Biochem 7 Oxygen Transporters

Hemoglobin structure

2 a subunits and 2 b subunits -Protein globin: 2 alpha and 2 beta chains -Heme pigment bonded to each globin chain Each subunit comes together Adult Hm is alpha 2 and beta 2 dimer and alpha 1 and beta 1 dimer Dimer of alpha beta subunits Hydrophobic pcket Prxoimal his to be ligand

Sickling is made worse by decreased oxygen

A hydrophobic hole is formed in both HbA and HbS when in the deoxy form A hydrophobic knob is present on the b subunit of HbS in both forms Thus The deoxy form of HbS can form long polymers Deoxy is bad -so knob fits into pocket so you get long polumers tha tbind to each other

case 3 Young child in rural area presents with "blue baby syndrome" Baby is fed with formula made up with well water that has not been tested for nitrate levels Patient does not respond to ascorbate and methylene blue Sequencing of the beta globin gene shows a switch from the liganding histidine to a tyrosine Upon oxygenation, the iron present in the heme changes size and moves into the middle of the heme. It is attached to a histidine that is known as the "proximal" histidine. Mutation of this histidine to another residue, such as tyrosine, causes

Answer: inherited methemoglobinemia has this sort of residue replacement' Methemoglobinemia - Fe in +3 state Acquired - no genetic changes, just of Fe being oxidized Inherited - a mutation in the polypeptides making hemo Probably inherited methemoglbinemia

Treatment ofSickle Cell Anemia

Antibiotic therapy: (to prevent secondary infections) - treat symptoms Hydroxyurea (stimulates the production of HbF) to disrupt polymers Bone marrow transplantation (replaces HbS with HbA) someone else's HbA Gene therapy (Phase I/II) new Won't present at birth wince you have gamma chains

Click where bisphosphoglycerate (BPG) binds on hemoglobin

BPG binds in a pocket formed by the beta globin subunits. In HbA, BPG binds to a pocket formed by the b subunits BPG is also known as DPG (diphosphoglycerate) BPG is allowing hemo to release more oxygen (nto that much effect at high O2 concentrations) lets hemo to release more oxygen We have built in capacity for hemo Doesn't give up alll the o2 all the time we don't have high BPG all the time

At high altitudes, oxygen concentrations are lower in the lungs, but the same at the tissues. What is the primary mechanism by which hemoglobin adapts to deliver as much oxygen as possible to tissues?

BPG rises under conditions of oxygen deprivation BPG increases Body response occurs over several hours We don't know why BPG increases which allows better O2 delivery

The binding of BPG to adult hemoglobin (HbA)

Bisphosphoglycerate (BPG) lowers the affinity of hemoglobin for oxygen. BPG rises under low oxygen conditions, allowing oxygen to be efficiently delivered to tissues BPG- looks like myoglobin Vary BPG level means we can vary hemo tendency

The Bohr Effect

Both H+ and CO2 allow hemoglobin to deliver more oxygen to actively metabolizing tissues. They are allosteric effectors that bind to hemoglobin in a site distinct from heme Deoxygenation of the blood increases its ability to carry carbon dioxide; conversely, oxygenated blood has a reduced capacity for carbon dioxide a decrease in the amount of oxygen associated with hemoglobin and other respiratory compounds in response to a lowered blood pH resulting from an increased concentration of carbon dioxide in the blood.

Bohr effect on hemoglobin

Both H+ and CO2 reduce the O2 affinity of hemoglobin Recall that CO2 is in equilibrium with carbonic acid (H2CO3) and bicarbonate (HCO3-) CO2 binds to hemo and stabilize the T state so even weaker binder of o2 More hydrogen also reduces hemo affinity for oxygen enhances O2 release in respiring tissues

Poll Title: Actively metabolized tissue produces acid and carbon dioxide. What is the effect on the oxygen carrying ability of hemoglobin?

