Intrinsic Dissociation Constant Research Articles

Control Mechanisms for Gas Hydrate Production by Depressurization in Different Scale Hydrate Reservoirs

The methane hydrate was formed in a pressure vessel 38 mm in id and 500 mm in length. Experimental works on gas production from the hydrate-bearing core by depressurization to 0.1, 0.93, and 1.93 MPa have been carried out. The hydrate reservoir simulator TOUGH-Fx/Hydrate was used to simulate the experimental gas production behavior, and the intrinsic hydration dissociation constant (K(0)) fitted for the experimental data was on the order of 104 mol m(-2) Pa(-1) s(-1), which was one order lower than that of the bulk hydrate dissociation. The sensitivity analyses based on the simulator have been carried out, and the results suggested that the hydrate dissociation kinetics had a great effect on the gas production behavior for the laboratory-scale hydrate-bearing core. However for a field-scale hydrate reservoir, the flow ability dominated the gas production behavior and the effect of hydrate dissociation kinetics on the gas production behavior could be neglected.

Potentiometric Titrations of Maleic Acid Copolymers in Dilute Aqueous Solution: Experimental Results and Theoretical Interpretation

AbstractSummary: The paper reports a study on the flexibility of a family of 1:1 hydrolysed maleic anhydride (maleic acid, MA)–olefin copolymers in dilute aqueous solution. The copolymers were MA–ethene, MA–propene and MA–isobutene. The study was carried out in the absence and in the presence of monomonovalent salts and at different polymer concentrations. Experimental data showing the negative logarithm of the ‘apparent’ dissociation constant, pKa versus the degree of dissociation, α, experimental data were obtained from potentiometric titrations using NaOH, KOH or tetramethylammonium hydroxide as the base. The pKa data were fully described with calculated curves obtained using an extension of the counterion condensation theory of linear polyelectrolytes, in which a semiflexible model for the polymers was introduced. Under the present experimental condition, no relevant specificity of the monovalent counterions was apparently observed for the different copolymers. The calculated pKa versus α curves allowed the derivation of both the intrinsic dissociation constants of the first and the second dissociation steps for the different copolymers and the corresponding stiffness parameters built into the model. The agreement between the experimental and calculated data shows an appreciable success of the model. The results pointed to an increase of stiffness parallel to the increase of size of the olefin comonomer, in qualitative agreement with already published findings. Furthermore, for all copolymers the chain rigidity was larger in the α range of the first dissociation than in that of the second one. The former rigidity was attributed to the formation of intramolecular hydrogen bonds upon the first ionisation of the MA repeating units, followed by an increase of rotational freedom upon breaking of the H‐bond in the second dissociation step. Comparison of the rigidity parameters of the MA copolymers with the data obtained for other polyelectrolytes, both natural—poly(L‐glutamic acid) and pectic acid—and synthetic—poly(acrylic acid) and poly(methacrylic acid)—was also performed. magnified image

Biochemical analysis of a Ca2+‐dependent membrane–membrane interaction mediated by the sea urchin yolk granule protein, toposome

Toposome, a high molecular mass protein, is an abundant component of the yolk granule in the sea urchin egg and embryo. Toposome is composed of a 160 kDa polypeptide that is proteolytically processed into smaller species of 120 and 90 kDa during embryonic development. The exact biological function of toposome during early development is unknown. In this study we have examined calcium binding to toposome and the effect of this binding on the secondary and tertiary structural characteristics of the purified protein. Initially, we used equilibrium dialysis to quantify calcium binding to toposome. Monophasic binding of up to 600 M of calcium per mole of protein was detected with an intrinsic dissociation constant (calcium) of 240 microm. Increasing concentrations of calcium resulted in an increase in alpha helical content from 3.0 to 22.0%, which occurred with an apparent dissociation constant (calcium) of 25 microm. In parallel experiments, toposome binding to liposomes required similar concentrations of calcium; an apparent dissociation constant (calcium) of 25 microm was recorded. Endogenous tryptophan fluorescence measurements, both in the presence and absence of liposomes, demonstrated that the tertiary structure is sensitive to increasing concentrations of calcium with an apparent dissociation constant (calcium) of 240 microm. Toposome-driven, liposome aggregation assays revealed a similar calcium requirement. Collectively, these results define a two-step model for calcium modulation of toposome structure and function.

