Unperturbed dimensions of Carrageenans in different salt solutions

Commercial samples of kappa-, iota-, and lambda-carrageenans were studied by means of Size Exclusion Chromatography with dual detection, i.e. employing a Refractive Index (concentration sensitive) and Multiangle Light Scattering (size sensitive) detectors. The eluent was water containing 0.1 M concentration of different ionic salts, namely LiCl, NaCl, KCl and NaI, with the exception of kappa-carrageenan that aggregates in presence of KCl. Molecular weight distributions and averages, coefficients of the scaling law of molecular dimensions and unperturbed dimensions were thus obtained from a single polydisperse sample of each polymer. Measurements were performed at 25 degrees C and all the systems were above theta conditions with values of the q exponent of the scaling law ranging from 0.51 to 0.59. Extrapolation to unperturbed conditions provides values of the characteristic ratio C(N)=56+/-1 and 40+/-5 respectively for lambda- and iota-carrageenans regardless of the ionic salt employed. However, kappa-carrageenan gives C(N)=31, 35 and 59, respectively, in presence of LiCl, NaCl and NaI, which clearly indicates that this polymer behaves on a different way in presence of NaI than with the other two salts. A tentative explanation of this difference is provided.     

Configurational statistics of polysaccharide chains. Part II. Cellulose

The characteristic ratio of unperturbed cellulose chain was computed as a function of the angle τ at, the bridge oxygen atom and the degree of polymerization. Very high values of the order of 40 or more, depending on the angle at the bridge oxygen atom, have been obtained for this ratio, indicating that cellulose chains are highly extended. The average dimensions of cellulose chains are found to be sensitive even for small changes in the angle at the bridge oxygen, and these chains attain the character of a random coil in very high molecular weight range (degree of polymerization greater than 2000). The large differences in the unperturbed dimensions of cellulosic chains observed in different solvents have been attributed to the possible small changes in the angle τ caused by specific solvent, interactions.     

Hydrodynamic properties of a new plasma expander: polyhydroxyethylaspartamide

The intrinsic viscosity and molecular weight of several samples of polyhydroxyethylaspartamide with differing degrees of polymerization were determined and related, according to the Mark-Howink equation. The a exponent is consistent with a random-coil structure. The average unperturbed dimensions of the polymer, the dimensions in water, and the water–polymer interaction parameter were calculated according to Flory and Fox. The interaction of the polymer with water may explain its good efficacy as a plasma expander, as has previously been demonstrated in biological experiments. The behavior of the molecule in 6 M guanidine hydrochloride solution was also studied: this solvent has a very slight effect on the configuration of the polymer, in contrast with its normal effect on proteins.     

Cholinergic ligand-induced affinity changes in Torpedo californica acetylcholine receptor

Measurement of small-angle X-ray scattering from a sample of hyaluronic acid of high molecular weight in 0.05 m HNO₃ gave persistence length plots which agreed in form with theory and led to apparent persistence lengths of from 4 to 6 nm. Similar measurements in 0.2 m NaCl gave plots which deviated somewhat in form from theoretical expectation, but which could be interpreted to give a persistence length of 4 nm in this solvent. Data for intrinsic viscosity [η] as a function of molecular weight were in reasonable agreement with the Yamakawa-Fujii treatment of [η] for the worm-like chain model for a persistence length of about 4 nm in both 0.5 m NaCl and 0.1 m HCl, perhaps slightly higher in the latter. The values of persistence length estimated from [η] depend somewhat on the choice of chain parameters and the method of correction of experimental data to unperturbed solvent conditions. Experimental data for the sedimentation coefficient, while less definitive, were consistent within experimental uncertainty with the same parameters of the worm-like chain model. These calculated results are in substantial agreement with the values derived from small-angle X-ray scattering. A fraction of hyaluronic acid of low molecular weight in 0.05 m HNO₃ gave an estimated molecular weight of 2.7 × 10⁴ and a radius of gyration of 8 nm, in reasonable agreement with expected values based on the worm-like chain model for a persistence length of about 4 nm.     

