Biophysical chemistry

Publications in this journal

• Article: Effect of the interfacial tension and ionic strength on the thermodynamic barrier associated to the benzocaine insertion into a cell membrane.

  J J López Cascales, S D Oliveira Costa
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  ABSTRACT: The insertion of local anaesthetics into a cell membrane is a key aspect for explaining their activity at a molecular level. It has been described how the potency and response time of local anaesthetics is improved (for clinical applications) when they are dissolved in a solution of sodium bicarbonate. With the aim of gaining insight into the physico-chemical principles that govern the action mechanism of these drugs at a molecular level, simulations of benzocaine in binary lipid bilayers formed by DPPC/DPPS were carried out for different ionic strengths of the aqueous solution. From these molecular dynamic simulations, we observed how the thermodynamic barrier associated with benzocaine insertion into the lipid bilayers diminished exponentially as the fraction of DPPS in the bilayer increased, especially when the ionic strength of the aqueous solution increased. In line with these results, we also observed how this thermodynamic barrier diminished exponentially with the phospholipid/water interfacial tension.
  Biophysical chemistry 12/2013; 172C:1-7.
• Article: Characterization of the nucleation process of lysozyme at physiological pH: Primary but not sole process.

  Giovanna Navarra, Filippo Troia, Valeria Militello, Maurizio Leone
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  ABSTRACT: We report on a kinetic study of the heat-induced aggregation process of lysozyme at physiological pH. The time evolution of the aggregation extent and the conformational changes of the protein were followed by dynamic light scattering (DLS) and FTIR spectroscopy, respectively, whereas the morphology of the aggregates was observed by Atomic Force Microscopy (AFM). The conformational changes of the secondary and tertiary structures were simultaneous and distinct in time with respect to the formation of aggregates. Oligomer formation occurred through at least two different aggregation processes: a nucleation process and a homogeneous non-nucleative diffusion-controlled process. FTIR measurements showed that supramolecular aggregation proceeded without the formation of β-aggregated structures and AFM images revealed the presence of oligomers and amorphous aggregates; no fibrillar structures were observed.
  Biophysical chemistry 04/2013; 177-178C:24-33.
• Article: Binding of new cationic porphyrin-tetrapeptide conjugates to nucleoprotein complexes.

  Adám Orosz, Gábor Mező, Levente Herényi, Jan Habdas, Zsuzsa Majer, Beata Myśliwa-Kurdziel, Katalin Tóth, Gabriella Csík
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  ABSTRACT: Ongoing research on DNA binding of cationic porphyrin derivatives and their conjugates is a subject of growing interest because of their possible DNA binding and demonstrated biological properties. In this study nucleoprotein binding of tri-cationic meso-tri(4-N-methylpyridyl)-mono-(4-carboxyphenyl)porphyrin (TMPCP) and tetrapeptides conjugated TMPCP (TMPCP-4P) and bi-cationic meso-5,10-bis(4-N-methylpyridyl)-15,20-di-(4-carboxyphenyl)porphyrin (BMPCP-4P2) was investigated with comprehensive spectroscopic methods. The key observation is that tetrapeptide-conjugates of cationic porphyrins with two or three positive charges bind to encapsidated DNA in T7 phage nucleoprotein complex. The binding modes were analyzed by fluorescent energy transfer, fluorescent life time and CD measurements. Intercalative binding is most feasible when tricationic ligands complex with DNA, especially when it is in close connection with protein capsid. It was found that larger ligand BMPCP-4P2 binds externally to encapsidated T7 DNA, and complex externally as well as by intercalation when the DNA accommodate to relaxed B-conformation. In the case of TMPCP and TMPCP-4P the intercalation is the predominant binding form both in nucleoprotein (NP) and preheated complexes. Further, melting experiments revealed that bound porphyrins do not influence the capsid stability or protein-DNA interactions, but efficiently stabilize the double helical structure of DNA without respect to binding form. A good correlation was found between porphyrin/base pair ration and DNA strand separation temperature.
  Biophysical chemistry 04/2013; 177-178C:14-23.
• Article: Bioinformatic analysis of RecQ4 helicases reveals the presence of a RQC domain and a Zn knuckle.

