Sickle-Cell Anemia
Primary Author: Meg Loven,
Graduate Research Assistant

Tutorial Objectives

Using the Biology Workbench, we will examine hemoglobin and the sequence of amino acids that make up this protein. This tutorial will demonstrate the simple mutation in the hemoglobin protein that single-handedly causes a terrible disease. From this mutation, we will look at the three-dimensional structure of the hemoglobin protein using a molecular imaging program. The ability to visualize and manipulate hemoglobin adds a dimension of understanding to the mechanism that causes the characteristic sickle-shaped red blood cells of sickle cell anemia.

Overview of Sicke-Cell Anemia

Sickle cell anemia is a disease in which the patient's red blood cells have an abnormal shape much like that of a sickle. These sickled red blood cells are very fragile and the result is severe anemia, or decreased number of red blood cells. The disease causes many painful symptoms in patients.

The abnormal shape of the cells in individuals with sickle cell anemia comes from a defective protein within the blood cells themselves. This defective protein is hemoglobin. The normal hemoglobin protein is made up of four parts, and therefore called a tetramer. Each part of the tetramer has the ability to bind an oxygen molecule and carry it from the lungs to the tissues in which oxygen is needed. When the defective hemoglobin in sickle cell anemia, referred to as Hb S, does not have an oxygen molecule bound, it tends to form a precipitate made up of lots of hemoglobin proteins stuck to each other. This precipitate is what causes the red blood cells to become sickle-shaped.

Sickle cell anemia glossary. (Not updated to V.3.2!)

Using the Biology WorkBench

If possible, open a second web browser window and open the Biology Workbench 3.2 in it. The best way to proceed through the tutorial is to set up the two browser windows next to each other. Alternatively, we recommend printing the tutorial.

In the Biology Workbench window, log in (setting up a new account, if necessary) and click on the Protein Tools button.

Highlight the Ndjinn-- Multiple Database Search from the scrollbar menu. The NDjinn Search is a feature of the Biology Workbench that allows the user to find information on a topic of interest, using specific databases that may be useful. For example, if one is looking for three dimensional protein structures, the PDBFINDER (Protein Databank) database is useful. After highlighting the Search, click on the "Run" button.

Scroll down the page and click the small box to the left of the tags labeled PIR 1, 2, 3, and 4 and SWISSPROT for your databases. If you are working with a PC, then you will have to hold down the Control key while selecting if you choose to select more than one item. If you are working with a Macintosh, then hold the open apple key. The Protein Infomation Resource (PIR) database is useful for finding resources and amino acid compositions for different proteins. PIR can be accessed through the Biology Workbench NDjinn Multiple Database Search. SWISSPROT is a database in Switzerland that stores the amino acid sequences that make up different proteins. It can also be accessed through the Biology Workbench NDjinn Multiple Databank Search.



in the query space above the database selections, and for ease in the next step, select Show All Hits instead of the default parameter.

Submit your query (by clicking on the Search button).

CLUSTAL-W Sequence Alignments

In the scroll menu, you will see that a large number of sequences were found by your query.
Scroll through the list and highlight 5 or 6 interesting animals. This can be done by clicking on an animal name while holding the open apple button on a Macintosh, or Ctrl on a Windows machine, as mentioned earlier.

Highlight the sequences you chose and import them to the workbench (by clicking the button Import Sequences).

Select each sequence by clicking in the small boxes next to the sequences and align them using CLUSTALW-Multiple Sequence Alignment. This is accomplished by highlighting the CLUSTAL-W program in the scroll menu and clicking the Run button. The next screen presents an assortment of settings for the CLUSTALW program. Just use the default settings click the Submit button. These settings would be important to a scientist, but are not necessary for everyday uses. CLUSTAL-W is a tool on the Biology Workbench that is used to align a group of protein sequences by their common elements so that they can be compared. When the results are returned, import them to the workbench (by clicking the Import Alignments button).

Comparing the Sequences

Select the aligned sequences by clicking in the small box next to the set of sequences and determine their similarities using BOXSHADE. Highlight BOXSHADE in the scroll menu and click on theRun button. As with CLUSTAL-W, just use the default settings on the screen and click the Submit button.
BOXSHADE can be used on groups of proteins that have been aligned (as by CLUSTAL-W) to determine their similarities. It produces a color-coded output of the protein sequences.
Green is for amino acids that are the same (conserved) in all the proteins examined. Yellow is for amino acids that are the same in nearly all proteins examined. Cyan means that the amino acid has the similar structure and charge but is a different amino acid. Lastly an Unshaded region means a very high variability within comparisons.

