hemoglobin affinity for oxygen. In the presence of disease or other conditions that change the hemoglobin oxygen affinity and, consequently, shift the curve to the right or left, the P50 changes accordingly. An increased P50 indicates a rightward shift of the standard curve, which means that a larger partial pressure is necessary to maintain a 50% oxygen saturation. This indicates a decreased affinity. Conversely, a lower P50 indicates a leftward shift and a higher affinity.

The 'plateau' portion of the oxyhemoglobin dissociation curve is the range that exists at the pulmonary capillaries (minimal reduction of oxygen transported until the p(O2) falls 50 mmHg).

The 'steep' portion of the oxyhemoglobin dissociation curve is the range that exists at the systemic capillaries (a small drop in systemic capillary p(O2) can result in the release of large amounts of oxygen for the metabolically active cells).

To see the relative affinities of each successive oxygen as you remove/add oxygen from/to the hemoglobin from the curve compare the relative increase/decrease in p(O2) needed for the corresponding increase/decrease in s(O2). 69

The strength with which oxygen binds to hemoglobin is affected by several factors. These factors shift or reshape the oxyhemoglobin dissociation curve. A rightward shift indicates that the hemoglobin under study has a decreased affinity for oxygen. This makes it more difficult for hemoglobin to bind to oxygen (requiring a higher partial pressure of oxygen to achieve the same oxygen saturation), but it makes it easier for the hemoglobin to release oxygen bound to it. The effect of this rightward shift of the curve increases the partial pressure of oxygen in the tissues when it is most needed, such as during exercise, or hemorrhagic shock. In contrast, the curve is shifted to the left by the opposite of these conditions. This leftward shift indicates that the hemoglobin under study has an increased affinity for oxygen so that hemoglobin binds oxygen more easily, but unloads it more reluctantly. Left shift of the curve is a sign of hemoglobin's increased affinity for oxygen (e.g. at the lungs). Similarly, right shift shows decreased affinity, as would appear with an increase in either body temperature, hydrogen ions, 2,3-bisphosphoglycerate (2,3-BPG) concentration or carbon dioxide concentration.

Control factors Change Shift of curve
Acidity [H + ]

  • Left shift: higher O2 affinity
  • Right shift: lower O2 affinity
  • fetal hemoglobin has higher O2 affinity than adult hemoglobin primarily due to much-reduced affinity to 2,3-bisphosphoglycerate .

The causes of shift to right can be remembered using the mnemonic, "CADET, face Right!" for CO2, Acid, 2,3-DPG, [Note 1] Exercise and Temperature. [2] Factors that move the oxygen dissociation curve to the right are those physiological states where tissues need more oxygen. For example, during exercise, muscles have a higher metabolic rate, and consequently need more oxygen, produce more carbon dioxide and lactic acid, and their temperature rises.

PH Edit

A decrease in pH (increase in H + ion concentration) shifts the standard curve to the right, while an increase shifts it to the left. This occurs because at greater H + ion concentration, various amino acid residues, such as Histidine 146 exist predominantly in their protonated form allowing them to form ion pairs that stabilize deoxyhemoglobin in the T state. [3] The T state has a lower affinity for oxygen than the R state, so with increased acidity, the hemoglobin binds less O2 for a given PO2 (and more H + ). This is known as the Bohr effect. [4] A reduction in the total binding capacity of hemoglobin to oxygen (i.e. shifting the curve down, not just to the right) due to reduced pH is called the root effect. This is seen in bony fish. The binding affinity of hemoglobin to O2 is greatest under a relatively high pH.

Carbon dioxide Edit

Carbon dioxide affects the curve in two ways. First, CO2 accumulation causes carbamino compounds to be generated through chemical interactions, which bind to hemoglobin forming carbaminohemoglobin . CO2 is considered an Allosteric regulation as the inhibition happens not at the binding site of hemoglobin. [5] Second, it influences intracellular pH due to formation of bicarbonate ion. Formation of carbaminohemoglobin stabilizes T state hemoglobin by formation of ion pairs. [3] Only about 5–10% of the total CO2 content of blood is transported as carbamino compounds, whereas (80–90%) is transported as bicarbonate ions and a small amount is dissolved in the plasma. The formation of a bicarbonate ion will release a proton into the plasma, decreasing pH (increased acidity), which also shifts the curve to the right as discussed above low CO2 levels in the blood stream results in a high pH, and thus provides more optimal binding conditions for hemoglobin and O2. This is a physiologically favored mechanism, since hemoglobin will drop off more oxygen as the concentration of carbon dioxide increases dramatically where tissue respiration is happening rapidly and oxygen is in need. [6] [7]

2,3-BPG Edit

2,3-Bisphosphoglycerate or 2,3-BPG (formerly named 2,3-diphosphoglycerate or 2,3-DPG - reference?) is an organophosphate formed in red blood cells during glycolysis and is the conjugate base of 2,3-bisphosphoglyceric acid. The production of 2,3-BPG is likely an important adaptive mechanism, because the production increases for several conditions in the presence of diminished peripheral tissue O2 availability, such as hypoxemia, chronic lung disease, anemia, and congestive heart failure, among others. High levels of 2,3-BPG shift the curve to the right (as in childhood), while low levels of 2,3-BPG cause a leftward shift, seen in states such as septic shock, and hypophosphataemia. [4] In the absence of 2,3-BPG, hemoglobin's affinity for oxygen increases. 2,3-BPG acts as a heteroallosteric effector of hemoglobin, lowering hemoglobin's affinity for oxygen by binding preferentially to deoxyhemoglobin. An increased concentration of BPG in red blood cells favours formation of the T (taut or tense), low-affinity state of hemoglobin and so the oxygen-binding curve will shift to the right.

Temperature Edit

Carbon monoxide Edit

Hemoglobin binds with carbon monoxide 210 times more readily than with oxygen. [4] Because of this higher affinity of hemoglobin for carbon monoxide than for oxygen, carbon monoxide is a highly successful competitor that will displace oxygen even at minuscule partial pressures. The reaction HbO2 + CO → HbCO + O2 almost irreversibly displaces the oxygen molecules forming carboxyhemoglobin the binding of the carbon monoxide to the iron centre of hemoglobin is much stronger than that of oxygen, and the binding site remains blocked for the remainder of the life cycle of that affected red blood cell. [9] With an increased level of carbon monoxide, a person can suffer from severe tissue hypoxia while maintaining a normal pO2 because carboxyhemoglobin does not carry oxygen to the tissues.

