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111.
M. J. Tiera V. A. De Oliveira H. D. Burrows M. Graça Miguel M. G. Neumann 《Colloid and polymer science》1998,276(3):206-212
The binding of the cations thallium(I), calcium(II) and terbium(III) to methyl methacrylate– methacrylic acid copolymers
with different fractions of acid groups (x) has been studied in aqueous solution at various pH values using the fluorescence of covalently bonded 9-vinyl anthracene
as a probe. In all cases, the extent of binding increases as a function of the charge of the polymer with either increasing
fraction of carboxylic acids or of pH. However, differences are observed in the behavior of the three cations. With Tl(I),
quenching of the anthracene group fluorescence is observed, indicating that the thallium(I) approaches the probe and suggesting
that the alkylanthracene is probably in a relatively polar region. Binding constants have been determined from anthracene
quenching data and from studies with the fluo-rescent-probe sodium pyrenetetra-sulfonate. Good agreement is obtained between
the two methods, and values for the binding constants increase from 250 to 950 M-1 as x increases from 0.39 to 1. It is suggested that the cation is held in the polyelectrolyte domain, partly by Debye–Hückel effects
and partly by more specific interactions. Stronger binding is found with calcium(II) and terbium(III), and in this case increases
in fluorescence intensity are observed on complexation due to the anthracene group being in a more hydrophobic region, probably
as a result of conformational changes in the polymer chain. In the former case the stoichiometry of the interaction was determined
from the fluorescence data to involve two carboxylate groups bound per calcium. Association constants were found using murexide
as an indicator of free calcium to vary from 8400 to 37 000 M-1 as x increases from 0.39 to 1. It is suggested that in this case specific calcium(II)–carboxylate interactions contribute to the
binding. With terbium(III), a greater increase in the probe fluorescence intensity was observed than with calcium, and it
is suggested that the interaction with the polymer is even stronger, leading to a more pronounced conformational change in
the polymer. It is proposed that the terbium(III) interacts with six carboxylic groups on the polymer chain, with three being
coordinated and three attracted by electrostatic interactions.
Received: 10 June 1997 Accepted: 24 October 1997 相似文献
112.
The influence of simulation methods, cutoff based and particle mesh Ewald (PME) on the accuracy by which experimentally derived nuclear Overhauser effect (NOE) data are reproduced, has been investigated using 500-ns-long molecular dynamics simulations on a model -sheet peptide in explicit solvent. The structural and conformational features under the different conditions were evaluated in terms of flexibility, secondary structure content, hydrogen-bonding pattern and percent of native contacts as a function of time. It was found that the different simulation methods strongly influence the dynamics of the peptide, confirming previous observations based on ideal peptide models simulated for much shorter times. Moreover, the results of our simulations prove once more that it is necessary to reach extremely long time scales to obtain enough statistics to accurately reproduce experimental NOE restraints even in the case of the PME method, despite its tendency to the stabilization of conformations which are structurally closely related to the ones derived through experiment. Possible implications regarding the stabilization and folding mechanisms, together with their relationship to the experimental study of peptide models, are discussed. 相似文献
113.
Yohannes G Wiedmer SK Tuominen EK Kinnunen PK Riekkola ML 《Analytical and bioanalytical chemistry》2004,380(5-6):757-766
Lipid membranes are well recognized ligands that bind peripheral and integral proteins in a specific manner and regulate their function. Cytochrome c (cyt c) is one of the partner peripheral protein that binds to the lipid membranes via electrostatic and hydrophobic interactions. In this study, asymmetrical flow field-flow fractionation (AsFlFFF) was used to compare the interactions of cyt c with the acidic phospholipid 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG), oleic acid (OA), and sodium dodecyl sulfate (SDS). The influence of pH and the cyt c–lipid molar mass ratios were evaluated by monitoring the diffusion coefficients and particle diameter distributions obtained for the free and lipid-bound protein. The hydrodynamic particle diameter of cyt c (pI 10) was 4.1 nm at pH 11.4 and around 4.2 nm at pH 7.0 and 8.0. Standard molar mass marker proteins were used for calibration to obtain the molar masses of free cyt c and its complexes with lipids. AsFlFFF revealed the binding of cyt c to DMPG and to OA to be mainly electrostatic. In the absence of electrostatic interactions, minor complex formation occurred, possibly due to the extended lipid anchorage involving the hydrophobic cavity of cyt c and the hydrocarbon chains of DMPG or SDS. The possibility of the formation of the molten globule state of cyt c, induced by the interaction between cyt c and lipids, is discussed.Electronic Supplementary Material Supplementary material is available for this article at 相似文献
114.
