10.05.16 (Structure & Function of Myoglobin and Hemoglobin)

What gene deletion presents as a thalassemia without symptoms, but the person is a silent carrier for the deletion?

1-alpha gene locus.

What gene is deleted in beta-thalassemia minor?

1-beta gene locus.

Where is 2,3-DPG synthesized and how does it regulate O2 binding to hemoglobin?

1. 2,3-di-phosphoglycerate (2,3-DPG) is an isomer of 1,3-bis-phosphoglycerate and is produced in RBCs when [glucose] is abundant. 2. 2,3-DPG binds in the cleft between the beta-subunits of Hb and stabilizes the Taut (deoxy) state. Through this stabilization, Hb-bound O2 dissociates and supplies O2 to tissues.

What are the main physiological modulators that affect the binding of oxygen to hemoglobin (*3*) and what is their most common effect.

1. 2,3-diPhosphoglycerate 2. Low pH (acidity) 3. High carbon dioxide (CO2) These 3 physiological modulators of O2 binding to Hb most often decrease the affinity of Hb for O2, allowing a release of more O2 over a wider range of pO2.

What types of hemoglobin do we produce before and after birth?

1. After birth, we shift to producing Hb in the bone marrow (as opposed to the yolk sac, liver, or spleen) and we all increase our production of beta-hemoglobin, while decreasing our production of gamma, and maintaining our production of alpha.

What are *4* physiological changes that result in 2,3-DPG level changes?

1. COPD 2. High Altitude 3. Chronic Anemia 4. Pregnancy

What are *4* substances that compete for the iron-binding site of heme in HbA?

1. Cyanide (CN-) 2. carbon monoxide (CO) 3. nitrogen dioxide (NO2) 4. hydrogen sulfide (H2S) *All of these compounds inhibit oxygen binding but they do not change the oxidation status of iron, it remains ferrous (Fe2+)*

Describe what P50 means in terms of myoglobin versus hemoglobin:

1. Fifty percent saturation with O2 for myoglobin means that on average half of myoglobin molecules (in our muscles) have O2 bound at their heme and the other half do not. 2. Fifty percent O2 binding for hemoglobin means that the average number of hemes binding O2 is 2 per hemoglobin molecule in our blood.

What are the *2* main physiological roles of hemoglobin and what are they dependent upon?

1. Hb binds O2 in the alveoli of the lungs and delivers this O2 to tissues throughout the body. 2. Hb assists in maintaining acid-base balance in the body by binding some CO2 produced by metabolism and releasing CO2 when Hb reaches the lungs. Both functions of Hb are dependent upon partial pressure gradients of O2 or CO2.

What is HbA, what is it composed of, and how does its glycosylation prove useful?

1. HbA is the primary/major hemoglobin molecule in adults (97-98.5% of total Hb). 2. It is composed of two alpha and two beta chains. 3. It's glycosylation is a marker for chronically elevated blood sugar.

How is oxygen a positive allosteric regulator for the binding of itself (or more oxygen) on hemoglobin?

1. Hemoglobin exists in two different conformations: T (for taut) and R (for relaxed). 2. The T conformation favors the deoxygenated form (O2 release) while the R conformation favors the oxygenated form (O2 binding). 3. The binding of the initial oxygen affects the equilibrium between these two forms and facilitates the binding of the next O2 molecule.

Describe the structure of hemoglobin in *5* characteristics:

1. Hemoglobin, unlike myoglobin, is composed of four polypeptide chains; 2-alpha and 2-beta chains. 2.Each polypeptide chain folds similar to myoglobin. 3. There is one single heme molecule covalently bound to each subunit; so Hb can bind up to 4 O2 at one time. 4. Extensive strong hydrophobic interactions between the alpha-1 & beta-1 subunits and between the alpha-2 & beta-2 subunits; 5. There are weaker polar interactions between the heterodimers: alpha-1/beta-1 and alpha-2/beta-2.

What is methemoglobinemia and what mutations cause it?

1. Methemoglobinemia is characterized by cyanosis and brown colored blood. 2. Mutations that change any of the 3 amino acids on the distal side of heme, thus preventing oxygen release, result in hereditary methemoglobinemia.

