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1.
The translational diffusion coefficient (D) of H-meromyosin in actin (F-actin) and ATP solution was measured under conditions wherein the actin-activated ATPase activity is close to its maximal value at a very low electrolyte concentration. The results were compared with similar data obtained with 0.1 M KCl, where H-meromyosin and actin were almost completely dissociated. With 0.1 M KCl, it was found that there was no dependence of the D of H-meromyosin on actin concentration. On the other hand, at a very low electrolyte concentration, it was found that the D of H-meromyosin did depend on actin concentration; at a rather high actin concentration (and activation of ATPase), it was slightly larger than at low or zero actin concentrations. This behavior of D at a low electrolyte concentration is interpreted on the assumption that even in solution, H-meromyosin molecules can actively slide on actin filaments due to the ATPase activity. 相似文献
2.
The diffusion coefficient and viscosity of lysozyme solutions were measured at 25°C in various buffers with and without sodium
chloride. Measurements were made over the entire concentration range in each case and were extended into the supersaturated
region. The results show that diffusion coefficient behavior depends strongly on the buffer used and the ionic strength of
the solution, which means the amount of sodium chloride used in buffer solution. Viscosity measurements indicate a small degree
of time dependence, with the viscosity increasing with solution age. 相似文献
3.
A method has been developed measuring the diffusion coefficient of KCl in amylose films. The films were soaked in potassium chloride solutions, then immersed in pure water and conductivity measured as a function of time. Different concentrations of the soaking solution were used and the measurements were made at several temperatures. The diffusion coefficient of KCl was found to be independent of the soaking solution KCl concentration, but found to increase with increasing temperature. The diffusion coefficient values were about one quarter of those found in water and varied from 4.8×10−10 to 11×10−10 m2 s−1. The activation energy of diffusion was close to that found in water. Two values for the activation energy were obtained, 20.1 and 14 kJ mol−1, indicating a change in the film structure at 45 °C. Amylose films swelled equally in KCl-solutions and water. The thickness of amylose films doubled and the increase in mass was 100–200% corresponding the decrease of amylose content from about 87 to 37%, when the conditions changed from normal humidity conditions to water. 相似文献
4.
Interaction of macromolecules in aqueous salt‐containing solution with a hydrophobic adsorbent is studied by adsorption equilibrium measurements and by independent isothermal titration calorimetry. The macromolecules are native as well as mono‐, di‐, and tri‐PEGylated lysozyme and four pure PEGs. The hydrophobic adsorbent is Toyopearl PPG‐600M. The salt is sodium chloride. The sodium chloride concentration in the aqueous 25 mM sodium phosphate buffer is varied from 2000 to 4500 mM at pH 7.0 and 25°C. PEGylation of the lysozyme is carried using 5 and 10 kDa PEG chains. The molar enthalpy of adsorption is calculated from the adsorption equilibrium and the calorimetric data. The results show that the adsorption of the PEGylated lysozyme is caused by both the interaction of the lysozyme and the interaction of the PEG chains with the adsorbent, respectively, but the interaction of the lysozyme is stronger than that of PEG. The comparison of the results of the present study on the influence of sodium chloride with a corresponding study on the influence of ammonium sulfate shows that the adsorption mechanism changes upon the variation of the salt. The knowledge of the adsorption mechanisms supports the systematic development of chromatographic purification steps. 相似文献
5.
