scholarly journals The macromolecular properties of blood-group substances. Sedimentation-velocity and viscosity measurements

1967 ◽  
Vol 105 (3) ◽  
pp. 1135-1145 ◽  
Author(s):  
J. M. Creeth ◽  
C. G. Knight
Keyword(s):  
Secondary Structure ◽  
Blood Group ◽  
Sodium Dodecyl Sulphate ◽  
Molecular Conformation ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Sedimentation Velocity ◽  
Random Coil ◽  
Partial Specific Volume ◽  
Blood Group Substances

1. Sedimentation-velocity, intrinsic-viscosity and partial-specific-volume measurements on a typical blood-group-specific glycoprotein are reported for a range of environmental conditions. 2. The sedimentation coefficients, S, are strongly concentration-dependent, and follow the reciprocal law; the limiting values at 2°, 25° and 45°, after correction to 25°, show slight dependence on temperature. 3. The intrinsic viscosities, [η], at 25° and 45° show more marked temperature-dependence, and correspond to a very asymmetric or very expanded molecular conformation. 4. From the value of the ratio K/[η], where K=S0.d(1/S)/dc, it is concluded that the molecular conformation is roughly spherical; application of the Einstein viscosity equation then suggests an expansion factor of about 60, compatible with a flexible configuration approaching that of a random coil. 5. The sedimentation coefficient is not affected by variation of ionic strength in the range 0·01–0·50, nor by pH in the range 3–10. 6. Sodium dodecyl sulphate at 1·5% produces a small decrease in S; the effect is greater than would be expected from the observed extent of binding, but is too small to correspond to a significant change in secondary structure; the serological activity is unaffected by sodium dodecyl sulphate. 7. All the properties observed indicate the absence of any secondary structure in blood-group substances.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

Germin: physicochemical properties of the glycoprotein which signals onset of growth in the germinating wheat embryo

1987 ◽  
Vol 65 (12) ◽  
pp. 1039-1048 ◽  
Author(s):  
William C. McCubbin ◽  
Cyril M. Kay ◽  
Theresa D. Kennedy ◽  
Byron G. Lane
Keyword(s):  
Circular Dichroism ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Helical Structure ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Cross Linking ◽  
Wheat Embryo ◽  
Circular Dichroïsm

The size and structure of germin, the homooligomeric glycoprotein which marks the onset of growth in germinating wheat embryos, has been examined by gel filtration, ultracentrifugation, electron microscopy, chemical cross-linking, and optical techniques (circular dichroism). Germin has a sedimentation coefficient (S20,w) of 7.3S, and a Stokes' radius (RS) of 4.5 nm, the latter value being compatible with the dimensions of the particle observed by negative staining in the electron microscope. By three methods (sedimentation equilibrium, sodium dodecyl sulphate (SDS) – polyacrylamide electrophoresis, S20,w/RS), the mean particle mass of the two closely related forms of germin (G and G′) is ca. 130 kilodaltons (kDa). Cross-linking with dimethyl suberimidate indicates that the oligomer is homopentameric, compatible with the molecular mass of the protomer (ca. 26 kDa) as determined by SDS–polyacrylamide gel electrophoresis. Using the Provencher and Glockner analysis to interpret circular dichroism measurements (in the far ultraviolet), both forms of germin contain about 10–20% α-helical structure, 50–60% β-sheet/turn structure, and 20–30% random coil. In a structure-inducing environment (45% trifluoroethanol), the α-helical structure increases to a value (35–40%) similar to that predicted by Chou–Fasman analysis of the protein sequence deduced by cDNA sequencing.

scholarly journals Physical properties and subunit structure of l-asparaginase isolated from Erwinia carotovora

1972 ◽  
Vol 126 (2) ◽  
pp. 361-379 ◽  
Author(s):  
K. A. Cammack ◽  
D. I. Marlborough ◽  
D. S. Miller
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Molecular Conformation ◽  
Sodium Dodecyl ◽  
Erwinia Carotovora ◽  
Optical Rotatory Dispersion ◽  
Spherical Particles ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Equilibrium Ultracentrifugation

