Bioscavengers are molecules able to neutralize neurotoxic organophosphorus compounds (OP) before they can reach their biological target. Human butyrylcholinesterase (hBChE) is a natural bioscavenger each molecule of enzyme neutralizing one molecule of OP. The amount of natural enzyme is insufficient to achieve good protection. Thus, different strategies have been envisioned. The most straightforward consists in injecting a large dose of highly purified natural hBChE to increase the amount of bioscavenger in the bloodstream. This proved to be successful for protection against lethal doses of soman and VX but remains expensive. An improved strategy is to regenerate prophylactic cholinesterases (ChE) by administration of reactivators after exposure. But broad-spectrum efficient reactivators are still lacking, especially for inhibited hBChE. Cholinesterase mutants capable of reactivating spontaneously are another option. The G117H hBChE mutant has been a prototype. We present here the Y124H/Y72D mutant of human acetylcholinesterase; its spontaneous reactivation rate after V-agent inhibition is increased up to 110 fold. Catalytic bioscavengers, enzymes capable of hydrolyzing OP, present the best alternative. Mesophilic bacterial phosphotriesterase (PTE) is a candidate with good catalytic efficiency. Its enantioselectivity has been enhanced against the most potent OP isomers by rational design. We show that PEGylation of this enzyme improves its mean residence time in the rat blood stream 24-fold and its bioavailability 120-fold. Immunogenic issues remain to be solved. Human paraoxonase 1 (hPON1) is another promising candidate. However, its main drawback is that its phosphotriesterase activity is highly dependent on its environment. Recent progress has been made using a mammalian chimera of PON1, but we provide here additional data showing that this chimera is biochemically different from hPON1. Besides, the chimera is expected to suffer from immunogenic issues. Thus, we stress that interest for hPON1 must not fade away, and in particular, the 3D structure of the hPON1 eventually in complex with OP has to be solved.
Title: Preparation and characterization of methoxy polyethylene glycol-conjugated phosphotriesterase as a potential catalytic bioscavenger against organophosphate poisoning Jun D, Musilova L, Link M, Loiodice M, Nachon F, Rochu D, Renault F, Masson P Ref: Chemico-Biological Interactions, 187:380, 2010 : PubMed
Bioscavengers are considered as promising antidotes against organophosphate poisoning. We focused on a bacterial phosphotriesterase (PTE) expressed in Escherichia coli. The main disadvantage of this non-human catalytic bioscavenger is its relatively short half-life in the organism and strong immunogenicity after repeated administration. Therefore, we prepared different methoxy polyethylene glycol (MPEG)-conjugated recombinant PTE as a potential catalytic bioscavenger with the aim to improve its biological properties. Enzyme was modified with two linear monofunctional MPEG derivatives with reactive aldehyde group of molecular weight 2 kDa and 5 kDa. We optimized reaction conditions (reagent ratios, temperature and duration of modification reaction) and we prepared homogeneous population of fully modified recombinant PTE with molecular weight around 52 kDa and 76 kDa, respectively. Modified PTE was characterized using SDS-PAGE and MALDI-TOF and by determining K(m) and V(max). We also investigated thermal stability of modified enzyme at 37 degrees C. Based on our results, for future in vivo evaluation of pharmacokinetics and pharmacodynamics properties, we selected recombinant PTE modified with 5 kDa MPEG aldehyde for its superior thermal stability.
Title: Integrative analytical approach by capillary electrophoresis and kinetics under high pressure optimized for deciphering intrinsic and extrinsic cofactors that modulate activity and stability of human paraoxonase (PON1) Renault F, Carus T, Clery-Barraud C, Elias M, Chabriere E, Masson P, Rochu D Ref: Journal of Chromatography B Analyt Technol Biomed Life Sciences, 878:1346, 2010 : PubMed
Paraoxonase (PON1) is working in vivo in a particular dynamic environment including HDL particles and associated molecules. To decipher the respective and/or concomitant role of the different cofactors involved in this molecular organization, an approach using multiple experimental techniques based on capillary electrophoresis and classical kinetics or kinetics under high pressure was implemented. The effects of calcium and phosphate as protein or plasma cofactor, of human phosphate binding protein (HPBP) as enzyme chaperone, and of a PON1 inhibitor as an active site stabilizer, on the catalytic activities and functional oligomerization of PON1 were scrutinized. PON1 displays two distinct catalytic behaviors, one against esters and lactones, the other against organophosphorus compounds; its functional states and catalytic activities against these substrates are differently modulated by the molecular environment; PON1 exists under several active multimeric forms; the binding of HPBP amends the size of the oligomeric states and exerts a stabilizing effect on the activities of PON1; PON1 functional properties are modulated by HPBP, calcium and phosphate. This integrative approach using several optimized analytical techniques allowed performing comparison of catalytic properties and oligomeric states of functional PON1 in different enzyme preparations. Relevance of these data to understand in vivo physiological PON1 functioning is mandatory.