  Francesca Marino, Alessandro Vindigni, Silvia Onesti
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  ABSTRACT: RecQ helicases play essential roles in the maintenance of genome stability and contain a highly conserved helicase region generally followed by a characteristic RecQ-C-terminal (RQC) domain, plus a number of variable associated domains. Notable exceptions are the RecQ4 helicases, where none of these additional regions have been described. Particularly striking was the fact that no RQC domain had been reported, considering that the RQC domain had been shown to play an essential role in the catalytic mechanism of most RecQ family members. Here we present the results of detailed bioinformatic analyses of RecQ4 proteins that identify, for the first time, the presence of a putative RQC domain, including some of the key residues involved in DNA binding and unwinding. We also describe the presence of a novel "Zn knuckle" domain, as well as an additional Sld2-homology region, providing new insights into the architecture, function and evolution of these enzymes.
  Biophysical chemistry 03/2013; 177-178C:34-39.
• Article: The role of APC/C inhibitor Emi2/XErp1 in oscillatory dynamics of early embryonic cell cycles.

  P K Vinod, Xin Zhou, Tongli Zhang, Thomas U Mayer, Bela Novak
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  ABSTRACT: The early embryonic Xenopus cell cycles are characterized by alternating oscillations of Cyclin-dependent kinase-1 (Cdk1) and Anaphase Promoting Complex/Cyclosome (APC/C) activities. The early cycles before midblastula transition lack significant inhibitory Cdk1 phosphorylations and are driven by periodic accumulation of Cyclin B before M phase and its degradation by APC/C at the end of M phase. Both experiments and mathematical modelling suggest that while Cdk1:CycB phosphorylation activates APC/C, it inhibits its co-activator Cdc20 (Fizzy). These interactions create an amplified negative-feedback loop which is at the heart of all cell cycle oscillations. Recent experiments find that the APC/C inhibitor, Emi2/XErp1 is essential for large amplitude and short period Cyclin B oscillations during early divisions in the intact Xenopus embryo. This finding is counter-intuitive since larger amplitudes should come with slower cycle times. We explain this paradox by analysing the amplified negative feedback model extended with APC/C inhibition by Emi2. We show that Emi2 interferes with the intrinsic time-delay in APC/C activation and inactivation to increase the amplitude as well as shorten the period of Cyclin B oscillation.
  Biophysical chemistry 03/2013; 177-178C:1-6.
• Article: Light scattering methods for tracking gold nanoparticles aggregation induced by biotin-neutravidin interaction.

  Ines Delfino
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  ABSTRACT: Dynamic and Static Light Scattering (DLS and SLS), Resonance Light Scattering (RLS) and angular-ratiometric methods have been used for investigating the aggregation of biotinylated bovine serum albumin gold nanoparticles induced by the interaction of biotin with its partner neutravidin. All the approaches have shown to be sensitive to the presence of neutravidin in solution at nM-concentrations. Changes in scattered light intensity dependence on the scattering angle (studied by SLS) and on the wavelength of the incident light (by RLS) have been observed. Predictions from Mie theory have enabled to connect these changes to the increase in the hydrodynamic size of the aggregates, quantified by DLS. The results here reported confirm the potentials of light scattering approach for realizing methods for analyte quantification and offer a good starting point for evaluating the limits and advantages of each method, which would further widen the use of light scattering approach in biosensing.
  Biophysical chemistry 03/2013; 177-178C:7-13.
• Article: Reaction-complexation coupling between an enzyme and its polyelectrolytic substrate: Determination of the dissociation constant of the hyaluronidase-hyaluronan complex from the hyaluronidase substrate-dependence.

