Hemoglobin structure and function

Dr. Niveen M. Daoud Ass. Prof. clinical pathology

Outcomes
Enlist the heme containing proteins in human body Enumerate the functions of proximal & distal histidine

residues of globin chain Describe the quaternary structure of hemoglobin Differentiate b/w the tense T & relaxed R forms of hemoglobin Name the compounds which can bind to hemoglobin State the functions of hemoglobin in blood Enlist the composition of HbA, HbA2, Hb Gower 1 & HbF Discuss the formation & clinical importance of glycosylated hemoglobin HbA1c Describe the structural deformities of hemoglobin molecule in Hb S, Hb C & Hb SC diseases

 Proteins that contain an iron-porphyrin, or heme, prosthetic

heme containing proteins in human body

group resembling that of hemoglobin. Hemoglobin Myoglobin Both are examples of hemeprotein that are essential in the storing and transports of oxygen in mammals. Hemoglobin is a quaternary protein that occurs in the red blood cell, whereas, myoglobin is a tertiary protein found the muscle cells of mammals. Although they might differ in location and size, their function are similar. Being hemeproteins, they both contain a prosthetic group (tightly-bound cofactors ). NP nitrophorin Cytochromes Catalase peroxidase

What are the compounds can bind to Hb

Carbon monoxide (CO)

Carbon dioxides (CO2)

Oxygene (O2) Nitric oxide (NO) Hydrogen sulfate (H2S) Fe+3 2,3-BPG

Hemoglobin
Hemoglobin (Hb) is the protein molecule

responsible for transporting respiratory gases. The normal hemoglobin value for adult males is 14-18 grams per deciliter of whole blood.

Quaternary structure of Hemoglobin

In adult humans, the most common

hemoglobin type is a tetramer (which contains 4 subunit proteins) called hemoglobin A, consisting of two and two subunits they made of 141 and 146 amino acid respectively. This is denoted as 22. The subunits are structurally similar and about the same size..

 Hemoglobin has a complex quaternary

structure The four polypeptide chains are bound to each other by salt bridges, hydrogen bonds, and have hydrophobic effect. Each of the 4 globulin protein subunits contains a single nitrogenous pigmented molecule of heme. Each heme molecule contains a single iron ion which associates with oxygen to form oxyhemoglobin (a bright red pigment). Oxygen easily dissociates from the iron ion to become deoxyhemoglobin (a darker

Structure of hemoglobin

Hb tetramer is two identical dimers

Hemoglobin
and chain of Hb consist of

seven and eight helical regions ( from A through H). An invariant histidine in all hemoglobins, called F8 or the proximal histidine, binds heme. . F8 refers to the eighth residue of F helix

Structure of monomer of Hb

the functions of proximal & distal histidine residues of globin chain

His-F8 of the hemoglobin known as the

proximal histadine is covalently bonded to the 5th coordination position of the iron. And as an Oxygen binds to the 6th coordination position of the iron, a distal histidine (a histidine that doesn't bond directly with the Iron), HisE7 of the hemoglobin binds to the oxygen that is now covalently bonded to the iron. The same occurs for the myoglobin.

T and R status of Hb
The structure of Hb differs in the oxygenated and

deoxygenated states. The quaternary structure of deoxy. Called T or taut form which has low O2 affinity. Oxygenated Hb called R or relaxed form which has high O2 affinity. Hemoglobin exists in two forms, a taut form (T) and a relaxed form (R). Various factors such as low pH, high CO2 and high 2,3 BPG 2,3,bisphosphoglycerate at the level of the tissues favor the taut form, which has low oxygen affinity and releases oxygen in the tissues as well as contains

Oxy versus deoxy hemoglobin

T and R states of Hb(conti)

Differences in quaternary structure of

the R and T states are produced by the movement of F8 histidine as oxygen binds or dissociates from heme. The F8 histidine alters the position of the F and E helics and leads to altered contacts between the subunits.

Hb Tense & Relaxed States

Difference between oxy and deoxy Hb

Oxyhemoglobin

Oxyhemoglobin is formed during physiological

respiration when oxygen binds to the heme component of the protein hemoglobin in red blood cells. This process occurs in the pulmonary capillaries adjacent to the alveoli of the lungs. The oxygen then travels through the blood stream to be dropped off at cells where it is utilized in glycolysis and in the production of ATP by the process of oxidative phosphorylation. It does not, however, help to counteract a decrease in blood pH. Ventilation, or breathing, may reverse this condition by removal of carbon dioxide, thus causing a shift up in pH.

 Deoxygenated hemoglobin Deoxygenated hemoglobin is the form of

hemoglobin without the bound oxygen. The absorption spectra of oxyhemoglobin and deoxyhemoglobin differ. The oxyhemoglobin has significantly lower absorption of the 660 nm wavelength than deoxyhemoglobin, while at 940 nm its absorption is slightly higher. This difference is used for measurement of the amount of oxygen in patient's blood by an instrument called pulse oximeter. This difference also accounts for the presentation of cyanosis, the blue to purplish color that tissues develop during hypoxia

Methemoglobinemia
Normally, methemoglobin levels are <1%, as measured by

the co-oximetry test. Elevated levels of methemoglobin in the blood are caused when the mechanisms that defend against oxidative stress within the red blood cell are overwhelmed and the oxygen carrying ferrous ion (Fe2+) of the heme group of the hemoglobin molecule is oxidized to the ferric state (Fe3+). This converts hemoglobin to methemoglobin, resulting in a reduced ability to release oxygen to tissues and thereby hypoxia. This can give the blood a bluish or chocolatebrown color clinically called (black mouth). In this case the distal His (E7) was substituted by Tyr (E7)which results in the formation of a 5- coordination Fe (III) complex, and phenolate ion of the mutant tyr E7 displacing the imidazole ring of His F8. The phenolate ion in these methemoglobin so stabilize the Fe (III) oxidation

Hemoglobin Structure and Function

Methgb - occurs when iron is oxidized to the +3 (ferric) state. In order for hgb to carry O2 the iron must be in the +2 (ferrous) state. In the body Normally (2% ) small amount of Met-HB is continuously present but an enzymes systems in the RBCs called NADHmethemoglobin reductase Change Met-HB back to HB to maintain its conc. Stable. Increases above 2% can occur with the ingestion of strong oxidant drugs or As a result of enzyme deficiency.