H S Rollema S H de Bruin L H Janssen G A van Os 《The Journal of biological chemistry》1975,250(4):1333-1339
The normal and differential titration curves of liganded and unliganded hemoglobin were measured at various KCl concentrations (0.1 to 2.0 M). In this range of KCl concentrations, the curves for deoxyhemoglobin showed no salt-induced pK changes of titratable groups. In the same salt concentration range oxyhemoglobin showed a marked change in titration behavior which could only be accounted for by a salt-induced increase in pK of some titratable groups. These results show that the suppression of the alkaline Bohr effect by high concentrations of neutral univalent salt is not caused by a weakening of the salt bridges in deoxyhemoglobin but is due to an interaction of chloride ions with oxyhemoglobin. Measurements of the Bohr effect at various KCl concentrations showed that at low chloride ion concentration (5 times 10-3 M) the alkaline Bohr effect is smaller than at a concentration of 0.1 M. This observation indicates that at a chloride ion concentration of 0.1 M, part of the alkaline Bohr effect is due to an interaction of chloride ions with hemoglobin. Furthermore, at low concentrations of chloride ions the acid Bohr effect has almost vanished. This result suggests that part of the acid Bohr effect arises from an interaction of chloride ions with oxyhemoglobin. The dependence of the Bohr effect upon the chloride ion concentration can be explained by assuming specific binding of chloride ions to both oxy- and deoxyhemoglobin, with deoxyhemoglobin having the highest affinity. 相似文献
6.
David J. Wedlock Glyn O. Phillips Endre A. Balazs 《International journal of biological macromolecules》1981,3(6):384-388
A study of the sedimentation behaviour of lysozyme in sodium hyaluronate (Na-HA) solution and of the Na-HA medium itself, has been carried out to determine whether the strongly basic enzyme lysozyme forms complexes with Na-HA at physiological ionic strength. At typical physiological salt concentration, 0.146 m NaCl, and also in 0.100 M NaCl, lysozyme sedimentation in an Na-HA solution can be adequately described as independent sedimentation of a slightly associated protein through a three-dimensional network acting partially as a macromolecular sieve. The s20,w of lysozyme when determined in 0.146 M NaCl, indicated partial aggregation of the enzyme at this salt concentration. Decreases in sedimentation coefficients of lysozyme with increase in Na-HA concentration show a pronounced sieving effect by the equality of observed sedimentation coefficient of lysozyme and Na-HA at higher Na-HA concentrations, but typically individual sedimentation coefficients when the macromolecular mixture was diluted approximately ten-fold. 相似文献
7.
Time dependence of aggregation in crystallizing lysozyme solutions probed using NMR self-diffusion measurements
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The time dependence of aggregation in supersaturated lysozyme solutions was studied using pulsed-gradient spin-echo NMR diffusion measurements as a function of lysozyme concentration at pH 6.0 and 298 K in the presence of 0.5 M NaCl. The measurements provide estimates of the weight-averaged diffusion coefficient of the monomeric to intermediate molecular weight lysozyme species present in the solution (very large aggregates and crystals are excluded from the average due to the NMR relaxation-weighting effects inherent in the method). The results show that the average molecular weight of the various lysozyme aggregates changed with sigmoidal kinetics and that these kinetics were strongly influenced by the initial lysozyme concentration. The visualization of the time dependence of the protein aggregation afforded by this method provides a deeper understanding of how the crystallizing conditions (especially the initial protein concentration) are related to the resulting crystals. 相似文献
8.
"Substrate inhibition", which has been described earlier for myosin Ca-ATPase in low ionic strength KCl solution [1], is found to take place also at high KCl concentration and under partial modification of enzyme thiol groups with p-CMB. "Substrate inhibition" disappeared when increasing Ca2+ concentration up to 25-40 mM. These kinetic properties are characteristic for fresh isolated enzyme and myosin preparations stored in 0.5 M KCl. They may change under storage of enzyme preparations at higher KCl concentrations: no "substrate inhibition" is observed after 6-8-day storage of myosin preparations in 3 M KCl at the presence of 4-5 mM CaCl2. The data on optical rotation dispersion and analytical ultracentrifugation have shown that the storage of myosin in 3 M KCl is accompanied by structural changes of the protein. 相似文献
9.