Determination of proteins with Alizarin Red S by Rayleigh light scattering technique 总被引:1,自引:0,他引:1
A new protein determination method by enhanced Rayleigh light scattering (RLS) technique has been developed. In acid condition (pH=3.60), RLS of 1,2-dihydroxyanthraquinone-3-sulfonate (Alizarin Red S) can be greatly enhanced by addition of proteins, resulting in two characteristic peaks, 360 and 505 nm, respectively. The new protein assay is based on the RLS enhancement and spectrum change. The optimum condition for the reaction was investigated. The linear range is 0.20-24.9 μg ml−1 for BSA and 0.20-15.5 μg ml−1 for HSA. The detection limits (S/N=3) are 9.59 ng ml−1 for BSA and 9.51 ng ml−1 for HSA. The results of determination for human serum samples were comparable to those obtained by Bradford method. The binding stoichiometry was determined. 相似文献
115.
The interactions between proteins and Ponceau 4R (PR) in aqueous solution have been studied by the techniques of resonance light scattering (RLS) spectroscopy, the absorption spectroscopy, zeta potential assay and circular dichroism (CD) spectrum. The dry PR can assemble on the surface of protein via electrostatic and hydrophobic forces to produce an associated compound of protein-PR, this compound can enhance the RLS of protein. Based on this fact, a simple, rapid, and sensitive method has been developed for the determination of proteins at nanogram level by RLS technique with a common spectrofluorimeter. Under optimum conditions, the linear range is 0.10-39.2 μg mL−1 for the determination of both bovine serum albumin (BSA) and human serum albumin (HSA). The detection limits (S/N = 3) are 6.96 ng mL−1 for BSA and 5.71 ng mL−1 for HSA, respectively. There is almost no interference from amino acids, most of the metal ions, and other coexistent substances. The method has been satisfactorily applied to the direct determination of the total protein in human serum. 相似文献
116.
Petra A W Van Den Berg Koert Grever Arie Van Hoek Willem J H Van Berkel Antonie J W G Visser 《Journal of Chemical Sciences》2007,119(2):123-133
Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate
(analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations;
the ‘in’ conformation with the isoalloxazine ring located in the active site, and the ‘out’ conformation with the isoalloxazine
ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions
for the ‘in’ and ‘out’ conformations. The reason for this is twofold. First, the active site of PHBH contains various potential
fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222
is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including
a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these
quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute
to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred
by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching.
The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes. 相似文献
117.
Synthesis, characterization and protein adsorption behaviors of PLGA/PEG di-block co-polymer blend films 总被引:1,自引:0,他引:1
A series of poly(D,L-lactic-co-glycolic acid) (PLGA)/poly(ethyleneglycol) (PEG) di-block copolymers were synthesized by ring-opening polymerization of D,L-lactide and glycolide with different molecular weights of monomethoxy polyethyleneglycol (mPEG) 750, 2000 and 5000 as an initiator. The bulk properties of these co-polymers were characterized by using 1H NMR spectroscopy, gel permeation chromatography, differential scanning calorimetry (DSC). Electron spectroscopy for chemical analysis (ESCA) results, in which the blend films with the di-block copolymers showed increasing surface oxygen atomic percentage with increasing PEG chain length, indicate that PEG chain segment in the di-block copolymers is surface oriented and enriched onto the surface of the blend films. The extent of protein adsorption onto the surface of these blend films was studied, using iodine radio-labeled human serum albumin, gamma globulin and human growth hormone. The protein adsorption amount was reduced for the blend films prepared with PLGA/PEG 750 and 2000 di-block copolymers, but increased to a great extent for PLGA/PEG 5000 di-block copolymer. This is due to the increased water uptake capacity of the blend film, which absorbed more protein molecules into a swollen polymer matrix in addition to surface adsorption. 相似文献
118.