Encapsulation of Fluorescent Molecules by Functionalized Polymeric Nanocontainers: Investigation by Confocal Fluorescence Imaging and Fluorescence Correlation Spectroscopy

Nanocontainers (NCs) were prepared from amphiphilic triblock copolymers, having an average molecular weight of around 8000 g/mol, by using previously published preparation methods consisting of dispersing the polymer in an aqueous buffer solution containing molecules for encapsulation. A small molecular weight fluorophore, sulforhodamine B, as well as the fluorescent protein avidin labeled with Alexa 488 were encapsulated, and the resulting nanocontainers were characterized using fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS). Nanocontainer size determination by FCS is very robust and compares well with results obtained from photon correlation spectroscopy: the measured diameters of the polymeric nanocontainers vary between 140 and 172 nm. Encapsulation of fluorescent molecules was determined by evaluating the molecular brightness of nanocontainers with an encapsulated fluorescently labeled protein (avidin-Alexa 488). Results indicate that the number of encapsulated avidin-Alexa 488 molecules corresponds well with the initial concentration of the fluorescently labeled protein and the encapsulated volume. A nanocontainer binding assay was developed using biotinylated fluorescently labeled nanocontainers. Binding of biotinylated nanocontainers to fluorescently labeled streptavidin was followed by fluorescence cross-correlation spectroscopy. The intrinsic dissociation constant, K(d), of labeled streptavidin to the ligand-modified nanocontainers is 1.7 +/- 0.4 x 10(-8) M, and about 1921 +/- 357 molecules of labeled streptavidin are bound to each nanocontainer.

Hydrogen ion titration of alkyldimethylamine oxides by 13C and 1H NMR and conventional methods

In the hydrogen ion titration of micelles, the degree of ionization of the micelle at a given pH has to be evaluated to obtain a p K a value of micelles ( K a being the proton dissociation constant) at the pH. We compared the degree of ionization obtained from 13C and 1H NMR spectra with that obtained from the stoichiometric method. We used dodecyldimethylamine oxide (C12DMAO) and hexyldimethylamine oxide (C6DMAO) to examine the titration behavior of micelles and monomers, respectively. We determined p K a values of amine oxides both in H 2O and D 2O. As to the monomer (C6DMAO), the degree of ionization from NMR, α NMR, coincided with that from the conventional stoichiometric method α. The difference of p K 1 of amine oxide monomer between D 2O and H 2O was about 0.5: p K 1(D) ≈ p K 1(H) + 0.5. The difference was about the same as that for carboxylic acids. As to the C12DMAO micelle, α NMR did not coincide with α over a considerable range of α. The NMR chemical shift might be influenced by micellar structure changes induced by the ionization, such as the sphere-to-rod transition. The intrinsic logarithmic dissociation constants of the micelle were 5.9 ± 0.1 for H 2O, and 6.5 ± 0.1 for D 2O.

Titration of hydrophobic polyelectrolytes using Monte Carlo simulations

The conformation and titration curves of weak (or annealed) hydrophobic polyelectrolytes have been examined using Monte Carlo simulations with screened Coulomb potentials in the grand canonical ensemble. The influence of the ionic concentration pH and presence of hydrophobic interactions has been systematically investigated. A large number of conformations such as extended, pearl-necklace, cigar-shape, and collapsed structures resulting from the subtle balance of short-range hydrophobic attractive interactions and long-range electrostatic repulsive interactions between the monomers have been observed. Titration curves were calculated by adjusting the pH-pK(0) values (pK(0) represents the intrinsic dissociation constant of an isolated monomer) and then calculating the ionization degree alpha of the polyelectrolyte. Important transitions related to cascades of conformational changes were observed in the titration curves, mainly at low ionic concentration and with the presence of strong hydrophobic interactions. We demonstrated that the presence of hydrophobic interactions plays an important role in the acid-base properties of a polyelectrolyte in promoting the formation of compact conformations and hence decreasing the polyelectrolyte degree of ionization for a given pH-pK(0) value.

