Average molecular weight and molecular weight distribution of agarose and agarose-type polysaccharides

A procedure to determine the absolute weight-average molecular weight and molecular weight distribution of agarose and agarose-type polysaccharides by aqueous size-exclusion chromatography coupled with low-angle laser light scattering is described. The molecular weights of the majority of the commercial samples investigated were between 80 000 and 140 000 with a polydispersity lower than 1·7. In contrast, most of the laboratory-extracted agarose-type polysaccharides had lower molecular weights.     

Flow field-flow fractionation/multiangle light scattering of sodium hyaluronate from various degradation processes

Sodium hyaluronate (NaHA) is an ultrahigh molecular weight polysaccharide that is found in body tissues, synovial fluid, the vitreous humor, and the umbilical cord, and the size characterization of NaHA is important in pharmaceutical applications. On-line field-flow fractionation/multiangle light scattering/differential refractive index (FlFFF/MALS/DRI) has been applied for the study of degradation efficiency of sodium hyaluronate (NaHA). A NaHA raw sample was degraded by different chemical or physical methods and the degraded NaHA samples were separated using field-programming FlFFF, in which separation is achieved by differences in diffusion coefficients or hydrodynamic diameters. Separation was followed by serial detection using MALS and DRI. Molecular weight distribution (MWD) and information relating to the radius of gyration of the NaHA samples were examined for the raw and degraded NaHA samples. Samples studied include: two different products of ultrasonic degradation, two products of alkaline degradation, and four different products of enzymatic degradation. While alkaline degradation showed a moderate degradation compared to ultrasonic and enzymatic methods in reducing average MW, the latter two degradation methods showed significant changes in average molecular weight and in conformation of NaHA.     

Low molecular weight derivatives of different carrageenan types and their antiviral activity

A number of low molecular weight (LMW) fractions of carrageenans with different structural types were obtained by free radical depolymerization (H₂O₂), mild acid hydrolysis (HCl), and a specific enzyme. Three samples of carrageenans were depolymerized: kappa-carrageenan from Chondrus armatus, kappa-carrageenan from Kappaphycus alvarezii, and kappa/beta-carrageenan from Tichocarpus crinitus with initial molecular weights of 250, 390, and 400 kDa, respectively. The chemical depolymerization by two methods resulted to LMW derivatives of carrageenans with molecular weight from 1.2 to 3.5 kDa. Oligosaccharides of kappa- and kappa/beta-carrageenans with molecular weight of 2.2 and 4.3 kDa, respectively, were obtained after enzymatic depolymerization by recombinant kappa-carrageenase from Pseudoalteromonas carrageenovora. It was shown that the antiviral activity of high molecular weight carrageenans against tobacco mosaic virus was higher than that of their LWM derivatives independently on the depolymerization method. The method of depolymerization had some influence on the antiviral activity of carrageenan. LMW derivatives of kappa- and kappa/beta-carrageenans obtained by mild acid hydrolysis showed higher antiviral activity than the products of free radical depolymerization. The oligosaccharides prepared by enzymatic degradation possessed the lowest activity.     

Size and structure of antigen-antibody complexes. Electron microscopy and light scattering studies

Size parameters of model antigen-antibody (Ag-Ab) complexes formed by the interaction of bovine serum albumin (BSA) and pairs of monoclonal anti-BSA antibodies (mAb) were evaluated by quasielastic light scattering, classical light scattering, and electron microscopy (EM). Mean values for the hydrodynamic radius, radius of gyration, and molecular weight were determined by light scattering. Detailed information regarding the molecular weight distribution and the presence of cycles or open chains was obtained with EM. Average molecular weights were calculated from the EM data, and the Porod-Kratky wormlike chain theory was used to model the conformational behavior of the Ag-mAb complexes. Ag-mAb complexes prepared from three different mAb pairs displayed significantly different properties as assessed by each of the techniques employed. Observations and size parameter calculations from EM photomicrographs were consistent with the results from light scattering. The differences observed between the mab pairs would not have been predicted by idealized thermodynamic models. These results suggest that the geometric constraints imposed by the individual epitope environment and/or the relative epitope location are important in determining the average size of complexes and the ratio of linear to cyclic complexes.     

