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To get more insight into the mechanisms of cold gelation of beta-lactoglobulin (beta-lg), macroscopic and molecular structural changes during Fe(2+)-induced gelation of beta-lg were investigated using Fourier transform-infrared (FTIR) spectroscopy and rheological methods. The FTIR spectroscopy results show that, upon the preheating treatment (first step of gel process), native globular proteins are denatured and aggregated molecules are found in solution. The spectra are similar to those of gels obtained in the second step of the process upon incorporation of Fe, which suggests that aggregated molecules formed during the preheating treatment constitute the structural basis of the aggregation. However, the rheological data show that the aggregation is achieved via two molecular mechanisms, both of which are modulated by the iron concentration. At 30 mM of iron, gel formation is essentially controlled by van der Waals interactions, while at 10 mM of iron, hydrophobic interactions predominate. At the two concentrations, disulfide bonds contribute to gel consolidation, the effect being more pronounced at 10 mM of iron. These mechanisms lead to the formation of gels of different microstructures. At the highest iron concentration, a strong and rapid decrease in the repulsion forces is produced, resulting in random aggregation. At the lowest iron concentration, the iron diminishes the superficial charge of both molecules and aggregated molecules, facilitating the interaction among hydrophobic regions and leading to the growth of the aggregation in the preferential direction and to filamentous gel formation. This study provides a comprehensive view of the different modes of gelation. 相似文献
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Sonicated calf-thymus DNA (200 ± 30 base pairs) spontaneously forms viscoelastic gels over a wide range of concentration, temperature, and buffer conditions. Quasielastic light scattering (QLS) can be used to monitor this process, because the ratio of dynamic-to-static scattering intensity decreases dramatically as gelation occurs. Using QLS, we have explored the effects of DNA concentration and mono- and divalent cations on the thermal stability of DNA gels. We found that the gel–sol transition temperature (Tgel) varies linearly with [DNA] from 7.5 to 17 mg/mL. Both Na+ and Mg2+ strongly stabilize the gel state. The sharpness of the transition increases with increasing ionic and DNA concentrations. Analysis of the Na+-dependent gelation indicates that the process requires the association of one Na+ per 118 base pairs. Mg2+ effectively stabilizes the gel at concentrations 10-fold below those required for Na+. The unexpectedly large effect of Mg2+ suggests that ion-specific interactions may play an important role in determining gel stability. 相似文献
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Kavanagh GM Clark AH Ross-Murphy SB 《International journal of biological macromolecules》2000,28(1):41-50
Heat-set gels and aggregates from beta-lactoglobulin (beta-Lg), one of the major globular proteins from milk, have been studied on a molecular distance scale using negative-staining transmission electron microscopy (TEM), wide-angle X-ray diffraction (WAXD), and Fourier transform infrared spectroscopy (FTIR). The microscopy showed long linear aggregates forming in solutions at pH 2 (and sometimes 2.5) after prolonged heating. While there appeared to be no differences in aggregates formed under these conditions in H(2)O as compared with D(2)O, at all other pH and pD values, and in the presence of added salt, much shorter linear aggregates were formed. These became slightly more extended the further the pH was removed from pI. Wide-angle X-ray diffraction (WAXD) showed a diffuse beta-sheet halo at 2θ=19 degrees in patterns for both dried native and aggregated protein (irrespective of pH) with only a small change (sharpening) of this feature on heat treatment. Solution FTIR spectra, measured at pD=2, 2.5, 3, and 7, during heating, indicated shoulder development at 1612 cm(-1) in the carbonyl-stretching Amide I region diagnostic of a modest increase in intermolecular beta-sheet. In terms of the shoulder size, no distinctions could be made between acid and neutral aggregate structures. At all pHs, beta-lactoglobulin showed only limited secondary and tertiary structural changes in aggregation, in contrast to previous studies of insulin aggregation, where highly ordered crystalline fibrils were indicated. The current work has implications both in structural studies of food biopolymers and in ongoing studies of pathological protein self-assembly in disease states, such as spongiform encephalopathies. 相似文献
