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.