Exploring Protein Structure and Function

Exploring Protein Structure and Function 

SUPPLEMENTARY INFORMATION FOR ACTIVITY 4 

(Aidan Budd, Francesca Diella – EMBL, Heidelberg) 

HEMOGLOBIN 

Hemoglobin, a transport protein found in red blood cells (erythrocytes), is the 

primary carrier of oxygen throughout the respiratory system of the vertebrate. 

The advent of aerobic respiration (many billions of years ago, when all life was 

still at the bacterial level of organization), which added the oxygen-utilising 

tricarboxylic acid cycle and electron transport system onto anaerobic glycolysis, 

allowed aerobic organisms to extract 18 times more energy from glucose in the 

form of ATP. 

THE HEMOGLOBIN MOLECULE 

The hemoglobin molecule is made up of four polypeptide chains, non-covalently 

bound to each other. Each chain holds a heme group containing one Fe++ atom. 

Figure 1: Most of the amino acids in hemoglobin form alpha helices, connected by short 

non-helical segments. Copyright: Rachel Casiday and Regina Frey. 

“Molecular Evolution in School” 

ELLS LearningLAB – 28-30 September, 2009 – EMBL, Heidelberg

The four polypeptides chains of the hemoglobin are: two alpha chains (Alpha 1 

and Alpha 2) of 141 amino acid residues each and two beta chains (Beta 1 and 

Beta 2) of 146 amino acid residues each. The alpha and beta chains have 

different sequences of amino acids, but fold up to form similar three-dimensional 

structures (Hemoglobin has no beta strands and no disulfide bonds.). The four 

chains are held together by non-covalent interactions. There are four binding 

sites for oxygen on the hemoglobin molecule, because each chain contains one 

heme group. In the alpha chain, the residue 87 is histidine and in the beta chain 

the residue 92 is histidine . A heme group is attached to each of the four 

histidines. The heme consists of an organic part and an iron atom. The iron atom 

in heme binds to the four nitrogens in the center of the protoporphyrin ring. The 

hemoglobin molecule is nearly spherical, with a diameter of 55 angstroms. The 

four chains are packed together to form a tetramer. The heme groups are located 

in crevices near the exterior of the molecule, one in each subunit. Each alpha 

chain is in contact with both beta chains. However, there are few interactions 

between the two alpha chains or between the two beta chains. 



TRANSPORT OF OXYGEN 

When the iron is bound to oxygen, the heme group is red in colour 

(oxyhameoglobin), and when it lacks oxygen (deoxygenated form) it is blue-red. 

As blood passes through the lungs, the hemoglobin picks up oxygen because of 

the increased oxygen pressure in the capillaries of the lungs, and can then 

release this oxygen to body cells where the oxygen pressure in the tissues is 

lower. In addition, the red blood cells can pick up the waste product, carbon 

dioxide, some of which is carried by the hemoglobin (at a different site from 

where it carries the oxygen), while the rest is dissolved in the plasma. An 

elemental oxygen molecule binds to the ferrous iron atom in the lungs where 

oxygen is abundant, and is released later in tissues that need oxygen. 

Figure 2: oxygen transport in the human body. Copyright Deborah Maizels, 2003 



TYPES OF HEMOGLOBIN 

Different types of hemoglobin are classified according to the type of protein 

chains they contain. Normal hemoglobin types include: 

Hb A - makes up about 95 - 98% of the Hb found in adults; Hb A contains two 

alpha (α) protein chains and two beta (ß) protein chains. 

Hb A 2 - makes up about 2 - 3.5% of Hb; has two alpha (α) and two delta (δ) 

protein chains. 

Hb F - makes up to 2%; has two alpha (α) and two gamma (γ) protein chains. 

This is the primary hemoglobin produced by the fetus during gestation. Its 

production usually falls to a low level within a year after birth. 

HEMOGLOBIN AND DISEASE 

Defects in the hemoglobin genes can produce abnormal hemoglobins and 

anemia, a condition termed "hemoglobinopathia". Abnormal hemoglobins are 

usually caused by a mutation in one of the hemoglobin genes. Two scenarios will 

arise: a) structural defects in the hemoglobin molecule and b) reduced production 

of one of the hemoglobin subunits. 

Sickle cell anemia was first described by Linus Pauling. Sickle hemoglobin 

differs from normal hemoglobin B by a single amino acid: valine replaces 

glutamate at position 6 on the surface of the beta chain (hemoglobin S). This 

creates a new hydrophobic spot. When deoxygenated, a small hydrophobic patch 

appears on the surface. The hydrophobic spots stick to each other (excluding 

water) causing deoxygenated hemoglobin to aggregate into chains. In a sickled 

red blood cell, the valine 6 (beta chain) binds to a different hydrophobic patch. 

The polymerized hemoglobin distorts red blood cells into an abnormal sickle 

shape. Heterozygotes have a mixture of normal hemoglobin A and mutant 

hemoglobin S. The hemoglobin A stops polymerization, preventing serious 

sickling. 

Sickle cell anemia was recognized to be the result of a genetic mutation, inherited 

according to the Mendelian principle of incomplete dominance and it represent a 

particularly interesting example of heterozygote superiority among humans. The 

HbS allele occurs in some African and Asian populations with a high frequency. 

This formerly was puzzling because the severity of the anemia, representing a 

strong natural selection against homozygotes, should have eliminated the 

defective allele. But researchers noticed that the HbS allele occurred at high 

frequency precisely in regions of the world where a particularly severe form of 

malaria, which is caused by the parasite Plasmodium falciparum, was endemic. 

