scholarly journals Properties of allophycocyanin II and its α- and β-subunits from the thermophilic blue–green alga Mastigocladus laminosus

1979 ◽  
Vol 181 (3) ◽  
pp. 577-583 ◽  
Author(s):  
Jürg R. Gysi ◽  
Herbert Zuber
Keyword(s):  
Gel Filtration ◽  
Fluorescence Emission ◽  
Potassium Phosphate ◽  
Difference Spectrum ◽  
Spectroscopic Properties ◽  
Sedimentation Equilibrium ◽  
Visible Absorption ◽  
Molecular Weights ◽  
Α And Β Subunits ◽  
Equilibrium Analyses

Purified allophycocyanin II and its subunits have been examined with respect to spectroscopic properties, sedimentation, reconstitution and isoelectric behaviour. In 0.02m-potassium phosphate buffer, pH8.0, and at a concentration of 0.25mg/ml, allophycocyanin II and its α- and β-subunits show visible absorption maxima at 650, 615 and 615nm respectively, whereas the fluorescence emission maxima were determined to be at 662, 640 and 630nm respectively. The absorption difference spectrum (dilution difference) of allophycocyanin II displays maxima at 650 and 590nm with a minimum at 610nm. The c.d. spectrum of allophycocyanin II showed only one positive-ellipticity band at 635nm, and a major negative-ellipticity band at 340nm. Oxidation of allophycocyanin II, low- and high-pH solutions (pH3.0 and 11.0), various ethanol concentrations as well as dialysis against distilled water induce a spectral change leading to phycocyanin-like characteristics. In most cases these shifts are reversible. Allophycocyanin II is thermostable over a period of 60min at temperatures up to 60°C. The isoelectric points of allophycocyanin II and its α- and β-subunits are 4.65, 4.64 and 4.82 respectively. Estimated molecular weights from sedimentation-equilibrium analyses were 102500 for allophycocycanin II, 16000 for the α- and 31500 for the β-subunit. Recombination of α- and β-subunits leads to allophycocyanin II, which is indistinguishable from native allophycocyanin with respect to its spectral form, to its gel-filtration and to its electrophoretic behaviour.

scholarly journals Studies on the structure of hyaluronic acid. Characterization of the product formed when hyaluronic acid is treated with ascorbic acid

1969 ◽  
Vol 114 (4) ◽  
pp. 819-825 ◽  
Author(s):  
David A. Swann
Keyword(s):  
Ascorbic Acid ◽  
Hyaluronic Acid ◽  
Sedimentation Equilibrium ◽  
Connective Tissues ◽  
Molecular Weights ◽  
The Matrix ◽  
Molecular Sizes ◽  
Equilibrium Analyses ◽  
Physical And Chemical

Physical and chemical methods were used to characterize hyaluronic acid before (fraction HAIIBI) and after (fraction HA-AA) treatment with ascorbic acid. Fraction HA-AA was recovered with an almost quantitative yield and was shown to be chemically identical with fraction HAIIBI by all the methods used. These two materials, however, differed markedly in their molecular sizes and degree of polydispersity. By using sedimentation, diffusion and sedimentation-equilibrium analyses, weight-average molecular weights of about 1·2×106 and 6·5×104 respectively were obtained for fractions HAIIBI and HA-AA. It is concluded from these results that hyaluronic acid has a molecular weight of about 65000 and that the polysaccharide chain of this molecule is not depolymerized by ascorbic acid. It is further proposed that hyaluronic acid molecules in the matrix of connective tissues are present either in an aggregated form or as subunits of heterogeneous macromolecules, and that it is the linkages responsible for the organization of these structures which are broken by ascorbic acid.

scholarly journals Isolation and Biophysical Characterisation of Bioactive Polysaccharides from Cucurbita Moschata (Butternut Squash)

Polymers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 1650
Author(s):  
Shahwar Imran Jiwani ◽  
Richard B. Gillis ◽  
David Besong ◽  
Fahad Almutairi ◽  
Tayyibe Erten ◽  
...  
Keyword(s):  
Galacturonic Acid ◽  
Gel Filtration ◽  
Monosaccharide Composition ◽  
Sedimentation Equilibrium ◽  
Gas Chromatography Mass Spectrometry ◽  
Cucurbita Moschata ◽  
Molecular Weights ◽  
Flexible Coil ◽  
Neutral Sugars ◽  
Pectin Polysaccharide

