Amylose optical rotation in some mixed solvent systems

This paper extends a previous study in which a discontinuity in the specific rotation of open chain α-l,4-linked glucopyranosides in the water–dimethyl sulfoxide (H₂O–DMSO) system was attributed to a symmetry change about a polymer chain segment. Optical rotation of amylose, cyclohexamylose, methyl β-maltoside, and dextran was measured in the following mixed solvent systems: formamide–dimethyl sulfoxide (F-DMSO), ethylenediamine–dimethyl sulfoxide (E–DMSO), and hexamethylphosphoramide–dimethyl sulfoxide (HMPA–DMSO). Refractive index measurements were used in an attempt to detect hydrogen bonding between solvent components. The specific rotation of amylose corrected for variation in refractive index (CSR), as a function of solvent composition, showed a discontinuity at solvent compositions corresponding to about 1 mole F to 2 moles DMSO and to 1 mole E to at least 8 moles DMSO. A discontinuity in the CSR function of amylose in the H₂O-DMSO mixed solvent that occurs at 25°C is not observed at 70°C. The CSR function of methyl-β-maltoside exhibits a discontinuity in solvent composition corresponding to mole ratios between 2F–DMSO and 3F–DMSO. Present results indicate that an amylose chain segment may undergo a symmetry change in solvent compositions corresponding to mole ratios between F–DMSO and F–2DMSO. Our CSR measurements of amylose and model compounds in E–DMSO and HMPA–DMSO do not permit us to distinguish between possible changes in amylose chain segment symmetry and solvent interactions that could affect symmetry properties of the glucopyranose ring.     

Preparative fractionation of lignin by gel-permeation chromatography

The combination of an agarose gel (Bio-Gel A) and a dioxane–water (1:1) solvent system allowed the fractionation, on a preparative scale, of a very polydisperse, non-derivatized lignin preparation (enzymatically liberated lignin prepared from sweetgum sapwood with Lenzites trabea). Three fractions differing markedly in molecular weight were obtained. A gel of crosslinked alkylated dextran (Sephadex LH-20) with the same solvent system allowed division of the lowest molecular weight fraction into two fractions. These materials were characterized by measurements of intrinsic viscosity and number-average molecular weights in dimethylformamide and dioxane–water. It was established that the two highest molecular weight fractions were associated in an average trimeric form in dioxane-water (1:1) as compared to the form (considered to be molecular) that occurred in dimethylformamide. Molecular size distributions and eluant volumes of the fractions were determined with a Sephadex G-100–formamide system, the latter being one of the most powerful nonaqueous solvents for lignin. Adsorption effects were known to be absent in this case, and the lignin molecules were considered to be unassociated in formamide. The four fractions were distinguishable with the formamide–G-100 system, thus indicating that the original fractionation was based on molecular size. The enzymatically liberated lignin contained molecules that comprised a continuum of molecular weights from approximately monomeric to molecules that were at the limit of the solvating power of dioxane–water (1:1) and dimethylformamide. Limited physicochemical data were consistent with a compact, approximately spherically symmetric shape of the lignin in solution.     

Serum protein determination by high-performance gel-permeation chromatography

A general high-performance gel-permeation chromatography (HPGPC) method was developed to determine protein in human serum with improved sensitivity and speed. The optimum UV wavelength for protein detection was found to be 210 nm, by comparing the protein values obtained by varying the UV wavelength of the HPLC detection system with the protein values obtained from spectrophotometric protein assays, i.e., the bicinchoninic acid (BCA) method and the biuret method. The analysis time was less than 1 min. Since this HPGPC serum protein assay method is simple and rapid, it is expected to be particularly well adapted for use in clinical laboratories.     