Effects of methemoglobinaemia Edit

Methemoglobinaemia is a form of abnormal hemoglobin where the iron centre has been oxidised from the ferrous +2 oxidation state (the normal form) to the ferric +3 state. This causes a leftward shift in the oxygen hemoglobin dissociation curve, as any residual heme with oxygenated ferrous iron (+2 state) is unable to unload its bound oxygen into tissues (because 3+ iron impairs hemoglobin's cooperativity), thereby increasing its affinity with oxygen. However, methemoglobin has increased affinity for cyanide, and is therefore useful in the treatment of cyanide poisoning. In cases of accidental ingestion, administration of a nitrite (such as amyl nitrite) can be used to deliberately oxidise hemoglobin and raise methemoglobin levels, restoring the functioning of cytochrome oxidase. The nitrite also acts as a vasodilator, promoting the cellular supply of oxygen, and the addition of an iron salt provides for competitive binding of the free cyanide as the biochemically inert hexacyanoferrate(III) ion, [Fe(CN)6] 3− . An alternative approach involves administering thiosulfate, thereby converting cyanide to thiocyanate, SCN − , which is excreted via the kidneys. Methemoglobin is also formed in small quantities when the dissociation of oxyhemoglobin results in the formation of methemoglobin and superoxide, O2 − , instead of the usual products. Superoxide is a free radical and causes biochemical damage, but is neutralised by the action of the enzyme superoxide dismutase.

Effects of ITPP Edit

Myo-inositol trispyrophosphate (ITPP), also known as OXY111A, is an inositol phosphate that causes a rightward shift in the oxygen hemoglobin dissociation curve through allosteric modulation of hemoglobin within red blood cells. It is an experimental drug intended to reduce tissue hypoxia. The effects appear to last roughly as long as the affected red blood cells remain in circulation.

Fetal hemoglobin (HbF) is structurally different from normal adult hemoglobin (HbA), giving HbF a higher affinity for oxygen than HbA. HbF is composed of two alpha and two gamma chains whereas HbA is composed of two alpha and two beta chains. The fetal dissociation curve is shifted to the left relative to the curve for the normal adult because of these structural differences.

Typically, fetal arterial oxygen pressures are lower than adult arterial oxygen pressures. Hence higher affinity to bind oxygen is required at lower levels of partial pressure in the fetus to allow diffusion of oxygen across the placenta. At the placenta, there is a higher concentration of 2,3-BPG formed, and 2,3-BPG binds readily to beta chains rather than to alpha chains. As a result, 2,3-BPG binds more strongly to adult hemoglobin, causing HbA to release more oxygen for uptake by the fetus, whose HbF is unaffected by the 2,3-BPG. [10] HbF then delivers that bound oxygen to tissues that have even lower partial pressures where it can be released.


Another molecule favoring the release of oxygen by hemoglobin is 2,3- bisphosphoglycerate (also called 2,3-BPG or just BPG - Figure 4.2.5). Like protons and carbon dioxide, 2,3-BPG is produced by actively respiring tissues, as a byproduct of glucose metabolism. The 2,3-BPG molecule fits into the &lsquohole of the donut&rsquo of adult hemoglobin. Such binding of 2,3-BPG favors the T-state (tight - low oxygen binding) of hemoglobin, which has a reduced affinity for oxygen. In the absence of 2,3-BPG, hemoglobin can more easily exist in the R-state (relaxed - higher oxygen binding), which has a high affinity for oxygen.

Figure 4.2.5: 2,3- bisphosphoglycerate

Carbon Monoxide

CO is a highly toxic gas without color and odor. It is commonly produced the partial combustion of carbon-containing compounds. It competes with oxygen for hemoglobin binding. Its binding affinity is

Fibrous proteins - secondary structure

Proteins whose cellular or extracellular roles have a strong structural component are composed primarily of primary and second structure, with little folding of the chains. Thus, they have very little tertiary structure and are fibrous in nature. Proteins exhibiting these traits are commonly insoluble in water and are referred to as fibrous proteins (also called scleroproteins). The examples described in this category are found exclusively in animals where they serve roles in flesh, connective tissues and hardened external structures, such as hair. They also contain the three common fibrous protein structures &alpha -helices (keratins), &beta-strands/sheets (fibroin & elastin) and triple helices (collagen). The fibrous proteins have some commonality of amino acid sequence. Each possesses an abundance of repeating sequences of amino acids with small, non-reactive side groups. Many contain short repeats of sequences, often with glycine.

Figure 2.56 - The horns of an impala are composed of keratin Wikipedia

The keratins are a family of related animal proteins that take numerous forms. &alpha-keratins are structural components of the outer layer of human skin and are integral to hair, nails, claws, feathers, beaks, scales, and hooves. Keratins provide strength to tissues, such as the tongue, and over 50 different keratins are encoded in the human genome. At a cellular level, keratins comprise the intermediate filaments of the cytoskeleton. &alpha- keratins primarily contain &alpha-helices, but can also have &beta-strand/sheet structures. Individual &alpha-helices are often intertwined to form coils of coiled structures and these strands can also be further joined together by disulfide bonds, increasing structural strength considerably. This is particularly relevant for &alpha-keratin in hair, which contains about 14% cysteine. The odor of burned hair and that of the chemicals used to curl/uncurl hair (breaking/re-making disulfide bonds) arise from their sulfurous components. &beta-keratins are comprised of &beta-sheets, as their name implies.

Figure 2.57 - The repeating amino acid sequence of fibroin

An insoluble fibrous protein that is a component of the silk of spiders and the larvae of moths and other insects, fibroin is comprised of antiparallel &beta-strands tightly packed together to form &beta- sheets. The primary structure of fibroin is a short repeating sequence with glycine at every other residue (Figure 2.57). The small R-groups of the glycine and alanine in the repeating sequence allows for the tight packing characteristic of the fibers of silk. Wikipedia link HERE Elastin As suggested by its name, elastin is a protein with elastic characteristics that functions in many tissues of the body to allow them to resume their shapes after expanding or contracting. The protein is rich in glycine and proline and can comprise over 50% of the weight of dry, defatted arteries.

