Low Angle Laser Light Scattering Research Articles

Salting out of aqueous proteins: Phase equilibria and intermolecular potentials

AbstractSalting‐out phase equilibria are reported for lysozyme and α‐chymotrypsin from concentrated ammonium‐sulfate solutions. Supernatant and dense‐phase protein concentrations and the resulting protein partition coefficients are given as a function of solution pH and ionic strength. Phase equilibria with a trivalent salt (sodium citrate) confirm that ionic strength, rather than salt concentration, is the appropriate variable describing phase equilibria. The salting‐out behavior of a mixture of an aqueous lysozyme and α‐chymotrypsin is independent of the presence of the other protein.Parameters for a molecular‐thermodynamic description of salting‐out behavior are obtained from low‐angle laser‐light scattering (LALLS). Osmotic second virial coefficients from LALLS are reported over a range of pH for dilute chymotrypsin concentrations in aqueous electrolyte solutions at 0.01 and 1.0 M ionic strengths. Effective Hamaker constants, regressed from experimental osmotic second virial coefficients, are determined for models of the protein – protein potential of mean force. In addition to excluded volume, dispersion, and shielded charge – charge potentials, the description of protein ‐ protein interactions includes attractive charge – dipole and dipole – dipole potentials as well as an osmotic‐attraction potential that becomes important at high salt concentrations. Protein dipole – dipole potentials are required to account for the observed pH dependence of osmotic second virial coefficients, especially at low ionic strength.

Effects of poly(vinyl acetate) and poly(methyl methacrylate) low‐profile additives on the curing of unsaturated polyester resins. II. Morphological changes during cure

AbstractThe effects of two low‐profile additives (LPA), poly(vinyl acetate) (PVAc) and poly(methyl methacrylate) (PMMA), on the morphological changes during the cure of unsaturated polyester (UP) resins at 110°C were investigated by an approach of integrated reaction kinetics‐morphology‐phase separation measurements by using a differential scanning calorimeter (DSC), scanning electron microscopy (SEM), optical microscopy (OM), and a low‐angle laser light‐scattering appartus (LALLS). For the UP resins cured at 110°C, adding LPA could facilitate the phase separation between LPA and crosslinked UP phases early in the reaction, and discrete microgel particles were thus allowed to be identified throughout the reaction. Microvoids and microcracks responsible for the volume shrinkage control could also be observed evidently at the later stage of reaction under SEM. Depending on the types of LPA and the initial molar ratios of styrene to polyester CC bonds, the morphological changes during the cure varied considerably. The progress of microstructure formation during reaction has been presented. Static ternary phase characteristics for the styrene–UP–LPA system at 25°C have also been employed to elucidate the resulting morphology during the cure in both the continuous and the dispersed phases. © 1995 John Wiley & Sons, Inc.

Conformation of [formula omitted] in aqueous solution

The conformation and dilute solution properties of (2→1)-β- d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights ( M w) from 1.49 × 10 4 to 5.29 × 10 6 were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M w < 4 × 10 4, the intrinsic viscosity [η] varies with M w 0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M w > 10 5, [η] exhibits an unusually weak dependence on M w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.

Molecular Mass Characterization of Poly(4-Vinylpyridine)

Abstract Poly(4-vinylpyridine) samples, synthesized by anionic and radical initiators, have been characterized by Size Exclusion Chromatography, Light Scattering and Intrinsic Viscosity. The mobile phase used in SEC characterization was 50% methanol and 50% 0.1M LiNO3. Broad molecular weight distribution samples of P4-VP1 characterized via Low Angle Laser Light Scattering, have been used in the calibration of the SEC system. The polymers show different molecular weight distribution and high polydispersity. Mark-Houwink constants for P4-VP in the SEC mobile phase have also been obtained.

Characterization of Polyether-Polyurethanes by Sec Coupled with Viscometry and Lalls

Abstract Two- and three-component polyether-polyurethanes were characterized by means of size exclusion chromatography (SEC)-multidetector methods. SEC was coupled with three or two detectors in series, a continuous viscometer (CVM), a low angle laser light scattering (LALLS) photometer and a differential refractometer (DR), or with CVM and DR. Molecular weight (MW) averages were determined independently by SEC-LALLS and by SEC using universal calibration (SEC-UC). MWs obtained by SEC-UC agreed with the corresponding SEC-LALLS MWs up to 60,000, but were higher for MWs over 60,000. This apparent increase in MW was attributed to the non-linearity of the calibration curve in the range of MW over 7.105. Due to high limiting viscosity number [η] of polyu-rethanes as compared to polystyrenes with the same MW, there was a big influence of concentration effects upon SEC when using calibration curves. For an accurate SEC-UC determination of the MW distribution, the parameter [η]c should be considered.

