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H D Ellerton

Publications and source records attributed to H D Ellerton.

10 recordsLinked to original sources
PubMed1989

The effect of temperature on the renaturation of alpha-crystallin.

The effects of variations in temperature and protein concentration on the renaturation of bovine alpha-crystallin have been examined using gel permeation chromatography, sedimentation analysis, fluorescence spectroscopy and electron microscopy. High protein concentration (3-53 mg/ml) were found to generate heterogenous populations of aggregates. It was concluded that concentrations above 3 mg/ml were inappropriate for renaturation of alpha-crystallin. Aggregates with molecular masses gradually increasing from 461,000 to 695,000 Da were produced with increasing temperature over the range 6-39 degrees C. Electron microscopy demonstrated that the reaggregates were composed predominantly of particles with circular cross-sections and mean diameters of 13-14 nm. As the renaturation temperature increased, increasing amounts of sheet-like structures were observed. Tryptophan accessibility to acrylamide quenching decreased in these aggregates as the size increased. These observations are consistent with the concept that there is no unique quaternary structure, or set of structures, for alpha-crystallin but that the protein can exist in a variety of forms containing different numbers of subunits.

Animals↗
PubMed1988

Specific dissociation of alpha B subunits from alpha-crystallin.

Exposure of bovine alpha-crystallin to 0.1 M glycine at pH 7 decreases the average molar mass of the protein from 700 to 420 kDa. When the pH is lowered to 2.5, in the same buffer, the alpha B chains specifically dissociate from the aggregates, leaving a particle of 290 kDa containing only alpha A chains. The decrease in the molar mass corresponds to the mass of the alpha B chains in the original aggregate. The pH-dependent dissociation is fully reversible. Similar changes were observed with rat and kangaroo alpha-crystallins but the dogfish protein was not affected. Sedimentation velocity analyses and fluorescence spectroscopy yielded a pK, for the dissociation, of 3.7 for alpha-crystallin and 4.0 for a homopolymer constructed from purified alpha B2 polypeptides. An alpha A2 homopolymer was virtually unaffected by the lowering of pH. The products from the dissociation were isolated and their properties studied by sedimentation analysis and acrylamide quenching of tryptophan fluorescence. The alpha B chains were found to be completely denatured, whereas the structure of the alpha A chains, in the 290 kDa, particle, were only slightly altered. Comparisons of the sequences of the various proteins examined suggested that decreased ionization of aspartic acid 127 in the alpha B chain was responsible for the specific dissociation of this polypeptide.

Animals↗
PubMed1982

The physico-chemical and functional properties of Abarenicola affinis affinis (Ashworth) extracellular haemoglobin (erythrocruorin).

The purified erythrocruorin from the Arenicolid polychaete, Abarenicola affinis affinis (Ashworth) has an S020,w of 57.5 S, and a molecular weight of 3.29 . 10(6) as determined by sedimentation equilibrium at pH 7.0. In the electron microscope it displays a two-tiered hexagonal array characteristic of other annelid erythrocruorins and chlorocruorins, with dimensions of approximately 25.5 nm diameter and 17.0 nm height. The isoelectric point of the molecule is at pH 4.63, and its amino acid composition and absorption spectrum are similar to those of other annelid erythrocruorins. The haem and iron contents are 2.77% and 0.23%, corresponding to minimum molecular weights of 22 250 and 24 800, respectively. In pH-induced dissociation studies, the erythrocruorin is found to be stabilized by Ca2+ up to pH 8.6, while in the presence of EDTA, dissociation begins at pH 7.8. In discontinuous gel electrophoresis, dissociated erythrocruorin gives subunits with molecular weights 263 000, 117 500, 68 400, 35 900 and 28 800, while, in detergent electrophoresis in the presence of 2-mercaptoethanol, polypeptide chains with molecular weights 13 700, 15 800, 24 700, 27 800 and 33 600 are obtained. Abarenicola erythrocruorin is also shown to bind oxygen cooperatively with a Hill coefficient h = 4.0 and P50 = 6.0 mmHg at pH 7.0 and 25 degrees C. Over the pH range of 7.0 to 7.8 the molecule lacks an alkaline Bohr effect.

Amino Acids↗
PubMed1982

The subunits of Abarenicola affinis affinis (Ashworth) extracellular haemoglobin (erythrocruorin).

The subunits of the erythrocruorin of Abarenicola affinis affinis (Ashworth) were investigated by various physicochemical methods. Gel chromatography at pH 9.0 in Sephadex G-200 gave three main protein peaks with molecular weights of 240 000, 100 000 and 31 000, respectively. Polyacrylamide gel electrophoresis showed considerable heterogeneity in the subunits, although the main components gave similar molecular weight ranges to the above. Anomalous results were also obtained in SDS-polyacrylamide gel electrophoresis. In the analytical ultracentrifuge, the subunit from the first peak showed a sedimentation coefficient of 10 S and Mw 250 000, and the second yielded s20,w = 5 S. Functionally, the 10 S subunit showed cooperative oxygen binding, but the Hill coefficient was lower than that observed for the native molecule. A possible model for the subunit structure of Abarenicola affinis affinis erythrocruorin is discussed.

Animals↗
PubMed1977

Hydrogen ion titration and amino acid analysis of hemocyanin from the spiny lobster Jasus edwardsii.

Potentiometric and spectrophotometric titrations, isoelectric focusing and amino acid analyses, have been made on the hemocyanin from Jasus edwardsii. Counts of acidic neutral and alkaline groups were made from the titrations, enabling comparisons to be made with the amino acid analysis. Thermodynamic analysis of the data indicated that changes in the native protein structure took place at pH 4.0, 8.4 and 10.7. These observations are discussed in terms of dissociation, shifts in pK and conformational changes in the protein.

Amino Acids↗
PubMed1976

The subunit structure of the hemocyanin from the crayfish Jasus edwardsii.

The hemocyanin from the crayfish Jasus edwardsii(=lalandii) has been studied using ultracentrifugation, viscosity, circular dichroism and oxygen binding techniques. Sedimentation velocity experiments at pH 7.0 indicated the presence of principal species with S 20w=16.4 S, and at higher pH the presence of a species with S20,w=5.2S. Sedimentation equilibrium experiments yielded molecular weights of 490 000 and 81 000 respectively, indicating that the larger unit is a hexamer of the monomer unit. However, preliminary experiments with gel filtration and electrophoresis under denaturing conditions indicate that more than one monomer species may be present with molecular weight in the range 76-100 000. Circular dichroism (CD) spectra are presented at pH 7.0,8.6,10.0 and 11.0 for oxy-, deoxy- and apo-hemocyanins. Slight differences were observed in the magnitude of the bands in the presence or absence of Mg++. Oxygen binding studies have been made at pH 6.1,7.0,8.8 and 10.6, in the presence of 0.01 M MgCl2. The extent of cooperative binding was indicated by a maximum value of n=3.7, and a pronounced bohr effect was observed.

Animals↗