Organophosphorus chemical warfare agents (nerve agents) are to be feared in military operations as well as in terrorist attacks. Among them, VX (O-ethyl-S-[2-(diisopropylamino)ethyl] methylphosphonothioate) is a low volatility liquid that represents a percutaneous as well as an inhalation hazard if aerosolized. It is a potent irreversible cholinesterase (ChE) inhibitor that causes severe signs and symptoms, including respiratory dysfunction that stems from different mechanisms. VX-induced pulmonary oedema was previously reported in dogs but mechanisms involved are not well understood, and its clinical significance remains to be assessed. An experimental model was thus developed to study VX-induced cardiovascular changes and pulmonary oedema in isoflurane-anaesthetized swine. In the course of this study, we observed a fast and unexpected rebound of plasma ChE activity following inhibition provoked by the intravenous injection of 6 and 12 microg kg(-1) of VX. In whole blood ChE activity, the rebound could stay unnoticed. Further investigations showed that the rebound of plasma esterase activity was neither related to spontaneous reactivation of ChE nor to VX-induced increase in paraoxonase/carboxylesterase activities. A bias in Ellman assay, haemoconcentration or severe liver cytolysis were also ruled out. All in all, these results suggest that the rebound was likely due to the release of butyrylcholinesterase into the blood stream from ChE producing organs. Nature of the organ(s) and mechanisms involved in enzyme release will need further investigations as it may represent a mechanism of defence, i.e. VX scavenging, that could advantageously be exploited.
While there is a consensus that human PON1 (paraoxonase-1) has a protective role, its primary biological function remains unclear. A protective role against poisoning by organophosphates [OPs (organophosphorus compounds)] drove earlier works. Clinical interest has recently focused on a protective role of PON1 against vascular diseases. PON1 resides mainly on HDL (high-density lipoprotein) particles, and converging recent works show that both its activities and stability dramatically depend on this versatile and dynamic molecular environment. The discovery that HPBP (human phosphate-binding protein) has a firm tendency to associate with PON1 has steered new directions for characterizing PON1 functional state(s). Storage stability studies provided evidence that HPBP is involved in maintaining physiologically active PON1 conformation(s). Thermal stability studies showed that human PON1 is remarkably thermostable and that its association with HPBP strongly contributes to slowing down the denaturation rate. A hybrid PON1, displaying mutations that stabilized recombinant enzyme expressed in Escherichia coli, was shown to be more thermostable than natural human PON1. Predictably, its stability was unaffected by the presence of HPBP. Synergistic efforts on characterizing natural PON1 and rPON1 (recombinant PON1) provide information for the design of future stable mutants of PON1-based bioscavengers to be used as safe and effective countermeasures to challenge OPs. Maintaining a stable environment for such administrable human rPON1 should, at least, preserve the anti-atherogenic activity of the enzyme.