Cucurbits are plants that have been used frequently as functional foods. This study includes the extraction, isolation, and characterisation of the mesocarp polysaccharide of Cucurbita moschata. The polysaccharide component was purified by gel filtration into three fractions (NJBTF1, NJBTF2, and NJBTF3) of different molecular weights. Characterisation includes the hydrodynamic properties, identification of monosaccharide composition, and bioactivity. Sedimentation velocity also indicated the presence of small amounts of additional discrete higher molecular weight components even after fractionation. Sedimentation equilibrium revealed respective weight average molecular weights of 90, 31, and 19 kDa, with the higher fractions (NJBTF1 and NJBTF2) indicating a tendency to self-associate. Based on the limited amount of data (combinations of 3 sets of viscosity and sedimentation data corresponding to the 3 fractions), HYDFIT indicates an extended, semi-flexible coil conformation. Of all the fractions obtained, NJBTF1 showed the highest bioactivity. All fractions contained galacturonic acid and variable amounts of neutral sugars. To probe further, the extent of glycosidic linkages in NJBTF1 was estimated using gas chromatography–mass spectrometry (GCMS), yielding a high galacturonic acid content (for pectin polysaccharide) and the presence of fructans—the first evidence of fructans (levan) in the mesocarp. Our understanding of the size and structural flexibility together with the high bioactivity suggests that the polysaccharide obtained from C. moschata has the potential to be developed into a therapeutic agent.

scholarly journals Molecular Weight Distribution Studies on Heparin

1977 ◽  
Author(s):  
Grant H. Barlow
Keyword(s):  
Molecular Weight ◽  
Molecular Weight Distribution ◽  
Weight Distribution ◽  
Gel Filtration ◽  
Equilibrium Analysis ◽  
Resolving Power ◽  
Sedimentation Equilibrium ◽  
Distribution Data ◽  
Molecular Weights ◽  
Distribution Studies

The determination of molecular weight distribution using the sedimentation equilibrium analysis developed for polymers by T. Scholte (J. Polymer Sei, 6, 111, 1968) has been adapted for heparin analysis. Pork mucosal heparin separated into molecular weight subfractions by gel filtration on Ultrogel AcA44 (L.K.B.) was used to test the validity and resolving power of the method. Results indicate that the method is able to differentiate molecular weight distribution satisfactorily. Comparisons have been made of molecular weight distribution of samples from different species, organs and manufacturers. Average molecular weights for most samples center around 15,000 Dal tons, but samples show considerable variation in their distribution data. Results suggest that variations between manufacturers is more pronounced than the specie and organ difference indicating the importance of the purification procedure.

The Isolation and Characterization of a Ternary Human Plasmin B-Chain-Streptokinase-Plasminogen Complex

1987 ◽  
Vol 58 (02) ◽  
pp. 772-777 ◽  
Author(s):  
Louis Summaria ◽  
Irena Boreisha ◽  
Grant H Barlow ◽  
Kenneth C Robbins
Keyword(s):  
Ternary Complex ◽  
Double Diffusion ◽  
Sedimentation Equilibrium ◽  
Molar Ratio ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Human Plasminogen ◽  
Equilibrium Analyses ◽  
Species Specific

SummaryA ternary equimolar human plasmin B-chain-streptokinase-plasminogen complex was isolated from a mixture of the plasmin B-chain-streptokinase complex and human plasminogen at 0° C and 37° C. A ternary complex which was shown to be species specific, was identified and characterized by ultracentrifugal, acrylamide gel electrophoretic, and agarose double diffusion analyses. When mixed at a 1:1 molar ratio at 0° C, 39.9% of the preparation existed as a plasmin B-chain-streptokinase-plasmino-gen complex; when mixed at 37° C, 86.d% existed as a complex, which was identified by electrophoretic analyses to be a plasmin B-chain-streptokinase-plasmin complex. Sedimentation velocity analyses gave s°2o,w values of 3.79 for the plasmin B-chain-streptokinase complex, 4.10 for Lys-plasmin, and 6.23 for the plasmin B-chain-streptokinase-plasmin complex. Sedimentation equilibrium analyses gave molecular weights of 73,900 for the plasmin B-chain-streptokinase complex, 82,900 for Lys-plasmin, and 153,100 for the plasmin B-chain-streptokinase-plasmin complex. The diisopropylphosphorofluoridatc (DFP)-inhibitcd and the p-nitrophenyl-p-guanidino-benzoate (NPGB)-inhibited plasmin B-chain-streptokinase complexes both retained their ability to form a ternary complex with human plasminogen, but this complex did not convert to a plasmin B-chain-streptokinase-plasmin complex. Thus, the active site serine residue is essential for the activator activity of the plasmin B-chain-streptokinase complex, but it is not necessary for the binding of the plasmin B-chain-streptokinase complex to plasminogen to form a ternary complex.