What are some other causes of methemoglobinemia?

1. Mutations in the Pentose Phosphate Pathway, especially G-6-phosphate dehydrogenase deficiency. 2. Failure of glutathione to reduce reactive oxygen species. 3. Mutations in cytochrome b5 reductase (NADH-dependent methemoglobin reductase) - enzyme uses NADH generated through glyceraldehyde phosphate dehydrogenase (glycolysis) to reduce Fe3+ in methemoglobin to Fe2+ thereby reversing formation of Fe3+.

What is myoglobin and what is its function?

1. Myoglobin is a protein that is synthesized inside muscle cells. It is a protein that binds oxygen, delivered by Hemoglobin, and *stores* it for later use at times of high metabolic demand. 2. Different muscle tissues have varying degrees of myoglobin.

Describe the structure of myoglobin in *4* characteristics:

1. Myoglobin is a single polypeptide chain. 2. It is ~80% alpha-helical. 3. It has a closely-packed tertiary structure (think of dropping a single rope on the ground). 4. It has one covalently bound heme molecule that allows it to bind one O2 molecule at a time.

How many Hb variants are known and what are they usuall caused by? Describe the most common Hb variant and what disease does it lead to?

1. Over 1100 Hb variants known. Most are the result of point mutations that change a single amino acid and are not associated with any clinically significant pathology. 2. The most common Hb variant associated with significant pathology in the U.S. is the beta-S (𝜷^S) variant caused by a mutation at position 6 of the 𝜷-globin gene which changes Glu^6 to Val^6. 3. Individuals homozygous for the 𝜷^S mutation, or heterozygous for the 𝜷^S and a 𝜷-thalassemia allele, produce significant amounts of HbS (𝜶2𝜷2^S) and have *sickle cell disease.* Deoxy HbS forms long polymers within the erythrocyte, which shortens the lifespan of RBCs.

Describe what is meant by shifting the hemoglobin/oxygen curve to the right or left? What causes the curve to shift right?

1. Shifting the hemoglobin/oxygen binding curve to the *right* means that at high pO2, hemoglobin molecules are less saturated, meaning that oxygen is being released. 2. Shifting the hemoglobin/oxygen binding curve to the *left* means that at lower pO2, hemoglobin molecules are holding on to their oxygens. 3. Curve is shifted right by: -low pH -high CO2 -2,3-DPG -high temperature

Where are the genes for the globin chains of hemoglobin located? How many alpha and beta gene loci do we have?

1. The clusters of the globin genes are located on C.16 (two alphas) and C.11 (beta) and they are aligned concurrent to their order of expression during development. 2. After inheriting one copy of each chromosome from our parents, we have two beta-globin genes and four alpha-globin genes. This is important as it explains thalassemia possibilities.

What gene deletion leads to detectable carrying of alpha-thalassemia trait?

2-alpha gene loci

What gene is deleted in beta-thalassemia major?

2-beta gene loci (aka Cooley anemia).

What genes are deleted in HbH disease?

3-alpha gene loci

What gene is deleted in HbBart's or hydrops fetalis disease?

4-alpha gene loci.

What are the causes of thalassemia?

Can be due to gene deletion, mutations that affect gene regulation or splicing. Very unstable hemoglobins can also lead to thalassemia-like symptoms in some cases.

What is carboxyhemoglobin?

Carboxyhemoglobin is a stable complex of carbon monoxide (CO) and heme in Hb. Carboxyhemoglobin forms more readily than does oxyhemoglobin (HbO2).

How does acidity regulate O2 binding to hemoglobin?

Decrease in pH (increasing acidity) favors O2 release to tissues. Raising pH (decreasing acidity) favors O2 binding to Hb.

How does HbF differ in structure and affinity for O2 when compared to adult HbA?

Fetal hemoglobin (HbF) has a different beta chain, and it has a higher affinity for oxygen than adult hemoglobin (HbA).

What is the structure of Hb𝛆 (epsilon) and when is it prevalent?

