Enthalpy Compensation Research Articles

Study on the thermodynamic characteristics between fluoroquinolone and bovine serum albumin

The binding reactions of the fluoroquinolone with bovine serum albumin (BSA) were investigated by microcalorimetry. The thermodynamic parameters were measured with the help of spectroscopy in a Tris–HCl buffer solution (pH 7.0, made isotonic with sodium chloride) at T = 298 K. Microcalorimetric measurements show that the molar change of enthalpy Δ r H m is insignificant for the reaction, which may suggest that the interaction is governed mainly by entropy, and the interaction between the protein and the drugs is stronger. The results also reveal an entropy–enthalpy compensation relationship of the interaction.

Enthalpies of DNA melting in the presence of osmolytes

The melting of DNA in the presence of osmolytes has been studied with the intention of obtaining information about how base pair stability is affected by changes in solution conditions. In previous investigations, the melting enthalpies were assumed to be constant as osmolalities change, but no systematic evaluation of whether this condition is true has been offered. This paper presents calorimetric data on the melting of two synthetic DNA samples in the presence of a number of common osmolytes. Poly(dAdT)·poly(dTdA) and poly(dGdC)·poly(dCdG) melting have been examined by differential scanning calorimetry in solutions containing ethylene glycol, glycerol, sucrose, urea, betaine, PEG 200 and PEG 1450 at increasing osmolalities. The results show small, but significant changes in the enthalpy of melting of the two polynucleotides that are different, depending on the structure of the cosolvent. The polyols, ethylene glycol, glycerol, PEG 200 and also urea all show decreases in melting enthalpy, while betaine and sucrose display increases with increasing concentration of cosolvent. The large stabilizing PEG 1450 shows no change within the experimental errors. Using concepts relating to preferential interactions of the cosolvents with the DNA base pairs, it is possible to interpret some of the observed changes in the thermodynamic properties of melting. The results indicate that there is strong entropy–enthalpy compensation upon melting base pairs, but entropy increases dominate to cause the decreases in stability with increased cosolvent concentration. Excess hydration parameters are evaluated and their magnitudes discussed in terms of changes in cosolvent interactions with the DNA base pairs.

Microcalorimetry of interaction of dihydro-imidazo-phenanthridinium (DIP)-based compounds with duplex DNA

Isothermal titration (ITC) and differential scanning calorimetry (DSC) have been used to screen the binding thermodynamics of a family of DNA intercalators based on the dihydro-imidazo-phenanthridinium (DIP) framework. All members of this DIP-based ligand family bind to both genomic (calf thymus and/or salmon testes) and a synthetic dodecamer d(CGCGAATTCGCG) duplex DNA with broadly similar affinities regardless of side chain size or functionality. Viscosity measurements confirm that binding satisfies standard criteria for intercalation. Binding is exothermic but with an additional favourable positive entropy contribution in most cases at 25 °C, although a significant negative heat capacity effect (Δ C p) means that both Δ H° and Δ S° decrease with increasing temperature. DIP-ligand binding to DNA also shows significant entropy–enthalpy compensation effects that are now almost standard in such situations, probably reflecting the conformational flexibility of macromolecular systems involving a multiplicity of weak non-covalent interactions. This ability to vary side chain functionality without compromising DNA binding suggests that the DIP framework should be a promising basis for more adventurous chemistry at the DNA level.