Estimation of the relative stiffness of the molecular chain in polyelectrolytes from measurements of viscosity at different ionic strengths

A method was developed that allowed comparison of the stiffness of the chain in different polyelectrolyte from measurements of the intrinsic viscosity at different concentrations of added monovalent (sodium) salt. The response to salt was quantitatively expressed as the slope of straight lines relating the intrinsic viscosity to the reciprocal of the square-root of the ionic strength. This slope increased considerably with increasing molecular weight of the polyelectrolyte, and could serve to characterize the response to salt of different substances only when comparison was made at a constant molecular weight. An empirical parameter, B, which is the slope corresponding to an intrinsic viscosity of 1.0 at an ionic strength of 0.1 M could be correlated to the unperturbed dimensions of the molecules. A method of extrapolation, enabling the determination of B from measurements of viscosity on only one sample of unknown molecular weight, was evaluated. The empirically found correlation between Band some well established parameters of stiffness did not contrast predictions from the “fuzzy-sphere model” of Fixman, provided that reasonable assumptions regarding ion-binding and the interaction between polymer and solvent were made.     

Dilute solution properties of proteoglycan fractions from bovine nasal cartilage

Fresh proteogycans (adult bovine nasal cartilage) isolated from the densest portion of a dissociative density gradient had a weight-average molecular weight of ca. 10⁶ in 4M guanidine hydrochloride (GdnHCI) by light scattering. Fractions of such material obtained by elution with 4M GdnHCI from 2% agarose gel, both normal and cross-linkd, has proteoglycan subunit molecular weights ranging from 0.8 to 2.6 × 10⁶ and root-mean-square radii ranging from 35 to 52 nm in the same solvent. The protein molecular weight per proteoglycan subunit was about 1.2 × 10⁵ and that of keratan sulfate about 1.8 × 10⁵, both independent of total molecular weight. A random-flight “graft copolymer” model having uniform side chains of chondroitin sulfate (40 disaccharides) and keratan sulfate (15 disaccharides) and a random-coil polypeptide back bone was used to estimate the unperturbed radius, whihc was about 19 nm for a mol wt of 1.5 × 10⁶. Experimental light-scattering data for fractions were fitted very well by theoretical curves for the particale scattering factor for both linear and appropriate branched polymers. Examination of coil expansion on the basis of perturbation calculations for branched polymer models suggested that expansion did not account for the experimentally observed radii in terms of unperturbed radii calculated from the model. A possible explanation is that substantial local stiffening of the polypeptide chain due to substitution of side-chain clusters increases the unperturbed radii. The intrinsic viscosity [η] is 4M GdnHCI ranged from 120 to 180 ml/g, and could be interpreted in terms of th eequivalent sphere model; the Flory number has approximately its normal value for flexible linear polymers. The treatment of the sedimentation coefficient by this is less successful, since the Man delkern-Flory parameter β apparently increases with increasing molecular weight; average value are similar to those for flexible polymers, but the variation in β makes this method useful only for rough estimation of molecular weight of proteoglycans. Molecular weights of purified proteoglycans are the same in 0.2M NaCI as in 4M GdnHCI, while crude preparations gave higher molecular weights in 0.2M NaCI, probably because of association due to incomplete removel of “linking” proteins.     

Development and Optimization of Micro/Nanoporous Osmotic Pump Tablets

Micro/nanoporous osmotic pump tablets coated with cellulose acetate containing polyvinylpyrolidone (PVP) as pore formers were fabricated. Propranolol hydrochloride was used as a model drug in this study. Formulation optimization based on USP 31 requirements was conducted following a central composite design using a two-level factorial plan involving two membrane variables (pore former and coating levels). Effect of molecular weight of pore former (PVP K30 and PVP K90) was also evaluated. Responses of drug release to the variables were analyzed using statistical software (MINITAB 14). Scanning electron microscopy and atomic force microscopy showed that the pores formed by PVP. The drug release was dependent on the molecular weight and concentration of PVP and the level of coating. The results showed that acceptable 12-h profile could be achieved with only specific range of PVP K30-containing membrane at the defined membrane thickness. However, satisfactory 24-h profile could be accomplished by both PVP K30 and PVP K90-containing membrane at the range and membrane thickness tested. Preparation and testing of the optimized formulation showed a good correlation between predicted and observed values.     

Ionic polysaccharides. IV. Free-rotation dimensions for disaccharide polymers. Comparison with experiment for hyaluronic acid

The root-mean-square end-to-end distance has been calculated for a model allowing free rotation about glycoside bonds for the general case of polysaccharides having a disaccharide repeating unit. Numerical estimates are given for several naturally occurring structures based on an idealized pyranose unit in the C1 chair conformation. Extrapolation procedures which make use of the intrinsic viscosity [η] in good solvents to obtain unperturbed dimensions do not represent, data for hyaluronic acid very well, especially at low molecular weights. However, order-of-magnitude estimates suggest that this polymer behaves similarly to other polysaccharides, and probably has stiffer local structure than typical non-ionic synthetic polymers. A double logarithmic plot of the product of [η] and M?_w, the weight-average molecular weight, against the degree of polymerization in the range for M?_w of 10⁴ to 2 × 10⁴ permits a straight-line fit of available data for all the glycosaminoglycans, including heparin and the chondroitin sulfates, as well as sodium carboxymethyl cellulose. This result suggests similarity of short-chain hydrodynamic behavior of these polymers.     