Figure 2.58 - Weaving of a silk sari Wikipedia

Figure 2.59 - Desmosine Wikipedia

is made by linking tropoelastin proteins together through lysine residues to make a durable complex crosslinked by desmosine. In arteries, elastin helps with pressure wave propagation for facilitating blood flow.

Figure 2.60 - Collagen&rsquos triple helix Wikipedia

Collagen is the most abundant protein in mammals, occupying up to a third of the total mass. There are at least 16 types of collagen. Its fibers are a major component of tendons and they are also found abundantly in skin. Collagen is also prominent in cornea, cartilage, bone, blood vessels and the gut.

Collagen&rsquos structure is an example of a helix of helices, being composed of three lefthanded helical chains that each are coiled together in a right-handed fashion to make the collagen fiber (Figure 2.60). Each helix is stretched out more than an &alpha-helix, giving it an extended appearance. On the inside of the triple helical structure, only residues of glycine are found, since the side chains of other amino acids are too bulky. Collagen chains have the repeating structure glycinem-n where m is often proline and n is often hydroxyproline (Figure 2.61).

Figure 2.61 - Repeating sequences in collagen

Collagen is synthesized in a pre-procollagen form. Processing of the pre-procollagen in the endoplasmic reticulum results in glycosylation, removal of the &lsquopre&rsquo sequence, and hydroxylation of lysine and proline residues (see below). The hydroxides can form covalent cross-links with each other, strengthening the collagen fibers. As pro-collagen is exported out of the cell, proteases trim it, resulting in a final form of collagen called tropocollagen.


Hydroxylation of proline and lysine side chains occurs post-translationally in a reaction catalyzed by prolyl-4-hydroxylase and lysyl-hydroxylase (lysyl oxidase), respectively. The reaction requires vitamin C. Since hydroxylation of these residues is essential for formation of stable triple helices at body temperature, vitamin C deficiency results in weak, unstable collagen and, consequently, weakened connective tissues. It is the cause of the disease known as scurvy. Hydrolyzed collagen is used to make gelatin, which is important in the food industry. collagens. Wikipedia link HERE

Figure 2.62 - Oxidation and cross-linking of lysine residues in tropocollagen. Only two strands of the triple helix are shown for simplicity Image by Aleia Kim

Lamins are fibrous proteins that provide structure in the cell nucleus and play a role in transcription regulation. They are similar to proteins making up the intermediate filaments, but have extra amino acids in one coil of the protein. Lamins help to form the nuclear lamin in the interior of the nuclear envelope and play important roles in assembling and disassembling the latter in the process of mitosis. They also help to position nuclear pores. In the process of mitosis, disassembly of the nuclear envelope is promoted by phosphorylation of lamins by a protein called mitosis promoting factor and assembly is favored by reversing the reaction (dephosphorylation).

Structural domains - tertiary structure

Every globular protein relies on its tertiary structure to perform its function, so rather than trying to find representative proteins for tertiary structure (an almost impossible task!), we focus here on a few elements of tertiary structure that are common to many proteins. These are the structural domains and they differ from the structural motifs of supersecondary structure by being larger (25-500 amino acids), having a conserved amino acid sequence, and a history of evolving and functioning independently of the protein chains they are found in. Structural domains are fundamental units of tertiary structure and are found in more than one protein. A structural domain is selfstabilizing and often folds independently of the rest of the protein chain.

Leucine zipper

Figure 2.63 - Leucine zipper bound to DNA Wikipedia

A common feature of many eukaryotic DNA binding proteins, leucine zippers are characterized by a repeating set of leucine residues in a protein that interact like a zipper to favor dimerization. Another part of the domain has amino acids (commonly arginine and lysine) that allow it to interact with the DNA double helix (Figure 2.63). Transcription factors that contain leucine zippers include Jun-B, CREB, and AP-1 fos/ jun.

Figure 2.64 - Leucine zipper structure. Leucines are indicated by orange and purple balls. Image by Penelope Irving

Zinc fingers

The shortest structural domains are the zinc fingers, which get their name from the fact that one or more coordinated zinc ions stabilize their finger-like structure. Despite their name, some zinc fingers do not bind zinc. There are many structural domains classified as zinc fingers and these are grouped into different families. Zinc fingers were first identified as components of DNA binding transcription factors, but others are now known to bind RNA, protein, and even lipid structures. Cysteine and histidine side chains commonly play roles in coordinating the zinc.

Src SH2 domain

Figure 2.65 - SH2 Domain Wikipedia

The Src oncoprotein contains a conserved SH2structural domain that recognizes and binds phosphorylated tyrosine side chains in other proteins (Figure 2.65). Phosphorylation is a fundamental activity in signaling and phosphorylation of tyrosine and interaction between proteins carrying signals is critically needed for cellular communication. The SH2domain is found in over 100 human proteins.

Helix-turn-helix domain

Figure 2.66 - Helix-Turn-Helix Domain of a Protein Bound to DNA Wikipedia

Helix-turn-helix is a common domain found in DNA binding proteins, consisting of two &alpha-helices separated by a small number of amino acids. As seen in Figure 2.66, the helix parts of the structural domain interact with the bases in the major groove of DNA. Individual &alpha-helices in a protein are part of a helix-turn-helix structure, where the turn separates the individual helices.

Pleckstrin homology domain

Pleckstrin Homology (PH) domains are protein domains with important functions in the process of signaling. This arises partly from the affinity for binding phosphorylated inositides, such as PIP2 and PIP3, found in Figure 2.66 - Helix-Turn-Helix Domain of a Protein Bound to DNA Wikipedia Figure 2.65 - SH 2 Domain Wikipedia biological membranes. PH domains can also bind to G-proteins and protein kinase C. The domain spans about amino acids and is found in numerous signaling proteins. These include Akt/Rac Serine/ Threonine Protein Kinases, Btk/ltk/Tec tyrosine protein kinases, insulin receptor substrate (IRS-1), Phosphatidylinositolspecific phospholipase C, and several yeast proteins involved in cell cycle regulation.