Look at your aligned sequences.

  • Which species have the most similar hemoglobins?
  • If you have a sheep and a goat, are they more similar than the sheep and a dolphin?
  • Why do you think the sequences are similar?

Now let's look at hemoglobin and the role it plays in people with sickle cell anemia.

Looking at the Beta Chains

Go back to "protein tools" in the Biology Workbench and select the NDjinn- Multiple Database Search again. This time select the PDBFINDER database and enter "hemoglobin" in the query.

When you receive the results, look through them and select the entries called "2hbs" and "1hab". On a Windows computer, use the "Ctrl" key to select two items from the list. Now view the records by clicking on the "Show Records" button.

If you scroll down the record listing, you'll find information about the two proteins that you selected. The 2hbs sequence is the hemoglobin sequence that contains the sickle cell anemia disease mutation. As you scroll down, you'll come to some white check boxes. Each of these boxes corresponds to a part of the hemoglobin sequence.

The hemoglobin protein is made up of 4 parts, each of which is referred to as a "chain". The four chains consist of two identical chains referred to as "alpha" and two other identical chains referred to as "beta". The sickle cell anemia mutation is in the beta chain of hemoglobin. We want to look at the beta chain from a sickle cell anemia hemoglobin (such as the 2hbs sequence) and compare it to a normal hemoglobin beta chain (such as the 1hab sequence).

How do the two sequences match up?

A Single Amino Acid Difference

To look at only one beta chain from each type of hemoglobin, check only the small white boxes for "Chain B" from both the 2hbs and 1hab files and then import them to the workbench.

Now select the two beta chains and use the CLUSTAL-W program again to align the sequences.

Look at the results of the CLUSTAL-W alignment.

  • What differences do you see between the sequences?

There is only one difference between the beta chain sequence of 1hab, which is the normal hemoglobin, and 2hbs, which is the sequence of the beta chain found in the hemoglobin of people who have sickle cell anemia. The change of a single amino acid in hemoglobin causes the sickle cell anemia disease.

How does this mutation cause sickle cell anemia?

One Mutation Causes the Disease

Now that you have seen the mutation in the hemoglobin sequence that causes sickle cell anemia, let's look at where that mutation is in the hemoglobin quaternary structure. For proteins, there are 4 levels of structure. The first, primary structure, is composed of the amino acid sequence. The secondary structure is how the amino acids next to each other in the sequence are organized. The tertiary structure is the folded 3-D structure of the protein that allows it to perform its functions. The quaternary structure is the total protein structure that is made when all the subunits of the protein are in place.

Getting set up:
On your own computer, open the Rasmol application. This is a molecular modeling program that was written by Roger Sayle. It is a free program. 3-D coordinates for visualizing proteins can be found in the PDBFINDER database on the Biology Workbench. Tutorials on using Rasmol can be found at, and a good manual for the latest version of Rasmol at (note that this is one large document - close to 200 kilobytes).

Hint: When using Rasmol, the PDB files and scripts we give for this exercise should be in the same folder, or the scripts will not work. Also, the files must have the same name that they do here. For example, the 2hbs.pdb file you download should be named as "2hbs.pdb" on your computer. Also, if you place the files in the Rasmol folder, it will probably be easier for the Rasmol application to find the files.

To download the necessary files:

On a PC or Unix machine with a 2 or 3-button mouse, you need to right-button click on each file name, and then choose "Save Link As...". Find the Rasmol folder and place the files there.

On a Mac (1-button mouse), you either need to option-click on each file name, or to hold your click down on the file name (click but keep the button pressed until a dialog box pops up) and choose "Save Link As...". Find the Rasmol folder and place the files there.

Normal hemoglobin 1hab.pdb
script 1hab.txt

Sickle cell mutated hemoglobin 2hbs.pdb
script 2hbs.txt

Now that you have all the files, arrange your windows so that you can read this tutorial and look at Rasmol simultaneously.

What does normal hemoglobin look like?

Structure of Normal Hemoglobin

In Rasmol, from the pulldown menu "File", select "Open" and choose 1hab.pdb. If it is not listed, you may have to do some navigating to find the where you stored the files you downloaded. You can also type "load 1hab.pdb" in the command line window, but it is possible that Rasmol isn't pointed at the folder where your PDB files and/or scripts are located, so using this method could be more difficult.

