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1.
CD spectra and melting curves were collected for a 28 base-pair DNA fragment in the form of a DNA dumbbell (linked on both ends by T4 single-strand loops) and the same DNA sequence in the linear form (without end loops). The central 16 base pairs (bp) of the 28-bp duplex region is the poly(pu) sequence: 5′-AGGAAGGAGGAAAGAG-3′. Mixtures of the dumbbell and linear DNAs with the 16-base single-strand sequence 5′-TCCTTCCTCCTTTCTC-3′ were also prepared and studied. At 22°C, CD measurements of the mixtures in 950 mM NaCl, 10 mM sodium acetate, 1 mM EDTA, pH 5.5, at a duplex concentration of 1.8 μM, provided evidence for triplex formation. Spectroscopic features of the triplexes formed with either a dumbbell or linear substrate were quite similar. Melting curves of the duplex molecules alone and in mixtures with the third strand were collected as a function of duplex concentration from 0.16 to 2.15 μM. Melting curves of the dumbbell alone and mixtures with the third strand were entirely independent of DNA concentration. In contrast, melting curves of the linear duplex alone or mixed with the third strand were concentration dependent. At identical duplex concentrations, the dumbbell alone melts ~20°C higher than the linear duplex. The curve of the linear duplex displayed a significant pretransition probably due to end fraying. On melting curves of mixtures of the dumbbell or linear duplex with the third strand, a low temperature transition with much lower relative hyperchromicity change (~ 5%) was observed. This transition was attributed to the melting of a new molecular species, e.g., the triplex formed between the duplex and single-strand DNA molecules. In the case of the dumbbell/single-strand mixture, these melting transitions of the triplex and the dumbbell were entirely resolvable. In contrast, the melting transitions of the linear duplex and the triplex overlapped, thereby preventing their clear distinction. To analyze the data, a three-state equilibrium model is presented. The analysis utilizes differences in relative absorbance vs temperature curves of dumbbells (or linear molecules) alone and in mixtures with the third strand. From the model analysis a straightforward derivation of fT(T), the fraction of triplex as a function of temperature, was obtained. Analysis of fT vs temperature curves, in effect melting curves of the triplexes, provided evaluation of thermodynamic parameters of the melting transition. For the triplex formed with the dumbbell substrate, the total transition enthalpy is ΔHT = 118.4 ± 12.8 kcal/mol (7.4 ± 0.8 kcal/mol per triplet unit) and the total transition entropy is ΔST = 344 ± 36.8 cal/K · mol (eu) (21.5 ± 2.3 eu per triple unit). The transition curves of the triplex formed with the linear duplex substrate displayed two distinct regions. A broad pretransition region from fT = 0 to 0.55 and a higher, sharper transition above fT = 0.55. The transition parameters derived from the lower temperature region of the curve are ΔHT = 44.8 ± 9.6 kcal/mol and ΔST = 112 ± 33.6 eu (or ΔH′ = 2.8 ± 0.6 kcal/mol and ΔS′ = 7.0 ± 2.1 eu per triplet). These values are probably too small to correspond to actual melting of the triplex but instead likely reveal effects of end fraying of the duplex substrate on triplex stability. Transition parameters of the upper transition are ΔHT = 128.0 ± 2.3 kcal/mol and ΔST = 379.2 ± 6.4 eu (ΔH′ = 8.0 ± 0.2 kcal/mol and ΔS′ = 23.7 ± 0.4 eu per triplet) in good agreement (within experimental error) with the transition parameters of the triplex formed with the dumbbell substrate. Supposing this upper transition reflects actual dissociation of the third strand from the linear duplex substrate this triplex is comparable in thermodynamic stability to the triplex formed with a dumbbell substrate. Even so, the biphasic melting character of the linear triplex obscures the whole analysis, casting doubt on its absolute reliability. Apparently triplexes formed with a dumbbell substrate offer technical advantages over triplexes formed from linear or hairpin duplex substrates for studies of DNA triplex stability. © 1993 John Wiley & Sons, Inc.  相似文献   

