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1.
The standard Gibbs free energy change of hydrolysis of α-d-ribose 1-phosphate has been measured at pH 7.0, ionic strength 0.1 m, and 25 °C by combining the corresponding values of the two following reactions: adenosine + H2O ág adenine + ribose (ΔG0′ = ?2.3 ± 0.1 kcal/mol), catalyzed by adenosine nucleosidase, and ribose 1-phosphate + adenine ág adenosine + PiG0′ = ?3.1 ± 0.1 kcal/mol), catalyzed by adenosine phosphorylase. The standard Gibbs free energy changes were calculated for both reactions from the equilibrium constant. A value of -5.4 ± 0.15 kcal/mol, comparable to that of other hemiacetal phosphoric esters, was obtained for the hydrolysis of ribose 1-phosphate.  相似文献   

2.
The enthalpy of the bioluminescent reaction
FMNH2 + RCHO + O2luciferase FMN + RCOO + H3O+ + hv
has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH2) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔHL = ?338.9 k.J (mol FMN)?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔGL = ?464.8 kJ (mol FMN)?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH2 + O2 ? FMN + H2O2; ΔHD = ?158.5 kJ (mol FMN)?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG1, and intrinsic enthalpy, ΔH1, which are functionally defined by the relations ΔGI = ΔGL ? uhvΔHI = ΔHL ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.  相似文献   

3.
We have studied the conformational stability of the two homologous membrane skeletal proteins, the erythroid and non-erythroid spectrins, in their dimeric and tetrameric forms respectively during unfolding in the presence of urea and guanidine hydrochloride (GuHCl). Fluorescence and circular dichroism (CD) spectroscopy have been used to study the changes of intrinsic tryptophan fluorescence, anisotropy, far UV-CD and extrinsic fluorescence of bound 1-anilinonapthalene-8-sulfonic acid (ANS). Chemical unfolding of both proteins were reversible and could be described as a two state transition. The folded erythroid spectrin and non-erythroid spectrin were directly converted to unfolded monomer without formation of any intermediate. Fluorescence quenching, anisotropy, ANS binding and dynamic light scattering data suggest that in presence of low concentrations of the denaturants (up-to 1M) hydrogen bonding network and van der Waals interaction play a role inducing changes in quaternary as well as tertiary structures without complete dissociation of the subunits. This is the first report of two large worm like, multi-domain proteins obeying twofold rule which is commonly found in small globular proteins. The free energy of stabilization (ΔGu H 2 0) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.  相似文献   

4.
The repair of double-stranded DNA breaks via homologous recombination involves a four-way cross-strand intermediate known as Holliday junction (HJ), which is recognized, processed, and resolved by a specific set of proteins. RuvA, a prokaryotic HJ-binding protein, is known to stabilize the square-planar conformation of the HJ, which is otherwise a short-lived intermediate. Despite much progress being made regarding the molecular mechanism of RuvA-HJ interactions, the mechanochemical aspect of this protein-HJ complex is yet to be investigated. Here, we employed an optical-tweezers-based, single-molecule manipulation assay to detect the formation of RuvA-HJ complex and determined its mechanical and thermodynamic properties in a manner that would be impossible with traditional ensemble techniques. We found that the binding of RuvA increases the unfolding force (Funfold) of the HJ by ~2-fold. Compared with the ΔGunfold of the HJ alone (54 ± 13 kcal/mol), the increased free energy of the RuvA-HJ complex (101 ± 20 kcal/mol) demonstrates that the RuvA protein stabilizes HJs. Interestingly, the protein remains bound to the mechanically melted HJ, facilitating its refolding at an unusually high force when the stretched DNA molecule is relaxed. These results suggest that the RuvA protein not only stabilizes the HJs but also induces refolding of the HJs. The single-molecule platform that we employed here for studying the RuvA-HJ interaction is broadly applicable to study other HJ-binding proteins involved in the critical DNA repair process.  相似文献   