Title: Capillary electrophoresis versus differential scanning calorimetry for the analysis of free enzyme versus enzyme-ligand complexes: in the search of the ligand-free status of cholinesterases Rochu D, Clery-Barraud C, Renault F, Chevalier A, Bon C, Masson P Ref: Electrophoresis, 27:442, 2006 : PubMed
Cholinesterases (ChEs) are highly efficient biocatalysts whose active site is buried in a deep, narrow gorge. The talent of CE to discover inhibitors in the gorge of highly purified preparations has fairly altered the meaning of a ChE ligand-free status. To attempt at a description of this one, we investigated the stability of Bungarus fasciatus acetylcholinesterase (AChE), alone or complexed with different inhibitors. Determination of mid-transition temperature for thermal denaturation, using differential scanning calorimetry (DSC) and CE, provided conflicting results. Discrepancies strongly question the reality of a ligand-free AChE state. DSC allowed estimation of the stability of AChE-ligands complexes, and to rank the stabilizing effect of different inhibitors. CE acted as a detector of hidden ligands, provided that they were charged, reversibly bound, and thus dissociable upon action of electric fields. Then, CE allowed quantification of the stability of ligand-free AChE. CE and DSC providing each fractional and nonredundant information, cautious attention must be paid for actual estimation of the conformational stability of ChEs. Because inhibitors used in purification of ChEs by affinity chromatography are charged, CE remains a leading method to estimate enzyme stability and detect the presence of bound hidden ligands.
Incoherent elastic neutron scattering experiments on members of the cholinesterase family were carried out to investigate how molecular dynamics is affected by covalent inhibitor binding and by differences in primary and quaternary structure. Tetrameric native and soman-inhibited human butyrylcholinesterase (HuBChE) as well as native dimeric Drosophila melanogaster acetylcholinesterase (DmAChE) hydrated protein powders were examined. Atomic mean-square displacements (MSDs) were found to be identical for native HuBChE and for DmAChE in the whole temperature range examined, leading to the conclusion that differences in activity and substrate specificity are not reflected by a global modification of subnanosecond molecular dynamics. MSDs of native and soman-inhibited HuBChE were identical below the thermal denaturation temperature of the native enzyme, indicating a common mean free-energy surface. Denaturation of the native enzyme is reflected by a relative increase of MSDs consistent with entropic stabilization of the unfolded state. The results suggest that the stabilization of HuBChE phosphorylated by soman is due to an increase in free energy of the unfolded state due to a decrease in entropy.
A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type.
Title: Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase: temperature- and pH-dependence Rochu D, Viguie N, Renault F, Crouzier D, Froment MT, Masson P Ref: Biochemical Journal, 380:627, 2004 : PubMed
Phosphotriesterase (PTE) detoxifies nerve agents and organophosphate pesticides. The two zinc cations of the PTE active centre can be substituted by other transition metal cations without loss of activity. Furthermore, metal-substituted PTEs display differences in catalytic properties. A prerequisite for engineering highly efficient mutants of PTE is to improve their thermostability. Isoelectric focusing, capillary electrophoresis and steady-state kinetics analysis were used to determine the contribution of the active-site cations Zn2+, Co2+ or Cd2+ to both the catalytic activity and the conformational stability of the corresponding PTE isoforms. The three isoforms have different pI values (7.2, 7.5 and 7.1) and showed non-superimposable electrophoretic titration curves. The overall structural alterations, causing changes in functional properties, were found to be related to the nature of the bound cation: ionic radius and ion electronegativity correlate with Km and kcat respectively. In addition, the pH-dependent activity profiles of isoforms were different. The temperature-dependent profiles of activity showed maximum activity at T < or =35 degrees C, followed by an activation phase near 45-48 degrees C and then inactivation which was completed at 60 degrees C. Analysis of thermal denaturation of the PTEs provided evidence that the activation phase resulted from a transient intermediate. Finally, at the optimum activity between pH 8 and 9.4, the thermostability of the different PTEs increased as the pH decreased, and the metal cation modulated stability (Zn2+-, Co2+- and Cd2+-PTE showed different T (m) values of 60.5-67 degrees C, 58-64 degrees C and 53-64 degrees C respectively). Requirements for optimum activity of PTE (displayed by Co2+-PTE) and maximum stability (displayed by Zn2+-PTE) were demonstrated.