Conformational Stabilization of an Engineered Binding Protein

We analyzed the thermodynamic basis for improvement of a binding protein by disulfide engineering. The Z(SPA)(-)(1) affibody binds to its Z domain binding partner with a dissociation constant K(d) = 1.6 microM, and previous analyses suggested that the moderate affinity is due to the conformational heterogeneity of free Z(SPA)(-)(1) rather than to a suboptimal binding interface. Studies of five stabilized Z(SPA)(-)(1) double cystein mutants show that it is possible to improve the affinity by an order of magnitude to K(d) = 130 nM, which is close to the range (20 to 70 nM) observed with natural Z domain binders, without altering the protein-protein interface obtained by phage display. Analysis of the binding thermodynamics reveals a balance between conformational entropy and desolvation entropy: the expected and favorable reduction of conformational entropy in the best-binding Z(SPA)(-)(1) mutant is completely compensated by an unfavorable loss of desolvation entropy. This is consistent with a restriction of possible conformations in the disulfide-containing mutant and a reduction of average water-exposed nonpolar surface area in the free state, resulting in a smaller conformational entropy penalty, but also a smaller change in surface area, for binding of mutant compared to wild-type Z(SPA)(-)(1). Instead, higher Z domain binding affinity in a group of eight Z(SPA)(-)(1) variants correlates with more favorable binding enthalpy and enthalpy-entropy compensation. These results suggest that protein-protein binding affinity can be improved by stabilizing conformations in which enthalpic effects can be fully explored.

Thermodynamics of monochlorophenol isomers and pyrite interfacial interactions in the activation state

Thermodynamic parameters of the activation state for phenol and three monochlorophenol (MCP) isomer–pyrite complexes, i.e., MCP isomers used were 2-chlorophenol (2-CP), 3-chlorophenol (3-CP), 4-chlorophenol (4-CP), have been derived from the temperature-dependent kinetic data. Both the initial rate and adsorption density values increased in the order phenol < 2-CP < 3-CP < 4-CP. This suggests that the presence of chlorine substituent on the aromatic ring results in enhanced CP adsorption on pyrite. The activation energy ( E a ) , Gibbs free energy ( Δ G # ) , entropy ( Δ S # ) , and enthalpy ( Δ H # ) of the activation stage for MCP adsorption on pyrite were calculated by Arrhenius and Eyring models. Always Δ S # values approximate to zero and − T Δ S # values are positive, which indicates that the activation state of MCP adsorption process is entropy-controlled, and the observed linear dependence of Δ H # on − T Δ S # signals an entropy–enthalpy compensation effect of the MCP adsorption process. The Γ MCP data were quantified well both by 1 − p K diffused double layer ( 1 − p K DLM) and Langmuir models.

Interactions of an acidic cyclooctapeptide with metal ions: microcalorimetric and fluorescence analyses

Abstract: The metal‐ion binding preferences of an acidic amphipathic cyclopeptide, cyclo[d‐Leu‐Leu‐d‐Leu‐Trp‐(d‐Glu‐Glu)2] (CP), was studied by isothermal titration calorimetry and steady‐state fluorescence spectroscopy. CP adopted a partial beta structure, and variable temperature circular dichroism showed small secondary structural changes over the temperature range from 5 °C to 95 °C. The peptide did not bind alkali or alkaline earth metal ions but exhibited selectivity for some divalent transition metal ions (with association constants KCu2+ 4.5 × 104 m−1, KZn2+ 1.6 × 105 m−1, KCd2+ 1.3 × 104 m−1, KHg2+ (1) 2.2 × 106 m−1, and KHg2+ (2) 6.5 × 103 m−1), for Pb2+ (2.0 × 105 m−1), and a trivalent Group III metal, Al3+ (1.6 × 105 m−1). The thermodynamic data show that the interaction between CP and these metal ions are spontaneous and entropically driven. A large range of binding enthalpies coupled with a smaller range of binding free energies of CP for these metal ions indicate an entropy–enthalpy compensation dependent on the ionic size of participating metal ions. The interaction of Pb2+, Hg2+, and Cu2+ with CP in aqueous solution specifically modulates the fluorescence emission properties of CP. The results of this study show that CP exhibits selectivity in metal‐ion binding, which is reflected in its fluorescence spectra. The observed trends can be useful for the design of heavy‐metal sensors based on fluorophore‐tagged acidic cyclopeptides.