2.
3.
The thermodynamics of ethidium ion binding to the double strands formed by the ribooligonucleotides rCA5G + rCU5G and the analogous deoxyribo-oligonucleotides dCA5G + dCT5G were determined by monitoring the absorbance versus temperature at 260 and 283 nm at several concentrations of oligonucleotides and ethidium bromide. A maximum of three ethidium ions bind to the oligonucleotides, which is consistent with intercalation and nearest-neighbor exclusion. For the ribo-oligonucleotide the binding mechanism is complex. Either two sites (assumed to be the intercalation sites at the two ends of the oligonucleotide) bind more strongly by a factor of 140 than the third site, or all sites are identical, but there is strong anticooperativity on binding (cooperativity parameter, 0.1). In sharp contrast, the binding to the same sequence (with thymine substituted for uracil) in the deoxyribo-oligonucleotide showed all sites equivalent and no cooperativity. For the ribo-oligonucleotides the enthalpy for ethidium binding is ?14 kcal/mol. The equilibrium constants at 25°C depend on the model; either K = 6 × 105M?1 for the two strong sites (4 × 103M?1 for the weak site) or K = 2.5 × 105M?1 for the intrinsic constant of the anticooperative model. For the equivalent deoxyribo-oligonucleotide the enthalpy of binding is -9 kcal/mol and the equilibrium constant at 25°C is a factor of 10 smaller (K = 2.5 × 104M?1).  相似文献   

4.
The kinetics of the coil-to-helix transition of (dG-dC)3 in M NaCl, 45 mM sodium cacodylate, pH 7, were measured in H2O, D2O, 10 mol % ethanol, 10 mol % urea, and 10 mol % glycerol. At 43°C in H2O the recombination rate is 1.3 ± 0.2 × 107 M?1 s?1; the dissociation rate is 68 ± 10 s?1. The destabilization of the helix in 10 mol % ethanol and 10 mol % urea relative to water is primarily due to a large increase in the helix-dissociation rate. In 10 mol % glycerol, the destabilization of the helix is due to a decrease in the recombination rate and an increase in the dissociation rate. Above 20°C, two exponential decays longer than 1 μs are observed after a temperature jump. The slower relaxation time is 4–10 times faster than the bimolecular component and is independent of oligomer concentration. We attribute this relaxation to a rapid equilibrium between two helical states. At low temperatures and oligomer concentrations of 1 mM or greater, the helices aggregate in 1M NaCl. Experimental data are presented under conditions where aggregation is unimportant and evidence is given that the ΔH-determined spectroscopically is unaffected by aggregation.  相似文献   

5.
Binding of ethidium to bacteriophage T7 and T7 deletion mutants   总被引:1,自引:0,他引:1  
Equilibrium binding of ethidium, quantitated by fluorescence enhancement, to DNA packaged in bacteriophage T7 and T7 deletion mutants has been compared with the binding of this dye to DNA released from its capsid (free DNA). During achievement of apparent equilibrium binding, no change in bacteriophage T7 structure occurred, by the criterion of agarose gel electrophoresis. However, excessive incubation with ethidium bromide caused detectable changes in bacteriophage structure, a possible explanation of disagreements in similar studies previously performed with T-even bacteriophages. Scatchard plots for packaged DNA had a curvature greater than the previously demonstrated [Bresloff, J. L. & Crothers, D. M. (1981) Biochemistry 20 , 3547–3553] curvature for free DNA. By treating plots for packaged DNA as though they were biphasic, it was found that binding to most sites occurred with an apparent association constant (Kap) 3.3–4.3 times lower than the Kap of free DNA. The number of these sites increased significantly as the density of packaged DNA was decreased by use of the deletion mutants. Values of ΔH° for these sites were negative and equal to the ΔH° for free DNA; values of ΔS° were positive and about half the ΔS° for free DNA. A second class of sites, roughly 1.2% of the total, had a significantly higher Kap and more negative ΔH° than those of the majority of sites.  相似文献   