Figure 2.67 - Pleckstrin homology domain of Btk tyrosine protein kinase. The protein is embedded in a membrane (above blue line) Wikipedia

Structural globular proteins

Enzymes catalyze reactions and proteins such as hemoglobin perform important specialized functions. Evolutionary selection has reduced and eliminated waste so that we can be sure every protein in a cell has a function, even though in some cases we may not know what it is. Sometimes the structure of the proFigure 2.68 - Relationship of basement membrane to epithelium, endothelium, and connective tissue tein is its primary function because the structure provides stability, organization, connections other important properties. It is with this in mind that we present the following proteins.

Basement membrane

The basement membrane is a layered extracellular matrix of tissue comprised of protein fibers (type IV collagen) and glycosaminoglycans that separates the epithelium from other tissues (Figure 2.68). More importantly, the basement membrane acts like a glue to hold tissues together. The skin, for example, is anchored to the rest of the body by the basement membrane. Basement membranes provide an interface of interaction between cells and the environment around them, thus facilitating signaling processes. They play roles in differentiation during embryogenesis and also in maintenance of function in adult organisms.

Figure 2.68 - Relationship of basement membrane to epithelium, endothelium, and connective tissue

Actin is the most abundant globular protein found in most types of eukaryotic cells, comprising as much as 20% of the weight of muscle cells. Similar proteins have been identified in bacteria (MreB) and archaeons (Ta0583). Actin is a monomeric subunit able to polymerize readily into two different types of filaments. Microfilaments are major component of the cytoskeleton and are acted on by myosin in the contraction of muscle cells (See HERE). Actin will be discussed in more detail in the next section HERE.

Intermediate Filaments

Figure 2.69 - Assembly of intermediate filaments

Intermediate filaments are a part of the cytoskeleton in many animal cells and are comprised of over 70 different proteins. They are called intermediate because their size (average diameter = 10 nm) is between that of the microfilaments (7 nm) and the microtubules (25 nm).

The intermediate filament components include fibrous proteins, such as the keratins and the lamins, which are nuclear, as well as cytoplasmic forms. Intermediate filaments give flexibility to cells because of their own physical properties. They can, for example, be stretched to several times of their original length.

There are six different types of intermediate filaments. Type I and II are acidic or basic and attract each other to make larger filaments. They include epithelial keratins and trichocytic keratins (hair components). Type III proteins include four structural proteins - desmin, GFAP (glial fibrillary acidic protein), peripherin, and vimentin. Type IV also is a grouping of three proteins and one multiprotein structure (neurofilaments). The three proteins are &alpha-internexin, synemin, and syncoilin. Type V intermediate filaments encompass the lamins, which give structure to the nucleus. Phosphorylation of lamins leads to their disassembly and this is important in the process of mitosis. The Type VI category includes only a single protein known as nestin.

A third type of filament found in cells is that of the microbutules. Comprised of a polymer of two units of a globular protein called tubulin, microtubules provide &ldquorails&rdquo for motor proteins to move organelles and other &ldquocargo&rdquo from one part of a cell to another. Microtubules and tubulin are discussed in more detail HERE.

Vimentin (Figure 2.70) is the most widely distributed protein of the intermediate filaments. It is expressed in fibroblasts, leukocytes, and blood vessel endothelial cells. The protein has a significant role maintaining the position of organelles in the cytoplasm, with attachments to the nucleus, mitochondria, and endoplasmic reticulum (Figure 2.70). Vimentin provides elasticity to cells and resilience that does not arise from the microtubules or microfilaments. Wounded mice that lack the vimentin gene survive, but take longer to heal wounds than wild type mice. Vimentin also controls the movement of cholesterol from lysosomes to the site of esterification. The result is a reduction in the amount of cholesterol stored inside of cells and has implications for adrenal cells, which must have esters of cholesterol.

Figure 2.70 - Vimentin in cells Wikipedia

Mucins are a group of proteins found in animal epithelial tissue that have many glycosyl residues on them and typically are of high molecular weight (1 to 10 million Da). They are gel-like in their character and are often used for lubrication. Mucus is comprised of mucins. In addition to lubrication, mucins also help to control mineralization, such as bone formation in vertebrate organisms and calcification in echinoderms. They also play roles in the immune system by helping to bind pathogens. Mucins are commonly secreted onto mucosal surfaces (nostrils, eyes, mouth, ears, stomach, genitals, anus) or into fluids, such as saliva. Because of their extensive mucosylation, mucins hold a considerable amount of water (giving them the &ldquoslimy&rdquo feel) and are resistant to proteolysis.

Figure 2.71 - Actin filaments (green) attached to vinculin in focal adhesion (red) Wikipedia

Vinculin (Figure 2.72) is a membrane cytoskeletal protein found in the focal adhesion structures of mammalian cells. It is found at cell-cell and cell-matrix junctions and interacts with integrins, talin, paxillins and F-actin. Vinculin is thought to assist (along with other proteins) in anchoring actin microfilaments to the membrane (Figure 2.71). Binding of vinculin to actin and to talin is regulating by polyphosphoinositides and can be inhibited by acidic phospholipids.

Figure 2.72 - Vinculin Wikipedia

Syndecans are transmembrane proteins that make a single pass with a long amino acid chain (24-25 residues) through plasma membranes and facilitate G proteincoupled receptors&rsquo interaction with Figure 2.71 - Actin filaments (green) attached to vinculin in focal adhesion (red) Wikipedia ligands, such as growth factors, fibronectin, collagens (I, III, and IV) and antithrombin-1. Syndecans typically have 3-5 heparan sulfate and chondroitin sulfate chains attached to them.

Heparan sulfate can be cleaved at the site of a wound and stimulate action of fibroblast growth factor in the healing process. The role of syndecans in cell-cell adhesion is shown in mutant cells lacking syndecan I that do not adhere well to each other. Syndecan 4 is also known to adhere to integrin. Syndecans can also inhibit the spread of tumors by the ability of the syndecan 1 ectodomain to suppress growth of tumor cells without affecting normal epithelial cells.

Figure 2.73 - Defensin monomer (top) and dimer (bottom) - Cationic residues in blue, hydrophobic residues in orange, and anionic residues in red Wikipedia

Defensins (Figure 2.73) are a group of small cationic proteins (rich in cysteine residues) that serve as host defense peptides in vertebrate and invertebrate organisms. They protect against infection by various bacteria, fungi, and viruses. Defensins contain between 18 and 45 amino acids with (typically) about 6- 8 cysteine residues. In the immune system, defensins help to kill bacteria engulfed by phagocytosis by epithelial cells and neutrophils. They kill 120 Figure 2.72 - Vinculin Wikipedia bacteria by acting like ionophores - binding the membrane and opening pore-like structures to release ions and nutrients from the cells.