Configuration-dependent properties of randomly coiling polynucleotide chains. II. The role of the phosphodiester linkage

The dependence of the unperturbed dimensions of randomly coiling polynucleotides on the rotations about the phosphodiester linkages of the chain has been examined in order to understand the conformational discrepancies, set forth in paper I, regarding these angles (ω′ and ω). Large values of the characteristic ratio 〈r²〉₀/nl² , which agree with the experimental behavior of the chain, are obtained only if a sizeable proportion of the polymer residues have trans ω′ values. The asymmetric torsional potential that is believed to arise from gauche effects associated with the P-O bonds has been approximated using a hard core model. The calculated characteristic ratio exhibits a strong dependence upon the magnitude of this torsional barrier (separating trans and gauche conformations) and shows agreement with experimental values for polyribonucleotides only if this energy difference is 1 kcal/mol or less.     

Configuration-dependent properties of randomly coiling polynucleotide chains. I. A comparison of theoretical energy estimates

Various theoretical estimates of the conformational energy associated with polynucleotides in solution have been compared with each other and also with the experimentally observed conformations found in X-ray crystallographic investigations of low-molecular-weight nucleic acid analogs. In view of the disparities between these data, certain configuration-dependent properties (i.e., the mean-square unperturbed end-to-end distance 〈r²〉₀ and the average vicinal nmr coupling constant 〈J〉) appropriate to randomly coiling polynucleotides described by either the energy estimates or by the crystallographically preferred conformations have also been calculated and compared with the known solution behavior of polynucleotide chains. Both the theoretical energy surfaces and the X-ray data show good agreement with the nmr coupling constant indications of the preferred rotations about the O-C and C-C bonds of the chain backbone. The principal discrepancies between the theoretical methods and X-ray data arise in their ability to predict successfully the preferred rotations about the two phosphodiester bonds of the chain backbone and the unperturbed dimensions of randomly coiling polynucleotide chains.     

Characterization and hydrodynamic behaviour of modified gelatin: I. Light scattering and viscometry studies

Commercial samples of gelatin modified by succinylation and currently used as plasma substitutes and fractionated samples obtained by diafiltration have been studied by viscometry, light scattering and osmometry. Viscometric results show that the aqueous medium containing potassium phosphate (0.1 ) and NaCl (0.12 ) at pH 3.3 behaves nearly like a theta solvent (a=0.48) for these modified gelatins. The Stockmayer-Fixman diagram reveals a negative slope attributed to a swelling of the macromolecules which decreases as the molecular weight _w increases. The Stokes radius R_H determined by quasielastic light scattering is independent of the pH of the medium in a range 7-3.3. The conformation of gelatins in solution has been characterized through the ratio _G· _H⁻¹, the radius of gyration _G being determined by viscometry. This ratio decreases as the molecular weight increases. The low molecular weight fractions have a more compact structure than the Gaussian chains in theta conditions. For high molecuar weight fractions, the values of _G· _H⁻¹ tend to those of an hard sphere.     

Ionic volumes in solution

The volumetric properties of electrolytes in solutions indicate the interactions of the constituent ions with their environment: the solvent and other ions. The interactions with the solvent alone are manifested at infinite dilution by the standard partial molar volume, V(infinity)(salt), obtained from density measurements. To study the interactions, it is necessary to split V(infinity)(salt) into the additive ionic contributions, V(infinity)(ion), using an extra-thermodynamic assumption. Values of V(infinity)(ion) for small ions depend cardinally on the electrostriction of the solvent that can be obtained from an iterative shell-by-shell calculation from a continuum model of the solvent. The solvent shrinkage per mol of ions is DeltaV(el)(ion)<0. Also, the molar electrostriction of the solvent S, DeltaV(el)(S)<0, is calculable. The ratio DeltaV(el)(ion)/DeltaV(el)(S)=n(infinity) is the solvation number of the ion in S at infinite dilution. The calculated V(infinity)(ion)(calc) are compared with the experimental values, showing good agreement for many univalent ions in both single solvents and in some binary solvent mixtures, where no appreciable preferential solvation takes place. Ion pairing sets in under certain circumstances of ionic charge and solvent permittivity. The difference DeltaV(ip)=V(ip)(infinity)-[V(infinity)(+)+V(infinity)(-)]>0 is obtained experimentally from the pressure derivative of the association constant. The ratio Deltan(ip)=DeltaV(ip)/DeltaV(el)(S) represents the number of solvent molecules released to the bulk on ion pairing by the diminution of the electrostriction.     

