Analysis of solvent-mediated conformational changes of insulin by radioimmunoassay (RIA) techniques

Abstract

Stationary phase radioimmunoassay (RIA) (i.e. antibodies bound to polystyrene test tubes) techniques are used as an analytical probe of secondary and tertiary structural changes of radiolabelled (125)I porcine insulin. The effects of temperature, buffer composition, pH and ionic strength and solvents on insulin binding are studied. Optimum insulin-antibody binding occurred at 22 degrees C, pH 6 and a buffer strength of 0.1 M or less. Results of experiments with three pH 6 buffers (0.005 M phosphate, 0.1 M acetate and 0.1 M Tris) showed no statistical difference in binding properties. For all solvents tested, increasing the solvent concentration decreased the amount of insulin binding. Comparison of the various solvents tested indicated that ethylene glycol and methanol are the least denaturant whilst 1-propanol and acetonitrile are among the most denaturant.