Describe the overall structure of hemoglobin, indicating the site of oxygen binding.
Hemoglobin is a ~68-kD tetramer comprised of 2 pairs of globin polypeptide chains: one pair of α-globin chains and one pair of non- α-globin (γ-, δ-, or β-) chains. Hemoglobin A1 (α2β2) – also called Hemoglobin A - is the predominant form of hemoglobin in the adult.A heme prosthetic group, consisting of a protoporphyrin ring bound to iron, is associated with each globin chain of the hemoglobin tetramer. It is the heme group that binds oxygen.
Explain the concepts of allostery and positive cooperativity as they relate to hemoglobin function.
The primary function of hemoglobin is to deliver oxygen from the lungs to the tissues. In order to carry out this function, the hemoglobin molecule must be able to easily pick up available oxygen in an environment where the oxygen concentration is relatively high (such as in the lung) while being able to easily unload it under conditions of lower oxygen availability (such as in the tissues). This is accomplished through allosteric regulation of hemoglobin, meaning that with binding to its substrate, oxygen, the hemoglobin molecule changes its configuration, altering its binding affinity to additional oxygen molecules. This phenomenon of binding to substrate leading to increased affinity for additional substrate (Changing from T to R state) is called positive cooperativity.
Compare the oxygenated and deoxygenated states of hemoglobin in relationship to taut (T) and relaxed (R) configurations of the molecule.
It turns out that under conditions where the oxygen concentration is low enough that none of the four sites are occupied, the binding affinity to oxygen is relatively low. In this situation, the hemoglobin is in a taut or T configuration, due to inter-and intra-salt bonds within the molecule.As oxygen becomes more available and one of the binding sites becomes occupied, the configuration of the molecule changes such that the other three sites have higher binding affinity and can more easily bind to additional oxygen molecules. As the number of occupied sites increases, the affinity for the remaining sites continues to increase. This occurs through sequential breaking of the salt bonds, converting the hemoglobin to the relaxed or R form.This phenomenon of binding to substrate leading to increased affinity for additional substrate is called positive cooperativity.
Draw a typical oxygen dissociation curve. Explain why it is sigmoidal in shape.Define the p50.Roughly estimate % oxygen saturation with pO2s of 100 mmHg, 60 mmHg, 40 mmHg, 30 mmHg, and 10 mmHg.
Because of cooperativity, when the % saturation of hemoglobin by oxygen is plotted as a function of the partial pressure of oxygen, the resulting curve turns out to be sigmoidal, or S-shaped.A way to quantify this difference in oxygen affinity is by determining the P50, which is defined as the partial pressure of oxygen at which the oxygen carrying protein is 50% saturated. A simple mnemonic to remember the basic shape of the hemoglobin O2 dissociation curve is “30-60, 60-90, 40-75”, meaning that at a partial pressure of 30 mmHg, the % saturation is ~60 %, at 60 mmHg it’s ~90% and at 40 mmHg it’s ~75%.Also, at 10 mmHg the % saturation is ~10 %. 100 looks like 100 to me as well.
Explain the effects of pH, CO2 concentration, temperature, and 2,3-BPG concentration on the hemoglobin oxygen dissociation curve.
Curve shifts right when pH decreases, temperature increases, and DPG (BPG) binding increases. This means that hemoglobin has lower affinity and releases more O2 to tissues in this case. Think about exercise to make physiological sense of this.
Compare oxygen dissociation curves for myoglobin and hemoglobin and explain the reason for the differences.
Because of cooperativity, when the % saturation of hemoglobin by oxygen is plotted as a function of the partial pressure of oxygen, the resulting curve turns out to be sigmoidal, or S-shaped.Compare the shape of this curve with that for myoglobin, which is a protein which stores oxygen in muscle cells and is very similar to hemoglobin except that it is a monomer rather than a tetramer and therefore does not undergo allosteric regulation or cooperativity.The myoglobin curve is shaped more like a hyperbola, giving the myoglobin molecule very high oxygen affinity at very low oxygen concentrations.
Describe the location and the general organization of the genes for alpha-like globin chains and beta-like globin chains.
Chromosome 16 contains the “α-like” genes, including two copies of the α-globin gene itself along with variants expressed early in embryonic development; therefore, the genome contains a total of 4 copies of the α-globin gene (2 paternal and 2 maternal).The “β-like” genes (genes for the γ-, δ-, and β-globin chains along with variants produced early in embryonic development) are products of a set of genes on chromosome 11; one copy of the gene set is inherited from each parent.
Describe how the structural differences between fetal hemoglobin and hemoglobin A lead to differences in oxygen affinity and why it's important.
After week 8 of gestation, fetal hemoglobin or hemoglobin F (α2γ2) predominates. The γ- chain differs from the β-globin chain by 39 amino acids. Fetal red cells have a higher oxygen affinity than adult red cells, primarily because hemoglobin F binds 2,3-BPG poorly, stabilizing the hemoglobin in the R state and shifting the oxygen dissociation curve to the left.The Bohr effect is also increased by 20% in fetal hemoglobin, so that as fetal blood passes through the intravillous spaces of the placenta, H+ ions are transferred to the maternal circulation and the pH rises, leading to increased oxygen affinity in the fetus and a further shift of the curve to the left. These changes favor transfer of oxygen from the maternal circulation to the fetal circulation.
Describe how unstable hemoglobins or hemoglobins with altered affinity can affect oxygen delivery to the tissues.