Solvation Properties of N-Substituted Cis and Trans Amides Are Not Identical: Significant Enthalpy and Entropy Changes Are Revealed by the Use of Variable Temperature 1H NMR in Aqueous and Chloroform Solutions and ab Initio Calculations

The cis/trans conformational equilibrium of N-methyl formamide (NMF) and the sterically hindered tert-butylformamide (TBF) was investigated by the use of variable temperature gradient 1H NMR in aqueous solution and in the low dielectric constant and solvation ability solvent CDCl3 and various levels of first principles calculations. The trans isomer of NMF in aqueous solution is enthalpically favored relative to the cis (deltaH(o) = -5.79 +/- 0.18 kJ mol(-1)) with entropy differences at 298 K (298 x deltaS(o) = -0.23 +/- 0.17 kJ mol(-1)) playing a minor role. The experimental value of the enthalpy difference strongly decreases (deltaH(o) = -1.72 +/- 0.06 kJ mol(-1)), and the contribution of entropy at 298 K (298 x deltaS(o) = -1.87 +/- 0.06 kJ mol(-1)) increases in the case of the sterically hindered tert-butylformamide. The trans isomer of NMF in CDCl3 solution is enthalpically favored relative to the cis (deltaH(o) = -3.71 +/- 0.17 kJ mol(-1)) with entropy differences at 298 K (298 x deltaS(o) = 1.02 +/- 0.19 kJ mol(-1)) playing a minor role. In the sterically hindered tert-butylformamide, the trans isomer is enthalpically disfavored (deltaH(o) = 1.60 +/- 0.09 kJ mol(-1)) but is entropically favored (298 x deltaS(o) = 1.71 +/- 0.10 kJ mol(-1)). The results are compared with literature data of model peptides. It is concluded that, in amide bonds at 298 K and in the absence of strongly stabilizing sequence-specific inter-residue interactions involving side chains, the free energy difference of the cis/trans isomers and both the enthalpy and entropy contributions are strongly dependent on the N-alkyl substitution and the solvent. The significant decreasing enthalpic benefit of the trans isomer in CDCl3 compared to that in H2O, in the case of NMF and TBF, is partially offset by an adverse entropy contribution. This is in agreement with the general phenomenon of enthalpy versus entropy compensation. B3LY/6-311++G** and MP2/6-311++G** quantum chemical calculations confirm the stability orders of isomers and the deltaG decrease in going from water to CHCl3 as solvent. However, the absolute calculated values, especially for TBF, deviate significantly from the experimental values. Consideration of the solvent effects via the PCM approach on NMF x H2O and TBF x H2O supermolecules improves the agreement with the experimental results for TBF isomers, but not for NMF.

Heat capacity-independent determination of differential free energy of stability between structurally homologous proteins

Under the assumption of equivalent heat capacity values, the differential free energy of stability for a pair of proteins midway between their thermal unfolding transition temperatures is shown to be independent of Δ C p up to its cubic term in Δ T m . For model calculations reflecting the nearly 30 °C difference in T m for the adenylate kinases from the arctic bacterium Bacillus globisporus and the thermophilic bacterium Geobacillus stearothermophilus, the resultant error in estimating ΔΔ G by the formula 0.5 [Δ S T m1 (1) + Δ S T m2 (2)] Δ T m is less than 1%. Combined with the analogous thermal unfolding data for the adenylate kinase from Escherichia coli, these three homologous proteins exhibit T m and Δ S T m values consistent with differential entropy and enthalpy contributions of equal magnitude. When entropy–enthalpy compensation holds for the differential free energy of stability, the incremental changes in T m values are shown to be proportionate to the changes in free energy.

Studies on interaction between gatifloxacin and human serum albumin as well as effect of copper(II) on the reaction

The binding characteristics of gatifloxacin (GTFX) and human serum albumin (HSA) have been studied by fluorescence spectroscopy in aqueous solution, and the interaction influenced by copper(II) was also explored in the paper. The results show that the two-reaction equilibrium constant and the number of binding sites were K = 1.16 × 10 5 l mol −1, n = 1.27 for GTFX and K = 1.62 × 10 5 l mol −1, n = 1.74 for GTFX–Cu 2+, respectively. The quenching mechanism of fluorescence of HSA by GTFX is a static quenching procedure. The binding distance between GTFX and HSA and the energy transfer efficiency are obtained based on the theory of Fōrster spectroscopy energy transfer. The effect of GTFX on the conformation of HSA was also been analyzed by using synchronous fluorescence spectroscopy. The interaction of GTFX and HSA has been studied by flow-mixed microcalorimetry in the absence and presence of copper(II) and their thermodynamic parameters were obtained. The enthalpy changes and the entropy changes were calculated to be Δ H ≈ 0, Δ S > 0 in the absence of copper(II),which indicated that static forces played major role in the interaction of GTFX and HSA, and to be Δ H ≈ 0, Δ S > 0 in the presence of copper(II),which indicated that the static forces also played major role on the reaction. The molar free energy changes of the two reactions are identical with each other because the entropy–enthalpy compensation happened between the two reactions.

