A complete set of hyaluronan fragments obtained from hydrolysis catalyzed by hyaluronidase: Application to studies of hyaluronan mass distribution by simple HPLC devices

Hyaluronan (HA) has different biological functions according to its molar mass; short HA fragments are involved in inflammation processes and angiogenesis, whereas native HA is not. Physicochemically, studies of native HA hydrolysis catalyzed by bovine testicular hyaluronidase (HAase) have suggested that kinetic parameters depend on HA chain length. To study the influence of HA chain length in more detail, and to try to correlate the physicochemical and biological properties of HA, HA hydrolysis catalyzed by HAase was used in a new procedure to obtain HA fragments of different molar masses. HA fragments (10-mg scale) with a molar mass from 800 to 300,000 g mol(-1) were prepared, purified using low-pressure size exclusion chromatography (SEC), lyophilized, and characterized in molar mass by either mass spectrometry or HPLC-SEC-multiangle laser light scattering. The polydispersity index of the purified fractions was less than 1.25. The complete set of HA standards obtained was used to calibrate our routine HPLC-SEC device using only a refractive index (RI) detector. We showed that the N-acetyl-d-glucosamine reducing end assay and the calibrated HPLC-SEC-RI gave equivalent kinetic data. In addition, the HPLC-SEC-RI furnished the mass distribution of the polysaccharide during its hydrolysis.     

A Review of: “Progress in Separation and Purification Vol. 3, E. S. Perry and C. J,van Oss,eds., Willy/Intersciencc,New York, 1970; xi + 316 pares; hard cover; 515.95”

The optimal culture conditions of exopolysaccharides (EPS) production in submerged culture medium by Pleurotus geesteranus 5 ^# were determined using an orthogonal matrix method. The optimal defined medium (per liter) was 60.0 g maltose, 5.0 g tryptone, 1 mM NaCl, 5 mM KH₂PO₄, and initial pH 6.0 at 28 °C. In the optimal culture medium, the maximum EPS production was 16.97 g/L in a shake flask. Two groups of EPSs (designated as Fr-I and Fr-II) were obtained from the culture filtrates by size exclusion chromatography (SEC), and their molecular characteristics were examined by a multiangle laser-light scattering (MALLS) and refractive index (RI) detector system. The approximate weight-average molar masses of the Fr-I and Fr-II of EPS were determined to be 3.263 × 10⁴ and 5.738 × 10³ g/mol, respectively. The low values of polydispersity ratio (1.176 and 1.124 for Fr-I and Fr-II, respectively) of EPSs mean that these EPS molecules exist much less dispersed in aqueous solution without forming large aggregates. Furthermore, the experiments in vitro indicated that P. geesteranus 5^# EPS exhibit high antitumor and antioxidative effects.     

Characterization of gelatine and acid soluble collagen by size exclusion chromatography coupled with multi angle light scattering (SEC-MALS)

Acid soluble collagen (ASC) and a cattle hide gelatine were analyzed by size exclusion chromatography (SEC) coupled with multi angle light scattering (MALS). The SEC system was calibrated with ASC and its cyan bromide cleavage products. The accuracy of calibration was confirmed by MALS by measuring the mass-average molar masses (Mw). ASC acted as a mixture of two polymer standards of Mw = 90 and 180 kg/mol, respectively. The elution behavior of the gelatine in SEC-MALS was similar to that of ASC. Therefore, the determination of the molar mass distribution of this gelatine was possible either by SEC, using a calibration curve, or by MALS by direct measurement of Mw. According to the scaling law (1/2) = KMalpha, alpha = 0.78 was determined for the gelatine. This alpha could reflect a structure in solution, which is more similar to an ellipsoid than to a random coil.     

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.     

Pyruvic-acid-containing polysaccharide in the cell wall of Bacillus polymyxa AHU 1385