6.
Melting parameters of 2U:1A complexes formed by polyuridylic acid [poly(U)] and three adenine dinucleotides, diribonucleoside monophosphonate ApA and diastereoisomers of dideoxyribonucleoside methyl phosphonate [(dApA)1 and (dApA)2], in 1M NaCl and at a number of dinucleotide concentrations were obtained from differential scanning microcalorimetric data and interpreted in terms of the theory of helix–coil equilibrium in oligonucleotide–polynucleotide systems. The apparent binding constant, 1/cm, at 39°C and melting temperatures, Tm, at 1 × 10?3 M dinucleotide concentration indicate the following order of thermodynamic stability of the complexes: 2 poly(U) · (dApA)2 (2.27 × 103M?1, 44.2°C) > 2 poly(U) · (dApA)1 (9.9 × 102M1, 39.2°C) > 2 poly(U) · (ApA) (5.9 × 102M?1, 35.8°C). Corresponding calorimetric enthalpies of melting, ΔHm: 13.5, 12.7, and 12.8 kcal/mol (UUA base triplets) were found to be considerably lower than the van't Hoff enthalpies, ΔHapp: 29.4, 16.2, and 16.2 kcal/mol, respectively, evaluated from the dependence of the melting temperatures on dinucleotide concentration. Self-association of dinucleotides and their simultaneous binding as monomers, dimers, and higher-order associated species is suggested as the most probable cause of the differences between ΔHm and ΔHapp values. The differences in thermodynamic properties of the complexes formed by (dApA)1 and (dApA)2 diastereoisomers are discussed in connection with their known conformational properties. The higher and essentially enthalpic stability of the 2 poly(U) · (dApA)2 complex correlates with a lower degree of intramolecular stacking of the (dApA)2 isomer. The hydrophobically enhanced strong self-association of the latter greatly influences the thermodynamics of its complex formation with poly(U) and results in ΔHappHm = 2.3.  相似文献   

7.
Values for the thermodynamic quantities, ΔH° = 11.8 ± 2.0 Kcal/mole and ΔS° = 43.6 ± 6.0 e.u., of the 3-13 helix–coil equilibrium of isolated S-peptide (19 residue N-terminal fragment of ribonuclease A) in aqueous solution (3 m M, 1M NaCl, pD 5.4) have been determined from a joint analysis of the Thr 3γ, Ala 6β, Phe 8meta, and Phe 8para 1H chemical shift vs temperature curves (?7 to 80°C) in several aqueous–trifluorethanol mixtures. Chemical shifts in the coil and in the helix have been determined for up to 16 protons belonging to the 3-13 fragment. Thermodynamic parameters have also been determined for C-peptide (13 residue fragment) and a number of S-peptide derivatives. From the variation of the values of the thermodynamic parameters at pD 2.5, 5.4, and 8.0, a quantitation of the two helix-stabilizing side-chain interactions can be made: (1) Δ(ΔH°) ? 5 Kcal/mole and Δ(ΔS°) ? 18 e.u. for the salt bridge Glu 2? … Arg 10+ and (2) Δ(ΔH°) ? 3 Kcal/mole and Δ(ΔS°) = 9 e.u. for the one in which the His 12+ imidazolium group is involved, presumably a partial stacking with the Phe 8 side chain.  相似文献   

8.
The thermotropic properties of bovine blood coagulation Factors IX and X, as well as the activation intermediates and products of these proteins, have been investigated by differential scanning microcalorimetry in the presence and absence of Ca2+. Bovine Factor IX displays a single thermal-denaturation transition characterized by a temperature midpoint (TM) of 54.5 ± 0.5 °C and a calorimetric enthalpy (ΔHc) of 105 ± 15 kcal/mol, in the absence of Ca2+. In the presence of Ca2+ concentrations sufficient to saturate its sites on Factor IX, the Tm value is increased to 57.0 ± 0.5 °C and the ΔHc is virtually unchanged. When the activation intermediate, Factor IXα, is similarly analyzed in the absence of Ca2+, a broad, diffuse thermogram was obtained which did not lend itself to calculation of thermodynamic parameters. In the presence of Ca2+, Factor IXα displayed thermograms characterized by a TM of 51.0 ± 0.5 °C and a ΔHc of 109 ± 10 kcal/mol. The activated product, Factor IXaα, in the absence of Ca2+ (the values in the presence of saturating Ca2+ are given in parentheses), undergoes thermal denaturation with a TM of 54.5 ± 0.5 °C (57.0 ± 0.5 °C) and a ΔHc of 158 ±10 kcal/mol (156 ± 10 kcal/mol). Similarly, the terminal-activation product, Factor IXaβ, displays a TM of 51.5 ± 0.5 °C (54.0 ± 0.5 °C) and a ΔHc of 85 ± 5 kcal/mol (126 ± 10 kcal/mol). Bovine blood coagulation Factor X has been analyzed in this same fashion, and shows very similar thermal properties to Factor IX. The thermal denaturation of Factor X is represented by a TM of 54.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔHc of 102 ± 10 kcal/mol (118 ± 10 kcal/mol), whereas its activated form, Factor Xaβ, possesses a TM of 55.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔHc of 92.0 ± 5 kcal/mol (136 ± 10 kcal/mol). These studies indicate that, for many of these proteins, Ca2+ induces a conformational alteration to a more thermally stable form, which also requires the absorption of greater amounts of heat for thermal denaturation.  相似文献   