scholarly journals The Isolation of Aggregates of Spectrin From Bovine Erythrocyte Membranes

1975 ◽  
Vol 28 (3) ◽  
pp. 259 ◽  
Author(s):  
GB Ralston
Keyword(s):  
Ionic Strength ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Sedimentation Equilibrium ◽  
Erythrocyte Membranes ◽  
Bovine Erythrocyte ◽  
Molecular Weights ◽  
Wide Range

Aggregated states of spectrin from bovine erythrocyte membranes can be detected in sedimentation velocity experiments. These aggregates have been isolated by means of gel filtration on columns of 4 % agarose. They appear to be stable over a wide range of pH and ionic strength, although they are dissociated by sodium dodecyl sulphate. Sedimentation equilibrium measurements yielded values of 960000 and 480000 for the molecular weights of the major aggregates, corresponding to a tetramer and dimer, respectively. The presence of different aggregated states in spectrin preparations may explain the wide variation in the reported physica~ properties of spectrin.

Hydrodynamic and optical properties of the wheat germ Em protein

1985 ◽  
Vol 63 (8) ◽  
pp. 803-811 ◽  
Author(s):  
William D. McCubbin ◽  
Cyril M. Kay ◽  
Byron G. Lane
Keyword(s):  
Circular Dichroism ◽  
Gel Filtration ◽  
Fluorescence Emission ◽  
Sedimentation Coefficient ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Partial Specific Volume ◽  
Fluorescence Emission Spectrum ◽  
Fluorescence Measurements ◽  
Circular Dichroïsm

The size and shape of the Em protein from wheat embryos have been examined by gel filtration, densitometry, ultracentrifugation, and viscosity. Circular dichroism and fluorescence measurements have also been made. Em has an intrinsic sedimentation coefficient, [Formula: see text], of 1.11S and a Stokes radius, RS, of 28.2 Å (1 Å = 0.1 nm) as determined by high performance liquid chromatography gel filtration on a TSK 2000SW column. The partial specific volume [Formula: see text] from density measurements is 0.683 mL/g, a much lower than typical value. The molecular weight from sedimentation equilibrium is 11 200, with no indication for protein aggregation. The intrinsic viscosity [η] of Em is 6.02 mL/g. Circular dichroism shows the molecule to be about 70% random coil. The fluorescence emission spectrum is typical for a tyrosine-containing protein. The hydrodynamic data indicates a poor fit to either a prolate or oblate ellipsoid model; excess hydration or flexibility of the polypeptide chain caused by the rather unusual amino acid composition may be a possible cause. The implications that the low value of [Formula: see text], the high value of RS, and the random-coil configuration of the Em protein may have on its ability to bind water and to contribute to the maintenance of a minimal level of hydration compatible with the sustained viability of the "dry" organism are subjects of an extended discussion. Briefly, it is suggested that Em may provide a matrix of bound water which opposes denaturation of proteins in the "desiccated" cytoplasm of dry plant embryos.

scholarly journals An Atypical Immunoglobulin

Blood ◽  
1968 ◽  
Vol 32 (2) ◽  
pp. 189-204 ◽  
Author(s):  
A. F. LEWIS ◽  
D. E. BERGSAGEL ◽  
A. BRUCE-ROBERTSON ◽  
R. K. SCHACHTER ◽  
G. E. CONNELL
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Gel Filtration ◽  
Sedimentation Equilibrium ◽  
Light Chains ◽  
Equilibrium Studies ◽  
Heavy Chains ◽  
Molecular Weights ◽  
Plasma Cell Neoplasm ◽  
Ig G