Three acidic polymer fractions with molecular masses of about 16 kDa, 35 kDa and 70 kDa were isolated from lysozyme digests of N-acetylated cell walls of Bacillus polymyxa AHU 1385 by ion-exchange chromatography and gel chromatography. These fractions, containing mannosamine, glucosamine and pyruvic acid in a molar ratio of about 1:1:1 together with glycopeptide components, were characterized as polysaccharide-linked glycopeptides with one, two and more polysaccharide chains. On the other hand, treatment of the cell walls with glycine/HC1 buffer, pH 2.5, at 100 degrees C for 10 min followed by separation of water-soluble products on ion-exchange chromatography gave three polysaccharide fractions, PS-I-III, which contained different amounts of pyruvic acid (0,0.6 and 0.9 residue/mannosamine residue) along with equimolar amounts of mannosamine and glucosamine. Pyruvate-free polysaccharides similar to PS-I were also obtained from PS-II, PS-III and polysaccharide-linked glycopeptides by treatment with 10 mM HC1 at 100 degrees C for 1 h. Results of analyses of these polysaccharide preparations by 1H-NMR and 13C-NMR measurement and methylation, together with data from characterization of fragments obtained by hydrogen fluoride hydrolysis, lead to the most likely structure, ----3)[4,6-O-(1-carboxyethylidene)]ManNAc(beta 1----4)GlcNac(beta 1----, for the acidic polysaccharide of this strain.     

Solution properties of viilian, the exopolysaccharide from Lactococcus lactis subsp. cremoris SBT 0495

The exopolysaccharide (EPS) "viilian" was isolated from a large-batch fermentation of Lactococcus lactis subsp. cremoris SBT 0495. After applying a newly developed purification procedure, pure viilian with a weight-averaged molar mass of 2.64 x 10(3) kg/mol was obtained in a yield of 0.6 g/L culture broth. The native EPS, as well as lower molar mass fractions obtained by sonication of the native polymer, were studied by capillary viscometry and size-exclusion chromatography (SEC) coupled to multiangle laser light scattering detection (MALLS). From the viscosity data at various ionic strengths, we extracted a Mark-Houwink-Kuhn-Sakurada exponent a = 0.79, and a Smidsrod B value of 0.03. By application of the Hearst, Bohdanecky, and Odijk models for stiff polymer coils, in connection to the experimental viscosity data, we established the characteristic ratio to be C(infinity) = 44 and the intrinsic persistence length q(0) = 11.5 nm. The rms radii of gyration predicted from each of the models were in good agreement with the experimental radii (e.g., (1/2)(w) = 162 nm for native viilian in 0.2M NaNO(3)), as determined by SEC-MALLS. In addition, the Odijk model predicts correct ionic strength-linear charge density dependence of the rms radius of gyration. From the combined viscosity and SEC-MALLS experiments we concluded that, in dilute aqueous solutions, viilian behaves as an intermediately stiff, random coil polyelectrolyte system.Copyright 2000 John Wiley & Sons, Inc.     

Macromolecular conformation of chitosan in dilute solution: A new global hydrodynamic approach

Chitosans of different molar masses were prepared by storing freshly prepared samples for up to 6 months at either 4, 25 or 40 °C. The weight-average molar masses, M_w and intrinsic viscosities, [η] were then measured using size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) and a “rolling ball” viscometer, respectively.The solution conformation of chitosan was then estimated from:

(a) the Mark–Houwink–Kuhn–Sakurada (MHKS) power law relationship [η] = kM_w^a and

(b) the persistence length, L_p calculated from a new approach based on equivalent radii [Ortega, A., & Garcia de la Torre, J. (2007). Equivalent radii and ratios of radii from solution properties as indicators of macromolecular conformation, shape, and flexibility. Biomacromolecules, 8, 2464–2475].

Both the MHKS power law exponent (a = 0.95 ± 0.01) and the persistence length (L_p = 16 ± 2 nm) are consistent with a semi-flexible rod type (or stiff coil) conformation for all 33 chitosans studied. A semi-flexible rod conformation was further supported by the Wales–van Holde ratio, the translational frictional ratio and sedimentation conformation zoning.     

Cloning of an EcoRI fragment carrying E. coli tufA gene.

Summary EcoRI fragments of the transducing phage fus3 DNA have been linked to the ColEl derivative plasmid RSF2124 (ColEl-Ap^r) DNA using bacteriophage T4 ligase. Among the plasmids formed, one designated pTUAl was found to contain the E. coli tufA gene. The proof for the presence of tufA gene in pTUAl is based on the following observations: (1) ability of pTUAl DNA and its EcoRI fragments to direct synthesis of EF-Tu in a cell-free protein synthesizing system; and (2) RNA·DNA hybridization of RNA transcribed from phage rif ^d18 carrying tufB with DNA from pTUAl.     