  Hélène Lenormand, Fériel Amar-Bacoup, Jean-Claude Vincent
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  ABSTRACT: Hyaluronan (HA) is catalytically hydrolyzed by hyaluronidase (HAase). Depending on pH, HA is able to form a non-productive electrostatic complex with HAase in addition to the classical enzyme-substrate complex. Experiments have shown the strong inhibition of the HA hydrolysis catalyzed by HAase when performed at high HA over HAase concentration ratio and low ionic strength. The substrate-dependence thus shows a non-classic inhibition of HAase at high substrate concentrations due to the sequestration of HAase by HA in the electrostatic complex. The modeling of the HA/HAase system is characteristic of a reaction-complexation coupling and it is very difficult to study reaction or binding, separately. Here, we have established the equation controlling the global system and shown that the substrate-dependence of such a system is a direct combination of a pure Michaelis-Menten equation associated with the reaction and a hyperbolic curve associated with the binding. At low substrate concentrations, the hyperbola, representing the relative part of HAase not sequestered by HA, can be assimilated to a straight line. We have established the relationship between the slope of that straight line and the dissociation constant of the electrostatic HA-HAase complex. Fitting the theoretical equation to the experimental data allowed us to determine, for the first time, the Kd value of the non-productive HA-HAase complex at low ionic strength.
  Biophysical chemistry 03/2013;
• Article: Structural landscape of the proline-rich domain of Sos1 nucleotide exchange factor.

  Caleb B McDonald, Vikas Bhat, Dmitry Kurouski, David C Mikles, Brian J Deegan, Kenneth L Seldeen, Igor K Lednev, Amjad Farooq
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  ABSTRACT: Despite its key role in mediating a plethora of cellular signaling cascades pertinent to health and disease, little is known about the structural landscape of the proline-rich (PR) domain of Sos1 guanine nucleotide exchange factor. Herein, using a battery of biophysical tools, we provide evidence that the PR domain of Sos1 is structurally disordered and adopts an extended random coil-like conformation in solution. Of particular interest is the observation that while chemical denaturation of PR domain results in the formation of a significant amount of polyproline II (PPII) helices, it has little or negligible effect on its overall size as measured by its hydrodynamic radius. Our data also show that the PR domain displays a highly dynamic conformational basin in agreement with the knowledge that the intrinsically unstructured proteins rapidly interconvert between an ensemble of conformations. Collectively, our study provides new insights into the conformational equilibrium of a key signaling molecule with important consequences on its physiological function.
  Biophysical chemistry 03/2013; 175-176C:54-62.
• Article: Protochlorophyllide in model systems - An approach to in vivo conditions.

  Beata Mysliwa-Kurdziel, Jerzy Kruk, Kazimierz Strzałka
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  ABSTRACT: Absorption and fluorescence properties of protochlorophyllide (Pchlide) monomers and aggregates in various model systems are presented in this study. The absorption and fluorescence maxima, and fluorescence lifetimes of Pchlide monomers were not dependent on liposome composition. Fluorescence quenching experiments using KI and SASLs as fluorescence quenchers, revealed that Pchlide molecules entered a lipid bilayer and were localized close to the polar lipid headgroup area. The process of Pchlide aggregation was evident for high (i.e. at least 9mol%) Pchlide content in liposomes prepared from galactolipids. To our knowledge, this is the first study of Pchlide aggregation in membrane-mimicking model systems. The aggregates showed absorption maxima at 480 and 650nm. Fluorescence of the aggregates measured for excitation at 480nm had a maximum at 656nm and was characterized with two fluorescence lifetime components, i.e. 0.1 and 1-2ns. Pchlide aggregates observed in the buffer had similar position of absorption and fluorescence bands to those observed in liposomes, although the overall fluorescence intensity was considerably lower. Some differences in the relative intensity of Soret absorption bands were observed. These results showed that the presence of liposomes decreased the efficiency of the process of Pchlide aggregation. Water bound at the interface region of AOT/isooctane/water reversed micelles induced disaggregation of the Pchlide aggregates indicating that Pchlide aggregates are buried into hydrophilic core of micelles. The results are discussed with respect to the role of lipids in Pchlide aggregation found in plant etioplasts and their significance for light-induced Pchlide photoreduction.
  Biophysical chemistry 02/2013; 175-176C:28-38.
• Article: Simplicity within the complexity: Bilateral impact of DMSO on the functional and unfolding patterns of α-chymotrypsin.