Solvation and the hidden thermodynamics of a zinc finger probed by nonstandard repair of a protein crevice

The classical Zn finger contains a phenylalanine at the crux of its three architectural elements: a beta-hairpin, an alpha-helix, and a Zn(2+)-binding site. Surprisingly, phenylalanine is not required for high-affinity Zn2+ binding, but instead contributes to the specification of a precise DNA-binding surface. Substitution of phenylalanine by leucine leads to a floppy but native-like structure whose Zn affinity is maintained by marked entropy-enthalpy compensation (DeltaDeltaH -8.3 kcal/mol and -TDeltaDeltaS 7.7 kcal/mol). Phenylalanine and leucine differ in shape, size, and aromaticity. To distinguish which features correlate with dynamic stability, we have investigated a nonstandard finger containing cyclohexanylalanine at this site. The structure of the nonstandard finger is similar to that of the native domain. The cyclohexanyl ring assumes a chair conformation, and conformational fluctuations characteristic of the leucine variant are damped. Although the nonstandard finger exhibits a lower affinity for Zn2+ than does the native domain (DeltaDeltaG -1.2 kcal/mol), leucine-associated perturbations in enthalpy and entropy are almost completely attenuated (DeltaDeltaH -0.7 kcal/mol and -TDeltaDeltaS -0.5 kcal/mol). Strikingly, global changes in entropy (as inferred from calorimetry) are in each case opposite in sign from changes in configurational entropy (as inferred from NMR). This seeming paradox suggests that enthalpy-entropy compensation is dominated by solvent reorganization rather than nominal molecular properties. Together, these results demonstrate that dynamic and thermodynamic perturbations correlate with formation or repair of a solvated packing defect rather than type of physical interaction (aromatic or aliphatic) within the core.

Mechanism for entropy–enthalpy compensation in the fusion of organic molecules

Contrary to Walden's Rule [P. Walden, Z. Elektrochem. 14 (1908) 713–728], the entropies of fusion of rigid organic molecules, Δ S f, are linearly related (entropy–enthalpy compensation) to the enthalpies of fusion, Δ H f, by an expression of the form Δ S f = a + bΔ H f where a and b are constants [A.S. Gilbert, Thermochim. Acta 339 (1999) 131–142]. The compounds that show phase transitions in the solid state yield a value of the intercept a close to that expected (about 8 J K −1 mol −1) for the communal entropy of the liquid state. However, most compounds do not possess solid state transitions and for these the value of a is found to be much higher at around 30 J K −1 mol −1. A simple statistical mechanical treatment has been applied to the way the external vibrational modes (translational and librational) might change on fusion and gives some success in describing the features of the compensation observed, in particular the values of the intercepts. This involves consideration of the enthalpy of vapourisation of the liquids, Δ H v, as well as Δ H f. It appears that the ratio between these two parameters, Δ H f/Δ H v is a major determining factor so that compensation occurs more properly with this ratio than with Δ H f alone. The entropy and enthalpy changes for transitions in the solid state themselves also appear to be compensated in the same way as for fusion.