5.
The observed equilibrium constants (Kobs) for the reactions of d-2-phosphoglycerate phosphatase, d-2-Phosphoglycerate3? + H2O → d-glycerate? + HPO42?; d-glycerate dehydrogenase (EC 1.1.1.29), d-Glycerate? + NAD+ → NADH + hydroxypyruvate? + H+; and l-serine:pyruvate aminotransferase (EC 2.6.1.51), Hydroxypyruvate? + l-H · alanine± → pyruvate? + l-H · serine±; have been determined, directly and indirectly, at 38 °C and under conditions of physiological ionic strength (0.25 m) and physiological ranges of pH and magnesium concentrations. From these observed constants and the acid dissociation and metal-binding constants of the substrates, an ionic equilibrium constant (K) also has been calculated for each reaction. The value of K for the d-2-phosphoglycerate phosphatase reaction is 4.00 × 103m [ΔG0 = ?21.4 kJ/mol (?5.12 kcal/mol)]([H20] = 1). Values of Kobs for this reaction at 38 °C, [K+] = 0.2 m, I = 0.25 M, and pH 7.0 include 3.39 × 103m (free [Mg2+] = 0), 3.23 × 103m (free [Mg2+] = 10?3m), and 2.32 × 103m (free [Mg2+] = 10?2m). The value of K for the d-glycerate dehydrogenase reaction has been determined to be 4.36 ± 0.13 × 10?13m (38 °C, I = 0.25 M) [ΔG0 = 73.6 kJ/mol (17.6 kcal/mol)]. This constant is relatively insensitive to free magnesium concentrations but is affected by changes in temperature [ΔH0 = 46.9 kJ/mol (11.2 kcal/mol)]. The value of K for the serine:pyruvate aminotransferase reaction is 5.41 ± 0.11 [ΔG0 = ?4.37 kJ/mol (?1.04 kcal/mol)] at 38 °C (I = 0.25 M) and shows a small temperature effect [ΔH0 = 16.3 kJ/ mol (3.9 kcal/mol)]. The constant showed no significant effect of ionic strength (0.06–1.0 m) and a response to the hydrogen ion concentration only above pH 8.5. The value of Kobs is 5.50 ± 0.11 at pH 7.0 (38 °C, [K+] = 0.2 m, [Mg2+] = 0, I = 0.25 M). The results have also allowed the value of K for the d-glycerate kinase reaction (EC 2.7.1.31), d-Glycerate? + ATP4? → d-2-phosphoglycerate3? + ADP3? + H+, to be calculated to be 32.5 m (38 °C, I = 0.25 M). Values for Kobs for this reaction under these conditions and at pH 7.0 include 236 (free [Mg2+] = 0) and 50.8 (free [Mg2+] = 10?3m).  相似文献   

6.
The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability ΔGU(H2O) = 7.2 ± 0.25 kcal mol−1 is in good agreement with hydrogen exchange stability ΔGop. While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed.  相似文献   

7.
Several derivatives of hemocyanin from Taiwan snails (Achatina fulica) have been prepared. The reconstituted protein (R-HcO2) has lower Cu content, lower circular dichroism intensity, and higher fluorescence intensity than native oxyhemocyanin (HcO2). The Co(II) derivative (CoHc) does not take up molecular oxygen and only 50% of the total sites for Cu in native hemocyanin is taken up by Co. The half-apo derivative (half-apo-Hc) contains a single Cu per active site. Divalent cations quench the tryptophan fluorescence in the hemocyanin species and also quench the fluorescence from Tb3+ bound to the protein. The collisional quenching constants decrease in the order Co2+ > Mn2+ > Ca2+. The static component is negligible. For carboxy hemocyanin (HcCO), fluorescence originates from a Cu(I) CO complex and was used to study reaction of Hc CO with CN.  相似文献   