D Slootmaekers M Mandel H Reynaers 《International journal of biological macromolecules》1991,13(1):17-25
The intensity correlation functions of kappa- and lambda-carrageenan in various salt solutions and at different concentrations have been determined with the help of dynamic light scattering. From the first cumulant of these correlation functions the values of the translational diffusion coefficients D have been derived. They increase with macromolecular concentration. The extrapolated values to infinite dilution of the diffusion coefficients increase with increasing salt concentration as expected from the salt concentration dependence of the r.m.s. radii of gyration determined previously by static light scattering. The translational diffusion coefficient of lambda-carrageenan in 0.1 M NaCl is smaller than the corresponding value for the kappa species. This is consistent with the difference in contour length and linear charge density of the two samples used. No satisfactory interpretation for the concentration dependence of the diffusion coefficient seems to be possible at present. Although current theories for the macromolecular and salt concentration dependence of D, taking into account charge effects, seem to be applicable, they do not allow for a consistent interpretation of the data. No specific difference between the solution behaviour of kappa- and lambda-carrageenan has been detected. 相似文献
10.
Interactions of lysozyme in concentrated electrolyte solutions from dynamic light-scattering measurements
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D.E. Kuehner C. Heyer C. Rämsch U.M. Fornefeld H.W. Blanch J.M. Prausnitz 《Biophysical journal》1997,73(6):3211-3224
The diffusion of hen egg-white lysozyme has been studied by dynamic light scattering in aqueous solutions of ammonium sulfate as a function of protein concentration to 30 g/liter. Experiments were conducted under the following conditions: pH 4-7 and ionic strength 0.05-5.0 M. Diffusivity data for ionic strengths up to 0.5 M were interpreted in the context of a two-body interaction model for monomers. From this analysis, two potential-of-mean-force parameters, the effective monomer charge, and the Hamaker constant were obtained. At higher ionic strength, the data were analyzed using a model that describes the diffusion coefficient of a polydisperse system of interacting protein aggregates in terms of an isodesmic, indefinite aggregation equilibrium constant. Data analysis incorporated multicomponent virial and hydrodynamic effects. The resulting equilibrium constants indicate that lysozyme does not aggregate significantly as ionic strength increases, even at salt concentrations near the point of salting-out precipitation. 相似文献
11.
1. The process of denaturation of the chicken muscle dimeric enzyme triosephosphate isomerase on addition of guanidinium chloride has been studied at pH 7.6, the pH at which the recovery of activity is optimal (100%) on removal of denaturant. Determinations of the sedimentation coefficient, intrinsic viscosity, molecular weight (by sedimentation equilibrium studies) and the absorption coefficient at 280 nm in various concentrations of guanidinium chloride concurred in showing a single, sharp transition at about 0.7 M guanidinium chloride at a protein concentration 1-5 mg/ml from the native enzyme to the dissociated, unfolded chains of the monomer. Relative fluorescent intensity measurements revealed a single transition at about 0.4 M guanidinium chloride at enzyme concentrations of about 0.05 mg/ml. 2. The process of denaturation in different guanidinium chloride concentrations was first order with respect to enzyme and about sixth order with respect to denaturant. 3. The rate of attainment of equilibrium during the renaturation obeyed second-order/first-order reversible kinetics. It was concluded that the rate-determining step in renaturation at pH 7.6 must be the association of two subunits. 相似文献
12.
The diffusion coefficient of caffeine through agar gels containing a hyaluronic acid–protein complex. A model system for the study of the permeability of connective tissues
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M. McCabe 《The Biochemical journal》1972,127(1):249-253
A hyaluronic acid-protein complex was embedded into agar gel. This gel complex resembles in some respects the physiological situation in connective tissue, but still permits precise physicochemical measurements to be made. The diffusion coefficient of caffeine into and from such gels has been measured as a function of both agar and hyaluronate concentration. The value for the diffusion coefficient of caffeine was also measured by using a Gouy type diffusiometer. From both types of measurement the value for D (Fick) for caffeine when extrapolated to zero caffeine and agar concentrations agreed at (6.79+/-0.01)x10(-6)cm(2).s(-1) at 25 degrees C. Although agar concentration had only a small effect on caffeine diffusion, hyaluronic acid caused a large decrease in caffeine diffusion co-efficient. The presence of the hyaluronic acid-protein complex within the gel tended to oppose gel syneresis, a concentration of 1.7mg/ml abolishing the effect and higher concentrations reversing it. The possible physiological implications of these results are discussed. 相似文献
13.