9.
Thermodynamics of the B to Z transition in poly(dGdC)   总被引:1,自引:0,他引:1  
The thermodynamics of the B to Z transition in poly(dGdC) was examined by differential scanning calorimetry, temperature-dependent absorbance spectroscopy, and CD spectroscopy. In a buffer containing 1 mM Na cacodylate, 1 mM MgCl2, pH 6.3, the B to Z transition is centered at 76.4°C, and is characterized by ΔHcal = 2.02 kcal (mol base pair)?1 and a cooperative unit of 150 base pairs (bp). The tm of this transition is independent of both polynucleotide and Mg2+ concentrations. A second transition, with ΔHcal = 2.90 cal (mol bp)?1, follows the B to Z conversion, the tm of which is dependent upon both the polynucleotide and the Mg2+ concentrations. Turbidity changes are concomitant with the second transition, indicative of DNA aggregation. CD spectra recorded at a temperature above the second transition are similar to those reported for ψ(–)-DNA. Both the B to Z transition and the aggregation reaction are fully and rapidly reversible in calorimetric experiments. The helix to coil transition under these solution conditions is centered at 126°C, and is characterized by ΔHcal = 12.4 kcal (mol bp)?1 and a cooperative unit of 290 bp. In 5 mM MgCl2, a single transition is seen centered at 75.5°C, characterized by ΔHcal = 2.82 kcal (mol bp)?1 and a cooperative unit of 430 bp. This transition is not readily reversible in calorimetric experiments. Changes in turbidity are coincident with the transition, and CD spectra at a temperature just above the transition are characteristic of ψ(–)-DNA. A transition at 124.9°C is seen under these solution conditions, with ΔHcal = 10.0 kcal (mol bp)?1 and which requires a complex three-step reaction mechanism to approximate the experimental excess heat capacity curve. Our results provide a direct measure of the thermodynamics of the B to Z transition, and indicate that Z-DNA is an intermediate in the formation of the ψ-(–) aggregate under these solution conditions.  相似文献   

10.
H P Hopkins  W D Wilson 《Biopolymers》1987,26(8):1347-1355
Enthalpy changes (ΔHB) for the binding of ethidium (a monocation) and propidium (a dication) to calf thymus DNA have been determined calorimetrically in piperazine-N, N′-bis(2-ethanesulfonic acid) buffer with the fluoride ion as the counterion. Heats of dilution for the fluoride salts of ethidium and propidium were substantially less than the corresponding values found for other halide salts of these cations. At a Na+ ion concentrations of 0.019, ΔHB = ?8.3 and ?7.9 ± 0.3 kcal mol?1 for ethidium and propidium, respectively. For these two cations, just as was observed for the naphthalene monoimide (monocation) and diimide (dication) [H. P. Hopkins, K. A. Stevenson, and W. D. Wilson, (1986) J. Sol. Chem. 15 , 563–579], ΔHB is within the same experimental error for both cations. Apparently, charge–charge interactions in DNA–cation complexes produce only small changes in the enthalpy for the system. In the concentration range 0.019–0.207, the ΔHB values for propidium did not depend appreciably on the Na+ ion concentration, and a similar pattern was shown to exist for ethidium. When these results were combined with ΔGB values for the binding of these cations to DNA, we found the variation of ΔSB with Na+ ion concentration to be remarkably close to the predictions of modern polyelectrolyte theory, i.e., propidium binding to DNA causes approximately twice as many Na+ ions to be released into the bulk solution as does the binding of ethidium. The much stronger binding of propidium, relative to ethidium, at low ionic strengths is thus seen to be primarily due to entropic effects.  相似文献   