  Tatyana Tretyakova, Mikhael Shushanyan, Tamar Partskhaladze, Maya Makharadze, Rudi van Eldik, Dimitri E Khoshtariya
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  ABSTRACT: New understanding of the fundamental links between protein stability, conformational flexibility and function, can be gained through synergic studies on their catalytic and folding/unfolding properties under the influence of stabilizing/destabilizing additives. We explored an impact of dimethyl sulfoxide (DMSO), the moderate effector of multilateral action, on the kinetic (functional) and thermodynamic (thermal unfolding) patterns of a hydrolytic enzyme, α-chymotrypsin (α-CT), over a wide range of additive concentrations, 0-70% (v/v). Both the calorimetric and kinetic data exhibited rich behavior pointing to the complex interplay of global/local stability (and flexibility) patterns. The complex action of DMSO is explained through the negative and positive preferential solvation motifs that prevail for the extreme opposite, native-like and unfolded states, respectively, implying essential stabilization of compact domains by enhancement of interfacial water networks and destabilization of a flexible active site by direct binding of DMSO to the unoccupied specific positions intended for elongated polypeptide substrates.
  Biophysical chemistry 02/2013; 175-176C:17-27.
• Article: Structural study of hNck2 SH3 domain protein in solution by circular dichroism and X-ray solution scattering.

  Yoshitaka Matsumura, Masaji Shinjo, Tsutomu Matsui, Kaoru Ichimura, Jianxing Song, Hiroshi Kihara
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  ABSTRACT: We have done conformational study of hNck2 SH3 domain by means of far-ultraviolet (far-UV) circular dichroism (CD) and X-ray solution scattering (XSS). The results indicated that the following: (1) hNck2 SH3 domain protein exhibited concentration dependent monomer-dimer transition at neutral pH, while the secondary structure of this protein was independent of the protein concentration. (2) The hNck2 SH3 domain also exhibited pH dependent monomer-dimer transition. This monomer-dimer transition was accompanied with helix-β transition of the secondary structural change. Moreover, the acid-induced conformation, which was previously studied by Liu and Song by CD and nuclear magnetic resonance (NMR), was found to be not compact, but the conformation of the protein at acidic pH was similar to the cold denatured state (C-state) reported by Yamada et al. for equine β-lactoglobulin. We calculated that a structure of the equilibrium helix-rich intermediate of the hNck2 SH3 domain by DAMMIF program.
  Biophysical chemistry 02/2013; 175-176C:39-46.
• Article: The structure of poly-l-lysine in different solvents.

  Andreja Mirtič, Jože Grdadolnik
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  ABSTRACT: Understanding the factors that affect the conformational stability of the polypeptide main chain provides insight not only into the molecular basis of unfolded states but also into the earliest event that occurs during the protein folding. The presented study was concentrated on finding the conformational distributions of poly-l-lysine (PLL) by applying infrared spectroscopy. We assigned the amide bands for different conformations of PLL in water. At low pH values PLL mainly possesses the PII and β structures while at higher pH values and low temperatures characteristic bands for the α-helical conformation are found. The increase in temperature induces the formation of β structures. The obtained assignment of the infrared bands for various conformations was used to determine the conformational populations of PLL in non-aqueous solvents. In TFE, PLL possesses an α-helix structure that is after heating partially transformed into the PII conformation. DMSO enables a uniform α-helical conformation of PLL. A similar uniform conformation (PII, 88%) was found for PLL dissolved in ethylene glycol, suggesting that the PII structure is not limited to the presence of water molecules or charged side chains. The role of intermolecular interactions between the solvent molecules and PLL in stabilizing the PII conformation is discussed.
  Biophysical chemistry 02/2013; 175-176C:47-53.
• Article: Common noise induced synchronous circadian oscillations in uncoupled non-identical systems.