Title: Capillary zone electrophoresis detects unwanted cholinesterase-bound hidden ligands that alter enzyme conformational stability. Rochu D, Renault F, Bon C, Masson P Ref: Cholinergic Mechanisms, CRC Press, :675, 2004 : PubMed
Title: Detection of unwanted protein-bound ligands by capillary zone electrophoresis: the case of hidden ligands that stabilize cholinesterase conformation Rochu D, Renault F, Masson P Ref: Electrophoresis, 23:930, 2002 : PubMed
Detection, identification and characterization of compounds present in purified proteins and biopharmaceuticals are of central interest. As well as chemical remedies, proteins of pharmacological interest have to exhibit their nakedness to become therapeutic drugs. Cholinesterases (ChE) are enzymes of major importance for detoxification of poisonous esters. Likewise, ChE are characterized by the high catalytic efficiency of an active site positioned at the bottom of a deep gorge. The gorge can be partially or fully occupied by ligands, i.e., substrates and inhibitors that are currently used in affinity chromatography purification steps. Accordingly, a suitable method allowing to analyse the presence of unwanted ligands and its influence on the functional conformation and stability of these enzymes was essential. We have developed CZE approaches for that purpose. The factors causing discrepancies between data for thermal unfolding of ChE by electrophoretic and by calorimetric methods were investigated. The presence of unwanted hidden ligands bound to purified enzymes was first demonstrated. The incidence of these ligands was discussed. Altogether, our results raised several questions concerning the real conformation of the native state of enzymes. Finally, CZE was proved to be a pertinent tool to validate the conformity of purified enzymes to a status of biopharmaceutical.
Title: Dual effect of high electric field in capillary electrophoresis study of the conformational stability of Bungarus fasciatus acetylcholinesterase Rochu D, Pernet T, Renault F, Bon C, Masson P Ref: Journal of Chromatography A, 910:347, 2001 : PubMed
The effect of high electric field in capillary zone electrophoresis (CZE) was evaluated for the study of the thermally induced unfolding of Bungarus fasciatus acetylcholinesterase. This monomer enzyme is characterised by two interdependent uncommon structural features, the asymmetrical distribution of charged residues and a relatively low thermal denaturation temperature. Both traits were presumed to interfere in the thermal unfolding of this enzyme as investigated by CZE. This paper analyses the effect of high electric field on the behaviour of the enzyme native state. It is shown that increasing the applied field causes denaturation-like transition of the enzyme at a current power which does not induce excessive Joule heating in the capillary. The susceptibility to electric field of proteins like cholinesterases, with charge distribution anisotropy, large permanent dipole moment and notable molecular flexibility associated with moderate thermal stability, was subsequently discussed.
Title: Pressure- and heat-induced inactivation of butyrylcholinesterase: evidence for multiple intermediates and the remnant inactivation process Weingand-Ziade A, Ribes F, Renault F, Masson P Ref: Biochemical Journal, 356:487, 2001 : PubMed
The inactivation process of native (N) human butyrylcholinesterase (BCHE) by pressure and/or heat was found to be multi-step. It led to irreversible formation of an active intermediate (I) state and a denatured state. This series-inactivation process was described by expanding the Lumry-Eyring [Lumry, R. and Eyring, H. (1954) J. Phys. Chem. 58, 110-120] model. The intermediate state (I) was found to have a K(m) identical with that of the native state and a turnover rate (k(cat)) twofold higher than that of the native state with butyrylthiocholine as the substrate. The increased catalytic efficiency (k(cat)/K(m)) of I can be explained by a conformational change in the active-site gorge and/or restructuring of the water-molecule network in the active-site pocket, making the catalytic steps faster. However, a pressure/heat-induced covalent modification of native BCHE, affecting the catalytic machinery, cannot be ruled out. The inactivation process of BCHE induced by the combined action of pressure and heat was found to continue after interruption of pressure/temperature treatment. This secondary inactivation process was termed 'remnant inactivation'. We hypothesized that N and I were in equilibrium with populated metastable N' and I' states. The N' and I' states can either return to the active forms, N and I, or develop into inactive forms, N(')(in) and I(')(in). Both active N' and I' intermediate states displayed different rates of remnant inactivation depending on the pressure and temperature pretreatments and on the storage temperature. A first-order deactivation model describing the kinetics of the remnant inactivation of BCHE is proposed.