Focal adhesions

In the cell, focal adhesions are structures containing multiple proteins that mechanically link cytoskeletal structures (actin bundles) with the extracellular matrix. They are dynamic, with proteins bringing and leaving with signals regarding the cell cycle, cell motility, and more almost constantly. Focal adhesions serve as anchors and as a signaling hub at cellular locations where integrins bind molecules and where membrane clustering events occur. Over 100 different proteins are found in focal adhesions.

Focal adhesions communicate important messages to cells, acting as sensors to update information about the status of the extracellular matrix, which, in turn, adjusts/ affects their actions. In sedentary cells, they are stabler than in cells in motion because when cells move, focal adhesion contacts are established at the &ldquofront&rdquo and removed at the rear as motion progresses. This can be very important in white blood cells&rsquo ability to find tissue damage.

Figure 2.74 - Ankyrin&rsquos membrane-binding domain

Ankyrins (Figure 2.74) are a family of membrane adaptor proteins serving as &ldquoanchors&rdquo to interconnect integral membrane proteins to the spectrin-actin membrane cytoskeleton. Ankyrins are anchored to the plasma membrane by covalently linked palmitoyl-CoA. They bind to the &beta subunit of spectrin and at least a dozen groups of integral membrane proteins. The ankyrin proteins contain four functional domains: an N-terminal region with 24 tandem ankyrin repeats, a central spectrin-binding domain, a &ldquodeath domain&rdquo interacting with apoptotic proteins, and a C-terminal regulatory domain that is highly varied significantly among different ankyrins.

Figure 2.75 - Spectrin and other proteins in the cytoskeleton Wikipedia

Spectrin (Figures 2.75 & 2.76) is a protein of the cellular cytoskeleton that plays an important role in maintaining its structure and the integrity of the plasma membrane. In animals, spectrin gives red blood cells their shape. Spectrin is located inside the inner layer of the eukaryotic plasma membrane where it forms a network of pentagonal or hexagonal arrangements.

Spectrin fibers collect together at junctional complexes of actin and is also attached to ankyrin for stability, as well as numerous integral membrane proteins, such as glycophorin.

Figure 2.76 - Spectrin (green) and nuclei (Blue) Wikipedia

Figure 2.77 - Integrin and its binding site (on top left) Wikipedia

In multicellular organisms, cells need connections, both to each other and to the extracellular matrix. Facilitating these attachments at the cellular end are transmembrane proteins known as integrins (Figure 2.77). Integrins are found in all metazoan cells. Ligands for the integrins include collagen, fibronectin, laminin, and vitronectin. Integrins function not only in attachment, but also in communication, cell migration, virus linkages (adenovirus, for example), and blood clotting. Integrins are able to sense chemical and mechanical signals about the extracellular matrix and move that information to intracellular domains as part of the process of signal transduction. Inside the cells, responses to the signals affect cell shape, regulation of the cell cycle, movement, or changes in other cell receptors in the membrane. The process is dynamic and allows for rapid responses as may be necessary, for example in the process of blood clotting, where the integrin known as GPIbIIIa (on the surface of blood platelets) attaches to fibrin in a clot as it develops.

Integrins work along with other receptors, including immunoglobulins, other cell adhesion molecules, cadherins, selectins, and syndecans. In mammals the proteins have a large number of subunits - 18 &alpha- and 8 &beta-chains. They are a bridge between its links outside the cell to the extracellular matrix (ECM) and its links inside the cell to the cytoskeleton. Integrins play central role in formation and stability of focal adhesions. These are large molecular complexes arising from clustering of integrin-ECM connections. In the process of cellular movement, integrins at the &ldquofront&rdquo of the cell (in the direction of the movement), make new attachments to substrate and release connections to substrate in the back of the cell. These latter integrins are then endocytosed and reused.

Integrins also help to modulate signal transduction through tyrosine kinase receptors in the cell membrane by regulating movement of adapters to the plasma membrane. &beta1c integrin, for example, recruits the Shp2 phosphatase to the insulin growth factor receptor to cause it to become dephosphorylated, thus turning off the signal it communicates. Integrins can also help to recruit signaling molecules inside of the cell to activated tyrosine kinases to help them to communicate their signals.

Figure 2.78 - Extracellular ectodomain of a cadherin

Cadherins (Figure 2.78) constitute a type-1 class of transmembrane proteins playing important roles in cell adhesion. They require calcium ions to function, forming adherens junctions that hold tissues together (See Figure 2.69). Cells of a specific cadherin type will preferentially cluster with each other in preference to associating with cells containing a different cadherin type. Caderins are both receptors and places for ligands to attach. They assist in the proper positioning of cells in development, separation of different tissue layers, and cell migration.

Figure 2.79 - Selectin bound to a sugar Wikipedia

Selectins (Figure 2.79) are cell adhesion glycoproteins that bind to sugar molecules. As such, they are a type of lectin - proteins that bind sugar polymers (see HERE also). All selectins have an N-terminal calcium-dependent lectin domain, a single transmembrane domain, and an intracellular cytoplasmic tail.

There are three different types of selectins, 1) E-selectin (endothelial) 2) L (lymphocytic and 3) P (platelets and endothelial cells. Selectins function in lymphocyte homing (adhesion of blood lymphocytes to cells in lymphoid organs), in inflammation processes, and in cancer metastasis. Near the site of inflammation, P-selectin on the surface of blood capillary cells interacts with glycoproteins on leukocyte cell surfaces. This has the effect of slowing the movement of the leukocyte. At the target site of inflammation, E- selectin on the endothelial cells of the blood vessel and L-selectin on the surface of the leukocyte bind to their respective carbohydrates, stopping the leukocyte movement. The leukocyte then crosses the wall of the capillary and begins the immune response. Selectins are involved in the inflammatory processes of asthma, psoriasis, multiple scleroris, and rheumatoid arthritis.

Laminins are extracellular matrix glycoproteins that a major components of the basal lamina and affect cell differentiation, migration, and adhesion. They are secreted into the extracellular matrix where they are incorporated and are essential for tissue maintenance and survival. When laminins are defective, muscles may not form properly and give rise to muscular dystrophy.

