Self-association of insulin. Its pH dependence and effect of plasma.

We investigated some self-association properties of monocomponent porcine insulin with a simple and novel procedure that for the first time permits the association properties of insulin to be studied in mixed protein solutions. With this semiquantitative procedure, the monomer form of insulin was separated from aggregates during centrifuge desalting on Sephadex G-25. The proportion of monomer in solutions of insulin was estimated by monitoring [125I]monoiodoinsulin recovery off the gel. The self-association properties of insulin were studied in plasma with this procedure over the concentration range of physiologically circulating levels to storage concentrations of the hormone. We estimated the equilibrium constant for the formation of dimers to be 1.5 X 10(4)/M in pooled human plasma at pH 7.4 and 37 degrees C. This estimate was indistinguishable from that obtained for insulin in a defined phosphate-albumin buffer under similar conditions. This result provides direct confirmation that insulin is not associated with a circulating serum binding protein(s) and that insulin is present predominantly as a monomer in plasma. We also used the method to investigate the self-association properties of insulin over a wide pH range. The equilibrium constant for the formation of dimers decreased only 6-fold over a 9.2 pH unit increase from pH 2 to 11.2 but almost 200-fold for the additional 1.5 pH unit increase from pH 11.2 to 12.7. We conclude that a residue of pK 12 is critical to the maintenance of the quaternary structure of insulin. We assign this role to the single B22-arginyl residue in insulin.