Effect of osmolytes on the interaction of flavin adenine dinucleotide with muscle glycogen phosphorylase b

The effect of three osmolytes, trimethylamine N-oxide (TMAO), betaine and proline, on the interaction of muscle glycogen phosphorylase b with allosteric inhibitor FAD has been examined. In the absence of osmolyte, the interaction is described by a single intrinsic dissociation constant (17.8 μM) for two equivalent and independent binding sites on the dimeric enzyme. However, the addition of osmolytes gives rise to sigmoidal dependencies of fractional enzyme-site saturation upon free inhibitor concentration. The source of this cooperativity has been shown by difference sedimentation velocity to be an osmolyte-mediated isomerization of phosphorylase b to a smaller dimeric state with decreased affinity for FAD. These results thus have substantiated a previous inference that the tendency for osmolyte-enhanced self-association of dimeric glycogen phosphorylase b in the presence of AMP was being countered by the corresponding effect of molecular crowding on an isomerization of dimer to a smaller, nonassociating state.

Binding of lead ion to bovine serum albumin studied by ion selective electrode.

The binding of Pb2+ to bovine serum albumin (BSA) at neutral pH was studied using lead ion selective electrode. The binding data was treated according to Scatchard Equation. The number of binding classes and the number of binding sites, intrinsic dissociation constants and stepwise binding constants for each class were determined. Two binding classes were found. Four binding sites in the first class and five binding sites in the second class were determined. Binding in the first class was stronger than in the second. Similar binding studies were carried out with heat treated BSA. It was found that not only the number of binding sites but also the strength of binding increases upon heat treatment.

Extension of Donnan theory to predict calcium ion exchange on phenolic hydroxyl sites of unbleached kraft fibers

The Donnan theory is extended to formulate a model for determination of the distribution coefficient of calcium in slurries of fully bleached and unbleached kraft fibers at different pH, taking into account the presence of both carboxylic and phenolic groups in the fibers. The intrinsic dissociation constants of the carboxylic acid groups and phenolic hydroxyl, which are the key inputs of the extended model, were determined by potentiometric titration. The extension improves the model prediction significantly, particularly when the presence of phenolic lignin in fibers becomes significant. However, when the pH exceeds 10, the model underestimates the distribution coefficient, suggesting that other factors may influence the fiber charge. The structural changes of the fiber wall at high pH and the presence of hydroxyl groups on the cellulose, which ionize at high pH, may be major factors.

Channel-opening kinetics of GluR6 kainate receptor.

GluR6 is an ionotropic glutamate receptor subunit of the kainate subtype. It plays an essential role in synaptic plasticity and epilepsy. We expressed this recombinant receptor in HEK-293 cells and characterized the glutamate-induced channel-opening reaction, using a laser-pulse photolysis technique with the caged glutamate (gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate). This technique permits glutamate to be liberated photolytically from the caged glutamate with a time constant of approximately 30 micros. Prior to laser photolysis, the caged glutamate did not activate the GluR6 channel, nor did it inhibit or potentiate the glutamate response. At the transmembrane voltage of -60 mV, pH 7.4 and 22 degrees C, the channel-opening and -closing rate constants were determined to be (1.1 +/- 0. 4) x 10(4) and (4.2 +/- 0.2) x 10(2) s(-1), respectively. The intrinsic dissociation constant of glutamate and the channel-opening probability were found to be 450 +/- 200 microM and 0.96, respectively. These constants are derived from a minimal kinetic mechanism of the channel activation involving the binding of two glutamate molecules. This mechanism describes the time course of the open-channel form of the receptor as a function of glutamate concentration. On the basis of the channel-opening rate constants obtained, the shortest rise time (20-80% of the receptor current response) or the fastest time by which the GluR6Q channel can open is predicted to be 120 micros. The open-channel form of the receptor determines the transmembrane voltage change, which in turn controls synaptic signal transmission between two neurons. The comparison of the channel-opening kinetic rate constants between GluR6Q and GluR2Q(flip), reported in the companion paper, suggests that at a glutamate concentration of 100 microM, for instance, the integrated neuronal signal will be dominated by a slower GluR6Q receptor response, as compared to the GluR2Q(flip) component.