Laminins are associated with fibronectin, entactin, and perlecan proteins in type IV collagen networks and bind to integrin receptors in the plasma membrane. As a consequence, laminins contribute to cellular attachment, differentiation, shape, and movement. The proteins are trimeric in structure, having one &alpha-chain, a &beta-chain, and a &gamma-chain. Fifteen combinations of different chains are known.


Vitronectin is a glycoprotein (75kDa) found in blood serum (platelets), the extracellular matrix, and in bone. It promotes the process of cell adhesion and spreading and binds to several protease inhibitors (serpins). It is secreted from cells and is believed to play roles in blood clotting and the malignancy of tumors. One domain of vitronectin binds to plasminogen activator inhibitor and acts to stabilize it. Another domain of the protein binds to cellular integrin proteins, such as the vitronectin receptor that anchors cells to the extracellular matrix.

Catenins are a family of proteins interacting with cadherin proteins in cell adhesion (Figure 2.69). Four main types of catenins are known, &alpha-, &beta-, &gamma-, and &delta-catenin. Catenins play roles in cellular organization before development occurs and help to regulate cellular growth. &alpha-catenin and &beta-catenin are found at adherens junctions with cadherin and help cells to maintain epithelial layers. Cadherins are connected to actin filaments of the cytoskeleton and catenins play the critical role. Catenins are important for the process whereby cellular division is inhibited when cells come in contact with each other (contact inhibition).

When catenin genes are mutated, cadherin cell adhesions can disappear and tumorigenesis may result. Catenins have been found to be associated with colorectal and numerous other forms of cancer.


Figure 2.80 - Glycophorin a

All of the membrane proteins described so far are notable for the connections they make to other proteins and cellular structures. Some membrane proteins, though, are designed to reduce cellular connections to proteins of other cells. This is particularly important for blood cells where &ldquostickiness&rdquo is undesirable except where clotting is concerned.

Glycophorins (Figure 2.80) are membrane-spanning sialoglycoproteins of red blood cells. They are heavily glycosylated (60%).and rich in sialic acid, giving the cells a very hydrophilic (and negatively charged) coat, which enables them to circulate in the bloodstream without adhering to other cells or the vessel walls.

Five glycophorins have been identified - four (A,B,C,and D) from isolated membranes and a fifth form (E) from coding in the human genome. The proteins are abundant, forming about 2% of the total membrane proteins in these cells. Glycophorins have important roles in regulating RBC membrane mechanical properties and shape. Because some glycophorins can be expressed in various nonerythroid tissues (particularly Glycophorin C), the importance of their interactions with the membrane skeleton may have a considerable biological significance.

Cooperativity and allosterism - quaternary structure

Figure 2.81 - Two polypeptide units of a protein interact in quaternary structure Wikipedia

Quaternary structure, of course describes the interactions of individual subunits of a multi-subunit protein (Figure 2.81). The result of these interactions can give rise to important biological phenomena, such as cooperative binding of substrates to a protein and allosteric effects on the action of an enzyme.

Allosteric effects can occur by a series of mechanisms, but a common feature is that binding of an effector to an enzyme subunit causes (or locks) the enzyme in either a Tstate (less activity) or an R-state (more activity). Effectors can be enzyme substrates (homotropic effectors) or non-substrates (heterotropic effectors). Allosterism will be covered in more depth in the Catalysis chapter HERE.

We begin our consideration of quaternary structure with a discussion of cooperativity, how it arises in the multi-subunit protein hemoglobin and how its properties contrast with those of the related, single subunit protein myoglobin.


Cooperativity is defined as the phenomenon where binding of one ligand molecule by a protein favors the binding of additional molecules of the same type. Hemoglobin, for example, exhibits cooperativity when the binding of an oxygen molecule by the iron of the heme group in one of the four subunits causes a slight conformation change in the subunit. This happens because the heme iron is attached to a histidine side chain and binding of oxygen &lsquolifts&rsquo the iron along with the histidine ring (also known as the imidazole ring).

Movie 2.3 - Hemoglobin&rsquos structural changes on binding oxygen Wikipedia

Since each hemoglobin subunit interacts with and influences the other subunits, they too are induced to change shape slightly when the first subunit binds to oxygen (a transition described as going from the T-state to the R-state). These shape changes favor each of the remaining subunits binding oxygen, as well. This is very important in the lungs where oxygen is picked up by hemoglobin, because the binding of the first oxygen molecule facilitates the rapid uptake of more oxygen molecules. In the tissues, where the oxygen concentration is lower, the oxygen leaves hemoglobin and the proteins flips from the R-state back to the Tstate.

CO2 transport

Figure 2.82 - Heme structure within hemoglobin Image by Aleia Kim

Cooperativity is only one of many fascinating structural aspects of hemoglobin that help the body to receive oxygen where it is needed and pick it up where it is abundant. Hemoglobin also assists in the transport of the product of cellular respiration (carbon dioxide) from the tissues producing it to the lungs where it is exhaled. Like the binding of oxygen to hemoglobin, binding of other molecules to hemoglobin affects its affinity for oxygen. The effect is particularly pronounced when comparing the oxygen binding characteristics of hemoglobin&rsquos four subunits with the oxygen binding of the related protein myoglobin&rsquos single subunit (Figure 2.83).

Different oxygen binding

Figure by Aleia Kim

Like hemoglobin, myoglobin contains an iron in a heme group that binds to oxygen. The structure of the globin protein in myoglobin is very similar to the structure of the globins in hemoglobin and hemoglobin is thought to have evolved from myoglobin in evolutionary history. As seen in Figure 2.83, the binding curve of hemoglobin for oxygen is S-shaped (sigmoidal), whereas the binding curve for myoglobin is hyperbolic. What this tells us is that hemoglobin&rsquos affinity for oxygen is low at a low concentration oxygen, but increases as the oxygen concentration increases. Since myoglobin very quickly saturates with oxygen, even under low oxygen concentrations, it says that its affinity for oxygen is high and doesn&rsquot change.

Figure 2.84 - Binding of oxygen at the heme center of hemoglobin Image by Aleia Kim

Because myoglobin has only a single subunit, binding of oxygen by that subunit can&rsquot affect any other subunits, since there are no other subunits to affect. Consequently, cooperativity requires more than one subunit. Therefore, hemoglobin can exhibit cooperativity, but myoglobin can&rsquot. It is worth noting that simply having multiple subunits does not mean cooperativity will exist. Hemoglobin is one protein that exhibits the characteristic, but many multisubunit proteins do not.