Why Zinc Fingers Prefer Zinc: Ligand-Field Symmetry and the Hidden Thermodynamics of Metal Ion Selectivity,

The zinc finger, a motif of protein-nucleic acid recognition broadly conserved among eukaryotes, is a globular minidomain containing a tetrahedral metal-binding site. Preferential coordination of Zn(2+) (relative to Co(2+)) is proposed to reflect differences in ligand-field stabilization energies (LFSEs) due to complete or incomplete occupancy of d orbitals. LFSE predicts that the preference for Zn(2+) should be purely enthalpic in accord with calorimetric studies of a high-affinity consensus peptide (CP-1; Blasie, C. A., and Berg, J. (2002) Biochemistry 41, 15068-73). Despite its elegance, the general predominance of LFSE is unclear as (i) the magnitude by which CP-1 prefers Zn(2+) is greater than that expected and (ii) the analogous metal ion selectivity of a zinc metalloenzyme (carbonic anhydrase) is driven by changes in entropy rather than enthalpy. Because CP-1 was designed to optimize zinc binding, we have investigated the NMR structure and metal ion selectivity of a natural finger of lower stability derived from human tumor-suppressor protein WT1. Raman spectroscopy suggests that the structure of the WT1 domain is unaffected by interchange of Zn(2+) and Co(2+). As in CP-1, preferential binding of Zn(2+) (relative to Co(2+)) is driven predominantly by differences in enthalpy, but in this case the enthalpic advantage is less than that predicted by LFSE. A theoretical framework is presented to define the relationship between LFSE and other thermodynamic factors, such as metal ion electroaffinities, enthalpies of hydration, and the topography of the underlying folding landscape. The contribution of environmental coupling to entropy-enthalpy compensation is delineated in a formal thermodynamic cycle. Together, these considerations indicate that LFSE provides an important but incomplete description of the stringency and thermodynamic origin of metal-ion selectivity.

Thermodynamics of the interaction of xanthine oxidase with superoxide dismutase studied by isothermal titration calorimetry and fluorescence spectroscopy

Xanthine oxidase (XO) and copper, zinc superoxide dismutase (Cu, Zn-SOD) are function-related proteins in vivo. Thermodynamics of the interaction of bovine milk XO with bovine erythrocyte Cu, Zn-SOD has been studied using isothermal titration calorimetry (ITC) and fluorescence spectroscopy. The binding of XO to Cu, Zn-SOD is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong entropy–enthalpy compensation and weak temperature-dependence of Gibbs free energy change. An unexpected, large positive molar heat capacity change of the binding, 3.02 kJ mol −1 K −1, at all temperatures examined suggests that either hydrogen bond or long-range electrostatic interaction is a major force for the binding. XO quenches the intrinsic fluorescence of Cu, Zn-SOD and causes a small red shift in the fluorescence emission maximum of the protein. A small salt concentration dependence of the binding affinity measured by fluorescence spectroscopy and a large unfavorable change in entropy for the binding measured by ITC suggest that long-range electrostatic forces do not play an important role in the binding. These results indicate that XO binds to Cu, Zn-SOD with high affinity and that hydrogen bond is a major force for the binding.

Electrostatically optimized Ras-binding Ral guanine dissociation stimulator mutants increase the rate of association by stabilizing the encounter complex.

Association of two proteins can be described as a two-step process, with the formation of an encounter complex followed by desolvation and establishment of a tight complex. Here, by using the computer algorithm PARE, we designed a set of mutants of the Ras effector protein Ral guanine nucleotide dissociation stimulator (RalGDS) with optimized electrostatic steering. The fastest binding RalGDS mutant, M26K,D47K,E54K, binds Ras 14-fold faster and 25-fold tighter compared with WT. A linear correlation was found between the calculated and experimental data, with a correlation coefficient of 0.97 and a slope of 0.65 for the 24 mutants produced. The data suggest that increased electrostatic steering specifically stabilizes the encounter complex and transition state. This conclusion is backed up by Phi analysis of the encounter complex and transition state of the RalGDS(M26K,D47K,E54K)/Ras complex, with both values being close to 1. Upon further formation of the final complex, the increased Coulombic interactions are probably counterbalanced by the cost of desolvation of charges, keeping the dissociation rate constant almost unchanged. This mechanism is also reflected by the mutual compensation of enthalpy and entropy changes quantified by isothermal titration calorimetry. The binding constants of the faster binding RalGDS mutants toward Ras are similar to those of Raf, the most prominent Ras effector, suggesting that the design methodology may be used to switch between signal transduction pathways.