Extended application of gel-permeation chromatography by spin column

Separation of small volumes of proteins from unbound ligands or reequilibration with buffer by passing through a 1-ml Sephadex G-50 column under mild centrifugal force is a popular technique. Here it has been demonstrated that other Sephadex matrix could similarly be used for complete or partial separation of protein molecules. Proteins to be eluted at void volume are recovered near quantitatively, while others are partly or almost completely retained depending on molecular size. Calibration curves using standard proteins of Mw 12.5 to 440 kDa with Sephadex G-50-G-200 representing recovery versus molecular weight show profiles as expected from the fractionation ranges of the column matrix. The procedure may be applied to follow protein association-dissociation reactions if the molecular weights of the species concerned are known and a proper matrix exists for separating them. Equilibrium unfolding transitions constructed with model proteins in presence of 0-8M urea using recovery as an index correspond to profiles obtained from other physical measurements. This may be a convenient approach to follow change of protein hydrodynamic volume quickly when a parallel methodology is not readily available.     

High-performance gel-permeation chromatography of chitosan samples

The conformational properties of chitosan, a copolymer of 2-acetamido-2-deoxy- -glucose and 2-amino-2-deoxy- -glucose, have been examined both in solution and in the solid state. Little has been reported previously on the determination of molecular weight using high-performance gel-permeation chromatography (HPGPC) and no attempt has been devoted to an examination of molecular weight distribution. An HPGPC method for evaluating the above-mentioned parameters for chitosan samples having different molecular weights and different degrees of acetylation was therefore developed. Calibration using sodium polystyrene sulfonate commercial standards of narrow molecular weight distribution could not be carried out in the solvent system used for chitosan. Calibration was therefore performed by means of chitosan samples obtained by depolymerization.     

Theory of preferential solvation of nonelectrolytes

The concepts of local compositions around a solute and preferential solvation of a solute are defined in terms of the Kirkwood-Buff integrals. The difference between the local and the bulk composition is a measure of the preferential solvation of a solute with respect to the various components of the solvent. A statistical mechanical theory is developed that leads to simple relationships between local compositions and experimentally measurable quantities. Some preliminary results on preferential solvation of methane in mixtures of water-ethanol and water-p-dioxane are presented.     

A statistical mechanical theory of unfolding of globular proteins induced by preferential binding of solvent components in water-organic solvent systems

A statistical mechanical model was developed for use in connection with the problem of preferential binding of solvent components to proteins and of conformational transition in water-organic solvent systems. The model is a statistical one for the conformational transition of globular proteins induced by the adsorption of solutes in the solution, considered as a nearest-neighbor problem in statistical mechanics. Although a few illustrative examples are given, the actual interpretations of the experimental data using this theory are reserved for a later paper.     

Preferential and absolute interactions of solvent components with proteins in mixed solvent systems

An equation is derived relating total and preferential interactions of solvent components with macromolecules in a three-component system. Application of this equation to literature data shows that binding of non-polar solvents to proteins parallels the unfolding of the latter. This observation is discussed in terms of local inter-residue and residue-solvent interactions.     

Relationship between preferential interaction of a protein in an aqueous mixed solvent and its solubility

The present paper is devoted to the derivation of a relation between the preferential solvation of a protein in a binary aqueous solution and its solubility. The preferential binding parameter, which is a measure of the preferential solvation (or preferential hydration) is expressed in terms of the derivative of the protein activity coefficient with respect to the water mole fraction, the partial molar volume of protein at infinite dilution and some characteristics of the protein-free mixed solvent. This expression is used as the starting point in the derivation of a relationship between the preferential binding parameter and the solubility of a protein in a binary aqueous solution. The obtained expression is used in two different ways: (1) to produce a simple criterion for the salting-in or salting-out by various cosolvents on the protein solubility in water, (2) to derive equations which predict the solubility of a protein in a binary aqueous solution in terms of the preferential binding parameter. The solubilities of lysozyme in aqueous sodium chloride solutions (pH=4.5 and 7.0), in aqueous sodium acetate (pH=8.3) and in aqueous magnesium chloride (pH=4.1) solutions are predicted in terms of the preferential binding parameter without any adjustable parameter. The results are compared with experiment, and for aqueous sodium chloride mixtures the agreement is excellent, for aqueous sodium acetate and magnesium chloride mixtures the agreement is only satisfactory.     

