Acta (1989),
The time, 1956, was right for working on the structure of an abnormal human haemoglobin, suspected to be the cause of sickle-cell anaemia. The disease was called a molecular disease by Pauling in 1949, the first time this notion had been applied, implying that an abnormal molecule was responsible for the symptoms. In that year, Linus Pauling, Harvey Itano, John Singer and Ibert Wells had shown that the haemoglobin in sicklecell anaemia carries about three more positive charges per molecule than the normal adult protein, but it was not known what caused this difference. Also in 1949, James Nee1 had proved that sickle cell anaemia was due to homozygosity in a recessive autosomal gene. It remained to put these ideas together by discovering the precise nature of the chemical difference between wildtype and sickle-cell protein. This might prove that a gene controls the structure of a protein and might show how it does so. The surprise was in the fact that only a single amino acid was substituted in the protein by the sickle-cell allele, showing amazingly detailed control of the gene over the structure of its protein. It was also unexpected that so small a change could cause so serious a disease.
The time was right in 1956, because new tools for examining protein structure had just been devised. The methods used by Walter Schroeder and by Herbert Scheinberg in earlier attempts at quantitative amino acid analysis of the whole protein with its more than 100 amino acids were not sufficiently sensitive to show why the abnormal protein had a different electric charge near neutral pH. Even the new column chromatographic methods for amino-acid analysis developed by Stanford Moore and Bill Stein did not have the resolution necessary for what eventually turned out to be a single amino-acid difference. Clearly, it was necessary to cut the protein into smaller, more manageable pieces and examine these by other methods.
It was known that proteolytic enzymes, particularly trypsin, will cleave a protein cleanly into manageable peptides. In the case of haemoglobin some 30 peptides