Application of integral‐equation theory to aqueous two‐phase partitioning systems

AbstractA molecular‐thermodynamic model is developed for representing thermodynamic properties of aqueous two‐phase systems containing polymers, electrolytes, and proteins. The model is based on McMillan‐Mayer solution theory and the generalized mean‐spherical approximation to account for electrostatic forces between unlike ions. The Boublik‐Mansoori equation of state for hard‐sphere mixtures is coupled with the osmotic virial expansion truncated after the second‐virial terms to account for short‐range forces between molecules.Osmotic second virial coefficients are reported from low‐angle laser‐light scattering (LALLS) data for binary and ternary aqueous solutions containing polymers and proteins. Ion‐polymer specific‐interaction coefficients are determined from osmoticpressure data for aqueous solutions containing a water‐soluble polymer and an alkali chloride, phosphate or sulfate salt.When coupled with LALLS and osmotic‐pressure data reported here, the model is used to predict liquid‐liquid equilibria, protein partition coefficients, and electrostatic potentials between phases for both polymer‐polymer and polymer‐salt aqueous two‐phase systems. For bovine serum albumin, lysozyme, and α‐chymotrypsin, predicted partition coefficients are in excellent agreement with experiment.

Molecular mass determination of low-molecular-mass heparins: Application of wide collection angle measurements of light scattering using a high-performance gel permeation chromatographic system equipped with a low-angle laser light-scattering photometer

A high-performance gel permeation chromatographic system with on-line low-angle laser light-scattering detection (BPGPC-LALLS) was used to determine molecular masses of low-molecular-mass heparins (LMMHs). Measurements at wide and narrow collection angles were compared and the application of the HPGPC-LALLS method to small molecules, with molecular masses in the range 1000–10000, was assessed. The molecular mass averages of fractionated heparins and commercially available LNMHs were also determined by ordinary HPGPC analysis using an LMMH molecular-mass calibrant, supplied by the National Institute for Biological Standards and Control for the calibration of columns. The LALLS intensity at the routinely used narrow collection angle (θ col = 1°) about doubled at the wide collection angle (θ col = 2°) and the signal-to-noise ratio was improved. The present study thus indicates that wide collection angle measurement of light scattering allows the application of the HFGPC-LALLS method to very small biopolymers of molecular mass < 10 000.

Mark-Houwink-Sakurada coefficients for conventional poly(methyl methacrylate) in tetrahydrofuran

Intrinsic viscosities [η] in tetrahydrofuran (THF) at 30°C are reported for ten narrow molecular weight distribution poly(methyl methacrylate) (PMMA) samples covering nearly two orders of magnitude in molecular weight. By combining the viscosity results with absolute weight-average molecular weights \(\left( {\bar M_{\text{w}} } \right)\) determined via low-angle laser light scattering (LALLS) experiments, we have accurately determined the Mark-Houwink-Sakurada (MHS) parameters for this polymer-solvent pair (K=7.56x105 and a=0.731, when [η] is in dL g-1).

Comparison of the properties of dextran and hydroxyethyl starch substituted with benzene tetracarboxylate in terms of their use in blood transfusion

Solutions of dextran and hydroxyethyl starch are used as plasma substitutes but are not capable of carrying oxygen in vivo. To transform these solutions into blood substitutes, it has been suggested that the natural oxygen-carrier protein, i.e. human haemoglobin, is bound to these polymers. However, the polymers have to be modified so that in the protein conjugate haemoglobin exhibits the appropriate oxygen-binding properties. Thus, covalent conjugates of oxyhaemoglobin and of dextran substituted with benzene tetracarboxylate have been used and appear effective vascular oxygen carriers. The same procedure was applied to hydroxyethyl starch but this polysaccharide, because of its branched nature, could not be substituted with benzene tetracarboxylate without being highly cross-linked as evidenced by NMR and low-angle laser light-scattering analyses. As a consequence, in the haemoglobin-hydroxyethyl starch covalent conjugates, the polymer-linked benzene tetracarboxylate groups are not easily accessible to the allosteric site of the protein as in the dextran conjugates, and therefore cannot improve its oxygen-binding properties.