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Human glycodelin A (GdA) is a glycoprotein that is highly homologous to bovine beta-lactoglobulin A (beta-LgA) because the amino acid sequences are 50-60% identical. The structural characteristics of human GdA and beta-LgA were compared in water and 2-propanol/water solutions. Circular dichroism spectra reveal that in water the two proteins have a very similar beta-sheet secondary structure. In the presence of 2-propanol/water mixtures (up to 50% v/v) the alpha-helix structure of both proteins increases. A further increase in the alcohol percentage of the solvent (up to 80% v/v 2-propanol) causes the formation of a new folded tertiary structure containing mainly beta-sheet features. Synchrotron radiation small angle X-ray scattering indicates that, in a neutral pH aqueous solution, GdA is a dimer. Its radius of gyration value (Rg), 25.1+/-0.4 A, is greater than that of beta-LgA (21.1+/-0.3 A), probably because of the contribution of polysaccharides bound to Asn-28 and Asn-63 residues of GdA. Conversely, small angle X-ray scattering and gel permeation chromatography data on GdA in 2-propanol have revealed a massive aggregation of the protein. 相似文献
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Physically cross-linked beta-lactoglobulin (BLG) protein gels containing theophylline and sulfamethoxazole low molecular weight drugs were prepared in 50% ethanol solution at pH 8 and two protein concentrations (6 and 7% (w/v)). Swelling behavior of cylindrical gels showed that, irrespective of the hydrated or dehydrated state of the gel, the rate of swelling was the highest in water. When the gels were exposed to water, they first showed a swelling phase in which their weight increased 3 and 30 times for hydrated and dehydrated gels, respectively, due to absorption of water, followed by a dissolution phase. The absorption of solvent was however considerably reduced when the gels were exposed to aqueous buffer solutions. The release behavior of both theophylline and sulfamethoxazole drugs from BLG gels was achieved in a time window ranging from 6 to 24 h. The drug release depended mainly on the solubility of the drugs and the physical state of the gel (hydrated or dry form). Analysis of drug release profiles using the model of Peppas showed that diffusion through hydrated gels was governed by a Fickian process whereas diffusion through dehydrated gels was governed partly by the swelling capacities of the gel but also by the structural rearrangements inside the network occurring during dehydration step. By a judicious selection of protein concentration, hydrated or dehydrated gel state, drug release may be modulated to be engineered suitable for pharmaceutical as well as cosmetics and food applications. 相似文献
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Kuwata K Shastry R Cheng H Hoshino M Batt CA Goto Y Roder H 《Nature structural biology》2001,8(2):151-155
We have defined the structural and dynamic properties of an early folding intermediate of beta-lactoglobulin known to contain non-native alpha-helical structure. The folding of beta-lactoglobulin was monitored over the 100 micros--10 s time range using ultrarapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR. An initial increase in Trp fluorescence with a time constant of 140 micros is attributed to formation of a partially helical compact state. Within 2 ms of refolding, well protected amide protons indicative of stable hydrogen bonded structure were found only in a domain comprising beta-strands F, G and H, and the main alpha-helix, which was thus identified as the folding core of beta-lactoglobulin. At the same time, weak protection (up to approximately 10-fold) of amide protons in a segment spanning residues 12--21 is consistent with formation of marginally stable non-native alpha-helices near the N-terminus. Our results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like alpha/beta core domain. 相似文献
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Structural changes accompanying pH-induced dissociation of the beta-lactoglobulin dimer 总被引:1,自引:0,他引:1
We have used NMR spectroscopy to determine the three-dimensional (3D) structure, and to characterize the backbone dynamics, of a recombinant version of bovine beta-lactoglobulin (variant A) at pH 2. 6, where the protein is a monomer. The structure of this low-pH form of beta-lactoglobulin is very similar to that of a subunit within the dimer at pH 6.2. The root-mean-square deviation from the pH 6.2 (crystal) structure, calculated for backbone atoms of residues 6-160, is approximately 1.3 A. Differences arise from the orientation, with respect to the calyx, of the A-B and C-D loops, and of the flanking three-turn alpha-helix. The hydrophobic cavity within the calyx is retained at low pH. The E-F loop (residues 85-90), which moves to occlude the opening of the cavity over the pH range 7.2-6.2, is in the "closed" position at pH 2.6, and the side chain of Glu89 is buried. We also carried out measurements of (15)N T(1)s and T(2)s and (1)H-(15)N heteronuclear NOEs at pH 2.6 and 37 degrees C. Although the residues of the E-F loop (residues 86-89) have the highest crystallographic B-factors, the conformation of this loop is reasonably well defined by the NMR data, and its backbone is not especially mobile on the pico- to nanosecond time scale. Several residues (Ser21, Lys60, Ala67, Leu87, and Glu112) exhibit large ratios of T(1) to T(2), consistent with conformational exchange on a micro- to millisecond time scale. The positions of these residues in the 3D structure of beta-lactoglobulin are consistent with a role in modulating access to the hydrophobic cavity. 相似文献