Met-hemoglobinemia (Hb M)

 Spontaneous formation of methemoglobin is

normally reduced (via electron donation) by protective enzyme systems, e.g., NADH methemoglobin reductase (cytochrome-b5 reductase) (major pathway), NADH + H+ + 2 ferricytochrome b5 = NAD+ + 2 ferrocytochrome b5 NADPH methemoglobin reductase (minor pathway) and to a lesser extent the ascorbic acid and glutathione enzyme systems. Disruptions with these enzyme systems lead to the condition.

Causes of Met- Hb
Genetic defect due to a deficiency of the enzyme

diaphorase I (NADH methemoglobin reductase). Aquired cause 1. Exposure to exogenous oxidizing drugs and their metabolites (such as benzocaine, dapsone and nitrates) may accelerate the rate of formation of methemoglobin up to one-thousandfold. 2. classical drug causes of methemoglobi include antibiotics (trimethoprim, sulfonamides and dapsone), local anesthetics (especially articaine and prilocaine. 3- Infants under 6 months of age are particularly susceptible to methemoglobinemia caused by nitrates ingested in drinking water (called blue-baby syndrome),

Functions of Hb
Transport of respiratory gases (O2 & CO2) Buffer Being proteins density of RBC viscosity

of blood

What is the primary factor that determines how much oxygen is actually bound to hemoglobin? is the partial pressure of oxygen (pO2)

External respiration
Oxygen in the alveoli under a pressure comes in contact with oxygen in the venous blood and the gas diffuses into the blood until the pressures are equal

internal respiration
Oxygen which has diffused into the blood is carried in the Hg now called oxyheamoglobin to the tissue

Copyright 2008 Pearson Education, Inc., publishing as Benjamin Cummings.

CO2 Exchange: Lungs

CO2 at pressure 46mmHg come in contact with alveolar CO2 at pressure of 40 mm Hg so the gas diffuse out of the blood into the alveoli.
.

Hb regulate blood PH

different compositions of Hb
Hemoglobin A (HbA) or adult hemoglobin is the

most common human hemoglobin tetramer, comprising over 97% of the total red cell hemoglobin. It consists of two alpha chains and two beta chains (2 2). Hemoglobin A2 (22) - chain synthesis begins late in the third trimester and in adults, it has a normal range of 1.5-3.5% Hemoglobin F (22) - In adults Hemoglobin F is restricted to a limited population of red cells called F-cells. However, the level of Hb F can be elevated in persons with sickle-cell disease and beta-thalassemia.

different compositions of Hb (cont.)

Glycosylated hemoglobin HbA1c) is a form of

hemoglobin that is measured primarily to identify the average plasma glucose concentration over prolonged periods of time. It is formed in a nonenzymatic glycation pathway by hemoglobin's exposure to plasma glucose. Normal levels of glucose produce a normal amount of glycosolated hemoglobin. As the average amount of plasma glucose increases, the fraction of glycosolated hemoglobin increases in a predictable way. This serves as a marker for average blood glucose levels over the previous months prior to the measurement i.e used to monitor the control of diabetes mellitus.

Composition of Hb molecules
Name Hb A Formula 2 2
2 2-glucose

Percentage 90

# of a.a. 141 + 146

Hb A1c
Hb A2

39
25

141 + 146
141 + 146

22

Hb F
Hb Gower 1 Hb Gower 2

22
22
Zeta & Epsilon

<2

141 + 146

22

Found in embryonic life

Structural deformities of Hb
Hemoglobin H (4) - A variant form of hemoglobin,

formed by a tetramer of chains, which may be present in variants of thalassemia Hemoglobin S (2S2) - A variant form of hemoglobin found in people with sickle cell disease. There is a variation in the -chain gene, causing a change in the properties of hemoglobin, which results in sickling of red blood cells. Hemoglobin C (2C2) - Another variant due to a variation in the -chain gene. This mutated form reduces the normal plasticity of host erythrocytes causing variant causes of a mild chronic hemolytic anemia. Hemoglobin SC disease - A heterozygous form causing Sickle cell trait with one adult gene and one sickle cell disease.

Point mutation in chain gene

abnormal chains (substitution of valine for glutamate)

Hgb S
Nasty!
Aggregates and polymerizes on deoxygenation Red cell becomes sickle shaped Sickles clog up vessels plus, they are fragile

Sickle cell anemia

Clinical importance
Sickle cell trait (or sicklemia) describes a condition

in which a person has one abnormal allele of the hemoglobin beta gene (is heterozygous), but does not display the severe symptoms of sickle cell disease that occur in a person who has two copies of that allele (is homozygous). Those who are heterozygous for the sickle cell allele produce both normal and abnormal hemoglobin (the two alleles are codominant). Sickle cell disease is a blood disorder in which the body produces an abnormal type of the oxygen-carrying substance hemoglobin in the red blood cells. Sickling and sickle cell disease also confer some resistance to malaria parasitization of red blood cells, so that individuals with sickle-cell trait

Qualitative Hb abnormalities
Hb S

Hb C

Qualitative Hb abnormalities
Hb SC