In 1958 and afterwards, Pauling liked to discuss the small difference between normal and sickle cell hemoglobin in order to mention work completed at Caltech. In 1957 Pauling and two of Caltech's research associates, Herbert S. Rhinesmith and W. A. Schroeder, announced that normal adult hemoglobin has four polypeptide chains of two different types, which they called alpha and beta.

After Ingram announced that one amino acid differs in normal and sickle cell hemoglobins and after Rhinesmith, Schroeder, and Pauling determined that hemoglobin has two types of polypeptide chains, many researchers tried to ascertain where the amino acid substitution occurred on the polypeptide. In 1958 Itano and Singer were unable to state the exact location of the replacement, but narrowed down the possibilities. They concluded that the replacement had to occur within five Angstroms from where the hemes are bound together in order to support the explanation of how sickle cell hemoglobin contorts into a crescent shape as described in "Sickle Cell Anemia, a Molecular Disease." In addition, they stated that if only one amino acid replacement occurs, then only one of the two types of polypeptide chains (either the alpha or the beta) differs between normal and sickle cell hemoglobin.

In 1959 two experiments showed that the replacement of glutamic acid with valine occurs on the beta-chains. Jerome R. Vinograd, W. D. Hutchinson, and Schroeder of Caltech showed that the beta-chains of sickle cell and normal hemoglobin differ, whereas their alpha-chains are identical. Ingram also demonstrated that the difference occurs in the beta-chains and, in addition, stated that the substitution occurs at the sixth position from the N-terminus.