The Molecular Biology of Sickle Cell Anemia
In Part I we learned that sickle cell anemia was recognized to be the result of a genetic mutation, inherited according to the Mendelian principle of incomplete dominance. Initially, you will recall, it was not clear what the actual defect was that caused sickling. Various experiments, as described at the end of Part I, indirectly narrowed down the site of the defect to the hemoglobin molecule.
The most direct evidence that mutation affected the hemoglobin molecule came from a then-new procedure known as electrophoresis, a method of separating complex mixtures of large molecules by means of an electric current. To view and electrphoresis apparatus in progress, click here.When hemoglobin from people with severe sickle cell anemia, sickle cell trait, and normal red blood cells was subjected to electrophoresis, the following interesting results were obtained
It was clear that the hemoglobin molecules of persons with sickle cell anemia migrated at a different rate, and thus ended up at a different place on the gel, from the hemoglobin of normal persons (diagram, parts a and b). What was even more interesting was the observation that individuals sickle cell trait had about half normal and half sickle cell hemoglobin, each type making up 50% of the contents of any red blood cell (diagram part c). To confirm this latter conclusion, the electrophoretic profile of people with sickle cell trait could be duplicated simply by mixing sickle cell and normal hemoglobin together and running them independently on an electrophoretic gel (diagram part d). These results fit perfectly with an interpretation of the disease as inherited in a simple Mendelian fashion showing incomplete dominance. Here, then, was the first verified case of a genetic disease that could be localized to a defect in the structure of a specific protein molecule. Sickle cell anemia thus became the first in a long line of what have come to be called molecular diseases. Thousands of such diseases (most of them quite rare), including over 150 mutants of hemoglobin alone, are now known.
B. Sickle Cell and Normal Hemoglobin
But what was the actual defect in the sickle cell hemoglobin? Although we will investigate this question in more detail in a later case study (Web Page on Protein Structure), for now it will be helpful at least to outline the background of the discovery of just what it was that made sickle cell hemoglobin different from normal hemoglobin. It is the story of one of the first identifications of the molecular basis of a disease.
Again, Linus Pauling at Caltech, one of the most productive and imaginative of twentieth-century biological chemists (with co-workers Harvey Itano, a graduate of St. Louis University Medical School, I.C. Wells and S.J. Singer) turned his attention to determining the actual difference between normal and sickle cell hemoglobin molecules. Breaking the protein molecules down into shorter fragments called peptides, Pauling and co-workers subjected these fragments to another separatory technique called paper chromatography.
When this procedure is applied to samples of normal and mutant (sickle) hemoglobin molecules (alpha and beta chains) that had been broken down into specific peptides, all the spots are the same -- except for one crucial spot (shown darkened in the final chromatogram below), which represents the difference between sickle cell and normal hemoglobin.
The fact that the spots migrate to different places on the chromatogram indicates their molecular structures must be somewhat different. Pauling and his colleagues were convinced that the difference might be no more than one or two amino acids, but it was left to biochemist Vernon Ingram at the Medical Research Council in London to demonstrate this directly. Taking the one aberrant peptide and analyzing it one amino acid at a time, Ingram showed that sickle cell hemoglobin differed from normal hemoglobin by a single amino acid, the number 6 position in the beta chain of hemoglobin. That one small molecular difference made the enormous difference in people's lives between good health and disease.
C. Discovering the Difference Between Normal and Sickle-Cell Hemoglobin
Royer Jr., W.E. "High-resolution crystallographic analysis of co-operative dimeric hemoglobin," J. Mol. Biol., 235, 657. Oxyhemoglobin PDB coordinates, Brookhaven Protein Data Bank.
In overall structure, as we have already learned, a complete hemoglobin molecule consists of four separate polypeptide chains (i.e., each a long string, or polymer, of amino acids joined together end-to-end) of two types, designated the alpha and beta chains. The two a chains are alike (meaning they have the exact same sequence of amino acids), while the two beta chains are also alike.
You can rotate the molecule around, by clicking on it and hold the mouse button .
Step 1: Highlight the heme
Make sure you can distinguish the four subunits (the two a and the two b chains). Note the relative positions of the a and the b chains to each other. Hemoglobin is called a tetramer because the molecule as a whole is made up of four subunits, or parts. Find the porphyrin-based heme group and note how it is "sheltered" in a kind of groove within each polypeptide chain.
Step 2: Remove outer parts of the molecule
You can also switch from one to the other of several conventional modes of representing molecular structure: the space filling, ball and stick, wire, and ribbon forms, by holding down the mouse button and choosing-Display. As you will learn later, each gives you a different kind of information about the molecule's overall shape and some of its specific structural features.