Asymmetrical-flow field-flow fractionation with on-line multiangle light scattering detection. 1. Application to wormlike chain analysis of weakly stiff polymer chains

Four samples of hyaluronan in the sodium form, ranging in weight-average molecular weight, M(w), from 6.67 x 10(5) to 4.23 x 10(6) were investigated by asymmetrical-flow field-flow fractionation coupled to multiangle light scattering (FlFFF-MALS) in 0.2 M aqueous NaCl at 25 degrees C. M(w) and z-average radii of gyration, R(G)(z)(), obtained via FlFFF-MALS showed a good agreement with the results obtained by conventional static light scattering. Furthermore, the molecular weight dependence of the radius of gyration for sodium hyaluronan obtained via FlFFF-MALS was analyzed on the basis of the Kratky-Porod model for unperturbed wormlike chains combined with the Yamakawa theory for radius expansion factor, and a sufficiently good agreement was observed between the theoretical prediction and experimental data. These results show the potential usage of FlFFF-MALS regarding size separation and molecular characterization even for weakly stiff chains.     

Dynamic light-scattering study of muscle F-actin. II

By dynamic light scattering, the intensity autocorrelation function, G2(tau) = B[1 + beta[g1(tau)[2], was obtained over the scattering angles (theta) from 30 to 130 degrees in steps of 10 degrees for semidilute solutions of muscle F-actin and of F-actin complexed with heavy meromyosin in the absence of ATP (acto-HMM), where B is the baseline and beta a constant. The main findings were: (1) A 0.5 mg/ml F-actin solution gave nonreproducible spectra at theta less than or equal to 40 degrees but quite reproducible spectra at theta greater than or equal to 50 degrees, with beta = 0.9-0.8 at all theta values. Nonreproducibility of spectra at low theta values was concluded to be due to restricted motions of very long filaments confined in cages or zig-zag tubing formed by a major fraction of filaments, where the very long filaments were those at a distant tail of an exponential length distribution and the major fraction of filaments were those with lengths around Ln-2Ln, Ln being the number-average length. Spectral widths were compared with theoretical ones for rigid rods averaged over the length distribution with Ln = 900 nm, and were suggested to be largely contributed at high theta values from bending motions of filaments. (2) Acto-HMM solutions at 0.5 mg/ml F-actin and at weight ratios of HMM to F-actin of 0.5-2 gave spectra which, with respect to theta, behaved very similarly to those of F-actin alone. The spectral widths, however, drastically decreased with the weight ratio up to unity and stayed virtually constant above unity. In contrast to a previous study (F.D. Carlson and A.B. Fraser, J. Mol. Biol. 89 (1974) 273), beta values of acto-HMM were as large as those of F-actin alone. Acto-HMM was concluded to travel a distance far greater than 1/K with a mobility smaller than that of F-actin, where K = (4 pi/lambda) sin(theta/2), lambda being the wavelength of light in the medium. These results suggest that acto-HMM gels are very soft even though they did not pour from an inverted cell. Based on several intuitive models which give a mutual relationship between the beta value and modes of motion of scatterers, we discuss the restricted motions responsible for nonreproducibility of spectra at low angles and large beta values of acto-HMM gels at all theta values and weight ratios so far studied.     