11.
The Raman spectra of guanylyl (3′-5′) guanosine (GpG) in solution in H2O and D2O at pH 3–7 have been recorded at various temperatures between 0 and 80°C. The results are consistent with the existence in the lower temperature range of stable aggregates formed by the stacking of GpG tetramers. The aggregates melt cooperatively near 60°C, which results in important changes in the spectra. Among these, a large increase in intensity of some of the bands assigned to the guanine residues shows that unstacking of the bases occurs at the melting. Also apparent in the spectra are changes in the intensity and frequency of band attributable to molecular groups involved in intermolecular hydrogen bonding between adjacent molecules in the complex. The melting temperature of GpG decreases by approximately 15°C upon lowering the concentration from 5 × 10?2 to 5 × 10?4M, as shown by Raman, calorimetric, CD, and uv measurements. The experimentally determined ΔH and ΔS for the melting transition are 9 Kcal/mol and 28 e.u./mol, respectively. The aggregation of GpG in 1.5 × 10?3M solutions was found to be very slow. The half-time of the process, which roughly follows first-order kinetics, is approximately 3 min at 10°C and 21 min at 35°C. The negative energy of activation associated with this reaction (?143 Kcal) indicated that the process involves intermediates whose concentrations decrease the temperatures raised, thus slowing down the overall process. The rate of disaggregation of GpG upon dilution to very low concentration is also extremely slow, indicating that the GpG aggregates, once formed, are very stable.  相似文献   

12.
Isotherms of the EtBr adsorption on native and denatured poly(dA)poly(dT) in the temperature interval 20–70°C were obtained. The EtBr binding constants and the number of binding sites were determined. The thermodynamic parameters of the EtBr intercalation complex upon changes of solution temperature 20–48°C were calculated: 1.0·106 M−1K≤1.4·106 M−1, free energy ΔG o=−8.7±0.3 kcal/mol, enthalpy ΔH o≅0, and entropy ΔS o=28±0.5 cal/(mol deg). UV melting has shown that the melting temperature (T m) of EtBr-poly(dA)poly(dT) complexes (μ=0.022,4.16·10−5 M EtBr) increased by 17°C as compared with the ΔT m of free homopolymer, whereas the half-width of the transition (T m) is not changed. It was shown for the first time that EtBr forms complexes of two types on single-stranded regions of poly(dA)poly(dT) denatured at 70°C: strong (K 1=1.7·105 M−1; ΔG o=−8.10±0.03 kcal/mol) and weak (K 2=2.9·103 M−1; ΔG o=−6.0±0.3 kcal/mol).The ΔG o of the strong and weak complexes was independent of the solution ionic strength, 0.0022≤μ≤0.022. A model of EtBr binding with single-stranded regions of poly(dA)poly(dT) is discussed.  相似文献   

13.
R L Ornstein  J R Fresco 《Biopolymers》1983,22(8):1979-2000
Tm values for 20 DNA duplexes with different repeating base sequences provided the data base for developing a rational and relatively simple methodology for computing apparent enthalpies for the helix → coil transitions of DNA helices, ΔH calc. The computational variables and their range of acceptable values were selected on the basis of physically plausible arguments. Over 350,000 different combinations of the variables were tested for degree of fit. It was therby possible to find a combination giving a high degree of linear fit between Tm and ΔH calc (correlation coefficient, 0.99), with Tm values deviating (on average) from the regression line by only ±2.17°C. Most of this uncertainty is attributed to experimental limitations, although computational approximations also contribute. With ΔH calc for the melting of each of the unique complementary dinucleotide fragments computed by the method developed, it is possible to estimate Tm and (relative) ΔH calc reliable for the melting of any particular DNA [with base pairs G(I)·C and A·T] given only its base sequence. The ΔHcalc values for the complementary dinucleotide fragments, together with statistical considerations, make it apparent that Tm of DNAs with repeating base sequence show only an approximate linear dependence on G·C content because A·T and G·G pairs do not contribute to helix stability independently of the base-pair sequence in which they occur. In fact, the nearestneighbor stacking interactions are so significant that certain complementary dinucleotide fragment sequences with 0,50, and 100% G·C content have the same stability.  相似文献   