Staging Is More Important than Perturbation Method for Computation of Enthalpy and Entropy Changes in Complex Systems

Entropy and enthalpy compensation are frequently observed in thermodynamic measurements. To understand the molecular details of these compensating changes, molecular dynamics or Monte Carlo methods may, in principle, be used. Yet, in practice, computational methods to evaluate changes in entropy and enthalpy usually have much greater (e.g. order of magnitude worse) error than relative free energy calculations. In this paper, we examine ways to improve the computational ability to determine changes in both enthalpy and entropy. Toward that end we consider five different perturbation schemes for enthalpy determination and also consider the relative importance of staging the computation (i.e., dividing the entire perturbation calculation into a series of consecutive smaller ones) along the reaction coordinate. Two model systems are used for evaluation: a system of Lennard-Jones spheres, where the computation is directed to the addition/deletion of a sphere, and the alchemical transformation of an anion in e...

The Thermodynamic Basis for Enantiodiscrimination: Gas-Phase Measurement of the Enthalpy and Entropy of Chiral Amine Recognition by Dimethyldiketopyridino-18-crown-6

Discrimination between the enantiomers of 1-phenylethylamine (PhEt) and α(1-naphthyl)ethylamine (NapEt) by the chiral ligand protonated dimethyldiketopyridino-18-crown-6 was studied using Fourier transform ion cyclotron resonance mass spectrometry to perform variable-temperature equilibrium (van't Hoff) experiments in the gas phase. The heterochiral complexes [(S,S)-ligand with (R)-amine, for example] have more-favorable enthalpy in both studied cases than the homochiral complexes; the differences are −6.7 ± 0.7 kJ mol-1 for the PhEt enantiomers and −10.0 ± 1.2 kJ mol-1 for the NapEt enantiomers. Entropy disfavors the heterochiral complexes by −14.8 ± 2.2 J mol-1 K-1 for PhEt and by −20.0 ± 3.9 J mol-1 K-1 for NapEt; entropy−enthalpy compensation is evident. These results suggest that enantiodiscrimination in these complexes is enthalpic and that locking of methyl rotors in the thermodynamically disfavored complexes is probably not important. Computational methods were also used to determine complex geome...

Thermodynamics of Transfer of Some Alkyl Acetates from Water to Water–Ethanol Mixtures

The solubility of methyl acetate (MeOAc), ethyl acetate (EtOAc), 1-propyl acetate(1-PrOAc), 1-butyl acetate (1-BuOAc), 2-methyl-1-propyl acetate (iso-BuOAc), 2-butyl acetate (sec-BuOAc), 2-methyl-2-propyl acetate (tert-BuOAc), 1-pentyl acetate (1-PeOAc), and 1-hexyl acetate(1-HeOAc) in 2.5, 5.0, 7.5, and 10.0 weight per cent of ethanol in water were determined at 298.2 K. The solubility of the same compounds, except for tert-BuOAc, 1-PeOAc, and 1-HeOAc, was determined as a function of temperature at 10.0 weight per cent of ethanol in water. From the solubility measurements the standard Gibbs energy (ΔG t 0 ), enthalpy (ΔH t 0 ), and entropy (ΔS t 0 ) of transfer were determined. The calculated thermodynamic functions show that the predominant factors in the transfer of alkyl acetate molecules are the transfer of the cavity and the hydrophobic interaction of the non-polar alkyl chain. Scaled particle theory calculations were used to determine the thermodynamics of cavity transfer, which were combined with the experimental total transfer quantities to obtain the corresponding interaction transfer quantities. It was found that the Gibbs energy of interaction for the transfer is negative, whereas the enthalpy and entropy of interaction for the transfer are positive; almost complete compensation of enthalpy and entropy components occurs.