The first identified high-affinity hemoglobin variant (Hemoglobin Chesapeake) was described in 1966 in an 81-year old man with erythrocytosis and an abnormal band on hemoglobin electrophoresis. The genetic defect was a single point mutation. Erythrocytosis, or an elevated red blood cell count, is generally found in people with high-affinity hemoglobins. This occurs because oxygen delivery to the tissues is reduced, leading to increased erythropoiesis and an increased red cell count. Affected people are generally well.Low-affinity hemoglobin mutants are associated with cyanosis (a gray or bluish tint to the skin and mucus membranes). There are fewer low-affinity than high-affinity hemoglobin variants. More oxygen is delivered to the tissues with low-affinity hemoglobins.
Describe what methemoglobinemia is, what causes it, how to diagnose it, and how to treat it.
The iron contained within hemoglobin needs to be in the reduced or ferrous (Fe2+) form, not the ferric (Fe3+) form. Methemoglobinemia can occur because of too much methemoglobin production or because of decreased methemoglobin reduction. It may be an acquired or genetic process.Acquired methemoglobinemia can occur with exposure to a number of different drugs and chemicals. There are several different hereditary causes of methemoglobinemia with different inheritance patterns (autosomal dominant or autosomal recessive).The diagnosis of methemoglobinemia is suspected when a person looks cyanotic but the arterial partial pressure of oxygen is normal on an arterial blood gas. With methemoglobinemia, the blood looks dark-red, chocolate or brown-blue and with oxygen exposure does not change whereas if the cyanosis is due to increased deoxygenated hemoglobin, the blood will turn bright red with addition of oxygen.Treatment depends on the cause. No treatment is needed for hemoglobin M. Cytochrome b5 deficient patients are only treated for cosmetic reasons with methylene blue or ascorbic acid.Acquired methemoglobinemia is treated by removing the inciting drug or chemical. Methylene blue given intravenously provides an artificial electron acceptor for the reduction of methemoglobin via the NADPH-dependent pathway. Response is within one hour.
Explain the pathophysiology of carbon monoxide poisoning and its treatment.
When one heme binds CO, an allosteric change occurs so the other 3 hemes download oxygen less well, increasing the affinity of hemoglobin for oxygen and decreasing delivery of oxygen to tissues.People with carbon monoxide poisoning usually present complaining of a headache. They may also have malaise, nausea and dizziness. If levels are high enough, seizures, coma, and myocardial infarction may ensue. 40% of affected people will have late neurologic deficits such as loss of cognition, personality change, and movement disorders.Treatment is with 100% oxygen, which will compete with CO for the binding sites on the heme moiety. Hyperbaric oxygen can also be considered, which decreases the half-life of CO hemoglobin from 40-80 minutes to 15-30 minutes.
Describe how a pulse oximeter works and what it measures. Describe situations where a pulse oximeter reading may inaccurately reflect a patient’s true oxygenation status.
A pulse oximeter probe is a photo detector and two light-emitting diodes, one emitting at 660 nm and the other at 940 nm. They are in the red band (660 nm) where deoxyhemoglobin absorbs light maximally and the 940 nm infrared wavelength where oxyhemoglobin absorbs most. The detector and emitter face each other through tissue, so the probe is usually placed on the finger.Only pulsatile flow, representing arterial blood flow, is measured. It is important to remember that a pulse oximeter measures oxygen saturation, not partial pressure of oxygen.Pulse oximetry may be inaccurate if the probe is not placed correctly, if only one of the 2 diodes is working, if there is too much motion due to shivering or seizing, if nail polish is present, and with deeply pigmented skin, anemia, shock, or abnormal hemoglobins. Carboxyhemoglobin absorbs at 660 nm at a similar level to oxyhemoglobin so will give a falsely high reading. Methemoglobin absorbs at 660 nm and 940 nm so it will also give inaccurate results.
Define anemia and discuss the laboratory tests used to determine its existence in an individual. Explain the influence of age and gender on the definition.
Anemia is defined as insufficient red cell mass to adequately deliver oxygen to peripheral tissues.Ways to test for anemia: (1) hemoglobin concentration (Hgb), grams/ml; (2) hematocrit (Hct), percent volume of red cells in blood; and (3) red blood cell count (cells x10^12/l).At birth, infants have a very high hemoglobin and hematocrit concentration which in the first 8 weeks decreases to lower levels. During childhood, the levels of Hgb and Hct are lower than adults until the onset of puberty after which the values reach adult levels. Menstruating women have lower Hgb and Hct values than men, in part, because of more tenuous iron stores.
Define reticulocyte count, absolute reticulocyte count, and reticulocyte index and discuss how these measurements are used in assessing the rate of RBC production.
Reticulocytes are formally counted as the percent of 1,000 red cells counted and the normal range is from 0.4-1.7%. Increased red cell production is associated with a 3.5 to 5-fold increase over this baseline normal range.The calculation of the absolute reticulocyte count (which is equal to the percent retics times the red count) is helpful in determining the relevance of the reticulocyte count; anything over 50,000/μl is considered an increase over baseline maintenance production of red cells. Reticulocyte index (RI) is another measurement of the production of red cells and is a way to correct the reticulocyte count for red cell concentration and stress reticulocytosis. The corrected reticulocyte count or the reticulocyte index provides a ratio of how many fold over baseline the production of red cells is. RI = Reticulocyte Count × Patient Hgb /Normal Hgb* 1/Stress factor. Stress factor = 1.5 (mild anemia ≥ 9 gm/dl hgb); 2.0 (mod. anemia 6.5-9); 2.5 (severe < 1 with anemia indicates decreased production of reticulocytes and therefore red blood cells. A retic index > 2 with anemia indicates loss of red blood cells (destruction, bleeding, etc) leading to increased compensatory production of reticulocytes to replace the lost red blood cells.