Human serum paraoxonase (PON1) is a calcium-dependent organophosphatase. To identify residues essential for PON1 activity, we adopted complementary approaches based on chemical modification and site-directed mutagenesis. To detect 45Ca2+ binding to native and chemically modified PON1, we performed nondenaturating gel electrophoresis. The environment of calcium-binding sites was probed using the Ca2+ analogue, terbium. Tb3+ binds to calcium-binding sites as shown by displacement of 45Ca2+ by Tb3+. Binding of Tb3+ is accompanied by a complete loss of enzyme activity. PON1 chemical modification with the Trp-selective reagent, N-bromosuccinimide, and the Asp/Glu-selective, dicyclohexylcarbodiimide, established that Trp and Asp/Glu residues are components of the PON1 active center and calcium-binding sites. Additional evidence for the presence of a Trp residue in the PON1 calcium-binding sites was a characteristic fluorescence emission at 545 nm from the PON1-Tb3+ complex and abolishment of that fluorescence upon modification by N-bromosuccinimide. The importance of aromatic/hydrophobic character of the residue 280 was demonstrated by site-directed mutagenesis: the W280F mutant was fully active while the W280A and W280L mutants had markedly reduced activity. Twelve amino acids among conserved His and Asp/Glu residues were found essential for PON1 arylesterase and organophosphatase activities: H114, H133, H154, H242, H284, D53, D168, D182, D268, D278, E52, and E194. Finally, the cysteines constituting the PON1 disulfide bond (C41 and C352) were essential, but the glycan chains linked to Asn 252 and 323 were not essential for PON1 secretion and activity.
Title: Differential effect of pressure and temperature on the catalytic behaviour of wild-type human butyrylcholinesterase and its D70G mutant Weingand-Ziade A, Renault F, Masson P Ref: European Journal of Biochemistry, 264:327, 1999 : PubMed
The combined action of temperature (10-35 degrees C) and pressure (0. 001-2 kbar) on the catalytic activity of wild-type human butyrylcholinesterase (BCHE) and its D70G mutant was investigated at pH 7.0 using butyrylthiocholine as the substrate. The residue D70, located at the mouth of the active site gorge, is an essential component of the peripheral substrate binding site of BCHE. Results showed a break in Arrhenius plots of wild-type BCHE (at Tt approximately 22 degrees C) whatever the pressure (dTt/dP = 1.6 +/- 1.5 degrees C.kbar-1), whereas no break was observed in Arrhenius plots of the D70G mutant. These results suggested a temperature-induced conformational change of the wild-type BCHE which did not occur for the D70G mutant. For the wild-type BCHE, at around a pressure of 1 kbar, an intermediate state, whose affinity for substrate was increased, appeared. This intermediate state was not seen for the mutant enzyme. The wild-type BCHE remained active up to a pressure of 2 kbar whatever the temperature, whereas the D70G mutant was found to be more sensitive to pressure inactivation (at pressures higher than 1.5 kbar the mutant enzyme lost its activity at temperatures lower than 25 degrees C). The results indicate that the residue D70 controls the conformational plasticity of the active site gorge of BCHE, and is involved in regulation of the catalytic activity as a function of temperature.
Title: Purification, molecular characterization and catalytic properties of a Pseudomonas fluorescens enzyme having cholinesterase-like activity Rochu D, Rothlisberger C, Taupin C, Renault F, Gagnon J, Masson P Ref: Biochimica & Biophysica Acta, 1385:126, 1998 : PubMed
An enzyme with a cholinesterase (ChE) activity, produced by Pseudomonas fluorescens, was purified to homogeneity in a three-step procedure. Analysis by non-denaturing and SDS-PAGE, and by isoelectric focusing, indicated that the enzyme was a monomer of 43 kDa, with a pI of 6.1. The N-terminal sequence, AEPLKAVGAGEGQLDIVAWPGYIEA, showed some similarities with proteins of the ChE family and a strong similarity with a protein from Escherichia coli with unknown structure and function. Cholinesterase activity at pH 7.0 and 25 degreesC was maximum with propionylthiocholine as substrate (kcat,app=670 min-1), followed by acetylthiocholine, and significantly lower with butyrylthiocholine. Catalytic specificity (kcat/Km) was the same for propionylthiocholine and acetylthiocholine, but was two orders of magnitude lower for butyrylthiocholine. Kinetics of thiocholine ester hydrolysis showed inhibition by excess substrate which was ascribed to binding of a second substrate molecule, leading to non-productive ternary complex (Km=35 microM, KSS=0.49 mM with propionylthiocholine). There was low or no reactivity with organophosphates and carbamates. The enzyme inhibited by echothiophate (kII=0.44x102 M-1 min-1) was not reactivated by pralidoxime methiodide. However, the P. fluorescens enzyme had affinity for procainamide and decamethonium, two reversible ChE inhibitors used as affinity chromatography ligand and eluant, respectively. Although similarity of the N-terminal amino acid sequence of the enzyme with an internal sequence of ChEs is weak, its catalytic activity towards thiocholine esters, and its affinity for positively charged ligands supports the contention that this enzyme may belong to the ChE family. However, we cannot rule out that the enzyme belongs to another structural family of proteins having cholinesterase-like properties. The reaction of the enzyme with organophosphates suggests that it is a serine esterase, and currently this enzyme may be termed as having a cholinesterase-like activity.