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The effect of sodium caprate (a fatty acid salt) on the formation of beta-lactoglobulin A gels was studied at constant temperature (30 or 35 degrees C) using ultrasonic spectroscopy. During incubation at these temperatures, ultrasonic attenuation increased with the addition of sodium caprate, and reached a plateau after 5-7 h of incubation. Comparing beta-lactoglobulin A with and without sodium caprate, a decrease in net ultrasonic velocity was observed. These results suggested that aggregation occurred during incubation with sodium caprate, and the sample showed an increase in compressibility. Transmission electron microscopy with negative staining showed the formation of filamentous aggregates of beta-lactoglobulin A at around 3-4.5 h of incubation with sodium caprate. These results demonstrated that sodium caprate induced the formation of structures with unique gel properties compared to those formed by heating beta-lactoglobulin in the presence of NaCl alone. 相似文献
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The shear rheology of a microcrystalline cellulose dissolved in a 9% NaOH aqueous solution was studied in the steady and oscillatory modes. The cellulose-(9% NaOH-H(2)O) mixtures show not to be true solutions. In the dilute regime, with cellulose concentration below 1%, the rheological behavior is typical of the one of suspensions. The formation of cellulose aggregates is favored when temperature is increased. In the semidilute regime, an irreversible aggregate-based gelation occurs, being faster with increasing temperature. 相似文献
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The mechanism of quenching by quinones of the lowest excited singlet state of chlorophyll has been investigated using picosecond laser spectroscopy. With chlorophyll alone, laser excitation resulted in immediate (< 10 ps) bleaching of the 665 nm band and production of new absorption bands in the regions 460–550 and 800–830 nm. The lifetimes of these changes were greater than 500 ps. Addition of 2,6-dimethyl-benzoquinone caused quenching of these absorbance changes. No indication of chlorophyll cation radical formation was obtained. Thus, the interaction between quinone and the chlorophyll excited singlet state results in energy dissipation without measurable formation of radical species having lifetimes longer than 10 ps. This is in marked contrast to the quenching of the chlorophyll lowest triplet state by quinones, during which easily detectable stable radical formation has been observed. 相似文献
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Structural stability of beta-lactoglobulin in the presence of kosmotropic salts. A kinetic and thermodynamic study 总被引:2,自引:0,他引:2
The thiol group of beta-lactoglobulin reacted very sluggishly with dithio-bis-nitro-benzoic acid as compared to that of glutathione at pH 6.85. The pKapp value of the thiol group of the protein was 9.35. In the presence of 3 M urea, the thiol group reacted completely with dithio-bis-nitrobenzoic acid at pH 6.85. Heating (from 50 degrees to 80 degrees) increased the exposure of the thiol by dissociating the dimer unit. From the pseudo-first order rate constants of heat-exposure of thiol, thermodynamic activation parameters, delta G++, delta H++, and delta S++, for the heat-dissociation of beta-lactoglobulin dimer were estimated to be 23,290 cal/mol, 31,160 cal/mol, and 22.9 e.u. (at 70 degrees), respectively. Addition of kosmotropic salts, chloride, tartrate, sulfate, phosphate, and citrate (0.2 M) decreased the heat-induced exposure of the thiol group (at 70 degrees), probably by decreasing the dissociation of the dimer at pH 6.85. The relative change in free energy of activation for the dissociation of the dimer, delta(delta G++dimer), in the presence of the salts was positive, suggesting that these additives increase the stability of the dimer against heat. These salts also increased the conformational stability of beta-lactoglobulin as revealed by an increase in -delta(delta G0conf) values in their presence. Both delta(delta G++dimer) and -delta(delta G0conf) values followed the order, chloride less than tartrate less than sulfate less than phosphate less than citrate. These salts seem to manifest their structure-stabilizing effect by increasing both inter- and intramolecular hydrophobic interactions via changes in structure of water. 相似文献