The isolated skin of the toad Bufo marinus ictericus when impaled from the outer surface by glass microelectrodes filled with 3 M KCl shows a voltage profile which is a continuous function of the depth of impalement. The superficial intraepithelial potential difference measured with reference to the external solution (PDi) is negative with NaCl-Ringer's solution on both sides of the skin, displaying a minimum of -26.7+/-3.6 mV at 6+/-2 mum. Null value is obtained at 19+/-3 mum, with positive values for deeper impalements. Indications of cell impalements (abrupt voltage and resistance jumps) were frequently observed at sites deeper than 25 mum from the outer surface. Measurements of the electrical resistance between the microelectrode and the external solution, made with single- and double-barreled microelectrodes, showed great discrepancies, which may be attributed to distinct pathways of different resistances in the stratum corneum. PDi measured at a depth of 5 mum was a logarithmic function of Na2SO4 or K2SO4 concentration in the external solution, increasing in negativity with a reduction in concentration. Substitution of Na by K in the external solution had only minor effects on PDi. Acidification of the external solution from pH 9 is accompanied by a reduction in the negative value of PDi. At pH 3 PDi was positive. PDi was interpreted as a diffusion potential at the tip of the microelectrode due to KCl diffusion from the electrode into the matrix of the stratum corneum. Differences in K and Cl mobilities, responsible for the origin of PDi, were attributed to fixed charges in the matrix of the stratum corneum, with density and polarity determined by their degree of proponation, controlled by the hydrogen ion concentration of the external solution. Skin potential, short-circuit current and their relationship to PDI were discussed. 相似文献
14.
Diffusion in immobilized-cell agar layers: influence of bacterial growth on the diffusivity of potassium chloride 总被引:1,自引:0,他引:1
Summary The diffusitivity of potassium chloride in composite agar slab/microporous membrane structures loaded with various amounts of Escherichia coli whole cells was determined using both time-lag and steady-state methods. The diffusion coefficient of KCl decreased linearly with the logarithm of the immobilized-cells content. The effect exerted by bacterial growth inside the immobilization matrices on KCl diffusivity was then investigated. The diffusion coefficient of KCl obtained by time-lag analysis decreased during incubation of the immobilized-cell structures, whereas less consitent results arose from the steady-state method. An apparent doubling time for immobilized E. coli, increasing with the initial cell content of the gel, was obtained from the calibration relationship between KCl diffusivity and the number of organisms in agar.
Offprint requests to: G.-A. Junter 相似文献
15.
The regeneration of a solid, crystallized cellulose solution in a N-methylmorpholine-N-oxide (NMMO)-water mixture was studied by measuring the diffusion coefficient of both the water uptake from the regenerating bath and the NMMO outflow to this bath. The diffusion coefficient of water going to the cellulose solution is about 10 times larger than the diffusion coefficient of NMMO leaving the solution. This difference expresses the strongly hygroscopic character of NMMO. None of these coefficients depends on cellulose molecular weight showing that no major rearrangement of cellulose chains occurs at the beginning of the regeneration. The diffusion coefficient of water is not influenced by the cellulose concentration, whereas the diffusion coefficient of NMMO decreases strongly when the cellulose concentration increases. Extrapolating the diffusion coefficient of NMMO versus cellulose concentration to zero shows that the maximal concentration of cellulose in NMMO-water is about 15%. Above this value, undissolved cellulose should be present. From the influence of the NMMO content in the water regenerating bath, it is possible to see that NMMO is removed from the solution if the bath has a NMMO content lower than 60%, to be compared with the 80% NMMO concentration in the solution. 相似文献
16.
