Biophysical Chemistry, 39 (1991) 205-213 Q 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0301-4622/91/$03.50 205 BIOCHE 01541 Insulin association in neutral solutions studied by light scattering Smen Hvidt Department of Chemutry, RIM Natronal Loboratory and Roskilde University, DK-4000 Roskilde, Denmark Received 9 March 1990 Accepted 20 September 1990 Insulin: Light scattering; Zinc binding: Association constant Molecular weights and weight distributions of sulfated, Zn-free, and 2Zn insulins have heen measured at pH 7.3 as a function of concentration from 0.1 to 2 mg/ml by use of a combination of light scattering, refractometry, and size-exclusion chromatography. Results show that sulfated insulin is monomeric over the studied concentration range. Weight average molecular weights between those of a monomer and a hexamer were found for both zinc-free and 2Zn insulins. Zinc stabilizes the hexamer, and the dimer-hexamer equilibrium constant is approx. 400-times higher in the presence of zinc than in its absence. An average hydrody- namic radius of 5.6 nm, close to the crystallographic size of the insulin hexamer, was determined from dynamic light scattering of 2Zn msulin solutions. 1. Introduction Insulin has been the subject of extensive inves- tigations with respect to its structure and function, as discussed in comprehensive reviews [1,2]. The biologically active form of the hormone appears to be the insulin monomer, which has a molecular weight of 5778 g/m01 for porcine insulin [1,2]. Insulin associates in solution and can crystalize as a 2Zn form, which consists of a hexamer with two bound Zn2’. Its crystal structure has been de- termined [l]. Information about the association behavior of insulin in solution at near neutral pH stems primarily from sedimentation equilibrium studies [3-81 and stopped-flow measurements [9]. These studies show that insulin without zinc forms dimers with a typical equilibrium constant 4 X lo4 M-‘, and that insulin associates further. Some data have been analyzed with a model involving monomer-dimer-hexamer and higher order aggre- gates [3,6], whereas other results favor a model Correspondence address: S. Hvidt, Dept of Chemistry, Rise National Laboratory and Roskilde University, DK-4000 Roskilde, Denmark. with a monomer-dimer equilibrium and a further indefinite association of either dimers [7] or mono- mers [8]. With zinc present the hexamer is stabi- lized, and aggregates of even higher molecular weights are inferred from sedimentation equi- librium studies [4,5]. Light scattering (LS) offers an alternative way to investigate the association of macromolecules in solution. Early LS studies of insulin focused on its properties in acidic solutions, where insulin is found as monomers and dimers [lo]. A few studies have been performed in solutions near neutral pH [ll-121. These results indicate a very complex time dependent association pattern and the formation of higher molecular weight aggregates. Insulin has a well-known tendency to precipi- tate on surfaces and interfaces [2,13], and tradi- tional LS is very sensitive to traces of dust or particles. These considerations prompted us to perform low-angle laser light scattering on insulin solutions using a flow system with a size-exclusion chromatography (SEC) column to ensure that any large particles were effectively removed from the insulin. An advantage of low-angle laser LS, be- sides the elimination of an extrapolation to