Skeletal keratan sulfate chain molecular weight calibration by high-performance gel-permeation chromatography

A method has been developed for the molecular sizing of skeletal keratan sulfate chains using an HPLC gel-permeation chromatography system. Keratan sulfate chains and keratanase-derived oligosaccharides were prepared from the nucleus pulposus of bovine intervertebral disc (6-year-old animals). A Bio-Gel TSK 30 XL column eluted in 0.2 M NaCl and at 30 degrees C was calibrated with keratan sulfate oligosaccharides of known size as well as 3H-end-labeled keratan sulfate chains to yield the relationship.     

Analysis of mucin-derived oligosaccharides by medium-pressure gel-permeation and amino-plate thin-layer chromatography conducted in conjunction

Oligosaccharides present in mucin were labeled by reduction with NaB3H4 and separated by gel-permeation chromatography with a Toyopearl HW-40S column using 0.1 M pyridine acetate, pH 5.0, as the solvent. Each fraction was further analyzed by thin-layer chromatography (TLC) on a Funagel AMP plate, a glass plate precoated with 3-aminopropyl-bonded silica. Acetonitrile/10 mM triethylamine acetate (3/2, by volume) served as the solvent. The sites of oligosaccharides on the TLC plate could be determined according to size, anionic charge, and sugar composition. They could thus be "mapped" on the plate. In this manner, the distribution of oligosaccharides on bovine submaxillary mucin and rat gastric mucin was determined. Each radiolabeled oligosaccharide in newly synthesized rat gastric mucin, metabolically labeled with [14C]glucosamine or [35S]sulfate, was also identified by this method.     

Alkaline solvent systems for thin-layer chromatography of bile acids

Thin-layer chromatographic separation of the common bile acids and their taurine and glycine conjugates in chloroform-methanol-ammonia is reported. An alkaline system offers advantages for the separation and nondestructive staining of bile acid conjugates.     

Structural thermodynamics of protein preferential solvation: osmolyte solvation of proteins, aminoacids, and peptides

Protein stability and solubility depend strongly on the presence of osmolytes, because of the protein preference to be solvated by either water or osmolyte. It has traditionally been assumed that only this relative preference can be measured, and that the individual solvation contributions of water and osmolyte are inaccessible. However, it is possible to determine hydration and osmolyte solvation (osmolation) separately using Kirkwood-Buff theory, and this fact has recently been utilized by several researchers. Here, we provide a thermodynamic assessment of how each surface group on proteins contributes to the overall hydration and osmolation. Our analysis is based on transfer free energy measurements with model-compounds that were previously demonstrated to allow for a very successful prediction of osmolyte-dependent protein stability. When combined with Kirkwood-Buff theory, the Transfer Model provides a space-resolved solvation pattern of the peptide unit, amino acids, and the folding/unfolding equilibrium of proteins in the presence of osmolytes. We find that the major solvation effects on protein side-chains originate from the osmolytes, and that the hydration mostly depends on the size of the side-chain. The peptide backbone unit displays a much more variable hydration in the different osmolyte solutions. Interestingly, the presence of sucrose leads to simultaneous accumulation of both the sugar and water in the vicinity of peptide groups, resulting from a saccharide accumulation that is less than the accumulation of water, a net preferential exclusion. Only the denaturing osmolyte, urea, obeys the classical solvent exchange mechanism in which the preferential interaction with the peptide unit excludes water.     