Interactive 2.2 - Hemoglobin in the presence (top) and absence (bottom) of oxygen

Storage vs. delivery

The lack of ability of myoglobin to adjust its affinity for oxygen according to the oxygen concentration (low affinity at low oxygen concentration, such as in tissues and high affinity at high oxygen concentration, such as in the lungs) means it is better suited for storing oxygen than for delivering it according to the varying oxygen needs of and animal body. As we shall see, besides cooperativity, hemoglobin has other structural features that allow it to deliver oxygen precisely where it is needed most in the body.

Figure 2.85 - Sequential model of binding. The sequential model is one way to explain hemoglobin&rsquos cooperativity. Squares represent no oxygen bound. Circles represent subunits bound with oxygen and rounded subunits correspond to units whose affinity for oxygen increases by interacting with a subunit that has bound oxygen. Image by Aleia Kim 131

Bohr effect

Figure 2.86 - The Bohr effect with respect to pH changes Image by Aleia Kim

The Bohr Effect was first described over 100 years ago by Christian Bohr, father of the famous physicist, Niels Bohr. Shown graphically (Figures 2.86, 2.87, and 2.88), the observed effect is that hemoglobin&rsquos affinity for oxygen decreases as the pH decreases and as the concentration of carbon dioxide increases. Binding of the protons and carbon dioxide by amino Figure 2.85 - Sequential model of binding. The sequential model is one way to explain hemoglobin&rsquos cooperativity. Squares represent no oxygen bound. Circles represent subunits bound with oxygen and rounded subunits correspond to units whose affinity for oxygen increases by interacting with a subunit that has bound oxygen. Image by Aleia Kim acid side chains in the globin proteins helps to facilitate structural changes in them. Most commonly, the amino acid affected by protons is histidine #146 of the &beta strands. When this happens, the ionized histidine can form an ionic bond with the side chain of aspartic acid #94, which has the effect of stabilizing the T-state (reduced oxygen binding state) and releasing oxygen. Other histidines and the amine of the amino terminal amino acids in the &alpha-chains are also binding sites for protons.

Figure 2.87 - Binding affinity of hemoglobin for oxygen under different conditions Image by Aleia Kim

Figure 2.88 - The Bohr effect physiologically - oxygen binding curves for resting muscle (blue), active muscle (green) and reference muscle (orange) with respect to pH, 2,3-BPG, and CO2 Image by Aleia Kim

Another molecule favoring the release of oxygen by hemoglobin is 2,3- bisphosphoglycerate (also called 2,3-BPG or just BPG - Figure 2.89). Like protons and carbon dioxide, 2,3-BPG is produced by actively respiring tissues, as a byproduct of glucose metabolism. The 2,3-BPG mole cule fits into the &lsquohole of the donut&rsquo of adult hemoglobin (Figure 2.89). Such binding of 2,3-BPG favors the T-state (tight - low oxygen binding) of hemoglobin, which has a reduced affinity for oxygen. In the absence of 2,3-BPG, hemoglobin can more easily exist in the R-state (relaxed - higher oxygen binding), which has a high affinity for oxygen.

Figure 2.89 - The structure of 2,3 bisphosphoglycerate (2,3-BPG)

Notably, the blood of smokers is higher in the concentration of 2,3-BPG than non-smokers, so more of their hemoglobin remains in the T-state and thus the oxygen carrying capacity of smokers is lower than non-smokers.Another reason why smokers&rsquo oxygen carrying capacity is lower than that of non-smokers is that cigarette smoke contains carbon monoxide and this molecule, which has almost identical dimensions to molecular oxygen, effectively outcompetes with oxygen for binding to the iron atom of heme (Figure 2.90). Part of carbon monoxide&rsquos toxicity is due to its ability to bind hemoglobin and prevent oxygen from binding.

Figure 2.90 - Binding of oxygen (left) and carbon monoxide (right) by a heme group of hemoglobin Image by Aleia Kim

Carbon dioxide

Figure 2.91 - Hemoglobin&rsquos hole of the donut for binding 2,3-BPG Wikipedia

Carbon dioxide binds to form a carbamate when binding the &alpha-amine of each globin chain. The process of forming this structure releases a proton, which helps to further enhance the Bohr effect. Physiologically, the binding of CO2 and H+ has significance because actively respiring tissues (such as contracting muscles) require oxygen and release protons and carbon dioxide. The higher the concentration of protons and carbon dioxide, the more oxygen is released to feed the tissues that need it most.

About 40% of the released protons and about 20% of the carbon dioxide are carried back to the lungs by hemoglobin. The remainder travel as part of the bicarbonate buffering system or as dissolved CO2. In the lungs, the process reverses itself. The lungs have a higher pH than respiring tissues, so protons are released from hemoglobin and CO2 too is freed to be exhaled.

Fetal hemoglobin

Figure 2.92 - Formation of bicarbonate from CO2 in blood

Adult hemoglobin releases oxygen when it binds 2,3- BPG. This is in contrast to fetal hemoglobin, which has a slightly different configuration (&alpha2&gamma2) than adult hemoglobin (&alpha2&beta2). Fetal hemoglobin has a greater affinity for oxygen than maternal hemoglobin, allowing the fetus to obtain oxygen effectively from the mother&rsquos blood. Part of the reason for fetal hemoglobin&rsquos greater affinity for oxygen is that it doesn&rsquot bind 2,3-BPG. Consequently, fetal hemoglobin remains in the R-state much more than adult hemoglobin and because of this, fetal hemoglobin has greater affinity for oxygen than adult hemoglobin and can take oxygen away from adult hemoglobin. Thus, the fetus can get oxygen from the mother.

Figure 2.93 - Comparison of oxygen binding of myoglobin (blue), fetal hemoglobin (orange), and adult hemoglobin (green) Image by Aleia Kim

Sickle cell disease

Figure 2.94 - Four normal red blood cells (right) and one sickled red blood cell (left) Wikipedia

Mutations to the globin genes coding for hemoglobin can sometimes have deleterious consequences. Sickle cell disease (also called sickle cell anemia) is a genetically transmitted disease that arises from such mutations. There are different forms of the disease. It is a recessive trait, meaning that to be afflicted with it, an individual must inherit two copies of the mutated gene.