8.
The extra- and intramitochondrial phosphorylation potentials (ΔGp(out) and ΔGp(in), respectively) generated by respiring Ehrlich ascites tumor mitochondria were determined, using succinate, pyruvate + malate, ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine, and ascorbate + ferrocyanide as substrate systems. Values of ΔGp(out) exceeding 15 kcal mol?1 (62.8 kJ mol?1) in post-ADP state 4 respiration were found with succinate as substrate, in agreement with data on normal rat liver mitochondria. ΔGp(out) values exceeding 15 kcal mol?1 (62.8 kJ mol?1) were also observed with ascorbate + TMPD or ascorbate + ferrocyanide as substrates. Slightly lower values of ΔGp(out) were found with the NAD-linked substrates pyruvate + malate. The intramitochondrial ΔGp(in) developed by respiring Ehrlich ascites tumor mitochondria respiring on succinate approached 12 kcal mol?1 (50.2 kJ mol?1), in agreement with reported values on rat liver mitochondria. The prior accumulation of Ca2+ and phosphate by the Ehrlich cell mitochondria did not lower the extramitochondrial ΔGp(out) developed after a subsequent addition of ADP. Although the rate of oxidative phosphorylation of Ehrlich ascites tumor cells is reduced by intramitochondrial Ca2+ and phosphate (Villalobo and Lehninger (1980) J. Biol. Chem., 255, 2457–2464) they are still capable of generating ATP in the suspending medium against a high thermodynamic gradient, as expressed by the [ATP]/[ADP][Pi]mass action ratio.  相似文献   

9.
The interaction of bioactive protoberberine alkaloids berberine, palmatine, and coralyne with the DNA triplex poly(dT)⋅(poly(dA)⋅poly(dT)) was studied using biophysical and calorimetric techniques. All three alkaloids bound the triplex cooperatively. Berberine and palmatine predominantly stabilized the triplex structure, while coralyne stabilized both triplex and duplex structures as inferred from optical thermal melting profiles. Fluorescence quenching, polarization, and viscometric studies hinted at an intercalative mode of binding for the alkaloids to the triplex, coralyne being more strongly intercalated compared to partial intercalation of berberine and palmatine. The overall affinity of coralyne was two order higher (2.29×107 M −1) than that of berberine (3.43×105 M −1) and palmatine (2.34×105 M −1). Isothermal titration calorimetric studies revealed that the binding to the triplex was favored by negative enthalpy change (ΔH=−3.34 kcal/mol) with favorable entropy contribution (TΔS = 4.07 kcal/mol) for berberine, favored by almost equal negative enthalpy (ΔH =−3.88 kcal/mol) and entropy changes (TΔS = 3.37 kcal/mol) for palmatine, but driven by large enthalpy contributions (ΔH =−25.62 kcal/mol and TΔS =−15.21 kcal/mol) for coralyne. These results provide new insights on the binding of isoquinoline alkaloids to the DNA triplex structure.  相似文献   

10.
The oxidation enthalpy of reduced flavin mononucleotide at pH 7.0 in 0.2 m phosphate buffer has been studied by determining the heat associated with the reaction: FMNH2 + 2 Fe(CN)?36 ? FMN + 2 Fe(CN)?46 + 2 H+. (a) (The quinone, semiquinone, and hydroquinone forms of FMN are represented as FMN, FMNH, and FMNH2, respectively.) Calorimetric experiments were performed in a flow microcalorimeter which was modified to prevent sample contamination by oxygen. The enthalpy observed for reaction (a), after correction for dilution and buffer effects, was ?39.2 ± 0.4 kcal (mole FMNH2)?1 at 25 °C. The potential difference, ΔE′, developed by reaction (a) was determined potentiometrically and corresponded to a free energy change, ΔG′, of ?30.3 kcal (mole FMNH2)?1. The resulting entropy change, ΔS′, was thus calculated to be ?29.8 e.u. Reaction (a) was also studied at temperatures of 7 °C and 35.5 °C. ΔCp′ for the reaction was calculated as ?155 ± 18 cal deg?1 (mole FMNH2)?1 at 20 °C. ΔH′ for the reaction (b), FMNH2 ? FMN + H2, (b) was calculated as +14.2 ± 0.7 kcal mole?1 at 25 °C, relative to the enthalpy of the hydrogen electrode being identically equal to zero at all values of pH and temperature. The free energy at pH 7.0 for reaction (b), calculated from the potential was found to be ?9.7 kcal mole?1, which resulted in an entropy for reaction (b) of 80.2 e.u. A thermal titration of reaction (a) was used to calculate the thermodynamic parameters for the formation of semiquinone dimer according to the reaction FMNH2 + FMN ? (·FMNH)2. (c) The free energy, enthalpy, and entropy changes for reaction (c) were estimated to be ?6.1 kcal mole?1, ?7 kcal mole?1, and ?3 e.u., respectively.  相似文献   