Both carbon dioxide and hydrogen ions reduce oxygen affinity

Sickle Cell anemia

Characterized by sickled appearance of red blood cells due to insoluble Hemoglobin (HbS). Result is clogging of capillaries and general organ damage causing pain. Individuals can either be homozygous (SS) or heterozygous (AS). 1 in 13 African-Americans are heterozygous. 1 in 365 have the disease 100,000 with disease in U.S. First described genetic disease. Sickle cell anemia is the most common autosomal recessive disorder and the most common cause of hemolytic anemia in African Americans. Strenuous activity that reduced oxygen levels may have precipitated sickling of his blood cells. This resulted in an acute painful episode, also known as a pain crisis, which is a consequence of microvascular occlusion of bones by sickled cells. The most common sites are the long bones of the arms, legs, vertebral column, and sternum. The mainstay treatment of pain crisis is hydration and pain control with nonsteroidal anti-inflammatory agents. It is also important to provide adequate oxygenation to reduce sickling. Also anemia, jaundice, frequent infections. Don't flow through cells well

t and r states

T unhappy state - we see conformational change , In equilibrium , can go either way Histidine, chains moving around, chain nudging other chains to go into R form

Haldane effect

Deoxygenation of the blood increases its ability to carry carbon dioxide; conversely, oxygenated blood has a reduced capacity for carbon dioxide Muscles creating co2 and acid, so tissue needs 02 so hg drops it off in the muscls and gets more in the lungs Deoxygenation of the blood increases its ability to carry carbon dioxide

HbF binds oxygen with ....... affinity than HbA

HIGHER HbF has fewer positive charges in its corresponding pocket than the pocket made by the two b subunits on HbA. HbF binds BPG less well and therefore binds oxygen with higher affinity. Fetus needs to get oxygen from mom blood So maternal oxyhemo send o2 to fetal deoxyhemo Has higher affinity for oxygen fetal

Methemoglobinemia part 2

Hb:Fe3+ (HbM) can not carry oxygen. Clinically present with shortness of breath, cyanosis, mental status changes Methemoglobinemia is diagnosed by the absorption spectrum of the blood (peak at 630nm) Reducing agents (such as Vitamin C and methylene blue) treat the disease Also present with headache, fatigue, exercise intolerance, dizziness and loss of consciousness (i.e. ascorbate, glutathione, cytochrome b5 reductase) Reducing agents - send back to normal ferrous form

Poll Title: The hemoglobin of a fetus (HbF) has higher affinity for oxygen than the hemoglobin of the mother (HbA)

HbF has lower affinity for BPG than HbA HBF wants to hold onto O2 so lower affinity for BPG than HbA Fetal is alpha 2 and gamma 2 the alpha are slightly less negatively charged

Case 1. A patient presents with chronic hypochromic microcytic anemia and hemolytic anemia which worsens in periods of oxidant stress. Analysis of the patient's hemoglobin reveals an alpha thalassemia, which means a deficiency of alpha chains, but beta chains are present.The resultant hemoglobin is called Hemoglobin H. Hemoglobin H comprises mostly beta chains and binds oxygen 10 times more tightly than normal adult hemoglobin (HbA) Why?

HbH has no Bohr effect or heme-heme interaction

: The molecular basis of red blood cell sickling in individuals with sickle cell anemia involves the aggregation of mutant (HbS) hemoglobin molecules. Which of the following best describes the aggregation?

HbS polymerizes under low oxygen conditions HbS under low O2 conditions You need to be homozygous to symptomatic

The heme of myoglobin and hemoglobin

Heme organic molecule Contains Fe ETC too Carries O2 Pirel part of heme is the center 1 2 3 4 bound in plane 5 is bound to protein 6 is free to bind

Hemo vs myo structure

Hemoglobin (HbA) is a dimer of ab units Myoglobin, the a globin subunit and the b globin subunit all contain heme and have similar sequences and structures Hemo is a weaker o2 binding than myoglobin

To which of the following molecular species will a molecule of oxygen bind most tightly?

Hemoglobin bound to one molecule of oxygen will be in the "R" state. Myoglobin bound to one molecule of oxygen will not bind another molecule. Binding of Carbon dioxide weakens binding of oxygen. Hemoglobin with nothing bound will be in the T state won't bind oxygen as one in the "R" state. Hemoglobin with Fe3+ does not bind oxygen.