Figure 2.95 - Movement of blood in capillaries. Top - normal red blood cells. Bottom - sickled red blood cells

The predominant form of hemoglobin in adults is hemoglobin A, designated HbA (two &alpha chains and two &beta chains). The mutant form is known as HbS. The most common mutation is an A to T mutation in the middle of the codon for the seventh amino acid (some counting schemes call it the sixth amino acid) of the &beta-chain. This results in conversion of a GAG codon to GTG and thus changes the amino acid specified at that position from a glutamic acid to a valine. This minor change places a small hydrophobic patch of amino acids on the surface of the &beta-globin chains.


Figure 2.96 - Pattern of expression of six different globins of hemoglobin - &alpha,&beta,&gamma,&epsilon,&delta, and &zeta Image by Aleia Kim

Under conditions of low oxygen, these hydrophobic patches will associate with each other to make long polymers of hemoglobin molecules. The result is that the red blood cells containing them will change shape from being rounded to forming the shape of a sickle (Figure 2.94). Rounded red blood cells readily make it through tiny capillaries, but sickleshaped cells do not.

Worse, they block the flow of other blood cells. Tissues where these blockages occur are already low in oxygen, so stopping the flow of blood through them causes them to go quickly anaerobic, causing pain and, in some cases, death of tissue. In severe circumstances, sickled red blood cells death may result. The disease is referred to as an anemia because the sickling of the red blood cells targets them for removal by the blood monitoring system of the body, so a person with the disease has chronically reduced numbers of red blood cells.

Heterozygote advantage

Interestingly, there appears to be a selective advantage to people who are heterozygous for the disease in areas where malaria is prominent. Heterozygotes do not suffer obvious ill effects of the disease, but their red blood cells appear to be more susceptible to rupture when infected. As a consequence, the parasite gets less of a chance to reproduce and the infected person has a greater chance of survival.

The protective effect of the mutant gene, though, does not extend to people who suffer the full blown disease (homozygotes for the mutant gene). Treatments for the disease include transfusion, pain management, and avoidance of heavy exertion. The drug hydroxyurea has been linked to reduction in number and severity of attacks, as well as an increase in survival time1,2. It appears to work by reactivating expression of the fetal hemoglobin gene, which typically is not synthesized to any significant extent normally after about 6 weeks of age.

Oxygen binding

Animals have needs for oxygen that differ from all other organisms. Oxygen, of course, is the terminal electron acceptor in animals and is necessary for electron transport to work. When electron transport is functioning, ATP generation by cells is many times more efficient than when it is absent. Since abundant ATP is essential for muscular contraction and animals move around a lot - to catch prey, to exercise, to escape danger, etc., having an abundant supply of oxygen is important.

This is particularly a concern deep inside tissues where diffusion of oxygen alone (as occurs in insects) does not deliver sufficient quantities necessary for long term survival. The issue is not a problem for plants since, for the most part, their motions are largely related to growth and thus don&rsquot have rapidly changing needs/demands for oxygen that animals have. Unicellular organisms have a variety of mechanisms for obtaining oxygen and surviving without it. Two other important oxygen binding proteins besides hemoglobin are myoglobin and hemocyanin.

Figure 2.97 - Myoglobin bound to oxygen

Myoglobin is the primary oxygen-storage protein found in animal muscle tissues. In contrast to hemoglobin, which circulates throughout the body, myoglobin protein is only found in muscle tissue and appears in the blood only after injury. Like hemoglobin, myoglobin binds oxygen at a prosthetic heme group it contains.

The red color of meat arises from the heme of myoglobin and the browning of meat by cooking it comes from oxidation of the ferrous (Fe++) ion of myoglobin&rsquos heme to the ferric (Fe+++) ion via oxidation in the cooking process. As meat sits in our atmosphere (an oxygen-rich environment), oxidation of Fe++ to Fe+++ occurs, leaving the brown color noted above. If meat is stored in a carbon monoxide (CO) environment, CO binds to the heme group and reduces the amount of oxidation, keeping meat looking red for a longer period of time.

High affinity

Myoglobin (Figure 2.97) displays higher affinity for oxygen at low oxygen concentrations than hemoglobin and is therefore able to absorb oxygen delivered by hemoglobin under these conditions. Myoglobin&rsquos high affinity for oxygen makes it better suited for oxygen storage than delivery. The protein exists as a single subunit of globin (in contrast to hemoglobin, which contains four subunits) and is related to the subunits found in hemoglobin. Mammals that dive deeply in the ocean, such as whales and seals, have muscles with particularly high abundance of myoglobin. When oxygen concentration in muscles falls to low levels, myoglobin releases its oxygen, thus functioning as an oxygen &ldquobattery&rdquo that delivers oxygen fuel when needed and holding onto it under all other conditions. Myoglobin holds the distinction of being the first protein for which the 3D structure was determined by X-ray crystallography by John Kendrew in 1958, an achievement for which he later won the Nobel Prize.

Figure 2.98 - Oxygen bound at heme of myoglobin Wikipedia

Figure 2.99 - Oxygen binding in hemocyanin Wikipedia

Hemocyanin is the protein transporting oxygen in the bodies of molluscs and arthropods. It is a coppercontaining protein found not within blood cells of these organisms, but rather is suspended in the circulating hemolymph they possess. The oxygen binding site of hemocyanin contains a pair of copper(I) cations directly coordinated to the protein by the imidazole rings of six histidine side chains.

Figure 2.100 - Hemocyanin (purple) in a red rock crab Wikipedia

Most, but not all hemocyanins bind oxygen non-cooperatively and are less efficient than hemoglobin at transporting oxygen. Notably, the hemocyanins of horseshoe crabs and some other arthropods do, in fact, bind oxygen cooperatively. Hemocyanin contains many subunit proteins, each with two copper atoms that can bind one oxygen molecule (O2). Subunit proteins have atomic masses of about 75 kilodaltons (kDa). These may be arranged in dimers or hexamers depending on species. Superstructures comprised of dimer or hexamer complexes are arranged in chains or clusters and have molecular weights of over 1500 kDa.

Watch the video: Effect of 2,3-BPG on Hemoglobin (October 2022).