Comparative studies on the conformational change and aggregation behavior of irradiated carrageenans and agar by dynamic light scattering

The conformational associative properties of kappa-, iota-, and lambda-carrageenan and agar with irradiation dose were studied by dynamic light scattering. The random scission of the carrageenans and agar by gamma irradiation resulted in the formation of polydispersed lower molecular weight fragments. At high doses, the system moves towards uniformity. Conformational change from coil to helix was observed in all carrageenans and agar at doses up to 100 kGy. The conformational change in lambda-carrageenan may be due to the irregular and hybrid structure of this polysaccharide. Only agar and lambda-carrageenan still undergo conformational transition at a high dose of 200 kGy. Gelation is observed for kappa-, iota-carrageenan up to a dose of 50 kGy while gelation is still observed at 100 kGy for agar. Increase in the hydrodynamic radius with decreasing temperatures for the non-irradiated carrageenans follows this order: lambda-carrageenan>kappa-carrageenan>iota-carrageenan. Slight increases in hydrodynamic radius were observed with irradiation.     

Molecular weight of T7 and calf thymus DNA by low-angle light scattering

A low-angle light-scattering instrument has been used to measure molecular weights of native calf-thymus and T7 DNA. Molecular weights obtained by extrapolation of angular data to 0° from measurements above 30° are less than molecular weights from extrapolation of data taken at low angles (below 30°). The low-angle molecular weights determined for calf-thymus DNA and for T7 DNA agree well with estimates of weight-average molecular weight obtained with other techniques. The low-angle light-scattering molecular weight for calf-thymus DNA is higher than previously reported values by light scattering at angles above 30°. A concentration dependence in the scattering from DNA solutions is also observed.     

The flexibility of low molecular weight double-stranded dna as a function of length: I. Isolation and physical characterization of seven fractions

Sonicated calf thymus DNA was fractionated by rate zonal centrifugation into seven fractions with weight average molecular weights ranging from 0.28 to 1.3 × 10⁶ daltons, as determined by sedimentation equilibrium and light scattering measurements (the latter are described in the accompanying paper). Electron microscopy and sedimentation equilibrium analysis revealed these fractions to be narrowly disperse with M_w/M_n ratios averaging about 1.06. Intrinsic viscosities and sedimentation rates were measured and found to vary linearly with molecular weight in double-logarithmic plots in fair agreement with previously published functions relating these parameters for low molecular weight DNA. The average value for β from the Mandelkern— Flory equation was 2.59 × l0⁶, also agreeing with reported estimates of this parameter for short DNA. These data will be used in the second paper of this series to calculate the persistence length of the DNA fragments in each of the seven fractions by light scattering and hydrodynamic theories for the Kratky—Porod worm-like coil.     

Physical chemical studies of short-chain lecithin homologues. II. Micellar weights of dihexanoyl- and diheptanoyllecithin

The micellar weights of dihexanoyl- and diheptanoyllecithin in aqueous solutions are calculated from light scattering and ultracentrifugation data. A monomer-micelle assocation model is used and corrections for the thermodynamic nonideality, on the basis of rigid noninteracting particles, are applied. A few experiments on the influence of high NaCI concentrations (up to 3 M) are described. Dihexanoyllecithin forms micelles with micellar weight of 15 000 to 20 000 and with rather narrow weight distributions. Diheptanoyllecithin micelles however, have broad size distributions with micellar weights of 20 000 up to about 100 000 in the concentration range studied. Micelles are assumed to be spherical or to have sphero-cylindrical shapes depending on the molecular weights. Two models are used: (1) a compact structure, where no attention is paid to the hydrocarbon-water contact (2) micelles with as little hydrocarbon-water contact as possible.     