Developmental Regulation of Hemoglobin

Hemoglobin is composed of different subunits at different stages of human development. Fetal hemoglobin (HbF) is a2g2. Adult hemoglobin (HbA) is a2b2. HbF and HbA are VERY similar in sequence, structure, and function, except in their ability to bind yet another allosteric effector of hemoglobin. A2b2 in adult At birth there is fetal hb (HbF) Gamma subunits Similar in structure sequence and function But do NOT bind BPG

three main sections

I. Myoglobin II. Hemoglobin structure and function III. Diseases of hemoglobin: methemoglobinemia & sickle-cell anemia

Increased or decreased 2,3-BPG allows Hb to be a "better" transporter

Increased Dark line - under normal BPG levels At sea levels you will deliver 50p of oxyge But at high altitude, there is less delta, starting off with lower number of 02 BUT you are better at delivering the o2- better transporter at conditions of lower 02

case 2 Young child in rural area presents with "blue baby syndrome" Baby is fed with formula made up with well water that has not been tested for nitrate levels Patient responds well to ascorbate and methylene blue , the remainder of his examination is normal. : Clinically, methemoglobin is detected by an absorption band at an unusual wavelength (630 nm). What is the state of the iron in methemoglobin?

Methemoglobin (=meta+hemoglobin, as in metastable) cannot carry oxygen, thus it is deoxygenated. By definition, it carries Ferric (Fe3+) ion.

Myoglobin is in muscle and hemoglobin is in blood

Myoglobin functions well in muscle tissue where the partial pressure of oxygen is very low (as low as 5 mm Hg, up to 30 mg Hg) Hemoglobin is very similar to myoglobin but oxygen binding is altered to be in the correct range (30 to 100 mg) and is regulated Less coopertivity Binds o2 more readily Myoglobin is well saturated at 40mmHg in working museles, partial pressure will go as low as 5 so you need release from myglobin to help the actively

The T state is more ..... than the R form because it contains ..... electrostatic and hydrogen bonds

STABLE MORE aka "tense" Hydrogen bonds broken in oxygenated form Deoxy a bad oxygen binder until you have bound at least 1 o2 Unlike myoglobin, deoxyhemoglobin is set up to be a weak oxygen binder, until one or more oxygen molecules are bound.

Many mutations in Hemoglobin A have been identified

Some mutations have no effect on hemoglobin function (a nonpathological substitution) Others, such Hemoglobin S, disrupt hemoglobin structure or function

Administration of hydroxyurea results in only a small increase in the amount of HbF that is produced but is effective in disrupting aggregates of HbS. Why? Hint: thinking about the effect of mutations of structural collagen in Ehlos-Danlos syndrome

The gamma subunit substitutes for the beta subunit; the hydrophobic patch is no longer consistently present and thus the polymer is disrupted Thought question Big change on overall HbS is a subunit protein Disrupting polymer but adding gamma subunits

blue baby syndrome

The illness that occurs when we have too much nitrates in our water, especially in children. Not an issue with reducers/iron levels

Hemoglobin exists in T and R forms

The initial trigger is the movement of the Fe2+ ion on oxygenation of a subunit leading to a conformational change. The subunit are in contact with each other, causing the other subunits to take on the oxygen-binding conformation. The T form is more stable than the R form in the absence of oxygen. Note that the R and T state differ in the arrangement of the subunits although the subunits themselves barely change structure (T state and R state doesn't change much for each individual subunit but DOES change a lot for arrangement of subunits)

The prosthetic group of myoglobin and hemoglobin, heme, is in a deep, non-polar pocket

This sequestering from water helps maintain the Fe in the +2 state Prosthetic groups of proteins are not removed upon purification; cofactors are often removed. Proximal and distal refer to distance relative to the heme Buried in hydrophobic content (hydro is yellow) There Is a pocket - water stays away from heme Maintains functional form of Fe which is the Fe +2