You should now have a wireframe structure of the normal (not mutated) hemoglobin in the Rasmol window. This is a type of visualization in Rasmol. It consists of a drawing of the protein using a wireframe for the amino acids in the protein. Try grabbing and rotating the molecule with the cursor.

It doesn't look like much, does it? If you'd like to try to find out more about the structure of hemoglobin on your own, try changing the various options in the display of Rasmol (read the manual for more description of the options).

Otherwise, type "script 1hab.txt" in the command line window. If the script takes several seconds to load, don't worry - that is normal.

Now the molecule actually looks like something. Hemoglobin is made up of 4 parts, each of which is referred to as a "chain". The four chains consist of two identical chains referred to as "alpha" and two other identical chains referred to as "beta".The alpha chains are yellow, and the beta chains are colored blue. The red disks are the heme groups. Heme groups are the part of hemoglobin that actually bind the oxygen molecules. Each heme group contains an iron atom that is held in the center of the heme. The iron is responsible for the oxygen-binding activities of hemoglobin.

The two amino acids colored green are the glutamate residues (glu 6) that are mutated in the disease sickle cell anemia. ("residue" is a term for "amino acid".) Glutamate is one of the 20 amino acids found in biological organisms. Its three letter abreviation is Glu, and its single-letter designation is E. Glutamate is has an acidic side chain, which means that at the pH in the body, it is negatively charged. The charge on glutamate means that it likes to be associated with water or other amino acids that have charges.

Try rotating the hemoglobin molecule to get a look at the overall structure.

Where are the glu 6 residues in relation to the rest of the molecule? Inside or outside?

How does this compare to sickle cell Hb?

Structure of Sickle Cell Hemoglobin

Close the current Rasmol window and open another. Or if you have a lot of window space, open another copy of the Rasmol application simultaneously.

To get the hemoglobin molecule with the sickle cell mutation, load the file "2hbs.pdb" by finding it with the "Open" command in the "File" menu (as before), or by typing "load 2hbs.pdb" in the command-line window.

You may notice that there are actually 2 hemoglobin tetramers in this Rasmol file. (A tetramer is a protein made up of four subunits.) We'll get to the reason for that soon.

Load the script by typing "script 2hbs.txt" in the command-line window.

The color-coding is the same here, for alpha and beta chains and the beta-chain residue number 6, but that residue is now a valine, rather than a glutamate. Valine is one of the smallest amino acids and is also uncharged. Since it is uncharged, it is hydrophobic, which means it does not like to be near water. It would rather be associated with other hydrophobic amino acids. The purple residues are the amino acids that interact with the mutated valine-6 on the beta chains of another hemoglobin tetramer.

Where are the two hemoglobins in relation to each other?

How are they "attached"?

Hemoglobin Attaches to Itself

We know that valine is a hydrophobic ("afraid of water") amino acid, and that it is located on the outside of the hemoglobin tetramer, surrounded by water. This is not an energetically favored condition--the hydrophobic amino acid does not like to be near water. Energetically favored is a term that refers to whether two molecules or amino acids are likely to stay near each other. The amino acids that are hydrophobic (don't like water) try to associate with each other because they are not charged while water is charged. This is why cooking oil and water do not mix. The hydrophobic oil trys to stick to itself and get away from the water. The oil staying in droplets on the water is energetically favored, but the oil mixing with the water is NOT energetically favored.

So the valine-6 is now a kind of "sticky patch" on the outside of the hemoglobin. In order to get away from the water molecules, valine-6 interacts with a phenylalanine and a leucine from another hemoglobin molecule.

This causes the association of 2 individual hemoglobin molecules. (Phenyalanine and leucine are both very hydrophobic.)

But what about the other mutated valine-6 residues? Where are they in the structure? On the outside or inside of the two hemoglobin molecules?

Those mutated residues are hydrophobic, too.

What do you think will happen?

Fibers of Hemoglobin Form in the Cells

One of the problems with sickle cell anemia is that hemoglobins tend to form long columns in the blood. These long fibers then cause other problems in the body.

Look for the micrograph of the fibers.

What Happens to the Other Mutated Amino Acids?

The Laboratory for Electron Micropscopy at the University of Chicago, directed by Dr. Robert Josephs, has created a video of the process.
If your modem connection is slow, try this static view instead.

