Univalent ions in phospholipid model membranes: Thermodynamic and hydration aspects

By means of differential scanning calorimetry, effects of systematic series of Group I and VII ions on the phase state of model multibilayer dimyristoylphosphatidylcholine (di(14:0)PC) membranes have been studied at a lipid/ion molar ratio of 3/1. The sign-changing correlations between the ionic radii of cations and temperature shifts of di(14:0)PC phase transition were obtained. For cosmotropic Li⁺ and Na⁺, the observed shifts were positive (LiCl: ΔT _m = 0.6°C; ΔT _p = 1.9°C), whereas chaotropic K⁺ and Rb⁺ presence resulted in negative shifts (RbCl: ΔT _m = ?0.3°C; ΔT _p = ?2.5°C). The anions (Cl^?, Br^?, I^?) showed a similar effect increasing with the ion chaotropicity. An essentially weaker effect of Cs⁺ as compared to other alkali metal ions (CsCl: ΔT _m ≈ 0°C; ΔT _p = ?0.1°C) can be one of the reasons of its accumulation in living organisms. Generalization of all available data allowed us to specify some important factors of lipid-ion interactions that should be taken into account in further investigations in this field.     

Thermodynamic parameters of stabilization of the Yersinia pestis Caf113-149 subunit in compact form

Several experimental methods (circular dichroism, viscosity, intrinsic fluorescence, and fluorescence labeling) were used to study the conformational folding/unfolding transitions in a compact monomeric form of the Caf1_13-149 subunit under the action of guanidine hydrochloride in the temperature range 5–45°C. It has been shown that transitions always occur between two major states (unfolded and compact). This has made it possible to determine all the main thermodynamic functions that characterize the compact state of the Caf1_13-149 subunit: stability temperature T _m, free energy of stabilization ΔG _st, enthalpy ΔH _tr, and heat capacity jump ΔC in collapse of the structure. These data have been confirmed by an independent experiment on melting of fluorescently labeled protein.     

Calorimetric study of 2U:1A three-stranded complexes formed between poly(U) and adenine dinucleotides: ApA and diastereoisomers of nonionic adenine dideoxyribonucleoside methyl phosphonate

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/c_m, at 39°C and melting temperatures, T_m, 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, ΔH_m: 13.5, 12.7, and 12.8 kcal/mol (UUA base triplets) were found to be considerably lower than the van't Hoff enthalpies, ΔH_app: 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 ΔH_m and ΔH_app 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 ΔH_app/ΔH_m = 2.3.     

Enthalpic switch-points and temperature dependencies of DNA binding and nucleotide incorporation by Pol I DNA polymerases

This study examines the relationship between the DNA binding thermodynamics and the enzymatic activity of the Klenow and Klentaq Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Both polymerases bind DNA with nanomolar affinity at temperatures down to at least 5 °C, but have lower than 1% enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding (ΔH_binding) crosses zero (T_H) and becomes favorable (negative). This T_H/activity upshift temperature is 15 °C for Klenow and 30 °C for Klentaq. The results indicate that a negative free energy of DNA binding alone is not sufficient to proceed to catalysis, but that the enthalpic versus entropic balance of binding may be a modulator of the temperature dependence of enzymatic function. Analysis of the temperature dependence of the catalytic activity of Klentaq polymerase using expanded Eyring theory yields thermodynamic patterns for ΔG^‡, ΔH^‡, and TΔS^‡ that are highly analogous to those commonly observed for direct DNA binding. Eyring analysis also finds a significant ΔCp^‡ of formation of the activated complex, which in turn indicates that the temperature of maximal activity, after which incorporation rate slows with increasing temperature, will correspond with the temperature where the activation enthalpy (ΔH^‡) switches from positive to negative.     

Determination of melting temperature and temperature melting range for DNA with multi-peak differential melting curves

Many factors that change the temperature position and interval of the DNA helix–coil transition often also alter the shape of multi-peak differential melting curves (DMCs). For DNAs with a multi-peak DMC, there is no agreement on the most useful definition for the melting temperature, T_m, and temperature melting width, ΔT, of the entire DNA transition. Changes in T_m and ΔT can reflect unstable variation of the shape of the DMC as well as alterations in DNA thermal stability and heterogeneity. Here, experiments and computer modeling for DNA multi-peak DMCs varying under different factors allowed testing of several methods of defining T_m and ΔT. Indeed, some of the methods give unreasonable “jagged” T_m and ΔT dependences on varying relative concentration of DNA chemical modifications (r_b), [Na⁺], and GC content. At the same time, T_m determined as the helix–coil transition average temperature, and ΔT, which is proportional to the average absolute temperature deviation from this temperature, are suitable to characterize multi-peak DMCs. They give smoothly varying theoretical and experimental dependences of T_m and ΔT on r_b, [Na⁺], and GC content. For multi-peak DMCs, T_m value determined in this way is the closest to the thermodynamic melting temperature (the helix–coil transition enthalpy/entropy ratio).     

