Publisher Summary This chapter reviews the physical, chemical, and biological properties of insulin in the light of the atomic arrangement found in insulin crystals. It also describes the relation of the three-dimensional arrangement of the atoms in the molecule of 2-zinc insulin crystal to the solution properties of insulin (particularly its states of aggregation), to the chemical reaction and chemical modification of the molecule, and to its primary biological activity. Normally the insulin crystals contain two zinc ions to every six molecules of insulin—a hexamer. The slow solution of the crystals provides a method of delaying the action of insulin that closely parallels the methods adopted in the pancreas itself for the storage and release of insulin. Within many β granules, grains can be seen that almost certainly contain zinc insulin hexamers packed in a crystalline array, and in experimental animals diabetes has been induced by chelating agents, such as EDTA, perhaps simply by interfering with normal insulin storage. It, therefore, seems plausible that ready crystallization of insulin in the presence of zinc is a reflection of the storage processes in the β cell.

Insulin: The Structure in the Crystal and its Reflection in Chemistry and Biology by

Role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts.

New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates.

X-ray analysis, circular dichroism, receptor binding and biological potencies of chemically modified insulins suggest that the conformation of the insulin molecule is critical to the formation of both the zinc insulin hexamer and the insulin–receptor complex. Results are consistent with an insulin receptor-binding region including many of the hydrophobic residues important to dimerisation in addition to more polar surface residues. There is a further possibility of formation of an antiparallel sheet structure between the insulin and receptor molecules in the complex similar to that between monomers in the insulin dimer.

Receptor-binding region of insulin

The subunit structure of the high affinity insulin receptor. Evidence for a disulfide-linked receptor complex in fat cell and liver plasma membranes.

The acetylation of the free amino groups of insulin was studied by reaction of the hormone with N-hydroxysuccinimide acetate at pH6.9 and 8.5. The products formed were separated by chromatography on DEAE-Sephadex and were characterized by isoelectric focusing, by end-group analysis, by the incorporation of [3H]acetyl groups in the molecule, and by treatment with trypsin that had been treated with 1-chloro-4-phenyl-3-toluene-p-sulphonamidobutan-2-one (`tosylphenylalanyl chloromethyl ketone'). Three monosubstituted products, two disubstituted products and one trisubstituted derivative were prepared. The α-amino groups of the terminal residues and the -amino group of the lysine-B29 were the sites of reaction. Acetylation of any of the free amino groups did not affect the biological activity of insulin. It was demonstrated, however, that substitution at the glycine-A1 amino group by the larger residues, acetoacetyl or thiazolidinecarbonyl, produced a decrease in biological activity. Modification of the lysine-B29 or phenylalanine-B1 amino groups with these larger reagents did not affect the biological activity. Modification of the phenylalanine-B1 amino group by any of the three substituents resulted in a large decrease in the affinity of insulin for anti-insulin antibodies raised in the guinea pig. Modification of the other two amino groups did not affect the reaction with antibody. These observations are correlated with the tertiary structure of insulin.

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The acetylation of insulin.

Insulin was treated with diketen at pH6·9. The reaction mixture was resolved into four components by DEAE-Sephadex chromatography. The first component was unchanged insulin. The second and third components were shown by end-group analysis to be substituted on phenylalanine B-1 and glycine A-1 respectively. The fourth component was disubstituted on both phenylalanine B-1 and glycine A-1. The ∈-amino group of lysine B-29 was not involved in the reaction at low reagent concentrations. The purity of these derivatives was checked by their electrophoretic behaviour and by measurement of the rate of their reaction with trinitrobenzenesulphonic acid. The hormonal activity of the derivatives was determined. The effect of the modifications on the hormonal activity and the tertiary structure of insulin is discussed.

D. G. Lindsay

Papers

The acetylation of insulin.

Acetoacetylation of insulin.