1. l-Asparaginases from Erwinia carotovora and Escherichia coli (EC2 enzyme) are both capable of inhibiting and eliminating certain types of tumour cells. The Er. carotovora enzyme is a more basic protein, however, and in contrast with the EC2 enzyme it contains neither tryptophan nor cystine, and disulphide bonds are therefore absent. The molecule is very stable in solution from pH3.0 to about pH12.0, and is somewhat more stable at alkaline pH than is the Esch. coli enzyme. Calculations based on a s020,w 7.43S and a sedimentation-equilibrium molecular weight of 135000±10000 give a frictional ratio (f/f0) of 1.08. The molecular conformation is therefore very compact in solution, and the electron microscope shows the negatively stained molecules as almost spherical particles with a diameter of 7.2±0.7nm. 2. Sedimentation-velocity and equilibrium ultracentrifugation, in 5–8m solutions of urea and guanidinium chloride, and also electrophoresis in sodium dodecyl sulphate–polyacrylamide gel, reveal a dissociation of the native protein molecule into four subunits of similar molecular weight in the range 32500–38000. The enzymically inactive subunits can be physically reassembled into an active tetramer when urea is removed by dialysis. Although the subunit structures of the Er. carotovora enzyme and the Esch. coli enzyme molecules are similar, the secondary bonding forces holding the subunits together in the tetramer are somewhat stronger in the Er. carotovora enzyme. 3. The optical-rotatory-dispersion (o.r.d.) parameters that characterize the Cotton effects arising from ordered structure in the molecule are [m′]233=−3522±74° and [m′]200=9096±1700°. These show very marked changes as the secondary structure is disrupted and the molecule dissociates into subunits. A correlation pathway was traced on the basis of o.r.d. parameters and enzyme activity as the polypeptide chains were denatured and renatured (and reconstituted) into active molecules after the dilution of solutions in urea. Subunits resulting from treatment with sodium dodecyl sulphate do not show the typically disordered o.r.d. profile, but nevertheless they are inactive.

scholarly journals Purification and properties of succinyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney

1978 ◽  
Vol 173 (3) ◽  
pp. 759-765 ◽  
Author(s):  
J A Sharp ◽  
M R Edwards
Keyword(s):  
Isoelectric Focusing ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Coenzyme A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Polyacrylamide Gel ◽  
Transferase Activity ◽  
Coa Transferase

CoA-transferase (succinyl-CoA-3-oxo acid CoA-transferase, EC 2.8.3.5) isolated from sheep kidney was purified to homogeneity. The purified enzyme has a specific activity of approx. 200 units/mg. A mol.wt. of 110000 was obtained by gel filtration on Sephadex G-200, and a lower mol.wt. of 102000 was determined by analytical ultracentrifugation. A sedimentation coefficient of 5.6S was also determined. A subunit mol.wt. of 56000 was obtained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing of sheep kidney extracts indicated the presence of a single band of CoA-transferase activity with pI9.0. However, isoelectric focusing of purified CoA-transferase showed the presence of two peaks of CoA-transferase activity with pI values of 8.7 and 8.4, suggesting the presence of proteolytic activity during purification. Evidence for sheep kidney CoA-transferase being a dimer of two identical subunits has been obtained from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the amino acid composition, peptide ‘mapping’ and N-terminal analysis.

scholarly journals The molecular size of N-methylglutamate dehydrogenase of Pseudomonas aminovorans

1977 ◽  
Vol 167 (2) ◽  
pp. 509-512 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Partial Specific Volume ◽  
Triton X

N-Methylglutamate dehydrogenase, purified to a specific activity of 0.29 unit/mg of protein, gave one band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a molecular weight of 130 000. Enzyme-Triton complexes were found to have a partial specific volume of 0.73 cm3/g, suggesting that the protein binds less than 0.1 g of Triton/g of protein. A molecular weight for the intact enzyme in the presence of 1% (w/v) Triton X-100 of 550 000 suggested that the enzyme may be a tetramer.

Determination of the Molecular Weight and the Hydrodynamic Properties of a Polypeptide from the Thylakoid Membrane by Sedimentation, Diffusion and Binding Measurements in Dodecyl Sulphate Solutions

1975 ◽  
Vol 30 (9-10) ◽  
pp. 615-621 ◽  
Author(s):  
Hans Craubner ◽  
Friederike Koenig ◽  
Georg H. Schmid
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Equilibrium Dialysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Partial Specific Volume ◽  
Hydrodynamic Properties ◽  
Lamellar System ◽  
Self Diffusion

The molecular weight and hydrodynamic properties of a polypeptide isolated from the lamellar system of Antirrhinum chloroplasts were determined in sodium dodecyl sulphate solution by measurement of sedimentation velocity, diffusion and effective partial specific volume. The polypeptide fraction exhibits a molecular weight of 25 000 which agrees with the apparent molecular weight found by polyacrylamide gel electrophoresis. The molecular weight of the polypeptidesodium dodecyl sulphate micelle was 54 000, with a friction ratio of 1.6 which indicates an effective asymmetric hydrodynamic shape. For binding measurements self-diffusion equilibrium dialysis with dodecyl [35S] sulphate was used. In this case, dialysis equilibrium was reached within about 10 hours, in contrast to the dialysis with initial concentration differences which requires much longer times. A binding value of δD = 1.15g sodium dodecyl sulphate per g polypeptide was obtained which corresponds to a molar binding ratio of 100 mol dodecyl sulphate bound per mol of polypeptide. After the removal of dodecyl sulphate the polypeptide is present in an aggregated state. In phosphate buffers of pH 6.8 and 7.5 the aggregates preponderantly have sedimentation coefficients of 11.7 and 6.8 Svedberg units respectively. Assuming equivalent spheres the molecular weights were calculated to be 340 000 and 150 000.