14.
Bovine brain hexokinase enhances the effect of Mn(II) on the longitudinal relaxation rate of water protons. Direct interaction of Mn(II) with the enzyme has been studied using electron spin resonance and proton relaxation rate enhancement methods. The results indicate that brain hexokinase has 1.05 ± 0.13 tight binding sites and 7 ± 2 weak binding sites with a dissociation constant, KD = 25 ± 4 μM and KD = 1050 ± 290 μM, respectively, at pH 8.0, 23 °C. The characteristic enhancement ?b) for hexokinase-Mn(II) complex evaluated from proton relaxation rate enhancement studies, gave ?b = 3.5 ± 0.4 for tight binding sites and an average ?b = 2.3 ± 0.5 per site for weak binding sites at 9 MHZ. The dissociation constant of Mn(II) for tight binding sites on the enzyme exhibits strong temperature dependence. In the low-temperature region (5–12 °C) brain hexokinase probably undergoes a conformational change. Frequency dependence of the normalized relaxation rate for bound water at various temperatures has shown that the number of exchangeable water molecules left in the first coordination sphere of bound Mn(II) is about one at 30 °C and about two at 18 °C. Binding of glucose 6-phosphate to hexokinase results in large-line broadening of the resonances of anomeric protons of the sugar. However, no such effect was observed in the case of glucose binding. These results suggest different modes of interaction of these two sugars to hexokinase. Line broadening of the C-(1) hydrogen resonances of glucose caused by Mn(II) in the presence of hexokinase suggests the proximity of the Mn(II) binding site to that of glucose. A lower limit of 1330 ± 170 s?1 for the rate of dissociation of glucose from enzyme-Mn(II)-glucose complex has been obtained from these studies.  相似文献   

15.
We measured by batch microcalorimetry the standard enthalpy change ΔH° of the binding of Mn2+ to apo-bovine α-lactalbumin; ΔH° = −90 ± 4kJ·mol−1. The binding constants, KMn2+, calculated from the calorimetric and circular dichroism titration curves, are (4.6±1) · 105M−1, respectively. Batch calorimetry confirms the competitive binding of Ca2+, Mn2+ and Na+ to the same site. The relatively small enthalpy change for Mn2+ binding compared to Ca2+ binding favours a model of a rigid and almost ideal Ca2+-complexating site, different from the well-known EF-hand structures. Cation binding to the high-affinity site most probably triggers the movement of an α-helix which is directly connected to the complexating loop.  相似文献   

16.
Methods including spectroscopy, electronic chemistry and thermodynamics were used to study the inclusion effect between γ-cyclodextrin (CD) and vitamin K3(K3), as well as the interaction mode between herring-sperm DNA (hsDNA) and γ-CD-K3 inclusion complex. The results from ultraviolet spectroscopic method indicated that VK3 and γ-CD formed 1:1 inclusion complex, with the inclusion constant Kf = 1.02 × 104 L/mol, which is based on Benesi–Hildebrand's viewpoint. The outcomes from the probe method and Scatchard methods suggested that the interaction mode between γ-CD-K3 and DNA was a mixture mode, which included intercalation and electrostatic binding effects. The binding constants were K θ25°C = 2.16 × 104 L/mol, and Kθ37°C = 1.06 × 104 L/mol. The thermodynamic functions of the interaction between γ-CD-K3 and DNA were ΔrHmθ = ?2.74 × 104 J/mol, ΔrSmθ = 174.74 J·mol?1K?1, therefore, both ΔrHmθ (enthalpy) and ΔrSmθ (entropy) worked as driven forces in this action.  相似文献   

17.
Robert E. Hurst 《Biopolymers》1978,17(11):2601-2608
The thermodynamics of the partition of chondroitin sulfate–hexadecylpyridinium complexes wee studied in order to gain further insight into the mechanisms responsible for the sensitivity of the relative solubility of these complexes in aqueous slat and butanol phases to small changes in slat concentration. The dependence of the partition coefficient was measured as a function of temperature at three different salt concentrations. Increasing the temperature was found to favor the form of the complex which was soluble n the aqueous phase. Although the transition could be induced by temperature changes, he transition occurred over a 20°C range in temperature. The transition from the aqueous phase to the butanol phase was strongly exothermic, with ΔH = ?22.3 kcal/mol polymer. The value of ΔS was found to be dependent on the salt concentration, ranging from ?72.7 e.u./mol polymer in 0.05125M NaCl to ?77.1 e.u./mol polymer in 0.05375M MaCl. When placed on a disaccharide basis, the corresponding values are ΔH = ?402 cal/mol and ΔS = ?1.31 to ?1.3 e.u./mol. The sharpness of he transition was found to be due t the similarity in magnitude of ΔH and TΔS, and on the dependence of the later upon the salt concentration.  相似文献   