A thermodynamic description of the self-association of flavin mononucleotide.

Systematic heat of dilution studies of the self-association of flavin mononucleotide (FMN) have been conducted as a function of ionic strength (0.05 – 2.0 m) and pH (5–9) in aqueous solution. The data are adequately described by the expression $\text{Q}{}_{\mathrm{T}}\text{= ΔH ? (}\text{ΔH}\text{K}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{Q}{}_{\mathrm{T}}\text{c}{}_{\mathrm{T}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for an isodesmic self-association. Q_T is the molar heat of dilution, ΔH and K are the derived enthalpy and equilibrium constants for the process FMN + (FMN)_i?1 ? (FMN)_i, and c_T is the concentration of FMN expressed in monomer units. Typical values derived for the various thermodynamic parameters at 25 °C are ΔG = ?3.56 kcal mol^?1, ΔH = ?3.72 kcal mol^?1, and ΔS = ?0.54 cal (mol · deg)^?1. These data, plus nuclear magnetic resonance evidence (Yagi, K., Ohishi, N., Takai, A., Kawano, K., and Kyogoku, Y., 1976, Biochemistry15, 2877–2880) argue in favor of an open-ended association of flavin molecules. The signs of the various thermodynamic parameters suggest that both hydrophobic and surface energy forces contribute significantly to the association, while the lack of any significant ionic strength dependence indicates the lack of any ionic centers in the association.     

Effect of lamellarity and size on calorimetric phase transitions in single component phosphatidylcholine vesicles

Nano-differential scanning calorimetry (nano-DSC) is a powerful tool in the investigation of unilamellar (small unilamellar, SUVs, or large unilamellar, LUVs) vesicles, as well as lipids on supported bilayers, since it measures the main gel-to-liquid phase transition temperature (T_m), enthalpies and entropies. In order to assign these transitions in single component systems, where T_m often occurred as a doublet, nano-DSC, dynamic light scattering and cryo-transmission electron microscopy (cryo-TEM) data were compared. The two T_ms were not attributable to decoupled phase transitions between the two leaflets of the bilayer, i.e. nano-DSC measurements were not able to distinguish between the outer and inner leaflets of the vesicle bilayers. Instead, the two T_ms were attributed to mixtures of oligolamellar and unilamellar vesicles, as confirmed by cryo-TEM images. T_m for the oligolamellar vesicles was assigned to the peak closest to that of the parent multilamellar vesicle (MLV) peak. The other transition was higher than that of the parent MLVs for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and increased in temperature as the vesicle size decreased, while it was lower in temperature than that of the parent MLVs for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and decreased as the vesicle size decreased. These subtle shifts arose due to small differences in the values of ΔH and ΔS, since T_m is determined by their ratio (ΔH/ΔS). It was not possible to completely eliminate oligolamellar structures for MLVs extruded with the 200 nm pore size filter, even after 120 passes, while these structures were eliminated for MLVs extruded through the 50 nm pore size filter.     

On the pH-dependence of the light-induced hydrogen ion gradient in spinach chloroplasts

The dependence of the light-induced H⁺ gradient in chloroplasts (ΔpH) on external pH was examined using the distribution of aniline, an amine of low pK_a. ΔpH was essentially independent of pH over the range of 7–8. It was previously reported that ΔpH, determined from the distribution of relatively polar amines of high pK_a, decreased as the pH was lowered below 8. It is suggested that, in the case of amines of high pK_a, ΔpH values determined at low external pH values are too low because the permeability of chloroplasts to the amine cation relative to that of the unprotonated form may be significant.     

Nucleic acid-solvent interactions: temperature dependence of the heat of solution of thymine in water and ethanol

The heats of solution of thymine in water and ethanol have been determined calorimetrically as a function of temperature. These data, along with solubility data, have been used to calculate the thermodynamic quantities (ΔG_t, ΔH_t, ΔS_t and ΔC_p,t) associated with the transfer of thymine from ethanol to water. Since ΔS_t = ?2 cal/mole deg and ΔC_p,t = 0, it has been concluded that hydrophobic bonding does not play an important role in the thermocynamic stability of nucleic acids. However, large heat capacities of solution of thymine are observed in both solvents (ΔC°_p2 = 45 ± 4 cal/mole deg). This is explained in terms of temperature variation in the degree of solvent–solute hydrogen bonding. It is our proposal that the components of macromolecules (i.e., nucleic acid bases and amino acids) do not make all possible hydrogen bonds with the solvent in the vicinity of room temperature. Thus the thermodynamic contribution of hydrogen bonding to the stability of macromolecules in aqueous solution must be reassessed.     

