Insulin In Aqueous Solution Research Articles

Thermodynamic studies on the interaction of n-alkyl sulfates with insulin in aqueous solution

Binding isotherms for the interaction of a homologous series of sodium n-alkyl sulfates (chain lengths 8–10 and 12) on interaction with bovine insulin at 25 °C, at pH 3.2 and 10.0, have been determined. The isotherms show two unusual features, first the binding ranges up to 60–70 surfactant molecules per insulin molecule and secondly it is only slightly dependent on the n-alkyl chain length. The enthalpy changes on surfactant–insulin interaction have been measured by microcalorimetry and have been discussed in terms of exothermic contributions from sulfate head-group interactions with cation amino acid residues in the protein and an endothermic unfolding contribution. The exothermicities of the head-group interactions increase with decreasing pH but are in excess of those predicted from model studies on surfactant–polypeptide interactions. The Gibbs energies of binding per surfactant molecule decrease with increasing binding to a limiting value of ca.– 15 kJ (mol surfactant)–1 independent of pH. The high binding levels coupled with the slight chain length dependence suggest a micellar-based structure for the insulin–surfactant complexes.

Conformational flexibility of aqueous monomeric and dimeric insulin: a molecular dynamics study

A series of molecular dynamics simulations have been used to investigate the nature of monomeric and dimeric insulin in aqueous solution. It is shown that in the absence of crystal contacts both monomeric and dimeric insulin have a high degree of intrinsic flexibility. Neither of the two monomer conformations of 2Zn crystalline insulin appears to be favored in solution nor is the asymmetry of the crystal dimer reduced in the absence of crystal contacts. A shift is observed in the relative positions of molecules 1 and 2 in the dimer compared with that found in the crystal, which may have consequences for the prediction of the effects of mutants in the monomer-monomer interface designed to alter the self-association properties of insulin.

High resolution 1H-NMR studies of Des-(B26–B30)-insulin; assignment of resonances and properties of aromatic residues

The assignments of 1H resonances of the eight aromatic residues of Des-(B26–B30)-insulin are reported, based on pH titration, selective spin decoupling and its 500 MHz 1H two-dimensional (2D)-COSY spectrum. The p K values of the three tyrosines A14, A19 and B16 are 10.84, 11.27 and 10.40, respectively. Tyrosine A19 is buried in a hydrophobic environment, while Tyrosine B16 is exposed in a relatively hydrophilic state. Among the three phenylalanines, the ring proton resonances of Phe-B25 undergo abnormal upfield shifts, probably due to the ring currents of the nearby Phe-B24 and Tyr-B16. From this study of the low-field region of 1H-NMR spectrum of Des-(B26–B30)-insulin, we conclude that this molecule probably maintains the major structural features of insulin in aqueous solution, but there are some readjustments of the peptide conformation.

Spektrophotometrische Untersuchungen zur Bindung von Viktoriablau 4R an oxidiertes Insulin

The empirical demonstration of insulin by the basic (cationic) dye Victoria Blue 4R (V4R; Ivić 1959) shows the character of a histochemical reaction (Wohlrab et al. 1985), while the chemistry+ of the reaction is not quite understood yet. Aim of this investigation: Will the spectral behaviour of V4R in visible light be influenced by oxidized or non-oxidized insulin? Dependent on the concentration, V4R shows in aqueous solution 2 absorption maxima (lambda = 597 and 558 nm), which represents monomers and dimers of the dye. With increasing dye concentration (200 mumol/l), V4R forms dimers and even higher polymers. Constant V4R concentration (200 mumol/l) results with increasing concentration of oxidized insulin (4 to 16 mumol/l) in a reduction of extinction, while the extinction at lambda = 558 nm (dimers) is more decreased than at lambda = 597 nm (monomers). Non-oxidized insulin has no remarkable influence on the absorption behaviour of V4R. Extinction measurements on V4R stained B-cells of islets of Langerhans after pre-oxidation of the section resulted in a main absorption maximum at lambda = 554 nm. Depending on the concentration, the dye V4R is associated with the oxidized insulin in aqueous solution, which is also indicated by the occurrence of an isobestic point in the curve behaviour. This is expressed by the establishment of a concentration-dependent equilibrium between the dye V4R and the oxidized insulin (change in the dissociation and aggregation behaviour respectively of the dye V4R). The determined main absorption maximum (lambda = 554 nm) in the biological material points in the same way to interactions between the stain and oxidized insulin.