It was hypothesized that the heterozygotes, HbAHbS, were resistant to malaria, 



whereas the homozygotes HbAHbA were not. In malaria-infested regions then 

the heterozygotes survived better than either of the homozygotes, which were 

more likely to die from either malaria (HbAHbA homozygotes) or anemia 

(HbSHbS homozygotes). This hypothesis has been confirmed in various ways, 

e.g. most hospital patients suffering from severe or fatal forms of malaria are 

homozygotes HbAHbA. 

Structure of Normal Hemoglobin 

Molecule (HbA) 

Structure of Sickle Cell Disease 

Molecule 

Hemoglobin in Persons with Sickle Cell 

Disease 

Hemoglobin in Persons with Sickle Cell 

Trait 

2 alpha and 2 beta chains 

2 alpha and 2 s chains 

All hemoglobin molecules consist of 2 

alpha and 2 s chains 

Half of the hemoglobin molecules 

consist of 2 alpha and 2 beta chains, 

and half of 2 alpha and 2 s chains 

Thalassemias are a heterogeneous group of inherited disorders of hemoglobin 

synthesis resulting in a reduction in the amount of the particular globin chain 

produced. The result of an unbalanced globin-chain synthesis is the production of 

an excess of the partner chain. As a consequence of this, unusual forms of 

hemoglobin will precipitate and accumulate in the red cells. The majority of the 

beta thalassemias are characterized by the over-synthesis of the fetal 

hemoglobin (HbF). The thalassemias are usually broadly classified by the type of 

globin chain (alpha and beta thalassemias) whose synthesis is reduced. The 

thalassemias are extremely heterogeneous at the molecular level: more than 200 

different mutations of the globin genes have been found, and the thalassemias 

are almost as varied. Every severely-affected population in the world has a few 

common mutations unique to a particular region, together with varying numbers 

of rare ones. 

Porphyrias is not a single disease but a group of inherited (or acquired) 

disorders of certain enzymes in the heme biosynthetic (porphyrin) pathway. Each 

step of the process is controlled by eight enzymes. If any one of the enzymes is 

deficient, the process is disrupted. As a result, porphyrin or its precursors— 

chemicals formed at earlier steps of the process—may build up in body tissues 

and cause illness (mostly it has effects on the nervous system or the skin). The 

term "porphyria" is derived from the Greek word "porphyrus" meaning purple. 

Urine from some porphyria patients may be reddish in color due to the presence 

of excess porphyrins and related substances in the urine, and the urine may 

darken after exposure to light. 



HEMOGLOBIN AND EVOLUTION 

We usually expect that, after a gene has duplicated, both copies of the gene will 

evolve (more or less) independently of each other i.e. changes in one copy of the 

gene will not tend to also be acquired by the other copy. 

In contrast, "concerted evolution" is a process in which related genes within a 

species undergo genetic exchange, causing their sequence evolution to be 

concerted over some period of time. This means that each gene locus in the 

family comes to have the same genetic variant. 

The family of the globin genes of primates, to which the hemoglobin belongs to, 

exists almost since the time life originated on earth, nearly four billion years ago 

and it well illustrates the principle - several copies of the gene lie very close to 

each other on the same chromosome, and are evolving concertedly. 

Figure 3: a phylogeny of the human globin genes. The genes multiplied by gene 

duplications, and the dates on the figure are the times of the duplications as inferred 

from the molecular clock. From Jeffreys et al. (1983). 

All primates have two alpha globins; we can therefore assume that the common 

ancestor of primates had two alpha globin genes. The sequence of each alpha 

globin gene differs between primate species; in the great apes any two species 

differ by about 2.5 amino acid substitutions in each gene. If one gene 

accumulates about 2.5 amino acid changes in the time between two species, 

then two different genes (alpha1 and alpha2) which have been separated for 

maybe 300 million years should have accumulated many more changes if they 

have been evolving independently. The conclusion is that they have not evolved 

independently - i.e. they are undergoing concerted evolution. 



Note that there are two ways for a gene to have a common ancestor: by gene 

duplication and by speciation. When two genes share a common ancestor due to 

a duplication event we call them paralogous (a-hemoglobin and ß-hemoglobin in 

human are paralogous). When two genes share a common ancestor due to a 

speciation event we call them orthologous (a-hemoglobin in human and a- 

hemoglobin in chimps). 

These globin proteins are widely distributed in many organisms, appearing in the 

cells of plants, animals and even bacteria. Some globin proteins found in this 

family are: 

• Myoglobin (used as a reserve supply of oxygen, and facilitates the movement of 

oxygen within muscles) 

• Neuroglobin (involved in oxygen transport in the brain) 

• Cytoglobin (involved in intracellular oxygen storage or transfer) 

• Leghemoglobin (provides oxygen to bacteroids, which is essential for symbiotic 

nitrogen fixation) 

• Erythrocruorin (giant hemoglobin of worms) 

• Plant hemoglobin (may act as an oxygen sensor) 

• Flavohemoglobin (involved in NO detoxification) 

Reference: 

Jobling, Hurles, Smith “Human evolutionary genetics” Garland Science (2003) 

http://www.rcsb.org/pdb/home/home.do 

http://en.wikipedia.org/wiki/Hemoglobin

Exploring Protein Structure and Function

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