Complex formation of protein with different water-soluble synthetic polymers

The formation of protein-polymer complexes was studied in an aqueous system using dynamic light scattering (DLS) and static light scattering (SLS) as the main experimental tools. Human serum albumin (HSA) was used as a protein and complexed with four representative water-soluble polymers: poly(N-isopropylacrylamide) (PNIPA), poly(ethylene glycol) (PEG), poly(vinyl pyrrolidone) (PVP), and poly(vinyl alcohol) (PVA). The first three molecular weights were within 420,000-540,000 and the last one was 270,000. The complexation was performed at 25 degrees C in 0.01 M NaCl solution adjusted to pH 3 with HCl as a function of mixing ratio (rm; molar ratio of polymer to HSA). From SLS experiments, we determined the molecular weight of the resulting complexes, from the value of which the number (nb) of bound proteins per polymer was estimated. It was found that each polymer forms an intrapolymer complex over a wide range of rm (1.2 > or = rm > or = 0.01). Then, a marked decrease in nb with increasing rm was found. Over the whole rm range, the HSA-PNIPA complex exhibited a large nb value, as compared with the other three complexes whose nb values at the same rm were close to one another. Both the hydrodynamic radius (Rh) by DLS and the radius of gyration (Rg) by SLS for the complexes of PNIPA, PVP, and PVA decreased and then reached a constant value as nb decreased with increasing rm. In the PEG system, however, there were a few changes in Rh and Rg with nb. The Rg/Rh ratio, as an indication of chain expansion, was found to increase with decreasing nb in the PNIPA system. The complexes of PVA and PVP displayed a similar tendency, although the magnitude of the increasing trend was smaller than that of the PNIPA complex. In contrast, the Rg/Rh ratio of the PEG complex hardly varied depending on nb. These results were discussed in connection with the differences of physicochemical properties among four water-soluble polymers.     

Crosslinking of methylolcellulose during acetylation in the dimethylacetamide-paraformaldehyde system

This paper describes the physicochemical characterization of methylcellulose acetate obtained by acetylation of methylcellulose. Dilute polymer solutions were investigated by means of light scattering and hydrodynamic techniques. Several samples were examined, with DS(COCH₃) = 2·5–2·7 and DS(CH₂O) = 1·3–2·2. The polymers are soluble in most common organic solvents, except for a certain amount of material (4–15%) which swells without dissolving. Light scattering measurements in acetone, acetonitrile, ethyl acetate, tetrahydrofuran and 2-butanone indicate no aggregation for the polymer in the examined concentration range. On the other hand, DP_W values are obtained 2 to 4 times higher than the values of the starting cellulose. These findings are interpreted in terms of crosslinking reactions to yield acetal derivatives between two or more cellulose chains.Viscosity measurements also indicate lack of aggregation, thus supporting the above hypothesis.     

Molecular weight determination of heparin and dermatan sulfate by size exclusion chromatography with a triple detector array

The determination of molecular weight (M) and molecular weight distribution (MD) of heparins by a novel approach, consisting of a high performance size exclusion chromatography (HP-SEC) combined with a triple detector array (TDA) is described. HP-SEC/TDA permits the evaluation of MD of polymeric samples through a combined and simultaneous action of three on-line detectors, right-angle laser light scattering (RALLS), refractometer (RI), and viscometer. The method does not require any chromatographic column calibration, thus overcoming also the difficulty to obtain adequate reference standards. It permits the size determination also of small molecules, even when scattering dissimmetry is not observable. Unfractionated heparins, eight fractions of a size fractionated heparin, and dermatan sulfates were analyzed by HP-SEC/TDA. The M values found for the heparin fractions were used to build up a calibration curve of a conventional HP-SEC system: the results obtained analyzing unfractionated heparin samples with both HP-SEC/TDA and HP-SEC were in excellent agreement, suggesting the possibility to use the TDA data to generate standard samples with known MD and intrinsic viscosity [eta]. Moreover, HP-SEC/TDA can successfully be employed also for the determination of the Mark-Houwink a and k parameters.     

