Frederick Sanger Sequences the Amino Acids of Insulin, the First of any Protein.

In 1955 English biochemist Frederick Sanger sequenced the amino acids of insulin, the first of any protein.

Sanger's work “revealed that a protein has a definite constant, genetically determined sequence—and yet a sequence with no general rule for its assembly. Therefore it had to have a code” (Judson, Eighth Day of Creation, 188).

"Sanger's first triumph was to determine the complete amino acid sequence of the two polypeptide chains of bovine insulin, A and B, in 1952 and 1951, respectively. Prior to this it was widely assumed that proteins were somewhat amorphous. In determining these sequences, Sanger proved that proteins have a defined chemical composition. For this purpose he used the "Sanger Reagent", fluorodinitrobenzene (FDNB), to react with the exposed amino groups in the protein and in particular with the N-terminal amino group at one end of the polypeptide chain. He then partially hydrolysed the insulin into short peptides, either with hydrochloric acid or using an enzyme such as trypsin. The mixture of peptides was fractionated in two dimensions on a sheet of filter paper, first by electrophoresis in one dimension and then, perpendicular to that, by chromatography in the other. The different peptide fragments of insulin, detected with ninhydrin, moved to different positions on the paper, creating a distinct pattern that Sanger called 'fingerprints'. The peptide from the N-terminus could be recognised by the yellow colour imparted by the FDNB label and the identity of the labelled amino acid at the end of the peptide determined by complete acid hydrolysis and discovering which dinitrophenyl-amino acid was there. By repeating this type of procedure Sanger was able to determine the sequences of the many peptides generated using different methods for the initial partial hydrolysis. These could then be assembled into the longer sequences to deduce the complete structure of insulin. Finally, because the A and B chains are physiologically inactive without the three linking disulfide bonds (two interchain, one intrachain on A), Sanger and coworkers determined their assignments in 1955. Sanger's principal conclusion was that the two polypeptide chains of the protein insulin had precise amino acid sequences and, by extension, that every protein had a unique sequence. It was this achievement that earned him his first Nobel prize in Chemistry in 1958. This discovery was crucial for the later sequence hypothesis of Crick for developing ideas of how DNA codes for proteins" (Wikipedia article on Frederick Sanger, accessed 11-20-2013).