Assessing accumulated solvent near a macromolecular solute by preferential interaction coefficients

Biological macromolecules are often studied in mixed solvents. To understand cosolvent-macromolecule interactions, the preferential interaction coefficient, Gamma(3), may help determine surface solvent compositions. Gamma(3) measures the amounts of water, B(1), and cosolvent, B(3), within the "local domain," the (possibly far-reaching) region surrounding the macromolecule where the solvent is non-bulk-like. The local domain's boundary is, however, vague and it is unclear which molecules are counted in B(i). It is useful to explore a simple model system to make B(i) more concrete and to understand which aspects of the surface solvent distribution, rho(x), are sampled by Gamma(3). We performed computer simulations on a two-dimensional (2D) system consisting of a hard-wall solute (the macromolecule) in a mixed solvent (hard disks of different radii). We simultaneously calculated Gamma(3) and rho(x). We found that 1) in practice, the local domain's boundary is demarked by the outer limit of the first cosolvent (not water) layer; B(i) mainly counts the solvent near the macromolecule; 2) assuming B(1) to count only the waters within the first water layer is a poor approximation; 3) when determining B(1) and B(3), water and cosolvent molecules must be counted from the same region of space. We speculate that these 2D results may serve as a first-order approximation for the dominant contributions to Gamma(3) even in three dimensions, so long as the cosolvent is not strongly excluded from the macromolecular surface and there is no significant long-ranged solvent structure.     

High pressure liquid chromatography solvent systems for studies of bile acid biosynthesis

Reversed phase high pressure liquid chromatography (HPLC) solvent systems have been developed for the separation of intermediates in the formation of bile acids and bile acid conjugates from cholesterol. Four different mobile phases (water-methanol, 10 mM acetate buffer (pH 4.37)-methanol, 30 mM trifluoroacetic acid (pH 2.9 with triethylamine)-methanol, and 50 mM potassium phosphate buffer (pH 7.0)-2-propanol) have been applied to obtain separation of all the main intermediates with use of the same reversed phase column (Zorbax ODS).     

Calcium-dependent changes in properties of human prothrombin: A study using high-performance size-exclusion chromatography and gel-permeation chromatography

High-performance size-exclusion chromatography using a TSK 3000 SW column and aqueous gel filtration with Sephacryl S-200 SF have been used to characterize the effects of calcium ions on the hydrodynamic properties of human prothrombin and prethrombin 1. The results suggest that the effective hydrodynamic radius of prothrombin is less in the presence than in the absence of calcium ions. In addition, when using the TSK-3000 SW column, Ca²⁺-dependent formation of a hydrophobic site in the fragment 1 region of prothrombin results in an apparent further decrease in hydrodynamic radius.     

A trace-enrichment technique for the loading of gel-permeation high-performance liquid chromatography columns

Gel-permeation high-performance liquid chromatography (GP-HPLC) columns provide rapid high-resolution separations but are frequently limited to analytical tasks because the injection volumes must be small. The reduction of volume required for the loading of solutes can often be impractical and lead to poor recoveries. We have developed a trace-enrichment technique to circumvent this problem. By placing a Waters Guard Pak within the loop of a Valco injector and connecting a pump to the injection port it is possible to concentrate proteins and peptides onto the guard column from relatively large volumes. Enrichment onto a reversed-phase guard column insert is achieved by loading solutes in an aqueous solution or one of low organic solvent concentration. Provided that the GP-HPLC is mean-while equilibrated with a solvent system of sufficiently high organic solvent concentration (i.e., 40% acetonitrile containing 0.1% trifluoroacetic acid) it is possible to elute material that has been loaded in this manner by simply placing the injection loop in line with the column. The solvent strength abruptly increases and the peptide or protein sample is loaded onto the column in a very small volume. We have applied this loading principle to both analytical and semipreparative problems. The amino-terminal fragment of pro-opiomelanocortin (POMC) has been extracted from a single human fetal pituitary (18 weeks gestation) and characterized in terms of its molecular weight. This study indicated that no proteolytic processing of the amino-terminal fragment of POMC takes place at this stage in development. In a larger scale application the amino-terminal fragment of POMC was purified from bovine anterior pituitaries.(ABSTRACT TRUNCATED AT 250 WORDS)