Title: Combined pressure/heat-induced inactivation of butyrylcholinesterase Weingand-Ziade A, Renault F, Masson P Ref: Biochimica & Biophysica Acta, 1340:245, 1997 : PubMed
The combined effects of pressure and temperature on the activity of butyrylcholinesterase (BuChE) were investigated in the pressure range from 10(-3) to 5 kbar and temperature range from -10 degrees C to 70 degrees C. Inactivation of the enzyme showed a complex dependence on pressure and temperature. Under moderate pressures (1-3 kbar) at temperatures 40-65 degrees C BuChE was resistant to heat inactivation; under other conditions of pressure and temperature, the action of both parameters was synergistic and caused inactivation. Results allowed to construct a pressure-temperature kinetic phase diagram for the enzyme inactivation. The elliptic diagram for the irreversible transition active-->inactive BuChE as a function of both pressure and temperature has a positive angular coefficient. This indicates that pressure acts as a stabilizer of BuChE against heat denaturation.
Title: Raman spectroscopic study of conjugates of butyrylcholinesterase with organophosphates Aslanian D, Grof P, Renault F, Masson P Ref: Biochimica & Biophysica Acta, 1249:37, 1995 : PubMed
Raman spectra of human butyrylcholinesterase (BCHE; E.C. 3.1.1.8) were analyzed in the native state and after conjugation with organophosphates (soman, DFP and paraoxon). The secondary structure of the native BCHE in Tris-HCl buffer (pH 7.5), determined from analysis of the amide I polypeptide vibration band, indicates 47% alpha-helices, 26% beta-sheets, 16% turns and 12% undefined structure. We obtained the same values for paraoxon-phosphorylated BCHE, but 39% helical structure, 31% beta-sheets, 17% turns and 13% undefined structure for 'aged' DFP-BCHE conjugates and 36% helical structure, 34% beta-sheets, 20% turns and 10% undefined structure for 'aged' soman-BCHE conjugates. The approximately 10% decrease of alpha-helical structure observed upon phosphorylation by DFP and phosphonylation by soman, probably corresponds to the 'aging' process, which does not take place in the case of paraoxon. Considerable differences have been observed between native, paraoxon inhibited and 'aged' BCHE in aromatic ring vibrations, suggesting that the dealkylation of organophosphate conjugates modifies the environment or the interactions of aromatic amino-acid residues. In the aliphatic side chains an increase of the number of gauche configurations has been observed in 'aged' DFP-BCHE and soman-BCHE
Title: Pressure-induced molten globule state of cholinesterase Clery C, Renault F, Masson P Ref: FEBS Letters, 370:212, 1995 : PubMed
The denaturing effect of pressure on the structure of human butyrylcholinesterase was examined by gel electrophoresis under pressure and by 8-anilino-1-naphthalene sulfonate (ANS) binding. It was found that the fluorescence intensity of bound ANS is increased by pressure between 0.5 and 1.5 kbar and that the hydrodynamic volume of the enzyme swells when pressures around 1.5 kbar are applied. These findings indicate that pressure denaturation of butyrylcholinesterase is a multi-step process and that the observed transient pressure-denatured states have characteristics of molten globules.
Title: Poster: Molecular characterization of the C5 human plasma cholinesterase variant Masson P, Froment MT, Audras JC, Renault F Ref: In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology, (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC:60, 1991 : PubMed