Unfolding thermodynamics of the Delta-domain in the prohead I subunit of phage HK97: determination by factor analysis of Raman spectra

An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Δ-domain secondary structure and to quantify the thermodynamic parameters of Δ-domain unfolding. The results are compared for the Δ-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the α-helicity (∼ 70%), median melting temperature (T_m = 58 °C), enthalpy (ΔH_m = 50 ± 5 kcal mol^− 1), entropy (ΔS_m = 150 ± 10 cal mol^− 1 K^− 1), and average cooperative melting unit (〈n_c〉 ∼ 3.5) of the in situ Δ-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Δ-domain, despite an enhanced helical secondary structure (∼ 90% α-helix), exhibits diminished thermostability (T_m = 40 °C; ΔH_m = 27 ± 2 kcal mol^− 1; ΔS_m = 86 ± 6 cal mol^− 1 K^− 1) and noncooperative unfolding (〈n_c〉 ∼ 1) vis-à-vis the in situ Δ-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Δ-domains. The present results clarify the key role of the gp5 Δ-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.     

Thermodynamic descriptors,profiles and driving forces in membrane receptor-ligand interactions

Extension of the (isothermal) Gibbs–Helmholtz equation for the heat capacity terms (ΔC_p) allows formulating a temperature function of the free (Gibbs) energy change (ΔG). An approximation of the virtually unknown ΔC_p temperature function enables then to determine and numerically solve temperature functions of thermodynamic parameters ΔH and ΔS (enthalpy and entropy change, respectively). Analytical solutions and respective numeric procedures for several such approximation formulas are suggested in the presented paper. Agreement between results obtained by this analysis with direct microcalorimetric measurements of ΔH (and ΔC_p derived from them) was approved on selected cases of biochemical interactions presented in the literature. Analysis of several ligand-membrane receptor systems indicates that temperature profiles of ΔH and ΔS are parallel, largely not monotonic, and frequently attain both positive and negative values within the current temperature range of biochemical reactions. Their course is determined by the reaction change of heat capacity: temperature extremes (maximum or minimum) of both ΔH and ΔS occur at ΔC_p?=?0, for most of these systems at roughly 285–305 K. Thus, the driving forces of these interactions may change from enthalpy-, entropy-, or enthalpy-entropy-driven in a narrow temperature interval. In contrast, thermodynamic parameters of ligand-macromolecule interactions in solutions (not bound to a membrane) mostly display a monotonic course. In the case of membrane receptors, thermodynamic discrimination between pharmacologically defined groups—agonists, partial agonists, antagonists—is in general not specified and can be achieved, in the best, solely within single receptor groups.     

Influencing factors of dsDNA dye (high-resolution) melting curves and improved genotype call based on thermodynamic considerations

High-resolution melting of dsDNA using suitable dyes is a simple and cost-effective alternative for mutation scanning. Analytical variation can result from salt and template concentration (C_T). To overcome this problem the van’t Hoff transition enthalpy ΔH_vH from dsDNA melting curves was estimated and used for robust genotype calling or mutation scanning. Model calculations show the effect of salt, C_T, and temperature resolution on (1) T_m, (2) difference plots, (3) melting peaks, and (4) calculated ΔH_vH. Using the LightCycler480, the influence of dye (ResoLight) and scanning speed was assessed. The model calculations show that only ΔH_vH is not influenced by salt and C_T. Higher amplicon enthalpy ameliorates the ability to discriminate mutations. Temperature resolution is important for peak- but not for curve-based genotyping. ResoLight increases T_m by 3.4 °C, while lowering ΔH_vH. Using a 4-bp deletion in a 200-bp amplicon as a model, the miscalling rate improved substantially, when using ΔH_vH instead of difference plots. Melting curves of duplex DNA are influenced by dye and salt and less so by duplex concentrations. As predicted from theory, ΔH_vH is a robust measure for mutation detection in two-state melting. The influence of dyes on enthalpy is of general impact for PCR assays.     