scholarly journals Covalent labelling of the lutropin binding site. Evidence for a single Mr 90000 sialoglycopolypeptide

1984 ◽  
Vol 219 (2) ◽  
pp. 583-591 ◽  
Author(s):  
M K Metsikkö
Keyword(s):  
Binding Site ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Binding Activity ◽  
Polyacrylamide Gel ◽  
Receptor Complex ◽  
Stokes Radius ◽  
Triton X

Membrane-associated sialoglycopolypeptides of rat ovaries were oxidized with NaIO4, reduced with NaB3H4 and solubilized with Triton X-100. The solubilized proteins carrying the 3H label were subjected to affinity chromatography on human choriogonadotropin coupled to agarose. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate followed by fluorography revealed a single component of apparent Mr 90000. This component was abolished when ovaries saturated with choriogonadotropin were used as starting material. The above result is identical to that obtained previously by conventional detection methods [Metsikk ö & Rajaniemi (1982) Biochem. J. 208, 309-316] and indicates that the 3H-labelled lutropin/choriogonadotropin sialoglycopolypeptide was observed. The affinity-purified 3H-labelled protein co-eluted with the choriogonadotropin-binding activity solubilized with Triton X-100 from rat ovarian particles, showed a Stokes' radius of 6.2 nm and sedimented as a single band with a sedimentation coefficient of 5.1 S. The sedimentation coefficient of this 3H-labelled protein was not significantly altered when boiled in 1% sodium dodecyl sulphate, indicating that non-covalently associated subunits were not present. The 3H-labelled protein cosedimented with the choriogonadotropin-binding activity solubilized with Triton X-100 from rat ovary. When 125I-choriogonadotropin-receptor complex was covalently crosslinked with glutaraldehyde, an Mr 130000 component was produced as detected by sodium dodecyl sulphate gel electrophoresis. This component was extracted from the polyacrylamide gel and subjected to sucrose-density-gradient centrifugation in 0.1% Triton X-100. A single band sedimenting at the position of the 125I-choriogonadotropin-receptor complex solubilized from a prelabelled ovary was observed, exhibiting a sedimentation coefficient of 6.5S. These data suggest that the lutropin-binding site is a single sialoglycopolypeptide of Mr 90000, which binds one molecule of hormone resulting in an apparent Mr 130000 complex. The large Stokes' radius (6.2 nm) of the binding site is accounted for by bound detergent.

scholarly journals A study of the effects of reaction with formaldehyde on some optical and physical properties of reticulocyte ribosomes

1969 ◽  
Vol 114 (4) ◽  
pp. 743-751 ◽  
Author(s):  
R A Cox
Keyword(s):  
Electron Microscopy ◽  
Absorption Spectrum ◽  
Ribosomal Rna ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Optical Rotatory Dispersion ◽  
Ultraviolet Absorption Spectrum ◽  
Optical Rotatory

1. The optical rotatory dispersion and ultraviolet-absorption spectrum of ribosomal RNA in situ appear to be unchanged when the ribosome is dissociated into its RNA and protein moieties. 2. Reaction with 0·05% formaldehyde at 20° for 2hr. ‘fixes’ ribosomes so that they remain intact in 1% sodium dodecyl sulphate. 3. The RNA moiety of the ribosome undergoes a conformational change when ribosomes in 8% formaldehyde are heated at 70° for 10min. and cooled to 20°. After this treatment no double-helical character can be detected, but neither the sedimentation coefficient nor the morphology of the ribosome determined by electron microscopy is altered. 4. It is concluded that the RNA moiety of reticulocyte ribosomes is freely accessible to formaldehyde.

scholarly journals The Isolation of Aggregates of Spectrin From Bovine Erythrocyte Membranes

1975 ◽  
Vol 28 (3) ◽  
pp. 259 ◽  
Author(s):  
GB Ralston
Keyword(s):  
Ionic Strength ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Sedimentation Equilibrium ◽  
Erythrocyte Membranes ◽  
Bovine Erythrocyte ◽  
Molecular Weights ◽  
Wide Range

Aggregated states of spectrin from bovine erythrocyte membranes can be detected in sedimentation velocity experiments. These aggregates have been isolated by means of gel filtration on columns of 4 % agarose. They appear to be stable over a wide range of pH and ionic strength, although they are dissociated by sodium dodecyl sulphate. Sedimentation equilibrium measurements yielded values of 960000 and 480000 for the molecular weights of the major aggregates, corresponding to a tetramer and dimer, respectively. The presence of different aggregated states in spectrin preparations may explain the wide variation in the reported physica~ properties of spectrin.

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