  Lin Ji, Xinping Yan, Nan Li, Hengyu Li, Qianshu Li
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  ABSTRACT: The effect of common noise on the collective behavior of circadian oscillation systems was studied in an elementary circadian clock model. It is shown that common noise could induce synchronous oscillations in two uncoupled non-identical systems in the deterministic stable steady state region. The synchronicity of common noise induced oscillations is suppressed by the internal noise, but is not remarkably decreased within a wide range of internal noise intensity. This demonstrates that the common noise induced synchronous oscillations are rather robust to internal fluctuations.
  Biophysical chemistry 02/2013; 173-174C:15-20.
• Article: Appraisal of sildenafil binding on the structure and promiscuous esterase activity of native and histidine-modified forms of carbonic anhydrase II.

  Hamid Mahdiuni, Nooshin Bijari, Masoud Varzandian, Seyyed Abolghasem Ghadami, Mozafar Khazaei, Mohammad Reza Nikbakht, Reza Khodarahmi
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  ABSTRACT: Sildenafil was investigated for its interaction with the native and modified human carbonic anhydrase II (hCA II). Modification of exposed histidine side chains with diethyl pyrocarbonate decreased esterase activity of the enzyme. The treatment of both native and modified CA with sildenafil revealed slight and moderate enzyme activation profiles, respectively. In addition, in the present study the effects of sildenafil on the structural properties of native and modified hCA II were investigated employing different computer simulation and spectroscopic techniques such as UV-vis, circular dichroism (CD), fluorescence spectroscopy and molecular dynamics. Fluorescence measurements showed that the sildenafil acts as a quencher of the native and modified enzyme fluorescence. Stern-Volmer analyses revealed the existence of one binding site on the native/modified enzyme for sildenafil. The thermodynamic parameters, enthalpy change (∆H) and entropy change (∆S) of drug binding were not also similar, which indicate that different interactions are responsible in CA-drug interaction. Calculation of the protein surface hydrophobicity (PSH), using 1,8-Anilinonaphtalene Sulfonate (ANS), indicated the increment of PSH of native and modified hCA II in the presence of sildenafil. Overall, sildenafil-CA interaction probably induces protein conformational changes and completes reorganization of both hydrogen bond networks within the active site cavity and hydration positions on the protein surface.
  Biophysical chemistry 02/2013; 175-176C:1-16.
• Article: His-Trp cation-π interaction and its structural role in an α-helical dimer of HIV-1 Vpr protein.

  Takayuki Kamiyama, Takashi Miura, Hideo Takeuchi
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  ABSTRACT: Vpr is a multifunctional accessory protein of HIV-1 virus and was previously proposed to assume an antiparallel helical dimer with the third helices HIII of different subunits facing each other. In this study, we have examined the structure and stability of the antiparallel dimer by using a fragment peptide, Vpr52-80, spanning the HIII region. The present analyses of fluorescence, circular dichroism, and UV absorption spectra have shown that a cation-π interaction takes place between protonated His71 and Trp54 located near the opposite ends of the two antiparallel helices. The cation-π interaction induces a small elongation of the HIII helix, an increase in thermal stability of the helical dimer, and a modification of the helix arrangement to produce a more compact form. The His71-Trp54 cation-π interaction may be utilized in stabilizing and tuning the dimeric structure of Vpr to achieve proper interactions with other proteins.
  Biophysical chemistry 02/2013; 173-174C:8-14.
• Article: A T(3)R(3) hexamer of the human insulin variant B28Asp.