Physicochemical studies on xylinan (acetan). I. Characterization by G permeation chromatography on sepharose Cl-2B coupled with static light scattering and viscometry

Laboratory-made samples of the polysaccharide xylinan (acetan) were fractionated on Sepharose Cl-2B using 0.1M NaCl as eluant. The weight average molar masses and intrinsic viscosities were estimated in the fractions by multiangle laser light scattering (off-line) and capillary viscometry, respectively. The Mark-Houwink-Sakurada plot was found to be indicative of semiflexible coils (a = 0.90). The angular dependence of scattered light was interpreted by fitting with theoretically calculated “Master Curves” in terms of a wormlike chain model. The ambiguity of the interpretation of scattering curves owing to the overlapping effects of chain stiffness and polydispersity is discussed in detail. The experimental data is found to be consistent with a persistence length of L_p = 100 nm. The main proportion consists of double-stranded chains (consistent with a robust double-helix), but single- and multistranded chains also are present. Our results suggest a fractionation according to the contour length rather than the molar mass. © 1996 John Wiley & Sons, Inc.     

Production of Humanized Fab Fragment against Human High Affinity IgE Receptor in Pichia pastoris

Binding of allergen-IgE complexes to the high affinity IgE receptor (FcεRI) on mast cells and basophils leads to the release of various mediaters such as histamine. Fab fragments prepared by the papain digestion of humanized antibody against human FcεRI inhibited the release of histamine from human basophils. Here we established an expression system to directly produce Fab fragments of the humanized anti-human FcεRI antibody in methylotropic yeast, P. pastoris. Fab fragments were efficiently secreted into the medium at a concentration of 10-40 mg/L using a signal sequence from the P. pastoris phosphatase gene. They were consisted of disulfide-linked light and heavy chains correctly starting from the first amino acid residues by proper cleavage of the signal peptides. The obtained Fab fragments inhibited the binding between IgE and FcεRI as efficiently as the counterpart prepared by papain digestion of the whole antibody.     

Acetylation and characterization of spruce (Picea abies) galactoglucomannans

Acetylated galactoglucomannans (GGMs) are the main hemicellulose type in most softwood species and can be utilized as, for example, bioactive polymers, hydrocolloids, papermaking chemicals, or coating polymers. Acetylation of spruce GGM using acetic anhydride with pyridine as catalyst under different conditions was conducted to obtain different degrees of acetylation on a laboratory scale, whereas, as a classic method, it can be potentially transferred to the industrial scale. The effects of the amount of catalyst and acetic anhydride, reaction time, temperature and pretreatment by acetic acid were investigated. A fully acetylated product was obtained by refluxing GGM for two hours. The structures of the acetylated GGMs were determined by SEC-MALLS/RI, ¹H and ¹³C NMR and FTIR spectroscopy. NMR studies also indicated migration of acetyl groups from O-2 or O-3 to O-6 after a heating treatment in a water bath. The thermal stability of the products was investigated by DSC-TGA.     

Extraction and characterization of an alginate from the brown seaweed <Emphasis Type="Italic">Sargassum turbinarioides</Emphasis> Grunow

Matrix polysaccharide from the brown algae Sargassum turbinarioides collected in the coastal waters of Nosy Be (Madagascar) in the Indian Ocean was isolated and its structure was studied by ¹H-NMR spectroscopy, FT-IR, SEC-MALLS and HPAEC. An alginate with a molecular weight of 5.528 × 10⁵ g mol⁻¹ was identified as sole polysaccharide. Values of the M/G ratio, F _GG, F _MM and F _GM (or F _GM) blocks were measured at respectively 0.94, 0.39, 0.36 and 0.25 and compared with those of alginates from other Sargassum species. This sodium alginate appeared similar to some of the other Sargassum alginates with M/G < 1, high values of homopolymeric blocks (η < 1) and significant polyguluronic block content.     

Molar mass distribution of hydroxypropyl cellulose by size exclusion chromatography with dual light scattering and refractometric detection

Physico-chemical properties of cellulose derivatives are of considerable interest in many technical applications, for example, in the food and drug industry. Efficient and careful characterisation of these properties is thus highly desirable. In this study, two different size exclusion chromatography (SEC) systems, connected on-line either to a low-angle laser light scattering detector (LALLS) or to a multi-angle laser light scattering detector (MALLS), are employed for size characterisation of three batches of hydroxypropyl cellulose (HPC) from different manufacturers. All three samples turned out to have a weight average molar mass around 100,000 g/mol, but considerable differences concerning conformational properties were found. Two of the samples contained compact components, presumably aggregates of HPC, which were clearly detected by both SEC-systems. The third sample, obtained from another manufacturer, did not show any indication of aggregation. Both SEC-MALLS and SEC-LALLS are proven to be efficient techniques for characterisation of complex polysaccharides like HPC containing mixtures of solvated polymer chains, as well as micelle-like aggregates.     