On the pH dependence of Cu(II) binding to insulin: ESR and electronic studies

The ESR and electronic absorption spectra have been used to investigate co-ordination of Cu(II) to insulin in aqueous solutions at different pH. Two series of complexes with low- and high copper content were examined and the values of the magnetic tensor components and the shape of the diffuse reflectance transitions suggested that these copper-insulin derivatives have tetragonal symmetry with Cu(II) in a (N xO y) ligand field, where oxygen donor groups are predominant at low pH and nitrogenous ligands at high pH. Such a trend was further supported by the presence of superhyperfine structure at pH = 13. Oxygen of the carboxylato groups, nitrogen of α- and ε-amino groups and of imidazoles, all contribute to the coordinations field. At very high pH only, a preferential binding site for Cu(II) is found, which probably involves deprotonated peptide nitrogens.

Some Considerations in the Preparation of Radioiodoinsulin for Radioimmunoassay and Receptor Assay

125I-insulins, prepared by iodination with chloramine T in marked excess or by stepwise, stoichiometric addition of the oxidizing agent, were compared with respect both to their molecular distribution of iodine and to their suitability for use in a cultured lymphocyte receptor assay. Iodination of insulin in aqueous solution results in the same distribution of iodine atoms, independent of experimental method and dependent only on the average iodine number. This distribution can be calculated on the basis of a Monte Carlo simulation, For insulin iodinated at an average of 0.8 I atoms per molecule, approximately 50 per cent of the radioactivity is in other than monoiodoninsulin. Purification methods that separate on the basis of charge, such as starch-gel electrophoresis, are then required, to obtain monoiodoinsulin. More highly iodinated insulin do bind to the lymphocyte receptor, although, as in radioimmunoassay, the overiodinated species are less satisfactory for use as tracers. The shelf life of iodinated insulin appears to be related better to the average iodine content than to any other factor, presumably because of decay catastrophe. There is no evidence to suggest that exposure to chloramine T in marked excess for a few seconds is deleterious to insulin.

Monosubstituted 2,2-dimethyl-3-formyl-L-thiazolidine-4-carbonyl-insulins.

The treatment of insulin in aqueous solution at pH 6.9 and 8.5, with the hydroxysuccinimide ester of 2,2‐dimethyl‐3‐formyl‐l‐thiazolidine‐4‐carboxylic acid leads to the incorporation of thiazolidine residues into the protein.Examination by DEAE‐Sephadex chromatography of the products formed shows the presence of three products. The relative amount of products formed depends upon the reaction conditions. Characterisation of these products by end‐group analysis, cellulose acetate electrophoresis, amino acid analysis of the performic acid‐oxidised chains and treatment with TPCK‐treated trypsin suggests that they are monosubstituted derivatives formed by reaction at each of the free amino groups of insulin.The effect of modification on the biological activity and crystallisation properties of the hormone is discussed as well as the application of the preparation of covalent heavy atom derivatives of proteins.

Rate of radiolysis of the disulphidic groups of insulin in aqueous solutions exposed to X-irradiation

Rate of radiolysis of the disulphidic groups of insulin in aqueous solutions exposed to X-irradiation

The sedimentation coefficient of insulin in acid solution.

Abstract 1. 1. The sedimentation coefficient of insulin in aqueous solution at pH 2, I = 1.0 in the presence of chloride ions has been measured over a concentration range of approx. 0.1 g/100 ml to 0.7 g/100 ml. The Rayleigh interference optical system of the ultracentrifuge and the transport method have been used to improve the precision of measurement of the sedimentation coefficient at low concentrations. 2. 2. A value of 1.2 S has been obtained for s20,w0 in agreement with the value measured by Fredericq under similar conditions. This implies that insulin dissociates to molecules of molecular weight 6000 in aqueous solution.

Molecular Weight of Insulin in N,N-Dimethylformamide

THE detailed investigations of Sanger and co-workers have established the molecular weight of insulin as some multiple of about 6,000 1. It is not clear, however, whether the fundamental covalently bonded unit is the 6,000 or 12,000 species. (The determination of the positions of the S—S linkages in insulin1 does not establish the fundamental unit as the 6,000 species. The methods of analysis do not preclude the possibility of a staggered arrangement of the S—S linkages such that the 12,000 species is covalently bonded together.) Several workers using a variety of techniques—osmotic pressure, sedimentation and light-scattering—achieved results indicating that the minimum molecular weight of insulin in aqueous solution is 12,000 2. However, other work has indicated a value of 6,000. Harfeneist and Craig3, by counter-current distribution methods in 2-butanol-dichloracetic acid systems, Fredericq4 by sedimentation-diffusion studies in alkaline solutions, and Kupke and Linderstrom-Lang5 by osmotic pressure determinations in aqueous guanidine chloride solutions, have all recently contributed data consistent with the value of 6,000. Osmotic pressure studies of insulin in N,N-dimethylformamide in this laboratory also indicate a molecular weight of 6,000 for the fundamental unit.