Methemoglobinemia

can be environmental or inherited Environmental: Ingestion of nitrates and nitrites can promote HbM. Defects can also arise from the lack of maintenance of reducing agent in red blood cells (HbA sequence is normal) Inherited: Mutations in heme-binding pocket of Hb can promote HbM Also present with headache, fatigue, exercise intolerance, dizziness and loss of consciousness (i.e. ascorbate, glutathione, cytochrome b5 reductase) Environmental switch you can have HbA and hten it flips to HbM Sequence normal but you are oxidizing Mutation in binding pocket means that your iron is not in th eright form and attaches water NOT oxygen a blood disorder in which an abnormal amount of methemoglobin, a form of hemoglobin, is produced

Where does BPG bind?

central cavity HbA, BPG binds to a pocket formed by the b subunit BPG is also known as DPG (diphosphoglycerate) Allossteric effector of hemo function - does nto bind to heme but binds to beta subpocket BPG (neg charge) so binds to + charged amino acid pocket

The molecular defect in HbS is a point mutation converting

glutamate to valine on the exterior of the b subunit Trait is change in amino acid changes charge All symptoms occur after birth & can be diagnosed using electrophoresis of hemoglobin Glutamate is negatively charged; valine is neutral

Hemoglobin

iron-containing protein in red blood cells that carries oxygen for delivery to cells

Myoglobin changes its structure

little when oxygen (O2) binds Fe changed it radius and goes through porphyrin plane With oxygen (purple) heme is oxygenated so it moves

Diseases of hemoglobin:

methemoglobinemia & sickle-cell anemia

myo vs hemo in body

muscle v blood Myoglobin functions well in muscle tissue where the partial pressure of oxygen is very low (as low as 5 mm Hg, up to 30 mg Hg) Hemoglobin is very similar to myoglobin but oxygen binding is altered to be in the correct range (30 to 100 mg) and is regulated Myo has hyperbolic curve - good o2 transporter in muscles , gives up as needed as muscle working But bad for blood stream In lungs venous blood is lower nad when it leaves it is 100 and then it has to deliver to muscles So hemo curve is sigmoidal Hill plot linear plot Myo =1 Hemo 2.8 + coopereativitiy So binding of 1 ligand promotes bonding of next ligand up to 4 for hemo

Myoglobin

oxygen transporter of muscle Myoglobin contains a prosthetic group called a heme molecule F8 corresponds to 93 when the sequence is expressed as 1-153 Heme is red, attached to protein, prosthetic group (if purified it comes with it where as cofactors are lost when purified) 1-153 labelled Labeled each helices by alphabet and when they got to F they saw his (F8= proximal histidine) direct ligand to iron

Individuals with heterozygous sickle cell anomia have

partial resistance to the parasite that causes malaria. Why? Because in <4 yrs old with heterozygous sickle cell anemia red blood cells turn over faster than normal. The malaria parasite matures within red blood cells, and sickle cells are destroyed before the parasite can complete this part of its life cycle.

allosteric

reduce affinity

Ascorbate and methylene blue are

reducers

Myoglobin is a monomer

the change in structure on oxygenation has little consequence Myoglodbin can only carry one and conformational change is so small that it doesn't matter that much

SUMMARY 3

•HbS (sickle) has a substitution in the b subunit; more sickling under low oxygen conditions • •Methemoglobin carries Fe3+, which cannot carry oxygen; can be in-born or acquired Nitrates - contaminated water with fertilziers - causes converision to ferric form of iron which is dangerous

SUMMARY 2

•Hemoglobin has 4 myoglobin-like units (2 each of a and b that touch each other resulting in Hill coeff >1) •CO2, H+, and BPG are negative allosteric effectors • •Fetal hemoglobin is a2g2 and binds BPG less well

SUMMARY 1

•Myoglobin is a monomeric helical protein that contains heme. It transports oxygen in muscle. • •Oxygenated heme is bright red, oxidized heme is brownish, deoxygenated heme is more blue TRUE FOR BOTH MYOGLOBIN AND HEMOGLOBIN • •The proximal histidine forms a ligand to the iron that moves on oxygenation (both Myoglobin and Hemoglobin) • •There is a minor change in the structure upon oxygenation