Heat does not come in different colours: entropy–enthalpy compensation, free energy windows, quantum confinement, pressure perturbation calorimetry, solvation and the multiple causes of heat capacity effects in biomolecular interactions

Modern techniques in microcalorimetry allow us to measure directly the heat changes and associated thermodynamics for biomolecular processes in aqueous solution at reasonable concentrations. All these processes involve changes in solvation/hydration, and it is natural to assume that the heats for these processes should reflect, in some way, such changes in solvation. However, the interpretation of data is still somewhat ambiguous, since different non-covalent interactions may have similar thermodynamic signatures, and analysis is frustrated by large entropy–enthalpy compensation effects. Changes in heat capacity (Δ C p) have been related to changes in hydrophobic hydration and non-polar accessible surface areas, but more recent empirical and theoretical work has shown how this need not always be the case. Entropy–enthalpy compensation is a natural consequence of finite Δ C p values and, more generally, can arise as a result of quantum confinement effects, multiple weak interactions, and limited free energy windows, giving rise to thermodynamic homeostasis that may be of evolutionary and functional advantage. The new technique of pressure perturbation calorimetry (PPC) has enormous potential here as a means of probing solvation-related volumetric changes in biomolecules at modest pressures, as illustrated with preliminary data for a simple protein-inhibitor complex.

Effects of environment on flavin reactivity in morphinone reductase: analysis of enzymes displaying differential charge near the N-1 atom and C-2 carbonyl region of the active-site flavin

The side chain of residue Arg238 in morphinone reductase (MR) is located close to the N-1/C-2 carbonyl region of the flavin isoalloxazine ring. During enzyme reduction negative charge develops in this region of the flavin. The positioning of a positively charged side chain in the N-1/C-2 carbonyl region of protein-bound flavin is common to many flavoprotein enzymes. To assess the contribution made by Arg238 in stabilizing the reduced flavin in MR we isolated three mutant forms of the enzyme in which the position of the positively charged side chain was retracted from the N-1/C-2 carbonyl region (Arg238 → Lys), the positive charge was removed (Arg238 → Met) or the charge was reversed (Arg238 → Glu). Each mutant enzyme retains flavin in its active site. Potentiometric studies of the flavin in the wild-type and mutant forms of MR indicate that the flavin semiquinone is not populated to any appreciable extent. Reduction of the flavin in each enzyme is best described by a single Nernst function, and the values of the midpoint reduction potentials (E12) for each enzyme fall within the region of −247±10mV. Stopped-flow studies of NADH binding to wild-type and mutant MR enzymes reveal differences in the kinetics of formation and decay of an enzyme–NADH charge-transfer complex, reflecting small perturbations in active-site geometry. Reduced rates of hydride transfer in the mutant enzymes are attributed to altered geometrical alignment of the nicotinamide coenzyme with FMN rather than major perturbations in reduction potential, and this is supported by an observed entropy–enthalpy compensation effect on the hydride transfer reaction throughout the series of enzymes. The data indicate, in contrast with dogma, that the presence of a positively charged side chain close to the N-1/C-2 carbonyl region of the flavin in MR is not required to stabilize the reduced flavin. This finding may have general implications for flavoenzyme catalysis, since it has generally been assumed that positive charge in this region has a stabilizing effect on the reduced form of flavin.

Retention properties of the fluorinated bonded phase on liquid chromatography of aromatic hydrocarbons

The unique characteristics of a bonded-fluoroalkylsilane column, Fluofix, are described by comparison with those of a bonded octylsilane column, C 8, in the reversed-phase chromatography of aromatic hydrocarbons. The selectivity of homologous aromatics on the fluorinated column varied with the methanol concentration in the eluent. At a larger proportion of methanol than 80:20 (v/v) the larger aromatics eluted faster than the smaller ones. However, with less methanol, the aromatics were eluted in order of their molecular size, as usually reported for a conventional hydrocarbonaceous column. A dependence of the retention mechanism on the composition of the eluent was suggested by the decrease in the entropy–enthalpy compensation temperature, β, with increase of methanol concentration in the eluent. In order to explain the concentration dependence of the retention, the molecular interaction energy in the retention process was calculated by computer simulation. The interaction energy between aromatics and the stationary ligand on the Fluofix column was smaller than that on the C 8 column and comparable to the interaction energy between the aromatics and methanol. At higher methanol concentrations, solute–fluorinated ligand complex formation was obstructed by the methanol molecules solvating the solute, reducing the retention of the larger aromatics.