Differences in the nature of the interaction of insulin and proinsulin with zinc.
1. The reversible interaction of zinc with pig insulin and proinsulin has been studied at pH7 by equilibrium dialysis (ultrafiltration) and by sedimentation equilibrium and velocity measurements in the ultracentrifuge. Binding values calculated from equilibria, where the ratio of free to bound zinc was varied in the range 0.01:1-10:1, indicated that proinsulin and insulin each contained two main orders of zinc binding with very different affinities for the metal. 2. In equilibria containing low concentrations of free zinc (free: bound ratios of 0.01-0.1:1) both insulin and proinsulin aggregated to form soluble hexamers containing firmly bound zinc (up to 0.284g-atom/monomer) with an apparent intrinsic association constant of 1.9x10(6)m(-1). 3. Higher concentrations of zinc (free: bound ratios of 0.1-10.0:1) resulted in a progressive difference in the zinc binding, aggregation and solubility properties of the metal complexes of insulin and proinsulin. At the highest concentration of free zinc, proinsulin bound a total of more than 5.0g-atom/monomer and aggregated to form a mixture of soluble polymers (mainly 5.1S). In contrast, insulin bound a total of only 1.0g-atom/monomer and was almost completely precipitated from solution. 4. These results would indicate that the presence of the peptide segment connecting the insulin moiety in proinsulin does not prevent the firm binding of zinc to the insulin moiety and the formation of hexamers of zinc-proinsulin. At the same time although the connecting peptide contains additional sites of lower affinity for zinc, which should facilitate inter- and intra-molecular cross-linking, the general conformation of the zinc-proinsulin hexamer must preclude the formation of very large and close-packed aggregates that are insoluble in solutions at equilibrium.
D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003956:Dialysis; D005374:Filtration; D006863:Hydrogen-Ion Concentration; D007328:Insulin; D007700:Kinetics; D046911:Macromolecular Substances; D008851:Micropore Filters; D008956:Models, Chemical; D009942:Organometallic Compounds; D011384:Proinsulin; D011485:Protein Binding; D011487:Protein Conformation; D012995:Solubility; D013057:Spectrum Analysis; D013552:Swine; D014461:Ultracentrifugation; D014462:Ultrafiltration; D015032:Zinc; D015033:Zinc Isotopes
D007328:Insulin; D046911:Macromolecular Substances; D009942:Organometallic Compounds; D015033:Zinc Isotopes; D011384:Proinsulin; D015032:Zinc