Body Mass Estimates for Eocene Eosimiid and Amphipithecid Primates Using Prosimian and Anthropoid Scaling Models

We estimated body masses for middle to late Eocene East Asian eosimiids and amphipithecids from the crown areas of cheek teeth. First, we calculated body mass estimate equations via an extant primate sample of 11 prosimian and 30 anthropoid species, and compared the reliability of the resulting body mass estimate regressions. M ^1–2 and M _1–2 are better body mass estimators, especially for fossils with few samples, because of their low intraspecific variations in dimensions. Moreover, body masses derived from M _1–2 tend to indicate lower estimate error than those from other cheek teeth. The relationships between tooth crown areas and body mass differ between prosimians and anthropoids; the estimated body mass from crown area of P ⁴ or any molar will be larger if anthropoids, instead of prosimians, are used as a reference taxon. Second, We applied the regressions to the fossil primates. The estimated body masses in kg are as follows: Eosimias centennicus, 0.16; E. sinensis, 0.14; Eosimiidae indet. from the Pondaung Formation, 0.41; Bahinia pondaungensis, 0.57; Myanmarpithecus yarshensis, 1.8; Amphipithecus mogaungensis, 6.8; Pondaungia cotteri, 5.9; Pondaungia savagei, 8.8; Siamopithecus eocaenus, 5.9. Eosimiids fit the prosimian model better than the anthropoid model. Amphipithecids do not fit one model particularly better than the other, as the estimates vary considerably according to the tooth used and the reference taxon. The anthropoid model gives smaller differences between upper- and lower-molar-based body mass estimates, but premolars are relatively much smaller in amphipithecids than in extant prosimians and anthropoids.     

Mechanistic investigation of a starch-branching enzyme using hydrodynamic volume SEC analysis

Two linear alpha-(1,4)-D-glucans substrates, of degrees of polymerization DP approximately 150 and 6000, were exposed to maize starch-branching enzyme IIa (mSBEIIa) in vitro. The resulting branched alpha-glucans and their constituent chains (obtained by debranching) were analyzed by nuclear magnetic resonance (NMR) and size-exclusion chromatography (SEC). SEC data for the debranched species are presented as chain-length distributions, while those for branched species are presented as hydrodynamic volume distributions (HVDs), which is the most meaningful way to present such data (because SEC separates by size, not molar mass, and a sample of branched polymers with the same size can have a range of molar masses). A rigorous interpretation of the HVDs of the substrate and its branched product show that at least part of the branching is an interchain transfer mechanism in both the short- and long-chain substrate cases. A bimodal HVD of the in vitro branched alpha-glucan derived from the short-chain substrate was observed, and it is postulated that the divergence of the two populations is due to very small chains being unable to undergo branching. In the case of the in vitro branching of the long-chain substrate, the formation of maltohexaose during the reaction and the presence of a monomodal HVD were observed, suggesting a distinct mode of action of mSBEIIa on this substrate. Quantification of the branching level by NMR showed the branched glucans from both substrates had substantial amounts of branching (2.1-4.5%), ascribed to the intrinsic nature of the action of mSBEIIa on the two substrates. It is postulated that differences in the degrees of substrate association affect the pattern of branching catalyzed by the enzyme, and a putative active site structure is proposed based on the appearance of maltohexaose. The molar mass distribution of the constituent chains of the in vitro branched alpha-glucans obtained by isoamylase treatment reveals the transfer of chains of specific size and supports the supposition given in the literature that mSBEIIa is responsible for short-chain branching in amylopectin. It is suggested that hydrodynamic volume SEC analysis should be used as a tool for the mechanistic investigation of SBEs, allowing SEC data of in vitro branched alpha-glucans to be both comparable and quantitative.     

A novel DTPA cross-linking of hyaluronic acid and metal complexation thereof

Macromolecular conjugates of a natural polysaccharide, hyaluronic acid, with diethylenetriaminepentaacetic acid (DTPA)—metal complexes were synthesized and characterized by FTIR, NMR, SEC-MALLS and ICP analysis. Several parameters of the cross-linking reaction as molecular weight of starting HA, temperature, equivalent of DTPA bis-anhydride, concentration of HA, presence of transacylation catalyst DMAP and reaction time were studied. The mechanism for the reaction was suggested and relationship between the molecular weight assigned by SEC-MALLS, reaction parameters and rheological properties of the final cross-linked products were investigated.     