18.
Δ53β hydroxysteroid dehydrogenase activity transforms biologically inactive Δ53β hydroxy steroids into the active Δ43-keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for Δ53β HSD we have analysed the initial velocity rates (Vo) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20α hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 μm) of the dioestrous and proestrous rat ovary. The results are mean ± S.E.M. The relationship between DHEA concentration (0 to 50 μM) and Δ53β HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/Vo versus 1/S plot, the x intercept being positive. Using a 1/Vo versus 1/S2 plot the Vmax was determined to be 1·0 ± 0·08 μmol min?1 mg?1 CL (n = 6). The Hill constant was 2·7 ± 0·02 (n = 6) suggesting a high degree of positive co-operativity for DHEA binding. The S concentration for half maximal activity was 17 ± 1 μmoles (n = 6). In the corpora lutea cells of the proestrous ovary, the Vmax for DHEA transformation was unchanged (0·95 ± 0·04 μmol min?1 mg?1, n = 3) whilst the S0·5 was significantly increased to 27 ± 0·1 (p < 0·01, n = 3). The Hill constant remained positive being 2·9 ± 0·2 (n = 3). NAD+ binding to 3β HSD in regressing corpora lutea of the proestrous ovary has been demonstrated previously to be hyperbolic and fit the classical Michaelis-Menten model.1 Extending the analysis of NAD+ binding to the regressing corpus luteum of the dioestrous rat ovary revealed similar kinetic characteristics to that seen with the proestrous enzyme, the apparent Vmax and Km being 0·84 ± 0·04 μmol min?1 mg?1 CL (n = 3) and 27 ± 7 μmol 1?1 (n = 3) respectively. The Hill constant was 1·1 ± 0·03 (n = 3), indicating no co-operativity of co-factor binding.  相似文献   

19.
Chlorogenic acid, 3’-O-caffeoyl D-quinic acid, is an inherent ligand present inHelianthus annuus L. The effect of pH on chlorogenic acid binding to helianthinin suggests that maximum binding occurs at pH 6.0. The protein-polyphenol complex precipitates as a function of time. The association constant of the binding of chlorogenic acid to helianthinin, determined by equilibrium dialysis, at 31°C has a value of 3.5 ± 0.1 × 104M−-1 resulting in a ΔG value of − 6.32 ± 0.12 kcal /mol. The association constantK ais 1.0 ± 0.1 × 104M−1 as determined by ultraviolet difference spectral titration at 25°C with ΔG° of -5.46 ± 0.06 kcal/mol. From fluorescence spectral titration at 28°C, theK avalue is 1.38 ± 0.1 × 1 0 4M−1 resulting in a ΔG of − 5.70 ± 0.05 kcal/mol. The total number of binding sites on the protein are 420 ± 50 as calculated from equilibrium dialysis. Microcalorimetric data of the ligand-protein interaction at 23°C suggests mainly two classes of binding. The thermal denaturation temperature,T mof the protein decreases from 76°C to 72°C at 1 × 10−3M chlorogenic acid concentration upon complexation. This suggests that the complexation destabilizes the protein. The effect of temperature onK aof chlorogenic acid shows a nonlinear increase from 10.2°C to 45°C. Chemical modification of both lysyl and tryptophanyl residues of the protein decreases the strength of binding of chlorogenic acid. Lysine, tryptophan and tyrosine of protein are shown to be present at the binding site. Based on the above data, it is suggested that charge-transfer complexation and entropically driven hydrophobic interaction are the predominant forces that are responsible for binding of chlorogenic acid to the multisubunit protein, helianthinin. Publication No. 324.  相似文献   

20.
The binding of [Dy(dmp)2Cl3(OH2)], where dmp is 2,9-dimethyl 1,10-phenanthroline, with Fish salmon DNA (FS-DNA) is investigated by absorption and emission spectroscopy, quenching studies, salt dependent, and gel electrophoresis. The binding constant (Kb) of the interaction is calculated as (1.27 ± .05) × 105 M?1 from absorption spectral titration data. The Stern–Volmer constant (KSV), thermodynamic parameters involves ΔG°, ?H°, and ?S° are calculated by fluorescent data and Van’t Hoff equation. The thermodynamic studies show that the reaction for the binding of the complex with FS-DNA is endothermic and entropically driven (Δ > 0, ΔH° > 0). The effect of the complex concentration on FS-DNA cleavage reactions is also investigated by gel electrophoresis. Furthermore, the Dy(III) complex has been screened for its antibacterial activity. The experimental results suggest that the Dy(III) complex binds significantly to FS-DNA by hydrophobic groove binding mode and the complex has more efficient antibacterial activity compared to its metal salt.  相似文献   

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