Size and shape of the repetitive domain of high molecular weight wheat gluten proteins. II. Hydrodynamic studies

This study describes the hydrodynamic properties of the repetitive domain of high molecular weight (HMW) wheat proteins, which complement the small-angle scattering (SANS) experiments performed in the first paper of this series. The sedimentation coefficients, s(0), and diffusion coefficients, D(0), were obtained from the homologous HMW proteins dB1 and dB4 that were cloned from the gluten protein HMW Dx5, and expressed in Escherichia coli. Monodisperse conditions for accurate determination of s(0) and D(0), were obtained by screening a series of buffers using dynamic light scattering. For the first time, hydrodynamic parameters were obtained on monodisperse samples that enabled the determination of the monomeric size and shape. The hydrodynamic values determined on dB1 and dB4 were used to test the worm-like chain (WLC) model that was proposed in the SANS studies. The successful matching of two separately obtained hydrodynamic parameters of dB1 and dB4 using the WLC model provides further evidence for the WLC model. The small discrepancy between the hydrodynamic and scattering data, possibly coming from the excluded volume effect, was compensated by a solvation layer of 1-2 water molecules thick around the protein in the WLC model. The solvation of the central domain is much higher than those of the terminal domains of the HMW subunits. This difference emphasizes the dual role of HMW wheat gluten proteins in water-binding and aggregation.     

Teaching light scattering spectroscopy: the dimension and shape of tobacco mosaic virus.

The tobacco mosaic virus is used as a model molecular assembly to illustrate the basic potentialities of light scattering techniques (both static and dynamic) to undergraduates. The work has two objectives: a pedagogic one (introducing light scattering to undergraduate students) and a scientific one (stabilization of the virus molecular assembly structure by the nucleic acid). Students are first challenged to confirm the stabilization of the cylindrical shape of the virus by the nucleic acid, at pH and ionic strength conditions where the coat proteins alone do not self-assemble. The experimental intramolecular scattering factor is compared with the theoretical ones for several model geometries. The data clearly suggest that the geometry is, in fact, a rod. Comparing the experimental values of gyration radius and hydrodynamic radius with the theoretical expectations further confirms this conclusion. Moreover, the rod structure is maintained over a wider range of pH and ionic strength than that valid for the coat proteins alone. The experimental values of the diffusion coefficient and radius of gyration are compared with the theoretical expectations assuming the dimensions detected by electron microscopy techniques. In fact, both values are in agreement (length approximately 300 nm, radius approximately 20 nm).     

Neutron and light-scattering studies of DNA gyrase and its complex with DNA

The solution structure of Escherichia coli DNA gyrase, an enzyme that catalyzes the ATP-dependent supercoiling of DNA, has been characterized by small-angle neutron scattering (SANS) and dynamic light-scattering (DLS). The enzyme and its complex with a 172 base-pair fragment of duplex DNA, in H2O or 2H2O solvent, were studied by contrast variation and the measurement of hydrodynamic parameters as a function of scattering angle. The complex was also measured in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP), a non-hydrolyzable ATP analog that is known to support limited supercoiling. The values of the radius of gyration, Rg = 67 A, from SANS and the hydrodynamic radius, Rh = 64 A, from DLS predict a larger than expected volume for the enzyme, supporting the notion of channels or cavities within the molecule. In addition, several classes of models were rejected based on SANS data obtained in 2H2O at larger scattering angles. The best fit to both the SANS and DLS data is obtained for oblate, inhomogeneous particles approximately 175 A wide and 52 A thick. Such particles provide a large surface area for DNA interaction. Both Rg and Rh values change very little upon addition of DNA, suggesting that DNA binds in a manner that does not significantly change the shape of the protein. No appreciable change in structure is found with the addition of ADPNP. However, the higher-angle SANS data indicate a slight rearrangement of the enzyme in the presence of nucleotide.     

