Abstract
The aggregation behavior of zinc-free insulin has been studied by small-angle neutron scattering as a function of pH and ionic strength of the solution. The pair distance distribution functions for the 12 samples have been obtained by indirect Fourier transformation. The results show that the diameter of the aggregates is 40 Å at pH 11 and 10 mM NaCl, independent of the protein concentration. The largest diameter of about 120 Å is found for pH 8, 100 mM NaCl, and a protein concentration of 10 mg/ml. Estimates of the pair distance distribution functions, free of inter-particle correlation effects, were obtained by an indirect Fourier transformation, omitting the data at small scattering vectors, which are influenced by these effects. By this procedure the weight-averaged molecular mass and the average radius of gyration were determined. These parameters vary from 1.3 times the monomer mass and 14 Å, to 6.8 times the monomer mass and 31 Å, respectively. The mass distribution between the oligomers was determined by a model based on the crystal structure of zinc-free insulin. The results from this model and the Fourier transformations have been compared to an equilibrium model recently introduced by Kadima et al. (1993). The neutron scattering results agree well with the predictions of this model except that broader mass distributions are suggested by neutron scattering.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Badgar J, Caspar DLD (1991) Water structure in cubic insulin crystals. Proc Natl Acad Sci, USA 88:622–626
Badger J, Harris MR, Reynolds CD, Evans AC, Dodson EJ, Dodson GG, North ACT (1992) Structure of the pig insulin dimer in the cubic crystal. Acta Crystallogr B47:127–136
Baker EN, Blundell TL, Cutfield JF, Cutfield SM, Dodson EJ, Dodson GG, Crowfoot Hodgkin DM, Hubbard RE, Isaacs NW, Reynolds CD, Sakabe K, Sakabe N (1988) The structure of 2 Zn pig insulin crystals at 1.5 Å resolution. Philos Trans R Soc London B319:369–456
Bevington BR (1969) Data reduction and error analysis for the physical sciences. McGraw-Hill, New York
Bohidar HB, Geissler E (1984) Static and dynamic light scattering from insulin solutions. Biopolymers 23:2407–2417
Coffman FD, Dunn MF (1988) Insulin-metal ion interactions: The binding of divalent cations to insulin hexamers and tetramers and the assembly of the insulin hexamers. Biochemistry 27: 6179–6187
Dodson EJ, Harding MM, Crowfoot Hodgkin (1966) The crystal structure of insulin: III. Evidence for 2-fold axis in rhombohedral zinc insulin. J Mol Biol 16:227–241
Dodson EJ, Dodson GG, Lewitowa A, Sabesan M (1978) Zinc-free pig insulin: crystallization and structure determination. J Mol Biol 125:387–396
Doty P, Gellert M, Rabinovitch B (1952) The association of insulin. 1. Preliminary investigations. J Am Chem Soc 74:2065–2069
Glatter O (1977) A new method for the evaluation of small-angle scattering data. J Appl Crystallogr 10:415–421
Glatter O (1979) The interpretation of real-space information from small-angle scattering experiments. J Appl Crystallogr 12:166–175
Goldman J, Carpenter FH (1974) Zinc binding, circular dichroism, and equilibrium sedimentation studies of insulin (bovine) and several of its derivative. Biochemistry 13:4566–4574
Guinier A, Fournet G (1955) Small-angle scattering of X-rays. Wiley, New York
Hansen JF (1991) The self-association of zinc-free human insulin and insulin analogue B13-glutamine. Biophys Chem 39:107–110
Hansen S, Pedersen J Skov (1991) A comparison of three different methods for analyzing small-angle scattering data. J Appl Crystallogr 24:514–518
Hill CP, Dauter Z, Dodson EJ, Dodson GG, Dunn MF (1991) X-ray structure of an unusual Ca2+ site and the roles of Zn2+ and Ca2+ in the assembly, stability, and storage of the insulin hexamer. Biochemistry 30:917–924
Jacrot B (1976) The study of biological structures by neutron scattering from solutions. Rep Progr Phys 39:911–953
Jeffrey PD, Coates JH (1966a) An equilibrium ultracentrifuge study of self-association of bovine insulin. Biochemistry 5:489–498
Jeffrey PD, Coates JH (1966b) An equilibrium ultracentrifuge study of the effect of ionic strength on the self-association of bovine insulin. Biochemistry 5:3820–3824
Kaarsholm N, Havelund S, Hougaard P (1990) Ionization behavior of native and mutant insulins: pK perturbation of B13-Glu in aggregated species. Arch Biochem Biophys 283:496–502
Kadima W, Roy M, Lee RW-K, Kaarsholm NC, Dunn MF (1992) Studies of the association and conformational properties of metal-free insulin in alkaline sodium chlorine solutions by one-and two-dimensional 1H NMR. J Biol Chem 267:8963–8970
Kadima W, Ogendal L, Bauer R, Kaarsholm N, Brodersen K, Hansen JF, Porting P (1993) The influence of ionic strength and pH on the aggregation properties of zinc-free insulin studied by static and dynamic laser light scattering. Biopolymers (in press)
Lawson CL, Hanson RJ (1974) Solving least squares problems. Prentice-Hall, Englewood Cliffs, NJ
Lord RS, Gubensek F, Rupley JA (1973) Insulin self-association. Spectrum changes and thermodynamics. Biochemistry 12:4385–4392
Palmieri R, Lee RW-K, Dunn MF (1988) 1H Fourier transform NMR studies of insulin. Coordination of Ca2+ to Glu (B13) site drives hexamer assembly and induces a conformation change. Biochemistry 27:3387–3397
Pedersen J Skov, Posselt D, Mortensen K (1990) Analytical treatment of the resolution function in small-angle scattering. J Appl Crystallogr 23:321–333
Pekar AH, Frank BH (1972) Conformation of proinsulin. A comparison of insulin an proinsulin self-association at neutral pH. Biochemistry 11:4013–4016
Pocker Y, Biswas (1981) Self-association of insulin and the role of hydrophobic bonding: a thermodynamic model of insulin dimerization. Biochemistry 20:4354–4361
Roy M, Lee RW-K, Kaarsholm NC, Thøgersen H, Brange J, Dunn MF (1990) Sequence-specific 1H-NMR assignments for the aromatic region of several biologically active, monomeric insulins including native human insulin. Biochim Biophys Acta 1053: 63–73
Sober HA (1970) Handbook of Biochemistry 2. edn. CRC Press
Steiner RF (1951) Reversible association processes of globular proteins: Insulin. Arch Biochem Biophys 39:333–354
Strazza S, Hunter R, Walker E, Darnall DW (1985) The thermodynamics of bovine and porcine insulin determined by concentration difference spectroscopy. Arch Biochem Biophys 238:30–42
Svergun DI, Pedersen J Skov (1993) On the estimation of error propagation in small-angle data treatment. J Appl Crystallogr (in press)
Author information
Authors and Affiliations
Additional information
Correspondence to: J. Skov Pedersen
Rights and permissions
About this article
Cite this article
Pedersen, J.S., Hansen, S. & Bauer, R. The aggregation behavior of zinc-free insulin studied by small-angle neutron scattering. Eur Biophys J 22, 379–389 (1994). https://doi.org/10.1007/BF00180159
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00180159