Change in oligomeric structure of solubilized Na + /K +-ATPase induced by octaethylene glycol dodecyl ether, phosphatidylserine and ATP

Membrane-bound Na + /K +-ATPase purified from dog kidney was solubilized with octaethylene glycol dodecyl ether (C 12E 8), and the resultant solubilized enzyme was chromatographed on a TSKgel G4000SW XL or G3000SW XL column equilibrated with elution buffers containing various ligands affecting oligomerization of the enzyme. Weight-averaged molecular weight ( M w ) values for the main protein components eluted were estimated by low-angle laser light-scattering photometry. With increasing concentration of C 12E 8 included in the elution buffer from 0.1 to 5 mg/ml, the M w decreased from 230 000 to 153 000, indicating that C 12E 8 induced dissociation of the enzyme. In contrast, the M w of the protein component increased up to 1.44 · 10 6 as the concentration of phosphatidylserine (PS) added to the elution buffer containing a fixed concentration of 0.3 mg/ml C 12E 8 was increased to 120 μg/ml. The association and/or aggregation were reversible by removal of the PS by rechromatography. Addition of PS to the elution buffer also allowed the solubilized enzyme to exhibit ATPase activity comparable to that of the membrane-bound enzyme during passage through the column. This was also the case with phosphatidylglycerol (PG) and phosphatidylinositol, but not with phosphatidylcholine or phosphatidylethanolamine. The specific refractive index increment d n / d c p ) of the solubilized enzyme was increased by addition of exogenous PG or PS, strongly suggesting that the phospolipid became bound to the enzyme, and that it induced association of the enzyme. The association induced by PS was inhibited by ATP and ADP, but not AMP. The concentrations for half-maximal inhibition were 0.44 mM for ATP and 0.88 mM for ADP. The PS-induced associated enzyme isolated by chromatography in the presence of 120 μg/ml PS was dissociated by ATP with K 0.5 of 0.16 mM. The dissociating effect of C 12E 8, ATP and ADP and the associating effect of PS on the solubilized enzyme are consistent with the reports that C 12E 8 mimics the effect of regulatory ATP at the low-affinity site on the conformationl transition from E 2 to E 1, and that phospholipids are essential for the reverse transition from E 1 to E 2. The results can be explained by assuming that the enzyme takes the form of a loosely associated diprotomer in the E 1 state and a tightly associated one in the E 2 state.

Extrudate swell of high density polyethylene. Part I: Aspects of molecular structure and rheological characterization methods

AbstractTwo high density polyethylene (HDPE) resins–samples 801 and 802–both nominally the same material, as they are taken from successive batches of the same commercial grade, are characterized for their molecular structure and rheological properties. Gel permeation chromatography (GPC) and low angle laser light scattering (LALLS) results must be interpreted in combination with rheological data to show the presence of somewhat more high molecular weight material in 802 that in 801. Small amplitude oscillatory shear, steady shear, and capillary shear measurements performed in different laboratories show consistently higher shear viscosity values at low shear rates for sample 802. Extensional viscosity measurements show similar results. The interpretation of rheological data in terms of molecular structure could be complicated by the possible presence of long chain branching (LCB). The zero shear viscosity and discrete relaxation spectrum is estimated for both samples. The small rheological difference between 801 and 802 forms the basic information for understanding their time dependent extrudate swell behavior, as will be described in Part II.

Heat-induced aggregation of recombinant erythropoietin in the intact and deglycosylated states as monitored by gel permeation chromatography combined with a low-angle laser light scattering technique.

Aggregate formation of recombinant human erythropoietin (r-EPO) on heat-treatment was followed by gel permeation chromatography combined with a low-angle laser light scattering technique under various conditions with respect to pH and salt concentration in order to provide basic knowledge about the change strictly required to be monitored for medicinal proteins. When heated at 60 degrees C at neutral pH, an aggregate with a limited size consisting of about 20 r-EPO molecules was formed. On heating at 50 degrees C at acidic pH, aggregation was unlimited. The aggregation proceeded non-covalently in acidic pH, but in alkaline pH covalent bond formation was also involved. Increase in salt concentration enhanced the aggregation. Deglycosylation of the N-linked oligosaccharides made r-EPO remarkably susceptible to aggregation on heat-treatment, indicating that the carbohydrate chains are essential to the stability of r-EPO.