The hydrodynamic radii of macromolecules and their effect on red blood cell aggregation

The effects of nonionic polymers on human red blood cell (RBC) aggregation were investigated. The hydrodynamic radius (Rh) of individual samples of dextran, polyvinylpyrrolidone, and polyoxyethylene over a range of molecular weights (1,500-2,000,000) were calculated from their intrinsic viscosities using the Einstein viscosity relation and directly measured by quasi-elastic light scattering, and the effect of each polymer sample on RBC aggregation was studied by nephelometry and low-shear viscometry. For all three polymers, despite their different structures, samples with Rh <4 nm were found to inhibit aggregation, whereas those with Rh >4 nm enhanced aggregation. Inhibition increased with Rh and was maximal at approximately 3 nm; above 4 nm the pro-aggregant effect increased with Rh. For comparison, the Rh of 12 plasma proteins were calculated from literature values of intrinsic viscosity or diffusion coefficient. Each protein known to promote RBC aggregation had Rh >4 nm, whereas those with Rh <4 nm either inhibited or had no effect on aggregation. These results suggest that the influence of a nonionic polymer or plasma protein on RBC aggregation is simply a consequence of its size in an aqueous environment, and that the specific type of macromolecule is of minor importance.     

Evolution of the diacylglycerol lipases

Diacylglycerol lipases (DGLs) mainly catalyze “on-demand” biosynthesis of bioactive monoacylglycerols (MAGs) with different long fatty acyl chains, including 2-arachidonoylglycerol (2-AG), 2-linoleoylglycerol (2-LG), 2-oleoylglycerol (2-OG) and 2-palmitoylglycerol (2-PG). Enzymatic characterization of DGLs, their expression and distribution, and functional features has been elucidated from microorganisms to mammals in some extent. In mammals, biosynthesis, degradation and metabolism of these bioactive lipids intertwine and form a complicated biochemical pathway to affect the mammal neuromodulation of central nervous system and also other physiological processes in most peripheral organs and non-nervous tissue cells, and yet we still do not know if the neuromodulatory role of mammal DGL and MAGs is similar to invertebrates. Tracing the evolutionary history of DGLs from microorganisms to vertebrates will be an essential method to infer DGL and MAG research in organisms. In this review, we give an exhaustive explanation of the ancestral origin, divergence and evolutionary pattern through systemic searching of DGL orthologs in different species. Finally, we also summarize our recent work on the structural and functional studies of DGL in order to explore usage of DGLs in industry and the development of inhibitors for clinical intervention.     

DNA condensation by polyamines: a laser light scattering study of structural effects.

Polyamines such as spermidine and spermine are abundant in living cells and are believed to aid in the dense packaging of cellular DNA. DNA condensation is a prerequisite for the transport of gene vectors in living cells. To elucidate the structural features of polyamines governing DNA condensation, we studied the collapse of lambda-DNA by spermine and a series of its homologues, H2N(CH2)3NH(CH2)n=2-12NH(CH2)3NH2 (n = 4 for spermine), using static and dynamic light scattering techniques. All polyamines provoked DNA condensation; however, their efficacy varied with the structural geometry of the polyamine. In 10 mM sodium cacodylate buffer, the EC50 values for DNA condensation were comparable (4 +/- 1 microM) for spermine homologues with n = 4-8, whereas the lower and higher homologues provoked DNA condensation at higher EC50 values. The EC50 values increased with an increase in the monovalent ion (Na+) concentration in the buffer. The slope of a plot of log [EC50(polyamine4+)] against log [Na+] was approximately 1.5 for polyamines with even number values of n, whereas the slope value was approximately 1 for compounds with odd number values of n. Dynamic light scattering measurements showed the presence of compact particles with hydrodynamic radii (Rh) of about 40-50 nm for compounds with n = 3-6. Rh increased with further increase in methylene chain length separating the secondary amino groups of the polyamines (Rh = 60-70 nm for n = 7-10 and >100 nm for n = 11 and 12). Determination of the relative binding affinity of polyamines to DNA using an ethidium bromide displacement assay showed that homologues with n = 2 and 3 as well as those with n > 7 had significantly lower DNA binding affinity compared to spermine and homologues with n = 5 and 6. These data suggest that the chemical structure of isovalent polyamines exerts a profound influence on their ability to recognize and condense DNA, and on the size of the DNA condensates formed in aqueous solution.     

