Scientific paper: The insulin molecule

Background
Frederick Sanger presented the determination of the insulin molecule as a landmark in understanding protein architecture. At the time, proteins were known as complex mixtures of amino acids but their linear order was unknown. Determining the exact sequence of a hormone as biologically central as insulin provided the first clear evidence that proteins possess a unique, definable primary structure encoded by biological systems.

Experimental approach
A rigorous chemical strategy underpinned the work, combining selective labeling, controlled peptide cleavage, and systematic analysis of peptide fragments. Sanger used dinitrofluorobenzene to label N-terminal amino groups, enabling identification of terminal residues, and applied a variety of hydrolytic and partial-degradation techniques to produce overlapping peptides. Strategic reduction and reformation of disulfide bonds allowed isolation of the two chains and mapping of interchain connectivity. Careful chromatographic separations and amino acid analyses of the resulting fragments made it possible to assemble the full linear sequences.

Structural findings
The insulin molecule was shown to consist of two distinct polypeptide chains linked by disulfide bridges. The A chain contains 21 residues and includes an internal disulfide bond, while the B chain comprises 30 residues; two interchain disulfide bonds join the chains into the biologically active form. The complete primary sequences of both chains were determined, revealing the precise order of amino acids and the locations of cysteine residues responsible for the disulfide architecture. These chemical details established insulin as a discrete, chemically definable entity rather than a heterogeneous aggregate.

Functional and conceptual implications
The fixed, specific sequence of insulin provided direct support for the idea that a protein's primary structure underlies its biological activity and higher-level folding. The finding suggested that subtle sequence variations could modulate function, stability, and antigenicity, pointing to a molecular basis for species differences and physiological regulation. The work also strengthened the view that genetic information must specify amino acid sequences, anticipating later insights into the relationship between genes, mRNA, and protein synthesis.

Techniques and methodological significance
Beyond the sequence itself, methodological advances demonstrated the power of chemical sequencing approaches for larger proteins. The combination of selective labeling reagents, controlled fragmentation, and meticulous analytical chemistry became a template for subsequent protein sequencing efforts. The mapping of disulfide bonds showcased how covalent cross-links could be located and their role in stabilizing tertiary structure inferred from primary sequence data.

Broader impact and legacy
The elucidation of insulin's primary structure marked a turning point in molecular biology and biochemistry. It provided a concrete example that proteins are defined molecules amenable to precise chemical description and comparison. This achievement catalyzed further research into protein structure–function relationships, fostered techniques for sequencing other proteins, and helped frame questions about how genetic information encodes the amino acid order. The work on insulin remains a foundational milestone illustrating how chemical analysis can reveal the detailed architecture of biologically essential macromolecules.