Thermotropic and barotropic phase transitions of N-methylated dipalmitoylphosphatidylethanolamine bilayers

In order to understand the effect of polar head group modification on the thermotropic and barotropic phase behavior of phospholipid bilayer membranes, the phase transitions of dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidyl-N-methylethanolamine (DPMePE), dipalmitoylphosphatidyl-N,N-dimethylethanolamine (DPMe₂PE) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were observed by differential scanning calorimetry and high-pressure optical methods. The temperatures of the so-called main transition from the gel (L_β) or ripple gel (P_β′) phase to the liquid crystalline (L_α) phase were almost linearly elevated by applying pressure. The slope of the temperature-pressure boundary, dT/dp, was in the range of 0.220-0.264 K MPa⁻¹ depending on the number of methyl groups in the head group of lipids. The main-transition temperatures of N-methylated DPPEs decreased with increasing size of head group by stepwise N-methylation. On the other hand, there was no significant difference in thermodynamic quantities of the main transition between the phospholipids. With respect to the transition from the subgel (L_c) phase to the lamellar gel (L_β or L_β′) phase, the transition temperatures were also elevated by applying pressure. In the case of DPPE bilayer the L_c/L_β transition appeared at a pressure higher than 21.8 MPa. At a pressure below 21.8 MPa the L_c/L_α transition was observed at a temperature higher than the main-transition temperature. The main (L_β/L_α) transition can be recognized as the transformation between metastable phases in the range from ambient pressure to 21.8 MPa. Polymorphism in the gel phase is characteristic of DPPC bilayer membrane unlike other lipid bilayers used in this study: the L_β′, P_β′ and pressure-induced interdigitated gel (L_βI) phases were observed only in the DPPC bilayer. Regarding the bilayers of DPPE, DPMePE and DPMe₂PE, the interdigitation of acyl chain did not appear even at pressures as high as 200 MPa.     

Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method: I. Autoassociation of 2-aminopurine

A general equation was derived, describing fluorescence quantum yield and lifetime of an autoassociating compound in liquid solutions. The autoassociation of 2-aminopurine in aqueous solution was examined within the range from 0 to 90°C. The compound seemed to associate cooperatively. The thermodynamic parameters of polymerization change with temperature, so that its free enthalpy ΔG = ?0.0797 T² + 45.4 T ?7893. The dimerization enthalpy and entropy are approximately temperature-independent (ΔH₂ = ?4.17 $\text{kcal}\text{mol}$, ΔS₂ = ?10.9 e.u.), although the function: ΔG₂ = ?0.0308 T² + 30.3 T - 7213 fits experimental points better. The observed dependences can be explained by the increasing role of the hydrophobic effect with temperature and size of the aggregates. The association rate constants were determined, and a two-step reaction mechanism was demonstrated. The first step is diffusion-controlled. The second is characterized by an activation energy of ~2 $\text{kcal}\text{mol}$ and an encounter distance of ~8.3 Å.     

Ice nucleation and supercooling behavior of polymer aqueous solutions

We determined the homogeneous nucleation temperature depression, ΔT_f,hom, the equilibrium melting point depression, ΔT_m, and the value λ, which can be obtained from the linear relationship ΔT_f,hom = λΔT_m, for aqueous solutions of PEG (200-20,000 g mol⁻¹), PVP (10,000, 35,000, 40,000 g mol⁻¹), and dextran (10,000 g mol⁻¹) in the concentration range 0-40 wt% using the emulsion method. The molecular weight dependence of T_f,hom, T_m, and λ in PEG aqueous solutions was found to change in the vicinity of Mw 600-1540 at all concentrations. In addition, it was confirmed that for all of the polymers studied, there was a good linear relationship between λ and the logarithmic value of the self-diffusion coefficient D₀ of the solute molecule. These results indicate that the parameters that describe non-equilibrium freezing, such as T_f,hom and λ, are dependent on solution properties such as viscosity and self-diffusion of solute molecules.     

Calorimetric and spectroscopic studies of aminoglycoside binding to AT-rich DNA triple helices

