Torben Graves Pedersen

Isolation and characterization of the folate-binding protein from cow's milk

The folate-binding protein from cows milk has been purified in milligramme scale by combination of ion-exchange chromatography and affinity chromatography. The molecular weight has been determined by sedimentation equilibrium ultracentrifugation and found to be 30,000±2,000. The amino acid composition is compatible with this value. The molecule contains six disulphide bridges and no free SH-groups. The three per cent carbohydrate content was accounted for by six glucosamine residues per mole of protein. About 50 per cent of the amino acid sequence has been delineated, including the N-terminal sequence and the C-terminal sequence. Isoelectric focusing gave rise to four major peaks with isoelectric points ranging from 8.5 to 7.6, but no heterogeneity was observed in the sequence.

Dependence of aggregation and ligand affinity on the concentration of the folate-binding protein from cow's milk

Ultracentrifugation and gel-filtration studies showed that the folate-binding protein from cows milk possessed a remarkable aggregation tendency at pH 7.4. Aggregation was enhanced in the presence of folate which suggested an interrelationship between the mechanisms of ligand binding and polymerization. The degree of polymerization increased with increasing concentrations of binding protein. Thus, while the monomer prevailed at 1 nM, a polymer composed of more thn 32 monomers was formed at 130 microM. Two characteristics of folate binding, i.e., Scatchard plots that were convex upward and a ligand affinity that was inversely proportional to the concentration of binding protein, could be interpreted in terms of ligand binding to a polymerizing system in which the polymerization equilibria affect the ligand affinity.

Aggregation of a folate-binding protein from cow's milk

Sedimentation equilibrium experiments of folate-binding protein from cows milk performed in the analytical ultracentrifuge at pH 5.0 showed a single molecular species with a molecular weight of 30,000. In contrast, at pH 7.4 sedimentation equilibrium indicated molecular weights ranging from about 30,000 to at least 300,000. Sedimentation velocity experiments at pH 5.0 showed a single symmetrical peak with s20,w=2.8 S in agreement with a molecular weight of about 30,000. At pH 6.0 was seen a faster moving boundary (s20,w∼6S) in addition to the slow one. At higher pH two peak maxima were seen: a slow one with sedimentation coefficients of about 5S and a fast one increasing from 9S to 11S with increasing pH. Addition of folate further increased the sedimentation rates. By assuming thatGilberts rapid monomer-n-mer equilibrium theory was valid also for a lower polymer in equilibrium with a higher polymer our data can be interpreted as an equilibrium between a tetramer and a polymer consisting of more than 16 monomers. In the case of folate addition the results may similarly be interpreted as an equilibrium between an octamer and a polymer composed of more than 32 monomers.

Volumetric properties of aqueous solutions of alkali halides

The density of aqueous solutions of LiF, NaF, or KF, at room temperature, is measured as a function of the concentration. The measurements, together with corresponding data available on other alkali halides, are used to illustrate the concentration dependence of the apparent molar volume of the electrolytes in aqueous solution.The data is in accordance with the conception that an essential, negative contribution to the volume is due to a progressive hydration of the ions upon dilution (i.e. with increasing water concentration).The peculiar behaviour of the Li+-salts, compared with the other alkali halides, is associated with the small size of the Li+-ion compared with a water molecule.

Fractionation of protein components from beer by density gradient centrifugation

A protein-rich beer fraction obtained by alcohol precipitation has been fractionated by cesium chloride density gradient centrifugation into three fractions. One with buoyant density 1.27 g·ml−1 contained essentially pure protein, while a fraction with buoyant density 1.37 g·ml−1 contained protein and some carbohydrate, probably covalently bound to the protein. A large fraction with a buoyant density of 1.60 g·ml−1 consisted of carbohydrate.Preparative isoelectric focusing of the protein-containing fractions revealed in both cases a protein peak with pI=4.7 which for the 1.37 g·ml−1 fraction coincided with a carbohydrate peak.