11.
Results of experiments on the quenching of singlet delta oxygen (SDO) O2(a 1Δ g ) in lean hydrogen-oxygen mixtures at temperatures of 500–1020 K and pressures of 26–90 Torr behind the shock front are analyzed theoretically. The processes affecting SDO quenching are simulated taking into account the temporal characteristics of the experiment and various mechanisms of energy transformation in an O2(a 1Δ g )-H2-H-HO2 system. It is demonstrated that the approximations of both fast and slow (in comparison with the vibrational relaxation time of the HO2 radical) quenching of the electronically excited state of the radical are in good agreement with the experimental data on the effective rate constant of SDO deexcitation at temperatures of up to 700 K. It is shown that the available data on the kinetics of reactions involving SDO in H2-O2-O2(a 1Δ g ) mixtures overestimate the SDO quenching rate in comparison with the experimental results obtained at temperatures above 850 K. The decrease in the rate constant of the reaction H + O2(a 1Δ g )→ products by one order of magnitude makes it possible to match the simulation results to the experimental data. The existence of the processes restoring SDO in the presence of atomic hydrogen that are not considered in the current models of the H2-O2-O2(a 1Δ g ) kinetics is supposed.  相似文献   

12.
Trifluoroacetamide was found to be a good quencher of tryptophan fluorescence, and the quenching was shown to proceed via both a dynamic and a static process. The respective quenching constants were determined by the measurement of the decrease of the fluorescence lifetime in the presence of the quencher. The static and the bimolecular rate quenching constants of N-acetyltryptophanamide are equal to 0.34 1·mol?1 and 1.9·109 1·mol?1·s?1, respectively. These values indicate that trifluoroacetamide is an efficient quencher of tryptophan fluorescence. This conclusion is also supported by a complete quenching of bovine serum albumin and wheat germ agglutinin fluorescence. In the case of lysozyme, trifluoroacetamide quenches the fluorescence of tryptophan residues which fluoresce with a maximum at 348 nm but not the buried tryptophan residues which fluoresce with a maximum at 333 nm. Trifluoroacetamide quenching of wheat germ agglutinin emission confirms the homogeneity and the high accessibility of emitting tryptophan residues, in agreement with a previous report (Privat, J.P. and Monsigny, M. (1975) Eur. J. Biochem. 60, 555–567). The tryptophan fluorescence decay of wheat germ agglutinin is biexponential even in the presence of the quencher; the static and bimolecular rate quenching constants are equal to 0.22 1·mol?1 and 092·109 1·mol?1·?1, respectively. In the presence of a specific lectin ligand, the methyldi-N,N′-trifluoroacetyl-β- chitobioside, the quenching of wheat germ agglutinin fluorescence involves a direct contact between tryptophan residues and trifluoroacetamido groups of the ligand and in contrast with the quenching induced by free trifluoroacetamide shows that the tryptophan fluorescence is not fully quenched.  相似文献   