Now you can understand how those long fibers of hemoglobin molecules form. The mutated valine-6 residues just keep adding on more hemoglobin molecules as they try to stabilize their structure. The amino acids in a protein form associations with each other that try to keep hydrophobic amino acids together and separate them from the water and hydrophilic amino acids. If a hydrophobic amino acid such as valine were surrounded by water, this would be a very unstable structure. So the valine amino acids try to stay away from the water by associating with other hydrophobic amino acids such as phenylalanine and leucine.

As the fibers form, they cause the shape of the red blood cell to become sickle-shaped. The long fibers push the cell membrane out of shape, causing the characteristic shape of the red blood cells in the disease. These cells can no longer move normally through the blood vessels, so normal delivery of oxygen to the body is interrupted. This is what causes the disease, sickle cell anemia.

"How does this disease relate to a person's genes?"

Hemoglobin and Genes

Whether a person has sickle cell anemia or not is determined by the person's genes. The DNA sequences you looked at for the normal and sickle cell hemoglobin are two versions of the gene for hemoglobin. However, it's not as simple as saying that if a person has the sickle cell hemoglobin gene, then they have the disease.

Since each person has two sets of genes, one from the mother and one from the father, there are two copies of the gene for hemoglobin. This is important because a person can have two of the sickle cell anemia gene, or a normal and a disease gene, or two normal genes. Each of these combinations results in a different situation for the person. If a person has two of the same genes, either two normal or two sickle cell genes, they are "homozygous" (homo=the same). If a person has two different genes for hemoglobin, then they are called "heterozygous" (hetero=different).

A person who is heterozygous for the hemoglobin gene will have a few sickle-shaped red blood cells, and a very mild case of sickle cell anemia. Meanwhile, a person who is homozygous for the sickle cell hemoglobin will have lots of sickled cells and have a full-blown case of the disease. A person who is homozygous for normal hemoglobin will have completely normal red blood cells.

The question is, if people who have full-blown sickle cell anemia die before they become adults and can't pass the gene on to any children, Why doesn't the disease go away?

The Disease Doesn't Die Out

In Africa, as many as 4 out of 10 people have the sickle cell anemia gene. In parts of Africa where lots of people had sickle cell anemia, malaria was also very common. Researchers found that people who had a single copy of the sickle cell hemoglobin gene(heterozygous) didn't die of malaria as often as people who were homozygous for the normal hemoglobin gene. So people who had two sickle cell hemoglobin genes died of sickle cell anemia, and people who two normal genes died of malaria, but those with one of each gene lived to pass on the sickle cell hemoglobin gene to their children. As a result, the disease continues.

Scientists don't know exactly how heterozygous people are protected from malaria, but they think the protection is due to faster destruction of infected red blood cells. Malaria is a disease in which parasites invade the body and live in red blood cells. It makes sense that if a few red blood cells (those that contained sickle cell hemoglobin) were deformed and destroyed, the malaria parasites living in those cells would be destroyed also. These few deformed red blood cells protect the heterozygous person from a really nasty case of malaria.

This phenomenon of a gene that is deadly in the homozygous form having very helpful effects in the heterozygous form is called "balanced polymorphism". Sickle cell anemia is just one example of balanced polymorphism. Other examples of balanced polymorphism exist for humans. Maybe with a little research you can find some others. Good luck! Where can I find more sickle cell anemia information on the web?


General Information
Research on sickle cell hemoglobin, including videos of the sickle process.
A Queens Library search for references and addresses for more information.
Basic to molecular information on sickle cell anemia and treatment.
General information on the biochemistry of hemoglobin.
Sickle cell anemia links to advanced and basic information.

Pictures of sickled red blood cells:
Micrographs of red blood cells in sickle cell anemia patients.

Personal interest sites:
A charity that helps people and families dealing with sickle cell anemia.
Information on a support group for sickle cell anemia.
A news article about a girl in Nigeria with sickle cell anemia.
The story of a woman with sickle cell anemia who was refused treatment.

Treatments of sickle cell anemia:
A report on Hydrea, an important drug in the treatment of sickle cell anmeia.
A technical report on hydroxyurea treatments.


"Portrait of an allosteric protein" in Biochemistry, 4th Ed. Lubert
Stryer, W.H.Freeman and Company, New York:1995, pp.170-3.

"Blood components" in Physiology, 3rd Ed. R. Berne and M. Levy,
Mosby-Year Book, Inc., St. Louis:1993, p.334.

This page created and maintained by Kristian N. Engelsen.
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