Solubilities of water-insoluble dyes in internal water of swollen Sephadex gels.

It was found that internal water of swollen Sephadex gels could dissolve appreciable amounts of water-insoluble dyes, azobenzene (AB) and dimethylaminoazobenzene (DMAB). The solubilities of AB and DMAB increased exponentially with the concentration of dextran chain in swollen Sephadex gels, i.e., plots of solubility (CG) vs. dextran-chain concentration (Cf) were well described by a family of quartic regression curves (CG=kcf4+C0,k and C0 being constants). The thermodynamic parameters pertaining to the transfer of the dyes from external water to internal water indicate that in water extensively hydrated by gel matrices, "iceberg" formation is markedly hindered and hence the hydrophobic free energy is correspondingly reduced, thereby increasing the solubilities of hydrophobic dyes. Since the solubilities of the dyes are proportional to the fourth power of the dextran-chain concentration in swollen Sephadex gels, it can be assumed that the dyes are accommodated in cooperatively hydrated water contained in unit cells made up of four dextran chains.

Thermodynamic analysis of the nonspecific interaction of sodium dodecyl sulfate with swollen Bio-Gel beads.

In frontal gel chromatography on Bio-Gel P-2, sodium dodecyl sulfate (SDS) at the concentrations below its critical micelle concentration (cmc) showed anomalously high partition coefficients (Kav obs) indicative of strong interactions with the swollen gel phase; further, Kav obs was found to increase with concentration and temperature. This preferential partition of SDS in the Bio-Gel phase was analyzed in terms of the transfer free energy of SDS from the mobile phase (0.1 M NaCl) to the swollen Bio-Gel phase. The results showed that the overall transfer process is primarily governed by hydrophobic free energy arising from the anomalous nature of hydrated water in the gel matrix; that is, in highly hydrated water "iceberg" formation is evidently limited and the hydrophobic free energy is accordingly lowered, resulting in the preferential partition of SDS in the swollen Bio-Gel phase. The increase in the negativity of transfer free energy with concentration, though relatively small, indicated a definite tendency for the formation of SDS clusters in the gel phase. Finally, a model illustrating the states of SDS molecules in the gel matrix is presented, which may also be pertinent to SDS-protein and SDS-amylose complexes.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bovine serum albumin oligomers produced by lipid peroxidation.

The oligomers of bovine serum albumin were produced by controlled reaction with peroxidizing linoleic acid to examine their possible utility as calibration proteins insodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polymerization was effected in reaction mixtures containing linoleic acid undergoing peroxidation in the presence of ascorbic acid, and conditions that yield soluble oligomers with a wide molecular weight distribution were established. The interaction of these soluble oligomers with sodium dodecyl sulfate exhibited a binding isotherm indistinguishable from that obtained with bovine serum albumin. Furthermore, sodium dodecy sulfate-polyacrylamide gel electrophoresis of the albumin oligomers conformed to the empirical relation of molecular weight to mobility that pertains to the use of these oligomers as standard molecular weight markers.

An osmotic effect operative in frontal gel chromatography.

The osmotic effect operative in frontal gel chromatography was quantitatively studied. When mixtures of a non-penetrating solute (Kav = 0) and a partially penetrating solute (0 less than Kav less than 1) were subjected to frontal gel chromatography, the latter formed a coextensive concentration gradient across the trailing boundary of the former, leading to the formation of a second plateau where the concentration exceeded that of the original solution plateau. It was shown that this anomaly, which we have previously predicted, was a direct consequence of osmotic perturbation of the bead size of the Sephadex gel and could be satisfactorily described by an equation based solely on the osmotic distention of the gel beads. Finally, the implications of the osmotic effect in the frontal chromatographic analysis of acceptor-ligand interactions is discussed and a method for correcting this effect is presented.

Macromolecular components of the vitelline membrane of hen's egg. III. Physicochemical properties of glycoprotein II.

A glycoprotein fraction (GP-II) has been isolated from the vitelline membrane of hen's egg and its physicochemical properties clarified. GP-II is composed of polypeptide (92%), neutral sugar (4%), hexosamine (3.3%), and sialic acid (0.6%). The constituent neutral sugars of this glycoprotein are fucose, mannose, and galactose, in a molar ratio of 2:6:5. An interesting feature of the amino acid composition of GP-II is the high proportion of proline. GP-II exists in an aggregated form and is hydrophobic in nature. Upon velocity sedimentation in 0.5% SDS solution, it showed a hypersharp boundary with an apparent sedimentation coefficient of 5.6 S. Reduction of GP-II, however, gave a single component of 3.6 S which seems to be a subunit of GP-II.

Additional evidence for hydrophobic bond formation in the adsorption of sulfanilamide on bio-gel beads.

We have previously suggested the involvement of both hydrogen binding and hydrophobic bonding in the adsorption of sulfanilamide on Bio-Gel beads. In the present study, we closely examined the concentration dependence of the binding curve and our proposed binding model has been corroborated. For comparison, binding parameters and thermodynamic data pertaining to the sulfanilamide-Sephadex system have been also evaluated.

Macromolecular components of the vitelline membrane of hen's egg. II. Physicochemical properties of glycoprotein I.