Abstract A protein of the Ig G family has been isolated from the serum of a patient with a tentative diagnosis of a plasma cell neoplasm. The protein has a lower sedimentation constant (5.4) and a lower molecular weight (125,000) than normal immunoglobulins of the G family. The protein has heavy-chain determinants of type G and light-chain determinants of the κ-type. Heavy and light chains have been prepared by reductive cleavage followed by gel filtration. The heavy-chain preparation is homogeneous in starch gels in acidic buffer containing urea but has a faster mobility than normal Ig G heavy chains. The light-chain preparation is resolved into two components in electrophoresis, and both have slower mobility than normal Ig G light chains. The heavy- and light-chain preparations cross react with normal Ig G heavy and light chains in immunodiffusion analysis. Sedimentation equilibrium studies suggest that both the heavy and light chains have lower molecular weights than their normal counterparts.

Purification and characterization of tonin

1976 ◽  
Vol 54 (9) ◽  
pp. 788-795 ◽  
Author(s):  
S. Demassieux ◽  
R. Boucher ◽  
C. Grisé ◽  
J. Genest
Keyword(s):  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Potassium Phosphate ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Sedimentation Equilibrium ◽  
Angiotensin I ◽  
Ph Optimum ◽  
Ammonium Sulphate Precipitation

Tonin was purified from rat submaxillary glands by differential centrifugation, ammonium sulphate precipitation, gel filtration on Sephadex G150, and by ion-exchange chromatography on DEAE-cellulose, phospho-cellulose, SP-Sephadex C25, and SP-Sephadex C50. Purified tonin was shown to be homogeneous by analytical electrophoresis and by analytical ultracentrifugation analysis. Purified tonin was very stable when stored in buffers of low pH values or when incubated at high temperatures in neutral solutions. The molecular weight estimated by sedimentation equilibrium was 28 700. The pH optimum was near 6.8 in a 0.1 M potassium phosphate buffer. The Michaelis–Menten constant for tonin using angiotensin I as substrate was about 4 × 10−5 M. Tonin activity was strongly inhibited by plasma. Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by plasma was of the non-competitive type.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

scholarly journals Terpene polyacrylate TPA5 shows favorable molecular hydrodynamic properties as a potential bioinspired archaeological wood consolidant

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Michelle Cutajar ◽  
Fabrizio Andriulo ◽  
Megan R. Thomsett ◽  
Jonathan C. Moore ◽  
Benoit Couturaud ◽  
...  
Keyword(s):  
Molar Mass ◽  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Axial Ratio ◽  
Sedimentation Equilibrium ◽  
Archaeological Wood ◽  
Molecular Hydrodynamic ◽  
Pine Trees ◽  
Minimal Concentration ◽  
Hydrogen Bonding Potential

AbstractThere is currently a pressing need for the development of novel bioinspired consolidants for waterlogged, archaeological wood. Bioinspired materials possess many advantages, such as biocompatibility and sustainability, which makes them ideal to use in this capacity. Based on this, a polyhydroxylated monomer was synthesised from α-pinene, a sustainable terpene feedstock derived from pine trees, and used to prepare a low molar mass polymer TPA5 through free radical polymerisation. This polymer was extensively characterised by NMR spectroscopy (chemical composition) and molecular hydrodynamics, primarily using analytical ultracentrifugation reinforced by gel filtration chromatography and viscometry, in order to investigate whether it would be suitable for wood consolidation purposes. Sedimentation equilibrium indicated a weight average molar mass Mw of (4.3 ± 0.2) kDa, with minimal concentration dependence. Further analysis with MULTISIG revealed a broad distribution of molar masses and this heterogeneity was further confirmed by sedimentation velocity. Conformation analyses with the Perrin P and viscosity increment ν universal hydrodynamic parameters indicated that the polymer had an elongated shape, with both factors giving consistent results and a consensus axial ratio of ~ 4.5. These collective properties—hydrogen bonding potential enhanced by an elongated shape, together with a small injectable molar mass—suggest this polymer is worthy of further consideration as a potential consolidant.

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