13.
The observed equilibrium constants (Kobs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg2+]. Using Σ and square brackets to indicate total concentrations Kobs = Σ L-serine][Σ Pi]Σ L-phosphoserine]H2O], K = L-H · serine±]HPO42?][L-H · phosphoserine2?]H2O]. The value of Kobs has been found to be relatively sensitive to pH. At 38 °C, K+] = 0.2 m and free [Mg2+] = 0; Kobs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔGobs0 = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H2O] = 1). The effect of the free [Mg2+] on Kobs was relatively slight; at pH 7.0 ([K+] = 0.2 m) Kobs = 52.0 m at free [Mg2+] = 10?3, m and 47.8 m at free [Mg2+] = 10?2, m. Kobs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔGobs0 = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H2O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 104, m [ΔGobs0 = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. Kobs = [Σ L-serine][Σ Pi][Σ L-phosphoserine][H2O], K = [L-H · serine±]HPO42?][L-H · phosphoserine2?]H2O. Values of Kobs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg2+], being calculated to be 668 [ΔGobs0 = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg2+] = 0; 111 [ΔGobs0 = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg2+] = 10?3, m; and 9.1 [ΔGobs0 = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg2+] = 10?2, m). Kobs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of Kobs are 4000 [ΔGobs0 = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg2+] = 0; and 97.4 [ΔGobs0 = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg2+] = 10?3, m. Combining Kobs for the l-phosphoserine phosphatase reaction with Kobs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of Kobs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. Kobs = [Σ L-serine][Σ NADH][Σ Pi][Σ α-ketoglutarate][Σ d-3-phosphoglycerate][Σ NAD+][Σ L-glutamat0] The value of Kobs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K+ as the monovalent cation) is 1.34 × 10?2, m at free [Mg2+] = 0 and 1.27 × 10?2, m at free [Mg2+] = 10?3, m.  相似文献   

14.
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   

15.
To understand the role of ATP underlying the enhanced amyloidosis of hen egg white lysozyme (HEWL), the synchrotron radiation circular dichroism, combined with tryptophan fluorescence, dynamic light-scattering, and differential scanning calorimetry, is used to examine the alterations of the conformation and thermal unfolding pathway of the HEWL in the presence of ATP, Mg2+-ATP, ADP, AMP, etc. It is revealed that the binding of ATP to HEWL through strong electrostatic interaction changes the secondary structures of HEWL and makes the exposed residue W62 move into hydrophobic environments. This alteration of W62 decreases the β-domain stability of HEWL, induces a noncooperative unfolding of the secondary structures, and produces a partially unfolded intermediate. This intermediate containing relatively rich α-helix and less β-sheet structures has a great tendency to aggregate. The results imply that the ease of aggregating of HEWL is related to the extent of denaturation of the amyloidogenic region, rather than the electrostatic neutralizing effect or monomeric β-sheet enriched intermediate.  相似文献   

16.
pKa1 values of 3-methoxy-N-desmethyldiazepam in acetonitrile and methanol containing various acid concentrations were determined by spectrophotometry to be 3.5 and 1.3, respectively. Temperature-dependent racemization of enantiomeric 3-methoxy-N-desmethyldiazepam in methanol containing 0.5 M H2SO4 was studied by circular dichroism spectropolorimetry and the racemization reactions were found to follow apparent first-order kinetics. Thermodynamic parameters of the racemization reaction were found to be: Eact = 18.8 kcal/mol, and at 25°C: ΔH? = 18.3 kcal/mol, ΔS? = ?14.8 entropy unit, and ΔG? = 22.7 kcal/mol, respectively. The racemization had an isotope effect (kH/kD) of 1.6 at 42°C. Based on the results of this report and those of earlier reports by other investigators, a nucleophilically solvated C3 carbocation intermediate resulting from either a P (plus) or an M (minus) conformation is proposed to be an intermediate and responsible for the stereoselective nucleophilic substitution and the subsequent racemization of 3-methoxy-N-desmethyldiazepam enantiomers. © 1993 Wiley-Liss, Inc.  相似文献   

17.
We present here a systematic investigation on the interaction between a water‐soluble alloyed semiconductor quantum dot and bovine serum albumin using various spectroscopic techniques i.e. fluorescence quenching, resonance light scattering and synchronous fluorescence spectroscopy. The analysis of fluorescence spectrum and fluorescence intensity indicates that the intrinsic fluorescence of bovine serum albumin (BSA) gets quenched by both static and dynamic quenching mechanism. The Stern‐Volmer quenching constants, energy transfer efficiency parameters, binding parameters and corresponding thermodynamic parameters (ΔH0, ΔS0 and ΔG0) have been evaluated by using van 't Hoff equation at different temperatures. A positive entropy change with a positive enthalpy change was observed suggesting that the binding process was an entropy‐driven, endothermic process associated with the hydrophobic effect. The intermolecular distance (r) between donor (BSA) and acceptor (CdSeS/ZnS quantum dots) was estimated according to Förster's theory of non‐radiative energy transfer. The synchronous fluorescence spectra revealed a blue shift in the emission maxima of tryptophan which is indicative of increasing hydrophobicity. Negative ΔG0 values implied that the binding process was spontaneous. It was found that hydrophobic forces played a role in the quenching process. Copyright © 2016 John Wiley & Sons, Ltd.  相似文献   