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Gaspare Barone Maria Michela Corsaro Cristina De Castro Rosa Lanzetta Lorenzo Mangoni Michelangelo Parrilli 《Carbohydrate research》1994,260(2):259-270
The polysaccharide fraction from Ceratozamia spinosa appears to be made up mainly by a chemically homogeneous polysaccharide but with a wide range of molecular weight. By NMR and chemical degradative methods, it is shown to consist essentially of a backbone of alternate → 4)-β-
-GlcpA-(1 → and → 2)--
-Manp-(1 → units. On the 4 position of the latter, β-
-GlcpA residues are linked. End units of -
-Ara f, β-
-Xylp, -
-Rhap, and -
-3-OMe-Rhap are linked to C-3 and/or C-4 positions of β-
-Glc pA residues. 相似文献
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Macroscopic and molecular structural changes during heat-induced gelation of beta-lactoglobulin, bovine serum albumin, ovalbumin, and alpha-lactalbumin aqueous dispersions were probed by the mechanical and CD spectroscopy, respectively. Aqueous solutions of the native globular proteins, except for alpha-lactalbumin, exhibited solid-like mechanical spectra-namely, the predominant storage modulus G' over the loss modulus G" in the entire frequency range examined (0.1-100 rad/s), suggesting that these protein solutions were highly structured even before gelation, possibly due to strong repulsions among protein molecules. Such solid-like structures were susceptible to nonlinearly large shear but recovered almost immediately at rest. During gelation by isothermal heating, major changes in the secondary structure of the globular proteins completed within a few minutes, while values of the modulus continued to develop for hours with maintaining values of tandelta (= G"/G') less than unity. As a result, a conventional criterion for mechanically defining the gelation point, such as a crossover between G' and G", was inapplicable to these globular protein systems. beta-Lactoglobulin gels that had passed the gelation point satisfied power laws (G' approximately G" approximately omega(n)) believed to be valid only at the gelation point, suggesting that fractal gel networks, similar to those of critical gels (i.e., gels at the gelation point), were formed. 相似文献
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Conformational transitions of alternating purine-pyrimidine DNAs in perchlorate ethanol solutions. 总被引:3,自引:1,他引:2
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M Vorlícková 《Biophysical journal》1995,69(5):2033-2043
Conformational transitions of poly(dA-dC).poly(dG-dT), poly(dA-dT).poly(dA-dT), and other alternating purine-pyrimidine DNAs were studied in aqueous ethanol solutions containing molar concentrations of sodium perchlorate, which is a novel solvent stabilizing non-B duplexes of DNA. Using CD and UV absorption spectroscopies, we show that this solvent unstacks bases and unwinds the B-forms of the DNAs to transform them into the A-form or Z-form. In the absence of divalent cations poly(dA-dC).poly(dG-dT) can adopt both of these conformations. Its transition into the Z-form is induced at higher salt and lower ethanol concentrations, and at higher temperatures than the transition into the A-form. Submillimolar concentrations of NiCl2 induce a highly cooperative and slow A-Z transition or Z-Z' transition, which is fast and displays low cooperativity. Poly(dA-dT).poly(dA-dT) easily isomerizes into the A-form in perchlorate-ethanol solutions, whereas high perchlorate concentrations denature the polynucleotide, which then cannot adopt the Z-form. At low temperatures, however, NiCl2 also cooperatively induces the Z'-form in poly(dA-dT).poly(dA-dT). Poly(dI-dC).poly(dI-dC) is known to adopt an unusual B-form in low-salt aqueous solution, which is transformed into a standard B-form by the combination of perchlorate and ethanol. NiCl2 then transforms poly(dI-dC).poly(dI-dC) into the Z'-form, which is also adopted by poly(dI-br5dC).poly(dI-br5dC). 相似文献
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The ability of water preparations of different composition to affect the generation of superoxide radicals has been compared.
The superoxide-generating reaction of adrenaline autooxidation with some modifications was used as a model system, which makes
it possible to reveal the pro/antioxidant properties of materials being tested. It was shown that samples of water from sources
having different specific electroconductivity and, accordingly, ionic composition differ in the ability to affect reactions
proceeding with the participation of ROS. The parameter measured, the pro/antioxidant activity of water, is a new informative
indicator, and the approach proposed enables one to perform a comparative estimation of the quality of water and aqueous solutions. 相似文献