The self-association of insulin: determinations based on a differential gel adsorption procedure.
Self-association properties of zinc-insulin were investigated using a differential gel adsorption procedure based on the microcentrifuge desalting method of E. Helmerhorst and G. B. Stokes (1980, Anal. Biochem. 104, 130-135). This differential gel adsorption procedure offered a quick semiquantitative method for examining insulin association under a wide range of different conditions using simple equipment. Nonspecific adsorption effects with the gel matrix at low insulin concentrations were minimized by the presence of 0.1% bovine serum albumin. This procedure was based on the observation that the monomer species of insulin was separated from insulin aggregates due to its interaction with the devoided gel matrix of Sephadex G-25 and remained on the column following centrifugation. A residue with an apparent pK of 11.1 +/- 0.1 was shown to be critically responsible for the adsorption of monomer insulin to the gel matrix, thus implicating the tyrosyl residue(s) of insulin in this interaction. The proportion of monomer was simply computed from the recovery of insulin off the gel using [125I] monoiodoinsulin to monitor recovery. Insulin association was studied over the range of insulin concentrations from physiologically circulating levels (nM) to storage concentrations (mM). Our data are in agreement with monomer being the predominant form of the hormone in vivo. Our estimates for the Gibbs free energy change, enthalpy, and entropy for the formation of dimers and the effects of pH, ionic strength, and temperature on the equilibrium constant for the formation of dimers were in good agreement with the literature. This differential gel adsorption procedure may have application to other protein-associating systems with suitable adsorption characteristics.
D002498:Centrifugation; D003201:Computers; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000032:analysis*; D046911:Macromolecular Substances; D008433:Mathematics; D008832:Microchemistry; D013816:Thermodynamics
D007328:Insulin; D046911:Macromolecular Substances