Size exclusion chromatography with evaporative light scattering detection: Method for the determination of polydimethylsiloxanes. II. Application of TSK-GEL H HR GMH HR -M column to determine and separate molecular weight of linear polydimethylsiloxanes

Issues concerned with molecular weight distribution analysis of linear polydimethylsiloxanes have not been extensively investigated and mastered, yet. Current publications do not provide detailed research data on the evaluation of the polymerization degree of polydimethylsiloxanes (PDMS) present in variable matrices: e.g. pharmaceuticals, cosmetics, foodstuffs nor indicate molecular weights of the polymer used. However, the information on molecular weight, i.e. viscosity, is of primary importance as it directly affects PDMS toxicity, absorption and migration in the living organism. The vast majority of currently applied methods prove to be insufficiently specific for PDMS of a particular molecular weight and therefore alternative analytical methods have to be further researched. In this paper the results of determination of molecular weights in linear polydimethylsiloxanes, using size exclusion chromatography with the evaporative light scattering detector are described. The column calibration curve obtained from low-dispersion standard polystyrene of molecular weights ranging 376-2,570,000 Da was used to determine PDMS molecular weights. Precision and accuracy of determination was obtained. For the mobile phase flow-rate of 0.3 ml/min relative standard deviation RSD ranged to 0.45% and the accuracy of measurement amounted to -0.42%, whereas for flow-rate of 1.0 ml/min RSD ranged to 0.38% and accuracy to +2.15%.     

Insulin association in neutral solutions studied by light scattering

Molecular weights and weight distributions of sulfated, Zn-free, and 2Zn insulins have been measured at pH 7.3 as a function of concentration from 0.1 to 2 mg/ml by use of a combination of light scattering, refractometry, and size-exclusion chromatography. Results show that sulfated insulin is monomeric over the studied concentration range. Weight average molecular weights between those of a monomer and a hexamer were found for both zinc-free and 2Zn insulins. Zinc stabilizes the hexamer, and the dimer-hexamer equilibrium constant is approx. 400-times higher in the presence of zinc than in its absence. An average hydrodynamic radius of 5.6 nm, close to the crystallographic size of the insulin hexamer, was determined from dynamic light scattering of 2Zn insulin solutions.     

Characterization of acid-denatured DNA by low-angle light scattering

Low-angle light scattering results reported previously demonstrated that measurements on high molecular weight native DNA must be made at angles below 30° in order to obtain correct molecular weights. Earlier light-scattering data obtained on denaturated DNA at angles above 30° showed no change in molecular weight upon denaturation, even though other techniques clearly showed that strand separation occurred. This paper reports low-angle measurements on solutions of calf thymus and T7 DNA denatured under acidic conditions. The results demonstrate that a halving of molecular weight consistent with strand separation is detected by light scattering only when low-angle data are used to obtain correct molecular weights for native material. As expected from theoretical considerations, the scattering from denatured DNA is a linear function of sin²(θ/2), where θ is the angle of observation. This result shows that anticipated experimental artifacts have no significant effect on the low-angle measurements and demonstrates that the curvature in the scattering envelope observed for native DNA below 30° is an inherent property of the native molecule.     

Combination of affinity chromatography and analytical polyacrylamide gel electrophoresis for rapid measurement of human serum high-density lipoprotein apoproteins

Heparin of an average molecular weight of 13,000 was fractionated on the basis of size into five fractions of different weight-average molecular weight ranging from 8500 to 20,000. The heparin was also degraded using microbial heparinase resulting in products ranging from a disaccharide of molecular weight 500 to an oligosaccharide of molecular weight 3100. These products were also size fractionated. The individual heparin fractions and products were tested for metachromatic activity with Azure A. The metachromatic activity of the heparin fractions was independent of molecular weight, while the metachromatic activity of the products was dependent on molecular weight. Metachromatic activity was found in a fragment as small as a tetrasaccharide. Anticoagulant activity was found in fragments of tetrasaccharide or larger by a Factor Xa clotting assay and in fragments of hexasaccharide or larger by a Factor Xa amidolytic chromogenic assay.     