Concentration dependence of NaCl salting of 0-1.5 mM lysozyme solution in 0.1 M sodium acetate buffer, pH 4.25, was investigated for NaCl concentration varying up to 0.9 M. Calorimetric experiments demonstrated that depending on the salt concentration the estimated number of the binding sites on the lysozyme surface varied in the range of 5 up to 13, and the increase of salt concentration caused the decrease of the number of accessible sites. The small, but significant, local maximum centered at 0.63 M NaCl concentration indicated the specific salting-out of the lysozyme accompanied by binding of approximately 2-3 chloride anions. Generalized McMillan and Mayer's approach reduced to the third-order virial coefficients demonstrates the domination of lysozyme aggregation upon salt addition (a(21)-h(xxy)) and salt organization on the lysozyme surface (a(12)-h(xyy)) processes. 相似文献
17.
E. B. Damon 《The Journal of general physiology》1938,21(3):383-409
The concentration effect with sea waters containing more than the normal amount of potassium has been studied in Valonia macrophysa. This was done by comparing the initial changes in P.D. across the protoplasm when natural sea water bathing the cell was replaced by various isotonic dilutions of KCl-rich sea waters. With small dilutions of KCl-rich sea waters, the P.D.-time curves are of the same form as with the undiluted solution, exhibiting the fluctuations characteristic of KCl-rich solutions. This indicates that with these solutions K+ enters Valonia protoplasm and plays an important part in the P.D. The value of the initial rise in P.D. decreases with increasing dilution. With high dilutions of KCl-rich sea waters, the P.D.-time curves are of quite different shape, resembling the curves with diluted natural sea water; the P.D. is practically independent of small changes in the concentration of potassium, and increases with increasing dilution. That is, with these higher dilutions, the sign of the concentration effect is reversed, becoming the same as with diluted natural sea water. The greater the concentration of KCl in the undiluted sea water, the higher is the critical dilution at which K+ ceases to influence the P.D. For a wide range of sea waters containing both KCl and NaCl, it is shown that the concentration effect above the critical dilution is determined solely by the activity of NaCl in the external solution. It is concluded that with dilute natural sea water and with high dilutions of KCl-rich sea waters we have to do with a diffusion potential, involving only the Na+ and Cl- ions, which are diffusing out from the vacuole. A quantitative relation between the composition of the sea water and the critical dilution has been deduced from the classical theory of the diffusion of electrolytes. It is shown that with dilutions less than this critical value the diffusion of K+ in the outer non-aqueous layer of the protoplasm is directed inward; hence K+ enters the protoplasm from these solutions. With dilutions greater than the critical value, the diffusion of K+ in this layer is directed outward; hence K+ does not enter the protoplasm. Since the P.D. shows no evidence of this outward diffusion of K+, it is concluded that the amount of K+ ordinarily present in the protoplasm is too small to produce any lasting electrical effect, and that the outward diffusion of K+ from the vacuole is prevented by the mechanism responsible for the accumulation of KCl in the cell sap. 相似文献
18.