  Leonardo C Palmieri, Maely P Fávero-Retto, Daniela Lourenço, Luís Mauricio T R Lima
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  ABSTRACT: Insulin shows a complex equilibrium between monomers and hexamers, involving varying conformers and association states. We sought to perform a structural characterization of the fast-acting human insulin variant B28Asp ("aspart"). Small-angle X-ray scattering measurements reveal similar globular behavior in both the aspart and regular human insulin, with a R(g) of 19Å and a D(max) of approximately 50Å, indicating similar mean quaternary assembly distribution. Crystallographic assays revealed a T(3)R(3) assembly of the aspart insulin formed by the TR dimer in the asymmetric unit, with all the first 8 residues of the B chain in the R-state monomer in helical conformation and the participation of its B3Asn in the stabilization of the hexamer. Our data provide access to novel structural information on aspart insulin such as an aspart insulin dimer in solution, the aspart insulin in T conformation and a pure R-state conformer establishing a T(3)R(3) assembly, providing further insight on the stepwise conformational transition and assembly of this fast-insulin.
  Biophysical chemistry 02/2013; 173-174C:1-7.
• Article: Thermodynamic parameters of anion binding to halorhodopsin from Natronomonas pharaonis by isothermal titration calorimetry.

  Saori Hayashi, Jun Tamogami, Takashi Kikukawa, Haruka Okamoto, Kazumi Shimono, Seiji Miyauchi, Makoto Demura, Toshifumi Nara, Naoki Kamo
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  ABSTRACT: Halorhodopsin (HR), an inwardly directed, light-driven anion pump, is a membrane protein in halobacterial cells that contains the chromophore retinal, which binds to a specific lysine residue forming the Schiff base. An anion binds to the extracellular binding site near the Schiff base, and illumination makes this anion go to the intracellular channel, followed by its release from the protein and re-uptake from the opposite side. The thermodynamic properties of the anion binding in the dark, which have not been previously estimated, are determined using isothermal titration calorimetry (ITC). For Cl(-) as a typical substrate of HR from Natronomonas pharaonis, ΔG=-RT ln(1/K(d))=-15.9kJ/mol, ΔH=-21.3kJ/mol and TΔS=-5.4kJ/mol at 35°C, where K(d) represents the dissociation constant. In the dark, K(d) values have been determined by the usual spectroscopic methods and are in agreement with the values estimated by ITC here. Opsin showed no Cl(-) binding ability, and the deprotonated Schiff base showed weak binding affinity, suggesting the importance of the positively charged protonated Schiff base for the anion binding.
  Biophysical chemistry 02/2013; 172:61-7.
• Article: Single-molecule studies of disulfide bond reduction pathways used by human thioredoxin.

  Robert Szoszkiewicz
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  ABSTRACT: Disulfide bond reduction pathways used by human thioredoxin (hTrx) are studied at the single molecule level using a recombinant protein (I27(SS))(8). (I27(SS))(8) contains eight tandem repeats of identical immunoglobulin-like modules with one disulfide bond in each module. Single (I27(SS))(8) molecules are stretched at constant force applied by a cantilever in a force-clamp mode of atomic force microscopy (FC-AFM). Disulfide reduction events are accurately detected from stepwise increases in the end-to-end length of (I27(SS))(8). Earlier FC-AFM studies observed one disulfide reduction pathway used by hTrx and suggested an additional electron tunneling mechanism. Here, a very large set of unbiased FC-AFM data is collected in a range of clamping forces. By analyzing the data using exponential fits and dwell time histograms two disulfide reduction pathways used by hTrx are resolved. Based on previous studies one of these pathways is attributed to force-dependent Michaelis-Menten catalysis. The latter reduction pathway is weakly force-inhibited and occurs sporadically. Bimolecular nucleophilic substitutions (S(N)2) and electron tunneling (ET) mechanisms are discussed to explain the second pathway. Direct S(N)2 and ET mechanisms cannot be discounted, but a hypothetical E2-S(N)2 mechanism involving a hydride reducing a disulfide bond provides an interesting alternative, which needs to be verified in future experiments.
  Biophysical chemistry 01/2013;
• Article: The role of activity coefficients in bioreaction equilibria: Thermodynamics of methyl ferulate hydrolysis.