The flexibility of low molecular weight double-stranded dna as a function of length: II. Light scattering measurements and the estimation of persistence lengths from light scattering,sedimentation and viscosity

In the preceding paper are described the isolation and physical characterization of seven narrowly disperse fractions of calf thymus DNA in the molecular weight range 0.3 to 1.3 × 10⁶ daltons. Herein, we have determined by light scattering the molecular weights and root mean square radii of these fractions in a solvent comprising 0.2 M NaCl, 2 mM EDTA, 2m MNa-PO₄, pH 7. Measurements were made in a modified Wippler—Scheibling photometer to a 20° lower limit of scattering angle on solutions rendered virtually dust-free by procedures described. The optical aniso tropics of the DNA fractions were measured permitting the experimental molecular weights and root mean square radii to be corrected to their true values. From these values, with appropriate polydispersity corrections, we calculate a Kratky—Porod persistence length, a, of 54.0 ± 5.6 nm which is invariant over the molecular weight range examined. From the sedimentation coefficients (preceding paper) and the theory of Yamakawa and Fujii, we calculate a to be 66 nm, a value found to apply equally well to several DNA samples of various origins whose sedimentation rates are known in the molecular weight range from about 4 × 10⁴ to 10⁸ daltons. Similarly, from the intrinsic viscosities and the theory of Yamakawa and Fujii, we calculate a to be 59 nm, which again adequately applies to a number of DNA samples whose viscosities have been measured by other workers in the molecular weight range 3 × 10⁵ to 10⁸ daltons. The Flory—Mandelkern parameter, β, was found to vary with molecular weight in the manner predicted by the theory of Yamakawa and Fujii. The average value of a from the three sets of measurements is 60 ± 6 nm, which we believe applies to double-stranded DNA molecules, independent of chain length, over the whole range of molecular weights for which reliable data exist.     

A polydisperse linear random coil model for the quaternary structure of pig colonic mucin

The distribution of molecular weights for polymeric colonic mucus glycoprotein or ``mucin' isolated and solubilised in the presence of protease inhibitors from pig colons is shown to be considerably greater than its ``subunit' (thiol reduction product) and papain digested forms using the technique of size-exclusion chromatography coupled to multi-angle laser light scattering, and confirmed by sedimentation equilibrium measurements. The conformation of this mucin is probed by examining the molecular weight – intrinsic viscosity relationship in terms of the Mark-Houwink-Kuhn-Sakurada analysis for its polymeric (or ``whole'), reduced and papain-digested forms: an exponent ``a' of (1.1±0.1) is obtained indicating a linear random coil conformation consistent with other mucins. Size-exclusion chromatography coupled to multi-angle laser light scattering is shown to provide a relatively simple complementary technique to sedimentation equilibrium for the molecular weight distribution analysis of polydisperse materials. Received: 29 November 1996 / Accepted: 2 December 1996     

Size-Exclusion Chromatography with On-Line Light-Scattering, Absorbance, and Refractive Index Detectors for Studying Proteins and Their Interactions

Techniques of using size-exclusion chromatography (SEC) with on-line light-scattering, uv absorbance, and refractive index detectors to characterize the polypeptide molecular weights of simple proteins or glycoproteins or to determine the stoichiometry of protein complexes are described. Two unique advantages of this approach over conventional SEC are that the molecular weight measurement is independent of elution position and can exclude the contributions from carbohydrates. When a protein or complex contains no carbohydrates, a two-detector method, i.e., light scattering combined with refractive index, can be used to calculate the molecular weight. When a protein contains carbohydrates, a three-detector method is used to calculate the molecular weight of polypeptide alone. Finally, a self-consistent three-detector method is used to determine the stoichiometry of a protein complex containing carbohydrates. Example applications for all these methodologies are described.     

Degradation of ethylene glycol and polyethylene glycols by methanogenic consortia.

Methanogenic enrichments capable of degrading polyethylene glycol and ethylene glycol were obtained from sewage sludge. Ethanol, acetate, methane, and (in the case of polyethylene glycols) ethylene glycol were detected as products. The sequence of product formation suggested that the ethylene oxide unit [HO-(CH2-CH2-O-)xH] was dismutated to acetate and ethanol; ethanol was subsequently oxidized to acetate by a syntrophic association that produced methane. The rates of degradation for ethylene, diethylene, and polyethylene glycol with molecular weights of 400, 1,000, and 20,000, respectively, were inversely related to the number of ethylene oxide monomers per molecule and ranged from 0.84 to 0.13 mM ethylene oxide units degraded per h. The enrichments were shown to best metabolize glycols close to the molecular weight of the substrate on which they were enriched. The anaerobic degradation of polyethylene glycol (molecular weight, 20,000) may be important in the light of the general resistance of polyethylene glycols to aerobic degradation.     