《The Journal of general physiology》1978,72(4):513-538
The rates of diffusion of tritiated water (THO) and [14C]sucrose across cat right ventricular myocardium were studied at 23 degrees C in an Ussing-type diffusion cell, recording the time-course of increase in concentration of tracer in one chamber over 4--6 h after adding tracers to the other. Sucrose data were fitted with a model for a homogeneous sheet of uneven thickness in which the tissue is considered to be an array of parallel independent pathways (parallel pathway model) of varying length. The volume of the sucrose diffusion space, presumably a wholly extracellular pathway, was 23% of the tissue or 27.4 +/-1.7% (mean +/- SEM; n=11) of the tissue water. The effective intramyocardial sucrose diffusion coefficient, D8, was 1.51 +/- 0.19 X 10(-6)cm2.s-1 (n=11). Combining these data with earlier data, D8 was 22.6 +/- 1.1% (n=95) of the free diffusion coefficient in aqueous solution D degrees 8. The parallel pathway model and a dead-end pore model, which might have accounted for intracellular sequestration of water, gave estimates of DW/D degrees W (observed/free) of 15%. Because hindrance to water diffusion must be less than for sucrose (where D8/D degrees 8=22.6%), this showed the inadequacy of these models to account simultaneously for the diffusional resistance and the tissue water content. The third or cell-matrix model, a heterogeneous system of permeable cells arrayed in the extracellular matrix, allowed logical and geometrically reasonable interpretations of the steady-state data and implied estimates of DW in the cellular and extracellular fluid of approximately 25% of the aqueous diffusion coefficient. 相似文献
19.
Laser correlation Spectroscopy was used to measure the mutual diffusion coefficient, D, of human cyanomethemoglobin (Fe+++:CN) at varying protein concentrations. These measurements were male at 20°C in a 0.1 M phosphate buffer solution at pH 7.0. For low protein concentrations we find D = (6.43 ± 0.26) × 10?7 cm2/S and that there is a near linear decrease from this value at higher concentrations. The linear relation between the diffusion coefficient and protein concentration allows us to deduce the value of the linear frictional volume fraction coefficient, Kf= 7.75. and to extrapolate to hemoglobin concentrations equivalent to that in the red blood cell where we estimate D = 4.25 × 10?7 cm2/s Various theoretical predictions of the dependence of the mutual diffusion coefficient on concentration are tested; we find that the generalized Stokes-Einstein relation can be made to fit our high concentration data if we assume a hard-sphere model and if we include a term involving a hydrodynamic interaction integral. 相似文献
20.
The rapid mixing/photocross-linking technique developed in our laboratory has been employed in the study of the mechanism of promoter binding by Escherichia coli RNA polymerase (RPase). We have previously reported on the quantitation of the one-dimensional diffusion coefficient (D1) for RPase along the DNA template (Singer, P. T., and Wu, C.-W. (1987) J. Biol. Chem. 262, 14178-14189). In this paper, we describe the effect of salt concentration and temperature on the kinetics of promoter search by RPase using plasmid pAR1319 DNA, which contains the A2 early promoter from bacteriophage T7, as template. Over a range of KCl concentrations from 25 to 200 mM, the apparent bimolecular rate constant (ka) for the association of RPase with the A2 promoter on this DNA template varied approximately 2-fold, achieving a maximal value between 100 and 125 mM KCl. More significantly, the transient distribution of RPase among nonspecific DNA binding sites changed markedly as a function of salt concentration, indicative of gross changes in the average number of base pairs covered by sliding during a nonspecific lifetime. Using the mathematical treatment outlined in our earlier report, the nonspecific dissociation rate constant (koff) was calculated from the binding curves for the nonspecific as well as promoter-containing DNA. The observed variations in ka as a function of monovalent cation concentration ([M+]) were due primarily to changes in koff, as D1 was found to be essentially independent of [M+]. Interestingly, D1 decreased by one-third as the concentration of magnesium was lowered from 10 to 1 mM. In addition, the dependence of koff (and consequently the nonspecific equilibrium association constant, keq) on [M+] agreed qualitatively with the results of deHaseth et al. (deHaseth, P.L., Lohman, T. M., Burgess, R. R., and Record, M. T., Jr. (1977) Biochemistry 17, 1612-1622), though we consistently measure a weaker Keq. The association rate constant was also measured between 4 and 37 degrees C, and was found to vary approximately 2-fold over that range. An activation energy for the bimolecular association of RPase to the A2 promoter was calculated to be 2.2 +/- 0.4 kcal/mol, while the activation energy for one-dimensional diffusion was 4.7 +/- 0.8 kcal/mol. 相似文献