In sickle cell hemoglobin the two alpha chains are normal; the effect of the mutation resides only in the # 6 position in the two beta chains (the mutant beta chains are referred to as "S" chains, as explained in the Terminology Box below). As mentioned above, each a and b polypeptide is folded around and shelters a special ring structure, the heme group, consisting of a porphyrin ring at whose center is an iron atom bound by four coordinate covalent bonds to four nitrogens of the porphyrin. It is this iron to which the oxygen binds (. The whole porphyrin structure is called the prosthetic group, a general term in protein chemistry to refer to non-polypeptide portions of the molecule that are usually the functionally active sites.
Click here for the heme group bound to histidine residue.
Sickle hemoglobin tutorial by Eric Martz of the University of Massachusetts
The chart below summarizes some of the terminology we have encountered in discussing the various kinds of hemoglobins and their clinical manifestations. Study this chart and learn the specific meanings of these terms. They will help you keep clear exactly what aspect of sickle cell anemia, or what component of the genetic or molecular system is being discussed.
The difference in the one amino acid in the b chains of sickle cell hemoglobin must affect the way the molecules interact with one another. Pauling made a remarkable prediction about this difference in 1949, when he wrote:
Many years later is was shown that the amino acid that is substituted in the # 6 position in the beta chain forms a protrusion that quite accidentally fits into a complementary site on the beta chain of other hemoglobin molecules in the cell, thus allowing the molecules to hook together likes pieces of the play blocks called legos. The result is, as Pauling predicted, that instead of remaining in solution sickle cell hemoglobin molecules will lock together (aggregate) and become rigid, precipitating out of solution and causing the RBC to collapse. Early electron micrographs taken at the time showed dramatically that in sickle-cell hemoglobin, the molecules line up into long fibers inside the cell (see Fig. 4) forming trapezoidal-shaped crystals that have much the same shape as a sickled cell. Why this happens when oxygen tension is low and the hemoglobin becomes deoxygenated, will be discussed later.
It is interesting to note that in vitro (using solutions of hemoglobin extracted from red blood cells) studies of deoxygenation and reoxygenation of sickle-cell hemoglobin indicate the process is reversible, that is, as oxygen concentration is lowered hemoglobin molecules polymerize and form crystals, but as oxygen concentration is increased again the hemoglobin molecules can depolymerize and return to their soluble state. This can be written as:
However when similar in vivo experimental tests are run on sickle-cell hemoglobin in whole red blood cells, the process was only reversible up to a certain duration of exposure time. After several hours, the process could no longer be reversed. The reasons for this relate back to our earlier question of what was the exact effect of the mutation on the red blood cell and its contents. When a long-term sickled cell is broken open and a "ghost" prepared, even with the hemoglobin extracted, the cell retains its sickled shape.
In-Text Question 5: What might you hypothesize to be the cause of this phenomenon and how would it relate to the earlier conclusion that hemoglobin, not other cell components, are the site of the mutation's effect?
The notion that sickle cell anemia results from a specific amino acid substitution in a polypeptide was given further support by discovery, around the same time, of other hemoglobin variants with distinct molecular and physiological properties. In the mid 1940s it was found that Hemoglobin F, or fetal hemoglobin, has an electrophoretic mobility and a different affinity (higher) for oxygen than adult hemoglobin (fetal hemoglobin is produced by the fetus during gestation, and is slowly replaced by synthesis of the adult form in the first few months of life; the higher affinity of fetal hemoglobin for oxygen facilitates the transfer of oxygen across the placenta from the mother's blood to that of the fetus). Hemoglobin F was also found to have a different amino acid sequence, indeed producing a distinctive chain, the g (gamma) chain instead of the b chain, during most of fetal life (for more details see Stryer, p. 154). Then, in the early 1950s two other hemoglobin-based conditions, designated Hemoglobin C and Hemoglobin D, were discovered by Harvey Itano in two separate families. These hemoglobins were also found to have different eletrophoretic mobilities and different amino acid sequences, as well as unique physiological effects (not as severe, however, as sickle cell hemoglobin).
To learn more about other hemoglobinopathies, click on the following website http://sickle.bwh.harvard.edu/hemoglobinopathy.html
Taken together, these examples all supported the general paradigm that mutations produced alterations in the amino acid sequence of proteins that, in turn, had significant effects on the protein's function. Such a conception, coming as it did at just about the time of the development of the Watson-Crick model of DNA in 1953, helped launch the revolution in molecular biology that we are still experiencing today.
We will also explore in a later case study how at the DNA level the genetic mutation for sickle cell hemoglobin alters the specific structure of the beta polypeptide chain.