18.
The use of steady-state fluorescence quenching methods is reported as a probe of the accessibility of the single fluorescent tryptophan residue of bovine growth hormone (bGH, bovine somatotropin, bSt) in four solution-state conformations. Different bGH conformations were prepared by using previous knowledge of the multi-state nature of the equilibrium unfolding pathway for bGH: alterations in denaturant and protein concentration yielded different bGH conformations (native, monomeric intermediate, associated intermediate and unfolded). Because the intramolecular fluorescence quenching which occurs in the native state is reduced when the protein unfolds to any of the other conformations, steady-state fluorescence intensity measurements can be used to monitor bGH unfolding as well as the formation of the associated intermediate. These steady-state intensity changes have been confirmed with fluorescence lifetime measurements for the different conformational states of bGH. Fluorescence quenching results were obtained using the quenchers iodide (ionic), acrylamide (polar) and trichloroethanol (non-polar). Analysis of the results for native-state bGH reveals that the tryptophan environment is slightly non-polar (in agreement with the emission maximum of 335 nm) and the tryptophan is more exposed to acrylamide than most native-state tryptophan residues which have been studied. The tryptophan is most accessible to all quenchers in the unfolded state, because no steric restrictions inhibit quencher interaction with the tryptophan residue. The iodide quenching results indicate that the associated intermediate tryptophan is not accessible to iodide, probably due to negative charges inhibiting iodide penetration. The associated intermediate tryptophan is less accessible to all three quenchers than the monomeric intermediate tryptophan, due to tight packing of molecules in the associated intermediate state.  相似文献   

19.
Thermodynamics of base interaction in (A)n and (A.U)n   总被引:2,自引:0,他引:2  
Using precision scanning microcalorimetry we studied (A)n and (A·U)n melting in highly diluted solutions (0.3 to 5.0 mm) with different Na+ activity. This permitted us to determine directly the thermodynamic functions of stacking interaction in (A)n and base-pairing in (A·U)n. For (A-A) stacking at (A)n melting temperature we obtained ΔH(A)nm = 12.6 kJ mol?1; ΔS(A)nm = 41 J K?1 mol?1. For A·U base-pairing at a standard temperature of 298 K and 0.1 m-Na+ we have: ΔH(A·U) = 34 kJ mol?1; ΔS(A·U) = 102 J K?1 mol?1ΔG(A·U) = ?3.5 kJ mol?1.  相似文献   

20.
The interaction of dextromethorphan hydrobromide (DXM) with bovine serum albumin (BSA) is studied by using fluorescence spectra, UV–vis absorption, synchronous fluorescence spectra (SFS), 3D fluorescence spectra, Fourier transform infrared (FTIR) spectroscopy and circular dichroism under simulated physiological conditions. DXM effectively quenched the intrinsic fluorescence of BSA. Values of the binding constant, KA, are 7.159 × 103, 9.398 × 103 and 16.101 × 103 L/mol; the number of binding sites, n, and the corresponding thermodynamic parameters ΔG°, ΔH° and ΔS° between DXM and BSA were calculated at different temperatures. The interaction between DXM and BSA occurs through dynamic quenching and the effect of DXM on the conformation of BSA was analyzed using SFS. The average binding distance, r, between the donor (BSA) and acceptor (DXM) was determined based on Förster's theory. The results of fluorescence spectra, UV–vis absorption spectra and SFS show that the secondary structure of the protein has been changed in the presence of DXM. Copyright © 2015 John Wiley & Sons, Ltd.  相似文献   

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