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The Amino-Acid Sequence in the Phenylalanyl Chain of Insulin (1951)

by Frederick Sanger

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Summary

This 1951 paper by Frederick Sanger reports the first complete determination of the amino acid sequence of a protein, specifically the phenylalanyl chain (B chain) of bovine insulin. The central thesis is that proteins have a unique, precisely defined sequence of amino acids, disproving the then-prevailing view that they were amorphous colloids with variable compositions. Sanger achieved this by using the reagent 1-fluoro-2,4-dinitrobenzene (FDNB, later called Sanger's reagent) to label the N-terminal amino acid, then partially hydrolyzing the chain with acid, separating the resulting peptide fragments via paper chromatography, and identifying their sequences by further hydrolysis and end-group analysis. The paper presents the full 30-residue sequence of the B chain, establishing that each position is occupied by a specific amino acid. A reader takes away the foundational proof that protein primary structure is ordered and determinable, a breakthrough that earned Sanger his first Nobel Prize in Chemistry in 1958.

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Key concepts

  • Sanger's reagent (FDNB) — 1-fluoro-2,4-dinitrobenzene, a chemical that selectively labels the N-terminal amino acid of a peptide chain, enabling identification of the first residue.
  • Partial acid hydrolysis — A method of breaking a protein into overlapping peptide fragments by controlled acid treatment, allowing reconstruction of the full sequence from fragment overlaps.
  • Paper chromatography — A technique used to separate and identify amino acids and small peptides based on their differential migration on filter paper in a solvent system.
  • N-terminal analysis — The determination of the amino acid at the free amine end of a polypeptide chain, crucial for establishing sequence directionality.
  • Phenylalanyl chain (B chain) — One of the two polypeptide chains of insulin, consisting of 30 amino acids, whose sequence was fully determined in this paper.
  • DNP-amino acid — Dinitrophenyl-amino acid, the stable yellow derivative formed when FDNB reacts with an N-terminal amino acid, used for identification after hydrolysis.