The synthesis of poly‐L‐amino acids and their characterization by size exclusion chromatography with low‐angle laser light scattering (SEC‐LALLS/UV/DRI)

AbstractThe synthesis and characterization of polyamino acids are described in this paper. Detailed polymerization descriptions of newly synthesized polyamino acids are discussed. The final polyamino acids were characterized by elemental analysis, infrared (IR) spectroscopy, mass spectra, and low‐angle laser light scattering (LALLS). LALLS was used to determine the weight average molecular weight (MW) indicating the extent of the polymerization procedure. Isocratic size exclusion chromatography (SEC) coupled to a tri‐detection system consisting of a LALLS, ultraviolet/visible detector (UV/VIS), and a modified differential refractive index detector (DRI) was used to calculate on‐line specific refractive index increment (dn/dc), MW, percent recovery, and polydispersity, all in one injection once one determines the on‐line molar absorptivity value of the synthesized polyamino acid. © 1992 John Wiley &amp; Sons, Inc.

Determination of persistence lengths and mark-houwink constants of aromatic polyesters from GPC-differential viscometric coupling

The introduction of lateral substituents and/or twisted biphenylylene units increase the solubility of aromatic para-linked polyesters. Two polyesters of this type of structure were characterized by gel permeation chromatography (GPC), low angle laser light scattering (LALLS) and viscometry. From the molecular weights and limiting viscosity numbers obtained by universal calibration and LALLS/viscometry the Mark-Houwink constants and persistence lengths were determined. The values of these parameters indicated that the polyesters can be described as wormlike chains in dilute solution.

Characterization of polysaccharide solutions by means of chromatography-multidetection

Size exclusion chromatography (SEC) combined with double detection, utilizing a differential refractive index (DRI)- and a low angle laser light scattering (LALLS) - detector represents a powerful experimental set-up in the field of chromatography-multidetection (C-MD). By means of these techniques high-quality information for profound polymer characterization is provided in a fast and semi-automatical (autosampler/PC-controlled) way. Molecular characteristics such as molecular weight distributions, molecular weight averages, tendencies for intermolecular interactions, can be obtained by these techniques. Successful handling of data from C-MD-techniques within reasonable time ranges requires adequate hardware and software. This is especially true if advanced data processing is applied, e.g. to yield polymer particle size/shape characteristics in terms of Staudinger/Mark/Houwink constants (K,a), distributions of sphere equivalent particle radii (RcD)-, sphere equivalent mass density of solved polymer coils (ρcD)- and intrinsic viscosity ([η]D). For aqueous solutions of polysaccharide samples (waxy maize starch hydrolyzate, hydroxyethyl-amylopectin from waxy maize hydrolyzate, dextran) data from SEC-DRI/LALLS and universal calibration experiments are utilized for a comprehensive physico-chemical characterization of these polymers.

Interfacing gradient elution ion-exchange chromatography and low-angle laser light-scattering photometry for analysis of proteins

Molecular weights (MWs) of different proteins were determined by interfacing gradient elution ion-exchange chromatography and low-angle laser light-scattering photometry (IEC-LALLS). A high-performance strong cation-exchange column was used to elute proteins using fast (5 min) and conventional (15-30 min) gradients. The eluted proteins were characterized on-line by determining their MWs using LALLS. The specific refractive index (RI) increment (d n/d c) and the RI of the solvent used over the gradient range were determined off-line and used to calculate the absolute weight-average MWs. Four proteins, ribonuclease A, α-chymotrypsinogen A, trypsinogen and β-lactoglobulin A (β-LACT) were studied. Accurate MWs were obtained for all the proteins using fast and conventional gradients, except for β-LACT, which aggregated as a function of the gradient employed. The degree of aggregation of β-LACT increased as the rapidity of the gradient was increased over a fixed gradient range. This study indicated that it is possible to separate and characterize proteins rapidly using IEC-LALLS.