Francisco Batista-Viera Mariela Barbieri Karen Ovsejevi Carmen Manta Jan Carlsson 《Applied biochemistry and biotechnology》1991,31(2):175-195
A new method for the reversible immobilization of thiol bimolecules, e.g., thiolpeptides and thiolproteins, to beaded agarose
and other solid phases is reported. The method consists of an activation and a coupling step. The activation is based on oxidation
of disulfides (or thiol groups via disulfides) present in a solid phase by hydrogen peroxide at moderately acidic pH. This
oxidation leads to disulfide oxides (thiolsulfinate groups of which the majority are further oxidized to thiolsulfonate).
The thiolsulfonate groups react easily with thiol compounds, which become immobilized via disulfide bonds. The pH range for
thiol coupling is wide (pH 5-8), but for most thiols the reaction seems to proceed faster at pH>7. The stability of the reactive
group to hydrolysis, especially at neutral and weakly acidic pH, is very high. The activated gel, therefore, can be stored
as a suspension at pH 5 for extended periods. The method has been used to reversibly immobilize glutathione, β-galactosidase,
alcohol dehydrogenase, urease, and papain, all with exposed thiol groups as well as thiolated bovine serum albumin and sweet-potato
β-amylase.
Depending on the thiol content of starting thiol-agarose, thiol-sulfonate-agarose derivatives with different binding capacities
can be obtained. Thus, up to 5.0 mg (16 μmol) glutathione and 15 mg thiol-protein/mL gel derivative have been immobilized. 相似文献
119.
Dupont-Gillain CC Jacquemart I Rouxhet PG 《Colloids and surfaces. B, Biointerfaces》2005,43(3-4):179-186
In the last years, adsorbed collagen was shown to form layers with a supramolecular organization depending on the substrate surface properties and on the preparation procedure. If the concentration of collagen and the duration of adsorption are sufficient, fibrillar collagen structures are formed, corresponding to assemblies of a few molecules. This occurs more readily on hydrophobic compared to hydrophilic surfaces. This study aims at understanding the origin of such fibrillar structures and in particular at determining whether they result from the deposition of fibrils formed in solution or from the building of assemblies at the interface. Therefore, type I collagen solutions with an increasing degree of aggregation were prepared, using the “neutral-start” approach, by ageing pH 5.8 solutions at 37 °C for 15 min, 2 or 7 days. The obtained solutions were used to investigate the influence of collagen aggregation in solution on the supramolecular organization of adsorbed collagen layers, which was characterized by X-ray photoelectron spectroscopy and atomic force microscopy. Polystyrene and plasma-oxidized polystyrene were chosen as substrates for the adsorption. The size and the density of collagen fibrils at the interface decreased upon increasing the degree of aggregation of collagen in solution. This is explained by a competitive adsorption process between monomers and aggregates of the solution, turning at the advantage of the monomers. More aggregated solutions, which are thus depleted in free monomers, behave like less concentrated solutions, i.e. lead to a lower adsorbed amount and less fibril formation at the interface. This study shows that the supramolecular fibrils observed in adsorbed collagen layers, especially on hydrophobic substrates, are not formed in the solution, prior to adsorption, but are built at the interface, through the assembly of free segments of adsorbed molecules. 相似文献
120.
Abstract The paper deals with the influence of high pressure treatment of fresh egg white on its properties and protein composition (individual amino-acids predicted as a function of pressure and time levels). The rheological properties are changed by high pressure from Newtonian to non-Newtonian behaviour, with increasing apparent viscosity as the pressure and time increased. The pH, whipping ability, foam stability, gel strength of heat induced gels after treatment and the whole protein content, were also predicted. The results showed that the foam stability is increased with increasing pressure and time of processing. The foam volume is also increased with pressure. The pH did not change with pressure or time of processing. Composition of proteins as indicated by individual amino-acids did not exhibit statistically important changes. Gel strength of heat induced gels prepared from previously pressured liquid whites showed no important change of values with pressure or time of treatment. The modulus of elasticity showed a decrease for samples pressured to 400 MPa for 5 up to 15 minutes. 相似文献