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.     

Structural parameters of the myelin transmembrane proteolipid in reverse micelles.

The Folch-Pi proteolipid is the most abundant structural protein from the central nervous system myelin. This protein-lipid complex, normally insoluble in water, requires only a small amount of water for solubilization in reverse micelles of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The characterization of the proteolipid-free and proteolipid-containing micelles was undertaken by light scattering and fluorescence recovery after fringe pattern photobleaching (FRAPP) experiments. Quasi elastic light scattering (QELS) was carried out at a high (200 mM) AOT concentration, at low water-to-surfactant mole ratio (Wo = 7) and at increasing protein occupancy. Two apparent hydrodynamic radii, differing tenfold in size, were obtained from correlation functions. The smaller one (RaH = 5.2 nm) remains constant and corresponds to that measured for protein-free micelles. The larger one increases linearly with protein concentration. In contrast, FRAPP measurements of self-diffusion coefficients were found unaffected by the proteolipid concentration. Accordingly, they have been performed at constant protein/surfactant mole ratios. The equivalent RH, extrapolated to zero AOT concentration for protein-free reverse micelles (2.9 nm) and in the presence of the proteolipid (4.6 nm), do not reveal the mode of organization previously suggested by QELS measurements. The complex picture emerging from this work represents a first step in the characterization of an integral membrane protein in reverse micelles.     

The hydrodynamic characterization of waxy maize amylopectin in 90% dimethyl sulfoxide–water by analytical ultracentrifugation, dynamic, and static light scattering

Static light scattering of high amylopectin waxy maize starch gently dispersed in 90% dimethyl sulfoxide–water yielded a weight average molecular weight M_w and radius of gyration R_g of 560×10⁶ g/mol and 342 nm, respectively. To obtain an independent hydrodynamic characterization of these solutions, we measured the sedimentation coefficient for the main component in an analytical ultracentrifuge. The value of s⁰, the infinite dilution sedimentation coefficient, was 199 S. The translational diffusion coefficient D⁰ in very dilute solutions was measured by dynamic light scattering at 90° and found to be 2.33×10⁻⁹ cm²/s. An effective hydrodynamic radius R_h was calculated from this diffusion constant using the Stokes–Einstein equation and found to be 348 nm. The structure-related parameter ρ=R_g/R_h was calculated to be 0.98. The weight average molecular weight calculated from the Svedberg equation using the values measured for s⁰ and D⁰ was 593×10⁶ g/mol. This result is in reasonable agreement with the light scattering results. As light scattering results are subject to experimental errors due to the possibility of dust contamination, the presence of microgel or aggregates, and the questionable applicability of light scattering theory to interpret results for macromolecular sizes approaching the wave length of light used as a source for scattering, it is advisable to have corroborating hydrodynamic data when possible to further validate light scattering results in this very high molecular weight range.     

Cylindrical poly(oligo-DNA)

Oligo-desoxyribonuleic acids (ODNs) having a sequence of 5'-TCC ATG ACG TTC-3' were modified at the 5' end by introduction of an amine group via a C6-amino linker. After subsequent reaction of the amine group with N-methacryloyloxysuccinimide, polymerizable ODNs were obtained. Free radical homopolymerization results in the formation of comb polymers, which possess an ODN side-chain at each repeating unit of the main chain. Mainly due to steric repulsion, the main chain has to adopt a semi-flexible wormlike shape instead of the otherwise preferred coiled structure. This leads to the formation of cylindrical poly(oligo-DNA) molecules. Characterization by static and dynamic light scattering of the poly(oligo-DNA) in aqueous solution gave a radius of gyration Rg,app = 67.8 nm, a hydrodynamic radius Rh,app = 44.6 nm, and a characteristic ratio of rho = Rg/Rh = 1.52, indicating the cylindrical shape in solution. In addition, the cylindrical poly(oligo-DNA) molecules were adsorbed onto mica and visualized by atomic force microscopy.     