Calorimetric and fluorescence techniques were used to characterize the binding of aminoglycosides-neomycin, paromomycin, and ribostamycin, with 5′-dA₁₂-x-dT₁₂-x-dT₁₂-3′ intramolecular DNA triplex (x = hexaethylene glycol) and poly(dA)·2poly(dT) triplex. Our results demonstrate the following features: (1) UV thermal analysis reveals that the T_m for triplex decreases with increasing pH value in the presence of neomycin, while the T_m for the duplex remains unchanged. (2) The binding affinity of neomycin decreases with increased pH, although there is an increase in observed binding enthalpy. (3) ITC studies conducted in two buffers (sodium cacodylate and MOPS) yield the number of protonated drug amino groups (Δn) as 0.29 and 0.40 for neomycin and paromomycin interaction with 5′-dA₁₂-x-dT₁₂-x-dT₁₂-3′, respectively. (4) The specific heat capacity change (ΔC_p) determined by ITC studies is negative, with more negative values at lower salt concentrations. From 100 mM to 250 mM KCl, the ΔC_p ranges from −402 to −60 cal/(mol K) for neomycin. At pH 5.5, a more positive ΔC_p is observed, with a value of −98 cal/(mol K) at 100 mM KCl. ΔC_p is not significantly affected by ionic strength. (5) Salt dependence studies reveal that there are at least three amino groups of neomycin participating in the electrostatic interactions with the triplex. (6) FID studies using thiazole orange were used to derive the AC₅₀ (aminoglycoside concentration needed to displace 50% of the dye from the triplex) values. Neomycin shows a seven fold higher affinity than paromomycin and eleven fold higher affinity than ribostamycin at pH 6.8. (7) Modeling studies, consistent with UV and ITC results, show the importance of an additional positive charge in triplex recognition by neomycin. The modeling and thermodynamic studies indicate that neomycin binding to the DNA triplex depends upon significant contributions from charge as well as shape complementarity of the drug to the DNA triplex Watson–Hoogsteen groove.     

The effect of calcium on the thermotropic properties of bovine blood coagulation factors IX and X and their activation intermediates and products

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 (T_M) of 54.5 ± 0.5 °C and a calorimetric enthalpy (ΔH_c) 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 T_m value is increased to 57.0 ± 0.5 °C and the ΔH_c 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 T_M of 51.0 ± 0.5 °C and a ΔH_c 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 T_M of 54.5 ± 0.5 °C (57.0 ± 0.5 °C) and a ΔH_c of 158 ±10 kcal/mol (156 ± 10 kcal/mol). Similarly, the terminal-activation product, Factor IXaβ, displays a T_M of 51.5 ± 0.5 °C (54.0 ± 0.5 °C) and a ΔH_c 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 T_M of 54.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c of 102 ± 10 kcal/mol (118 ± 10 kcal/mol), whereas its activated form, Factor Xaβ, possesses a T_M of 55.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c 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.     

Glycine betaine may have opposite effects on protein stability at high and low pH values

The compatible osmolyte glycine betaine (GB) is the most efficient osmoprotectant and best excluder from the protein surface. It can reverse protein aggregation and correct mutant protein defects and counter the harmful effects of urea and salts in vivo and in vitro. In this study we have investigated the pH dependence of the stabilizing effect of GB on three different proteins, namely, α-lactalbumin (α-LA), lysozyme and ribonuclease-A (RNase-A). We show here that (a) GB stabilizes RNase-A at all pH values, and (b) GB has opposite effects on two proteins at high pH and low pH values, namely, α-LA and lysozyme. This conclusion was reached by determining T_m (midpoint of denaturation), ΔH_m (denaturational enthalpy change at T_m), ΔC_p (constant-pressure heat capacity change) and ΔG_D^o (denaturational Gibbs energy change at 25 °C) of proteins in the presence of different GB concentrations. Another conclusion of this study is that ΔH_m and ΔC_p are not significantly changed in the presence of GB. This study suggests that other methylated glycine osmolytes may also behave in the same manner.     

Thermodynamic analysis of purified human placental alpha-L-fucosidase

The temperature dependence for the hydrolysis of both 4-methylumbelliferyl-α-l-fucoside and p-nitrophenyl-α-l-fucoside was determined for purified α-l-fucosidase (EC 3.2.1.51) from human placenta. The inhibition of the enzymatic reaction by l-fucose was also studied using the first of these two substrates at different temperatures. The thermodynamic parameters calculated from the pK_m were for the 4-methylumbelliferyl-conjugate ΔF = ?6.6 kcal/mol, ΔH = ?8.5 kcal/mol, and ΔS = ?6.3 e.u. and for the p-nitrophenylconjugate ΔF = ?5.6 kcal/mol, ΔH = ?12.2 kcal/mol, and ΔS = ?21.1 e.u. The thermodynamic parameters for l-fucose were ΔH = ?12.4 kcal/mol and ΔS = ?20.1 e.u. The lower exothermicity and negative entropy calculated for the 4-methylumbelliferyl substrate compared to the thermodynamic parameters calculated for the p-nitrophenyl substrate and l-fucose suggest the existence of a secondary hydrophobic binding site for the 4-methylumbelliferyl moiety on the enzyme. The difference in the enthalpy for both substrates is also reflected in a difference in activation energy, being 15.8 kcal/mol for the 4-methylumbelliferyl substrate and 20.7 kcal/mol for the p-nitrophenyl substrate. From these results it may be concluded that altered kinetic properties of the enzyme could be the result of the binding of the “aglycone” moiety of the fluorogenic substrate to the enzyme.