Light scattering and hydrodynamic properties of linear and circular bacteriophage lambda DNA

Low-angle light scattering, sedimentation velocity, and intrinsic viscosity measurements have been made on circular and linear forms of lambda (λ) bacteriophage DNA. Available equations, used to relate hydrodynamic parameters of both forms to the molecular weight, give relatively consistent values of particle weights which essentially agree with the light-scattering results. An average molecular weight of (34 ± 3) × 10⁶ for λ DNA was obtained in good agreement with literature values of (31–33) × 10⁶. The linear λ DNA has a larger root-mean-square radius than the circular molecule, when determined by light scattering, but the difference does not appear to be us large as expected from hydrodynamic data. The two forms also show significantly different angular distrbutions of scattered light intensities which agree only qualitatively with those derived from existing theory. The light-scattering results suggest that further experiments and modifications of available theories should be undertaken.     

Highly enantioselective hydrolysis of dl-menthyl acetate to l-menthol by whole-cell lipase from Burkholderia cepacia ATCC 25416

Bisphenol A was polymerized by Coprinus cinereus peroxidase in aqueous 2-propanol solution. Various polymerized products with different molecular weights and hydroxyl values were synthesized depending on the reaction compositions (the ratio of aqueous buffer to 2-propanol). Poly(bisphenol A), a polymer of bisphenol A, was mixed with a diazonaphthoquinone derivative to form a new type of photoresist. A thin photoresist film was formed on the silicon wafer and exposed to UV light for different lengths of time. Poly(bisphenol A) having a molecular weight of approximately 3000 yielded sharply contrasted patterns as compared with the other poly(bisphenol A)s having different molecular weights.     

A comparison of molecular mass determination of hyaluronic acid using SEC/MALLS and sedimentation equilibrium

Hyaluronic acid (HA) is a natural polysaccharide with importance in the pharmaceutical, medical and cosmetic industries. Determining factors in its final applications are its physicochemical properties, particularly molecular mass. A high molecular mass HA was degraded using five different hydroxyl free-radical starting concentrations chemically produced from ascorbic acid and hydrogen peroxide. The aims of the study were to investigate the effect of different hydroxyl free-radical concentrations on the chain length of HA and compare the molecular masses obtained from analytical ultracentrifugation using sedimentation equilibrium experiments and size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). The results indicated that their molecular masses varied, depending on the degree of hydroxyl free-radical starting concentration. Molecular mass values obtained from sedimentation equilibrium experiments for each sample showed the same trend as those obtained from the SEC/MALLS in the range of molecular masses studied. The molecular masses obtained from sedimentation equilibrium for high molecular mass samples from reciprocal plots of apparent weight average molecular mass against concentration gave values similar to those obtained by SEC/MALLS. In contrast, the molecular mass from conventional plots for high molecular mass samples were much lower than those from SEC/MALLS, even when high ionic strength buffers were used.     

Differential Degradation of Different Benzodiazepine Binding Proteins by Incubation of Membranes from Cerebellum or Hippocampus with Trypsin

When rat brain membranes were incubated with [3H]flunitrazepam in the presence of UV light, predominantly one protein (P51) was irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. On digestion of membranes with increasing concentrations of trypsin up to 40% of radioactivity irreversibly bound to proteins was removed from the membranes. In addition, P51 was nearly completely degraded to a peptide with apparent molecular weight 39,000 and this peptide was further degraded to a peptide with apparent molecular weight 25,000. In contrast, protein P55 was only partially degraded by trypsin and yielded two proteolytic peptides with apparent molecular weights 42,000 and 45,000 which seemed to be rather stable against further attack by trypsin. Membranes treated with trypsin still had the capacity to bind [3H]-flunitrazepam reversibly with an affinity similar to that of membranes not previously treated with trypsin. When these membranes were irradiated with UV light, the same proteolytic peptides were detected as in membranes first photolabeled and then digested with trypsin. These results suggest a close association between reversible and irreversible benzodiazepine binding sites and indicate that membrane-associated proteins P51 and P55 are differentially protected against degradation by trypsin.