Evaluation of hyperbranched poly(amino ester)s of amine constitutions similar to polyethylenimine for DNA delivery

New hyperbranched poly(amino ester)s were synthesized via A3 + 2BB'B' ' approach, represented by the Michael addition polymerization of trimethylol-propane triacrylate (TMPTA) (A3-type monomers) with a double molar 1-(2-aminoethyl)piperazine (AEPZ) (BB'B'-type monomer) performed in chloroform at ambient temperature. The results obtained by in situ monitoring the polymerization using NMR and MS indicated that hyperbranched poly(TMPTA1-AEPZ2) was formed via a A(B'B')2 intermediate, and the B' ' (the formed 2 degrees amine) was kept intact in the reaction. Therefore, poly(TMPTA1-AEPZ2) contained secondary and tertiary amines in the core and primary amines in the periphery similar to polyethylenimine (PEI). The chemistry of protonated poly(TMPTA1-AEPZ2) was further confirmed by 13C NMR, and the molecular weight, the radius of gyration (Rg), and the hydrodynamic radius (Rh) were determined using GPC, small-angle X-ray scattering (SAXS), and laser dynamic light scattering (LDLS), respectively. The ratio of Rg/Rh of ca. 1.1 verified the hyperbranched structure. Protonated hyperbranched poly(TMPTA1-AEPZ2) is degradable and less cytotoxic as compared with PEI (25 K). Gel electrophoresis reflected that stable complexes could be formed from protonated hyperbranched poly(TMPTA1-AEPZ2) and DNA, and the size and xi-potential of the complexes were characterized. Remarkably, protonated hyperbranched poly(TMPTA1-AEPZ2) showed transfection efficiency comparable to PEI (25 k) for in vitro DNA delivery.     

Novel self-associative and multiphasic nanostructured soft carriers based on amphiphilic hyaluronic acid derivatives

The purpose of the present study was to investigate the physicochemical properties in aqueous media of amphiphilic hyaluronic acid (HA) derivatives obtained by reaction of HA's hydroxyl groups with octenyl succinic anhydride (OSA). The self-associative properties of the resulting octenyl succinic anhydride-modified hyaluronic acid (OSA-HA) derivatives were studied by fluorescence spectroscopy using Nile Red as fluorophore. The morphology, size and surface charge of the OSA-HA assemblies were determined by transmission electron microscopy, dynamic light scattering and by measuring their electrophoretic mobility, respectively. OSA-HA was shown to spontaneously self-associate in aqueous media into negatively charged spherical and multiphasic nanostructures with a hydrodynamic diameter between 170 and 230 nm and to solubilize hydrophobic compounds such as Nile Red. This was a good indication that OSA-HA could be used as building block for the formulation of soft nanocarriers towards the encapsulation and controlled delivery of hydrophobic active ingredients or drugs.     