Physical mapping of the HindIII, EcoRI, Sal and Sma restriction endonuclease cleavage fragments from bacteriophage T5 DNA.

Summary The DNA of bacteriophage T5⁺ (molecular weight 76×10⁶ dalton) has been dissected by various specific endonucleases. The restriction enzymes HindIII and EcoRI produce 16 and 7 fragments respectively, whereas Sal and Sma produce 4 fragments each. Complete cleavage maps were established for the enzymes EcoRI, Sal and Sma and an almost complete map for HindIII. Furthermore the location and size of the deletions St 20, St 14, b3, St 0 and b1 were determined. The correlation of the genetic and functional map of the phage with the arrangement of fragments produced by the different enzymes has been established.This paper is the 4^rd publication in the series Structure and Function of the Genome of Coliphage T5.     

Cell Wall Polysaccharide Biosynthesis by Membrane Fragments from Streptococcus pyogenes and Stabilized L-Form

The formation and composition of a cell wall rhamnose-containing polysaccharide by membrane fragments from Streptococcus pyogenes and its stabilized L-form were compared. Also, the effect of prior treatment on the ability of coccal whole-cell and membrane fragments to incorporate radioactivity from thymidine diphosphate-¹⁴C-rhamnose, and the results of subsequent attempts to remove labeled polysaccharide from such membranes are given. L-form membrane fragments were capable of only 10% uptake of ¹⁴C-rhamnose from this nucleotide as compared with streptococcal membranes. However, once bound, both membrane fragments polymerized rhamnose to the same extent. These findings tend to negate the almost complete lack of polymeric rhamnose within the intact L-form as being due to the absence of membrane enzymes necessary for the transfer of rhamnose from a suitable precursor to membrane acceptor sites or enzymes responsible for rhamnose polymerization. Degradation of labeled rhamnose polysaccharide after isolation from coccal membranes by mild acid hydrolysis showed muramic acid and glucosamine to be attached. This same polysaccharide from L-form membrane fragments was devoid of amino sugars. These data suggest the possible involvement of amino sugars in the attachment of cell wall polymeric rhamnose to the streptococcal cytoplasmic membrane. The absence of attached amino sugars to rhamnose polysaccharide from L-form membrane fragments is discussed in terms of this organism's continued inability for new cell wall formation. The isolation, from streptococcal membrane fragments, of a polysaccharide containing rhamnose and amino sugars common to at least two different streptococcal cell wall-type polymers was demonstrated.     

Preparation of carboxylic acid-bearing polysaccharide nanofiber made from euglenoid β-1,3-glucans

This paper introduces a new strategy for creating surface modified polysaccharide nanofibers. To demonstrate proof of principle, the synthesis, structure, and self-assembly behavior of a carboxylic acid-bearing polysaccharide made from paramylon (β-1,3-glucan) and succinic anhydride were investigated. Examination by a combination of NMR, FT-IR, and SEC-MALLS confirmed that successful preparation of the desired succinylated paramylon without significant depolymerization. NMR, SEC-MALLS, visible absorption and CD spectroscopic analyses indicated that the paramylon derivative forms the triplex structure in solutions. SEM observation revealed that succinylated paramylon forms a nanofiber that has carboxylic acid on the surface.     

Covalent immobilization of pullulanase on alginate and study of its hydrolysis of pullulan

The immobilization of pullulanase from Klebsiella pneumoniae by grafting was investigated. Pullulanase was linked after activation of alginate via a covalent bond between the amine groups of the enzyme and the carboxylic acid groups of alginate. The immobilization yield was 60%. The activity of free pullulanase and immobilized pullulanase was followed by the quantification of reducing ends by colorimetric assay and the determination of the molar masses of the hydrolyzed pullulan by SEC/MALS/DRI. Compared to free pullulanase, the kinetics is largely slowed. The evolution of the weight average molar mass of pullulan leading to high production of shorter oligosaccharides during hydrolysis is not the same as that obtained with free enzyme. Immobilized pullulanase retained 75% and 30% of its initial activity after 24 h and 14 days of incubation at 60°C, respectively while free pullulanase lost its activity after 5 h of hydrolysis at the same temperature. The kinetic parameters of immobilized pullulanase were also investigated by isothermal titration calorimetry (ITC). The affinity of immobilized enzyme to its substrate was reduced compared to the free pullulanase due to steric hindrance and chemical links. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:883–889, 2015