Of the three major macromolecular components of the vitelline membrane of hen's egg, the lowest molecular weight component (previously designated component I) has been studied and its physicochemical properties clarified. The molecular weight of this component is 27,000 and its chemical composition is typical of a glycoprotein, consisting of protein (91%), total hexose (4.4%), hexosamine (glucosamine 2.3%; galactosamine 0.7%), and sialic acid (1.7%). Uronic acid was not found. The molar ratios of the constituent neutral sugars of this glycoprotein (GP-1) are as follows: fucose 3, mannose 5, galactose 5, glucose 1, and xylose 1. The amino acid profile shows a relatively high proportion of hydrophobic amino acids (39%), which may partly account for the insolubility of GP-I in water.

Thermodynamic characteristics of the adsorption of sulfanilamide, phenol, and n-butanol on bio-gel beads.

The thermodynamic properties of the adsorption of sulfanilamide, phenol and n-butanol on Bio-Gel beads have been studied. Bio-Gel was chosen as the adsorbent as it possesses both hydrophobic and hydrophilic sites on its surface. Adsorption of the former two adsorbates was found to be exothermic, and the relevant thermodynamic parameters at 20 degrees are in the ranges: deltaH degrees = -2.7 to -5.4 kcal/mole; deltaF degrees = -6.0 to -7.6 kcal/mole; deltaS degrees = +7.7 to +11.6 e.u. In the presence of urea, adsorption of sulfanilamide and phenol was partially disrupted. This, together with the large entropy gain of the process, indicates that both hydrogen bonding and hydrophobic bonding contribute cooperatively to the adsorption. On the contrary, adsorption of n-butanol, which was not susceptible to urea, was an endothermic process with the parameters, deltaH degrees = +5.8kcal/mole, deltaF degrees = -1.8 kcal/mole, and deltaS = +26.1 E.U. at 20 degrees. These data conform to the thermodynamic properties of hydrophobic bond formation. Finally, possible implications of these data in the structural assembly of lipoprotein molecules are discussed.

Macromolecular components of the vitelline membrane of hen's egg. I. Membrane structure and its deterioration with age.

The vitelline membrane of hen's egg has been successfully solubilized in sodium dodecyl sulfate (SDS), guanidine hydrochloride and urea solutions, and its macromolecular components examined. SDS-gel electrophoresis of the membrane solution revealed the presence of three major components designated I, II, and III, all containing carbohydrate and protein. The approximate molecular weights of components I and II were 32,000 and 260,000, respectively, and the sedimentation coefficients were 2.2S and 4.3S. Component III was in an aggregated form which disintegrated into smaller components upon reduction with 2-mercaptoethanol. It was found that component II (4.3S component) deteriorated during storage of the egg with the concomitant formation of degraded components. The loss of this component was accompanied by a gradual decrease of the neutral sugar content of the vitelline membrane. On the basis of these data, the membrane structure and its deterioration during storage are discussed.

Analysis of macromolecule-macromolecule interactions by the moving boundary method.

A constituent velocity equation has been derived which permits analysis of macromolecule (P)--macromolecule (A) interactions in which a number of complexes, PA, PA2, PA3,---,PAn, are in rapid equilibrium. In previous treatments, it was assumed that the velocity of complex PAi (i = 1,2,3,---,n) increased linearly with i, an assumption limiting the validity of the resulting equation. In the present treatment, the velocity of PAi is expressed in the form of a polynomial function in i and a generalized equation has been formulated.

Gel electrophoresis of amylose in the presence of sodium dodecyl sulfate.

It has been shown that sodium dodecyl sulfate (SDS) is capable of forming stable complexes with amylose and that fractionation of short-chain amyloses can be effected by SDS-gel electrophoresis. Using a well-defined smylose fraction (molecular weight 4,000), the thermodynamic parameters pertaining to SDS-amylose interaction have been evaluated by means of frontal gel chromatography. The results are as follows: association constant (K)=5.0 times 10-3-M-minus 1 at 25 degrees (pH 9.4); standard free energy change (delta G degrees)=-5.1 kcal/mole; standard enthalpy change (delta H degrees)=-5.8 kcal/mole; standard entropy change (delta Sdegrees)=-2.3(e.u.) and the maximum number of binding sites for SDS (n)=1. In the presence of 0.5--1 percent SDS, amylose migrates toward the anode upon gel electrophoresis, giving a compact band. High resolution of amylose fractions (released by treatment of amylopectin with debranching enzyme) has been attained using pore-size gradient gel electrophoresis.

The origin and consequences of concentration dependence in gel chromatography.

Concentration dependence of elution volume was determined for Blue Dextran 2000, Dextran 500, Dextran sulphate 500 and bovine serum albumin on columns of Sephadex G-100 equilibrated with sodium phosphate buffer, I 0.1, pH6.8. From the results for Dextran 500, it was shown that a linear relation exists between elution volume and the corresponding osmotic pressure calculated for the same concentration and incorporating the term containing the second virial coefficient. This relationship was used to predict the concentration dependence of elution volume for bovine serum albumin and myoglobin, proteins that partially penetrate Sephadex G-100. Possible consequences of osmotic effects are considered in relation to various types of column experiments, including differential chromatography.