Correlations between in vitro potency of polyethylene glycol-protein conjugates and their chromatographic behavior

Pegylation is the most widely used and accepted methodology for half-life extension of biopharmaceutical drugs that also improves physicochemical and biological characteristics of proteins considerably. Most of the positive pharmacological effects of pegylated proteins are believed to be related to an increased hydrodynamic volume and molecular size. To explore the size impact of polyethylene glycol (PEG) on in vitro potency, a series of well-defined conjugates of interferon α-2b (IFN) were prepared with PEGs of different lengths and shapes specifically attached to the N-terminal amino group of the protein. Specificity of the attachment was confirmed by peptide mapping and mass spectroscopy. When potency values determined by reporter gene assay were correlated with methods for molecular weight and size characterization, such as size exclusion chromatography and dynamic light scattering, rough parallels were found. Unexpectedly, the retention times on cation exchange chromatography showed much higher correlation with experimentally determined in vitro potency. It appears that in a series of N-terminally pegylated IFNs, their in vitro potency could be predicted from the retention times on the cation exchange chromatography columns, probably because both methods reflect not only the influence of molecular size but also the impact of protein masking exerted by attached PEG moiety.     

Chain conformation of water-insoluble hyperbranched polysaccharide from fungus

Water-insoluble polysaccharide (TM3a), extracted from sclerotia of Pleurotus tuber-regium, was identified as a hyperbranched beta-d-glucan from the results of one- and two-dimensional NMR and GC-MS analysis. The degree of branching of TM3a is 65.5%. TM3a was fractionated by using a non-solvent addition method into 14 fractions, and its solution properties in 0.25 M LiCl/dimethylsulfoxide (DMSO) solution were studied systematically by using static laser light scattering, dynamic light scattering, and viscometry at 25 degrees C. The dependences among the values of intrinsic viscosity ([eta]), radius of gyration (z 1/2), and hydradynamic radius (Rh) on weight-average molecular weight (Mw) were found as the following: [eta] = 0.46Mw0.30+/-0.01, z 1/2 = 4.79 x 10-2Mw0.43+/-0.04, and Rh = 5.01 x 10-2Mw0.41+/-0.02 in the Mw range from 1.94 x 105 to 2.06 x 107 for TM3a in a 0.25 M LiCl/DMSO solution at 25 degrees C. The current theory of polymer solution was applied to explain the relationship among the fractal dimension, ratio of geometric to hydrodynamic radius (rho = z 1/2/Rh), and MwA2/[eta] of TM3a. The results indicated that TM3a existed as a compact chain conformation with a sphere-like structure in LiCl/DMSO solution. Furthermore, by using transmission electron microscopy, we observed directly the spherical molecules with an average diameter of 23.0 +/- 1.8 nm.     

Solution studies of the quaternary structure and assembly of human von Willebrand factor

The reversible association of protomers of von Willebrand protein (vWF) was studied in order to analyze the forces and mechanism of vWF polymer assembly. At concentrations of vWF found in plasma (approximately 16 micrograms/mL), disulfide bond reduction with 50 mM 2-mercaptoethanol (2-ME) markedly reduced both vWF activity, as measured by ristocetin-dependent platelet agglutination, and average polymer size (Rh, the mean hydrodynamic radius) in solution, as determined by quasi-elastic light scattering (QLS) and by gel filtration chromatography. With increasing vWF concentration, activity and Rh increased despite reduction of interprotomer disulfide bonds. Changes in temperature after 2-ME treatment produced reversible changes in activity and Rh. Varying the total vWF concentration at any given temperature after 2-ME treatment changed Rh in a consistent and predictable fashion, so that estimates of the dissociation constant for vWF protomer-polymer equilibrium were obtained: Kd5 degrees C = 0.77 micrograms/mL, Kd25 degrees C = 2.4 micrograms/mL, and Kd37 degrees C = 7.7 micrograms/mL, where under the conditions of reduction presented here, the basic protomer of vWF is a dimer. Increasing ionic strength after 2-ME treatment with 1 M KCl did not change Rh, while approximately 100 microM sodium dodecyl sulfate (SDS) or approximately 300 microM sodium deoxycholate (DOC) reduced both Rh and activity compared with those of unreduced polymer. These data show that disulfide bonds are necessary to maintain vWF polymer size and activity at plasma concentrations but that noncovalent forces of association can maintain vWF polymer size and activity at higher concentrations. These forces of association may be important for polymer assembly during intracellular synthesis of vWF.     

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.