Buoyant titration of ovalbumin in four alkali halides. Hydration and ion binding

The buoyant titrations of ovalbumin in CsCl, RbCl, CsBr and RbBr were measured between pH 2 and 13. The buoyant densities were found to depend on the salt employed to generate the density gradient and the solution pH. At low pH, nearly identical buoyant densities were observed in solutions having a common anion while at high pH, salt solutions having the same cation produced nearly indentical buoyant densities. The buoyant density of ovalbumin in RbBr was found to decrease as the pH was increased from 2 to 6. This is the first demonstration of a drop in the buoyant titration curve for a biopolymer.A model based on an invariant partial specific volume, normal titration of all amino acid residues, the binding of a counter-ion to each ionized residue and all hydration associated with these salt pairs was constructed and satisfactorily accounted for all four buoyant titration curves. These results indicate that no water is bound to the neutral portion of this protein.

The partial molar volumes of six alkali halides and their ions as a function of concentration

The application of the technique of sedimentation equilibrium in density gradients to proteins requires a knowledge of the partial molar volumes of bound ions and water. Partial molar volumes of water and six alkali halides, CsCl, CsBr, CsI, RbCl, RbBr and RbI were computed as a function of concentration throughout the entire solubility ranges. These values were apportioned into partial molar volumes of the ions according to the cubes of Paulings ionic radii. These data are tabulated as a function of density and water activity. The partial molar volumes of all of the salts and the ions increase with increasing salt concentration. Unique triple points are observed in the plots of the partial molar volumes of both the rubidium and cesium ions as a function of density.

Buoyant densities of subtilisin Carlsberg in dissolved and crystalline states

The buoyant densities of subtilisin Carlsberg in dissolved and crystalline states, including glutaraldehyde crosslinked crystals, have been determined by equilibrium centrifugation at neutral pH in the analytical ultracentrifuge in concentrated solutions of CsCl. Due to the negligible diffusion of suspended crystals they form a sharp band at the position in the gradient corresponding to their density. Different crystal preparations may be separated in a single gradient when their densities differ by more than 0.005 g·ml1 and the results are reproducible to±0.001 g·ml-1. Glutaraldehyde stabilized crystals were found to have higher buoyant densities than crystals of unmodified subtilisin. The increase in density due to the glutaraldehyde cross-linking can be explained if a polymer composed of approximately 5 glutaraldehyde residues is bound per pair of blocked lysine residues. Unmodified subtilisin Carlsberg shows identical densities in the crystalline and soluble states in CsCl solutions. The observation is consistent with identical molar volumes of subtilisin Carlsberg in solution and crystals and no charged groups being involved in intermolecular binding in the crystals.

Volumetric effects due to ion-solvent interaction in aqueous electrolyte solutions

The apparent volume of a series of chlorides dissolved in water—including hydrochloric acid and chlorides of divalent cations—is calculated from available data on the density of the aqueous solutions.The concentration dependence of the volume of the electrolytes illustrates the long range electrostriction of water by the ions. The volume of the cations, at infinite dilution, reveals that the electrostriction observed, is composed of two opposite volume effects. The experimental data supports the view that bulky, “icelike” water structures predominate near the surface of a small ion, and dense structures, less organized than bulk water, are present at a distance from the ionic center.

Buoyant and potentiometric titrations of synthetic polypeptides III. Poly-l-lysine and poly-l-histidine in five salt solutions

The buoyant titrations of poly-l-lysine and poly-l-histidine in CsCl, RbCl, CsBr, RbBr and KBr were measured. Large differences in buoyant densities measured at low pH were observed for both polymers. Densities in the alkali chloride solutions are lower than for the bromides and the buoyant density increases as the size of the cation decreases in the halide series. All buoyant densities converge to a common value for each polymer at high pH. These data are interpreted in terms of salt-pair formation at low pH and preferential hydrations were computed both for this charged species at low pH and for the neutral polymers at high pH. The resulting data were correlated with the water activity in each buoyant solution. The variation of hydration with water activity for the charged species is found to be similar to that previously reported for bovine serum mercaptalbumin while the hydrations of the neutral polymers are found to be nearly independent of water activity. Approximate observed ionization constants were determined for each polymer in four salt solutions.