Practical on-line determination of biopolymer molecular weights by high-performance liquid chromatography with classical light-scattering detection

Most biopolymer molecules are much smaller than the wavelength of light used in classical light-scattering experiments ( ca. 500 nm), and thus the simple Rayleigh equation and a 90° light-scattering photometer are sufficient to determine their molecular weight. In combination with high-performance liquid chromatography (HPLC), it is demonstrated that a simple HPLC fluorimeter can be used as a 90° light-scattering detector for biopolymer molecular weight determinations. To simplify data handling, only relative molecular weights are measured. Three mathematical assumptions are adopted, and their validity for proteins is shown. To place the work in perspective, the relative advantages and limitations of this 90° light-scattering detector are compared with the more commonly used low-angle laser light-scattering detector. Two examples of protein molecular weight determinations are given to illustrate the broad utility of 90° classical light scattering to the study of biopolymer structures and interactions.

Characterization of carbonic anhydrase isozyme CA2, which is the CAH2 gene product, in Chlamydomonas reinhardtii.

From high-CO2 (5% CO2) grown unicellular green alga, Chlamydomonas reinhardtii, carbonic anhydrase (CA) was isolated by affinity chromatography and characterized. Isolated CA was identified as an isozyme (CA2) which is the product from the second gene CAH2 by peptide sequencing. The CA2 was inactivated by dithiothreitol. This treatment caused dissociation of CA2 into the large (38 kDa) and small subunits (4243 Da). The molecular mass of the CA2 holoenzyme measured by low-angle laser light-scattering photometry and precision differential refractometry combined with gel-filtration HPLC was 87.9 kDa. These results and gene structure indicate that CA2 is a heterotetramer consisting of two large and two small subunits linked by disulfide bonds like CA1, which is the CAH1 gene product. The specific activity of CA2 purified by anion-exchange HPLC was 3300 units per mg protein, which was approximately 1.6 times higher than that of CA1. Therefore, it was concluded that two structurally related isozymes, CA1 and CA2, are present in the wild type cells of C. reinhardtii and differentially regulated by the atmospheric CO2 concentration.

Association of macromolecular prodrugs consisting of adriamycin bound to poly(L‐glutamic acid)

AbstractSoluble macromolecular conjugates for the controlled delivery of the strongly hydrophobic antitumor drug adriamycin (ADR) were prepared. The association behaviour of two types of these amphiphilic conjugates consisting of ADR bound to poly(L‐glutamic acid) (PGA) in aqueous solution was investigated using absorbance and fluorescence spectroscopy as well as GPC and lowangle laser light scattering (LALLS) measurements. ADR was bound via the aminoribosyl moiety either directly with PGA side‐chain groups or via oligopeptide spacer groups. UV/VIS, GPC and LALLS data showed that in buffer solution both ADR conjugates associate intermolecularly. The data of the directly bound ADR conjugates are consistent with the presence of multimers of different degrees of association in equilibrium with single polymer chains. In contrast, spacer‐containing ADR conjugates associate to give stable uniform multimers. UV/VIS and fluorescence spectroscopy performed at very low conjugate concentration show that polymer‐bound ADR residues associate intramolecularly as well. The degree of intra‐ and intermolecular association of the conjugate‐bound ADR molecules depends on the type of conjugate, ADR load and conjugate concentration as well as on ionic strength of the solvent, the presence of organic cosolvents and temperature. The data indicate that hydrophobic domains in spacer‐containing conjugates are more stable compared with directly bound conjugates, probably due to enhanced flexibility and the presence of hydrophobic leucine residues. It is concluded that spacer‐containing conjugates of ADR may have a potential for effective drug delivery under in vivo conditions due to their solution properties in addition to the biodegradability of the drug‐polymer bond.

Continuous culture system for production of biopolymer levan using erwinia herbicola

The optimal production of the fructan biopolymer levan by the bacterium Erwinia herbicola was investigated, including variations in nitrogen, carbon and phosphorous sources, pH, incubation time, culture yields up to 19% by weight produced based on conversion of sucrose as the carbon source when grown in a continuous culture system and processed by tangential flow filtration. Product identity was confirmed with gas chromatography (GC) and (13)C nuclear magnetic resonance (NMR). Gel permeation chromatography (GPC) and low-angle laser light scattering (LALLS) determination of the molecular weight of the product showed a significant difference in molecular weight values dependent on the method of analysis. Analysis by GPC resulted in molecular weight one order of magnitude lower than LALLS independent of sample, underscoring the unusual nature of this biopolymer.