  Philip Hoffmann, Matthias Voges, Christoph Held, Gabriele Sadowski
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  ABSTRACT: The Gibbs energy of reaction (Δ(R)g) is the key quantity in the thermodynamic characterization of biological reactions. Its calculation requires precise standard Gibbs energy of reaction (Δ(R)g(+)) values. The value of Δ(R)g(+) is usually determined by measuring the apparent (concentration-dependent) equilibrium constants K, e.g., the molality-based Km. However, the thermodynamically consistent determination of Δ(R)g(+) requires the thermodynamic (activity-based) equilibrium constant Ka. These values (Km and Ka) are equal only if the ratio of the activity coefficients of the reactants to the activity coefficients of the products (Kγ) is equal to unity. In this work, the impact of Kγ on the estimation of Ka for biological reactions was investigated using methyl ferulate (MF) hydrolysis as a model reaction. The value of Kγ was experimentally determined from Km values that were measured at different reactant concentrations. Moreover, Kγ was independently predicted using the thermodynamic model ePC-SAFT. Both the experimentally determined and the predicted Kγ values indicate that this value cannot be assumed to be unity in the considered reaction. In fact, in the reaction conditions considered in this work, Kγ was shown to be in the range of 3<Kγ<6 for different reactant molalities (2<mmol MF kg(-1)<10). The inclusion of Kγ and thus the use of the thermodynamically correct Ka value instead of Km lead to remarkable differences (almost 40%) in the determination of Δ(R)g(+). Moreover, the new value for Δ(R)g(+) increases the concentration window at which the reaction can thermodynamically occur. The influence of additives was also investigated both experimentally and theoretically. Both procedures consistently indicated that the addition of NaCl (0 to 1molkg(-1) water) moderately decreased the value of Kγ, which means that the values of Km increase and that a higher amount of products is obtained as a result of the addition of salt. Additionally, Km was found to strongly depend on pH. A ten-fold increase in the Km values was observed in the pH range of 6 to 7; this increase corresponds to a change of more than 100% in the value of Δ(R)g(+).
  Biophysical chemistry 01/2013;
• Article: Variant of the Thermomyces lanuginosus lipase with improved kinetic stability: A candidate for enzyme replacement therapy.

  H Wang, J Hagedorn, A Svendsen, K Borch, D E Otzen
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  ABSTRACT: Lipases with high kinetic stability and enzymatic efficiency in the human gastro-intestinal tract may help against exocrine pancreatic insufficiency. Here we mimic gastric conditions to study how bile salts and pH affect the stability and activity of Thermomyces lanuginosus lipase (TlL) and its stabler variant StL using spectroscopy, calorimetry and gel electrophoresis. Both enzymes resist trypsin digestion with and without bile salts. Bile salts activate native TlL and StL equally well, bind weakly to denatured TlL and StL at lower pH and precipitate native TlL and StL at pH 4. StL refolds more efficiently than TlL from gastric pH in bile salts, regaining activity when refolding from pH as low as 1.8 and above while TlL cannot go below pH 2.6. StL also unfolds 10-40 fold more slowly in the denaturant guanidinium chloride and the anionic surfactant SDS. We ascribe StL's superior performance to general alterations in its electrostatic potential which makes it more acid-resistant. These superior properties make StL a good candidate for pancreatic enzyme replacement therapy.
  Biophysical chemistry 01/2013; 172C:43-52.