William G. Griffin

Molecular basis of thermal aggregation of bovine β-lactoglobulin A

On heating, bovine β-lactoglobulin A (β-lg A) dimers dissociate and then aggregate. The extent of aggregation of β-lg A after a heat–quench treatment was studied quantitatively using photon correlation spectroscopy (PCS); circular dichroism (CD) spectroscopy was also carried out on heated solutions of β-lg A to examine changes in the secondary and tertiary structure of the protein. In pH 7.0 solution the protein underwent a change in tertiary structure at 67 °C; by contrast, the secondary structure, containing ca. 50%β-sheet, showed very little change up to 90 °C. PCS showed that the extent of aggregation of β-lg A depended on pH and buffer concentration; under some conditions it was observed that there was a temperature above which the rate of aggregation decreased. The pH-dependence of the temperature of onset of aggregation correlated with the conformational stability of β-lg A. Detailed examination of the light scattering data suggested initial aggregation of the protein molecules to form linear, rod-like particles. It is proposed that, at later stages in the aggregation process, the rods themselves aggregate and start to form a network.

The attenuation of ultrasound in aqueous suspensions of casein micelles from bovine milk

The ultrasonic attenuation of skimmed milk, of the serum containing the soluble whey proteins and small casein aggregates and of ultrafiltered serum was measured in the frequency range 15–96 MHz. From these measurements the attenuation due to the colloidal casein micelles in serum was obtained. For the serum, α∝f1.86±0.02; for the soluble proteins, Δα∝f1.16±0.04; and for the casein micelles, Δα∝f1.06±0.05. The ultrasonic attenuation of the soluble proteins is similar to that of other protein solutions; however, ultrasonic attenuation of the casein micelles differed from that of the soluble proteins. Using the theory of Allegra and Hawley [J. Acoust. Soc. Am. 36, 1545–1564 (1972)] for the ultrasonic attenuation of suspensions of elastic spheres, the expected attenuation for a model of the casein micelle suspension was computed. The frequency‐dependent attenuation, α’(f ), of the spheres is taken as an unknown for this model and, by varying α’(f ), a fit between the experimental data and the predictions of ...

Time-dependent polydispersity of growing colloidal aggregates: predictions from dynamic light scattering theory

A theory is presented for the variation of the polydispersity index measured in a dynamic light scattering experiment performed on a suspension of aggregating colloidal particles. The analytical forms of this index, calculated by cumulants analysis, are described for various models of colloidal aggregation and numerical data is presented showing the dependence of the index, which contains contributions from the dynamics of internal aggregate motions, on aggregate size and shape for aggregates of different fractal structure. The value of this dynamic optical index as a diagnostic for aggregation pattern is discussed and the analysis is applied to the results of a light scattering study of the heat-induced aggregation of a globular protein.

Spin-label studies on the lipoamide residues of the pyruvate dehydrogenase multienzyme complex of escherichia coli

Abstract The catalytic mechanism of the pyruvate dehydrogenase multienzyme complex of Escherichia coli has been investigated using spin-labelling and synthetic inhibitors of the enzyme. The lipoamide residues covalently bound to the lipote acetyltransferase component of the complex were selectively modified by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidino-oxyl in the presence of pyruvate. The electron spin resonance spectrum of the spin-labelled enzyme displayed two components, one highly mobile (correlation time about 1 ns) and the other immobilized (correlation time much greater than 50 ns). The temperature-dependence of the signal intensities of the two components suggested that the lipoamide residues can occupy two alternative states, the proportion of spin-labeled lipaomide in the mobile state increasing as the temperature is raised. Pyruvate caused a fall in the intensity of the ESR signal from the spin-labelled lipaomide residues in the presence of thiamine pyrophosphate and Mg 2+ . Acetoin also led to loss of ESR signal, but, with both pyruvate and acetoin, the additio of thiamine thiazolone pyprophosphate prevented the loss of signal. The rate of loss of ESR signal intensity was consistent with an intramolecular reaction between lipaomide-bound nitroxide groups and hydroxyethylthiamine pyrophosphate. The similar rates of reduction by isoascorbate of nitroxide spin-lebel free in solution and bound to the lipoamide residues of the pyruvate dehydrogenase complex indicated free access of the isoascorbate to the enzyme-boynd nitroxide groups. 13 P-NMR of acetyl phosphonate, a competitive inhibitor of the enzyme, bound to native enzyme and to spin-labelled enzyme, showed a large chemical shift on binding, but the linewidths did not indicate any spin-spin interaction between the nitroxide radicals and the 31 P nuclei. These results agree with recent experimental data from kinetic studies and 1 H-NMR spectroscopy on the system.

Probe Studies of the Gelation of Gelatin using the Forced Rayleigh Scattering Technique

Publisher Summary This chapter focuses on the probe studies of the gelation of gelatin using the Forced Rayleigh Scattering Technique (FRS). It presents a historical background as well as the principle of the FRS technique. The technique measures the diffusion of the chromophoric groups and can be used, therefore, to measure the diffusion of one species in a complicated mixture. In the study described in the chapter, the diffusion of a dye-labeled protein molecule, Bovine Serum Albumin (BSA), has been measured in an aqueous buffer solution and in an aqueous solution of gelatin. The study has been able to probe the development of structure in the gelling mixture through changes in the diffusional characteristics of the BSA revealed by FRS. Aqueous solutions of BSA are completely miscible with gelatin solutions at 40-50 °C without any apparent aggregation of the BSA. It is, therefore, possible to mix dye-labeled BSA with gelatin solutions and carry out FRS measurements on the BSA in the gelatin + BSA mixture. The chapter reports a FRS study of the self-diffusion of dye-labeled BSA in gelatin during the process of gelation.

Fourth order correlations in dynamic light scattering from flexible polymer chains: agreement of scaling laws with numerical predictions

We present calculations of g(2)(k1,k2; t), the fourth order correlation function of the electric field amplitude of light scattered by a flexible polymer chain, with and without hydrodynamic interactions using the Rouse and Zimm models respectively. Calculations of the second order correlation function, g(1)(k, t) are also presented. The results are shown to be in excellent agreement with theoretical scaling predictions in the limit of large N, N being the number of subunits in the chain. Calculations made using the Hearst approximation of the Zimm model show that numerical values of the scaling indices and the initial slopes of g(1) and g(2) undergo a smooth transition from the free-draining to the non-free draining limiting values as the strength of the hydrodynamic interaction is increased.