|"Sickle Cell Anemia, a Molecular Disease"
In March 1949, Pauling and Itano first announced the experimental results from comparing
sickle cell anemia and normal hemoglobin with electrophoresis. Nine months later,
Pauling, Itano, Singer, and Wells published the more thorough article "Sickle Cell
Anemia, a Molecular Disease" in Science.
Their experimental method included a couple of tests analyzing carbonmonoxyhemoglobin
and ferrohemoglobin of sickle cell anemia patients and healthy adults. They treated
the compounds with buffers and examined the samples at various levels of pH and then
subjected the samples to electrophoretic analysis by putting them through Caltech's
Tiselius apparatus. They found that blood samples from sickle cell anemia patients
and normal adults reacted differently in two ways. First, when they graphed the curves
of mobility versus pH, the sickle cell hemoglobin curve followed the same path as
normal hemoglobin, but had a higher isoelectric point (the pH at which the solution
will not migrate).
Secondly, the authors had found that sickle cell and normal adult hemoglobin behave
differently when their carbon monoxide derivatives are subjected to electrophoresis
at a neutral pH. In their words: "At pH 7.0 sickle cell carbonmonoxyhemoglobin moves
as a positive ion while normal carbonmonoxyhemoglobin moves as a negative ion." The
Longsworth scanning diagrams of carbonmonoxyhemoglobin demonstrate that normal and
sickle cell anemia hemoglobins are homogenous substances because only one peak occurs.
In addition, the normal hemoglobin peak is to the left of the arrow and therefore
negative, whereas the peak for the sickle cell anemia hemoglobin is to the right of
the arrow denoting that it is positive. From this result, they postulated that sickle
cell anemia hemoglobin has two to four more positive charges than normal hemoglobin.
In their attempts to find out more about the difference in charge, they ascertained
that the globins are different and the hemes are identical in the two substances.
Wells performed the tests showing that the hemes are identical.
Besides analyzing the heme and the globin, they performed two other important experiments.
First, they established that healthy people of Caucasian and African descents have
"indistinguishable" hemoglobin. Second, they analyzed blood taken from sickle cell
trait patients. From Longsworth scanning diagrams, they found that the mobility of
sickle cell trait hemoglobin acted similarly to the mixture they made by combining
equal parts of sickle cell anemia and normal hemoglobin, as can be seen by the location
of peaks in relation to the arrow. However, the authentic sickle cell trait hemoglobin
had more normal hemoglobin than their manufactured mixture of sickle cell and normal
adult hemoglobin, as is seen by the height of the peaks. They performed additional
experiments and ascertained that the ratio of normal to sickle cell hemoglobin in
people with sickle cell trait is about sixty to forty.