Hb-epsilon is composed of either two zeta (𝛇) and two epsilon (𝛆) chains *or* two alpha and two epsilon chains, *or* two zeta and two gamma chains. It is prevalent as embryonic Hb, ~1 week post-conception until birth.

What is the structure of HbA2 and how much of it is in adult blood?

HbA2 is composed of two alpha and two delta chains and represents (1.5-3%) of total Hb in adult.

How does HbF differ from HbA with respect to 2,3 DPG?

HbF binds 2,3-DPG very poorly because the cavity for 2,3-DPG binding is not as positively charged.

What is the structure of HbF and when is it prevalent?

HbF or fetal hemoglobin is composed of two alpha and two gamma chains. It is the major form of Hb from ~1 month gestation until near birth; it then composes~40% of Hb at birth.

What class of pigments does heme belong to? What are other examples?

Heme Belongs To The Class Of Pigments Known As Porphyrins (Cyclic Tetrapyrroles): Heme, Chlorophyll, Cyano-cobalamin or Vitamin B12

What is the Bohr effect?

In RBCs, protons bind to Hb and favor the taut (T) state, favoring the release of O2.

What is the Haldane effect?

In the lungs, high pO2 drives binding of O2 to Hb, thus releasing of H+ and causing the formation of H2CO3 (carbonic acid). Carbonic acid equilibrium favors synthesis of CO2 and H2O.

What are porphyrias?

Mutations in the Heme biosynthesis pathway = porphyrias

What always has the highest affinity for oxygen?

Myoglobin

What are Frank's *5* High-Yield Points for hemoglobin?

Not only does Hb bind oxygen in the lungs and delivers it to various tissue beds and cells, it picks up CO2 and returns it to the lungs; Each of the 4 subunits of Hb bind a single oxygen molecule, but do so in a concerted effort, termed positive cooperativity; Oxygen-binding is complex and is performed under an allosteric mechanism, which requires 2,3-DPG; Bohr and Haldene effects describe how changing the pH and levels of CO2 and O2 alter the affinity of Hb-oxygen binding/delivery. Alterations in (i) Hb sequence, (ii) heme ring synthesis, or (iii) Fe valency can each lead to significant pathological consequences.

Where does O2 bind in myoglobin and hemoglobin?

O2 binds on the heme group in both Myoglobin and Hemoglobin

How does maternal HbA support HbF?

O2 released by maternal Hb A is bound by fetal Hb F and transported to fetal tissue.

Why is the hyrdophobicity of the pyrrole rings so important for heme groups?

Oxygen is not soluble in aqueous solution; thus, the center of heme is a "greasy" or hydrophobic spot where oxygen can bind.

What is P50?

P50 is the partial pressure of O2 at which 50% of O2 binding sites are occupied.

What is pO2?

PO2 (partial pressure of oxygen) reflects the amount of oxygen gas dissolved *in the blood.* It primarily measures the effectiveness of the lungs in pulling oxygen into the blood stream from the atmosphere.

What type of rings are featured in heme?

Pyrrole rings that form coordinate covalent bonds to Fe2+ (ferrous iron).

What does the (alpha) 𝛂^+ or (beta) 𝛃^+ classification, in describing thalassemias, mean?

Superscript "+" means that there is a reduced level of a particular type of globin chain.

What does the (alpha) 𝛂^0 or (beta) 𝛃^0 classification, in describing thalassemias, mean?

Superscript "0" means that there is no synthesis of that particular type of globin chain.

What is thalassemia?

Thalassemias are a diverse group of genetic blood diseases characterized by absent alpha or beta globin genes—leading to decreased production of normal hemoglobin chains, resulting in anemia of varying degree.

What type of chains are involved in heme?

The hydrophobic chains from the pyrrole rings interact with the surrounding α and β-globin chains to stabilize heme binding.

What are the contents of plasma?

Water, proteins, nutrients, hormones, etc.

Describe the shape of the myoglobin/oxygen binding curve?

binding curve is hyperbolic, indicating a single constant affinity for O2.

Describe the shape of the hemoglobin/oxygen binding curve?

binding curve is sigmoidal, indicating changing affinity for O2 over the binding curve.