Viscosity of locust bean, guar and xanthan gum solutions in the Newtonian domain: a critical examination of the log (ηsp)o-log c[η]o master curves

The viscosity in the low shear rate Newtonian domain of three biopolymers, locust bean gum, guar gum and xanthan gum was studied as a function of temperature and of polymer concentration in various aqueous solvents. The intrinsic viscosities [η]_o of both galactomannans are not modified in the presence of 10 or 40% sucrose. In this case, a master curve relating the Newtonian specific viscosity (η_sp)_o, to the reduced concentration c[η]_o is obtained and allows (in good agreement with theoretical conjectures), two critical concentrations C* and C** to be defined, from which the value of the expansion coefficient may be estimated. For xanthan, as expected for a polyelectrolyte, [η]_o depends strongly on salt concentration and on added sucrose and the results did not obey the above-mentioned master curve. However, it is shown that (η_sp)_o depends only on xanthan concentration whenC > C**, and then it is assumed that chain dimensions have attained their unperturbed values whatever the solvent. Considering that both types of chains, random coils (galactomannans) and semi-rigid (xanthan) should give the same (η_sp)_o-C[η]_o master curve for C > C** when [η]_o is replaced by its unperturbed counterpart [η]_θ, a method for estimating [η]_θ for the xanthan sample is proposed. In conclusion, the numerous exceptions to the widely accepted (η_sp)_o vs C[η]_o “universal” behaviour are mainly ascribed to significant differences in expansion coefficient values which depend on both the polymer and the solvent.     

Tyrosine and peptide reorientational mobility in polymer solutions: Time-dependent fluorescence anisotropy measurements

The ability of peptides to form biologically active conformations that bind to receptors is governed by their dynamics and their propensity to form stable structures. Such factors are consequently important in the design of peptide drugs. Moreover, the stability of such peptides depends on interactions of the peptide with the surrounding matrix. In this article, we study the effect of the polymer poly(vinyl pyrrolidone) (PVP) on the mobility and orientational dynamics of tyrosine and a model peptide, Val-Tyr-Pro-Asn-Gly-Ala (VYPNGA) in glycerol-water solutions. Orientational dynamics are studied experimentally by time-resolved fluorescence anisotropy decays of tyrosine. The presence of PVP leads to the possibility of a distribution of environments for the peptide. The orientational dynamics of tyrosine show that the probe molecule experiences two very different environments. In one, tyrosine rotational motion is weakly coupled to PVP, while in the other, tyrosine interacts strongly with PVP leading to much slower rotational times. The dynamics of VYPNGA are more complex. Fast intramolecular, localized reorientations of the tyrosine are detected. The temperature dependence of the reorientational dynamics of the tyrosine side chain reveal that these motions are shielded from solvent friction. In contrast, global motions of the peptide are severely restricted by PVP, suggesting the ability of the polymer to restrict peptide mobility.     

Estimation of mean molecular weight of Enterobacteriaceae lipopolysaccharides using gas chromatography for determination of fatty acids

In the paper, we propose a method for estimation of the mean molecular weight of lipopolysaccharide, which is important for accuracy of endotoxin activity investigation. In our study, it was assumed that lipid A portion in Enterobacterial lipopolysaccharide is substituted by four 3-hydroxytetradecanoic acid residues. Lipopolysaccharides of S, Ra, Rc and Re chemotypes being laboratory preparations as well as purchased from Sigma were investigated. Fatty acids were determined by of gas chromatography as methyl esters according to the procedure described by Wollenweber and Rietschel. Mean molecular weight was calculated by the formula: MMW = [formula: see text]. A high agreement between the estimated and the theoretical molecular weight values was demonstrated in the case of Salmonella minnesota R595 (Re) LPS preparation. As expected, LPS heterogeneity increase together with enlargement of polysaccharide chain length which is visible in electrophoregrams also. Except for LPS mean molecular weight estimation, the method allows its detection in various preparations and samples, distinguishing of R and S LPS forms as well as the determination of mean length of O-specific chain in lipopolysaccharides which structures are known.     

Electro-optical studies on synthetic polyelectrolytes: IV. Electric polarizability and conformation of poly-N-methyl-2-vinylpyridinium bromide in aqueous solution

Measurements of the relaxation time on aqueous solutions of the title polyelectrolyte as a function of the concentration and of the molecular weight show that its conformation at very high dilution can be better accounted for by a weakly bending rod or worndike chain model, with persistence length ranging from 200 to 400 Å. The analysis of the field strength dependence of the electric birefringence yields electric polarizability values which increase sharply with the dilution and are not significantly dependent upon the molecular weight. This has been tentatively interpreted on the basis of the theories derived by Oosawa and by Mendel and Van der Touw. The partially flexible model proposed by the latter authors allow to identify the electric polarizability calculated from electro-optical data to the specific dielectric increment measured in the high frequency range; both parameters appear to be molecular weight independent. The electric polarizability obtained from the Kerr effect would be originated in the induced dipoles caused by the delocalization of the bound counterions along rigid subunits whose length seems however to differ from the persistence length.