Competitive Adsorption of Hydrogen, Calcium, Potassium, Magnesium, and Aluminum on Banana Roots: Experimental Data and Modeling

Aluminum (Al) adsorption on root exchange sites is one of the mechanisms that can be responsible for Al toxicity. Experimental measurements of calcium (Ca), magnesium (Mg), potassium (K), and Al adsorption on the main root axes of 5 banana cultivars (Grande Naine, Agbagba, Obino l'Ewaï, Kayinja, and Igitsiri) were carried out at pH 3.75 and 4.25. The data obtained were used to calibrate a mathematical model describing ion exchange as arising from Donnan effects and specific surface‐ion interactions. Adsorption of Ca, K, Mg, and Al ions increased with their concentration in solution and with increasing pH. The depletion rate of Ca, Mg, and K in the presence of the low Al concentration was greater at pH 4.25 than at pH 3.75. Therefore, any model based on constant exchange selectivity would fail to describe such complex equilibria. The calibration of the model involves to fix values for the intrinsic dissociation constants (KR−X ;) of cations (X) with root exchange sites (R−). Values from the literature were used for H‐surface (KR−H ;=10−3.2) and Ca‐surface (K R2– Ca=10−1.1) complexes. No specific ion‐surface interaction was assumed for K (KR−K ;=106) and a value of K R2– Mg=1 was found adequate for Mg‐surface complexes. Non‐linear regression led to K R3– Al=10(−3.78± 0.024). Introducing possible R– Al2+, R2– Al+, R–Al(OH)2, and R2–Al(OH) complexes did not improve the fitting. Using one single set of parameters for all experiments on the 5 banana cultivars resulted in good fitting of Al and Mg adsorption, and slight underestimation of Ca adsorption, although good estimate was obtained with pure Ca solutions. We conclude that Al is very efficient in competing with other cations for root exchange sites despite the low Al concentrations usually occurring in soil solutions. Magnesium was confirmed to form only little association with fixed charged groups, so that Al loading on roots occurs mainly to the detriment of Mg.

Titration of Fatty Acids in Sugar-Derived (APG) Surfactants: A 13C NMR Study of the Effect of Headgroup Size, Chain Length, and Concentration on Fatty Acid pKa at a Nonionic Micellar Interface

The pK(a) of a C-13-enriched fatty acid probe, tetradecanoic acid, was investigated in a variety of sugar-derived (APG) surfactants by using C-13 NMR to monitor the chemical environment of the probe during titration with base. There was an increase of approximately 2 pK(a) units for the probe within the micellar systems compared to the value of acetic acid in water. Analysis shows that the interfacial environment of alkylglucoside surfactants is similar to, although slightly more polar than, that of ethylene glycol-derived (EO) nonionic surfactants, and there is no indication that the alkylglucoside surfactants are deprotonated at pH levels between 2 and 10. The average pK(a) of the fatty acid probe in the APGs is measured to be 6.3, and it is argued that this value s an appropriate choice for pK(a)(0), the intrinsic dissociation constant for an acid at a hydrophobic/hydrophilic interface, when calculating the surface potential of charged systems. (Less)

COMPARISON OF ION EXCHANGE AND DONNAN EQUILIBRIUM MODELS FOR THE PH-DEPENDENT ADSORPTION OF SODIUM AND CALCIUM IONS ONTO KRAFT WOOD PULP FIBERS

ABSTRACT Management of nonprocess element (NPE) accumulation in pulp washing operations requires equilibrium models that predict the distribution of metals between the wash liquor and the pulp fibers. The overall goal of this study was to assess models for predicting the multi-component adsorption of hydrogen ions (H+), sodium ions (Na+), and calcium ions (Ca+2) onto bleached and unbleached kraft pulp fibers over a pH range of 2.7–11. As part of this study, binary equilibrium constants for hydrogen and metal ion exchange on carboxylate sites in bleached pulp (0.041 meq/g dry pulp) were measured at 25°C, with log K Na/Ca = −1.604 ± 0.119, log K H/Ca = 0.633 ± 0.087, and intrinsic dissociation constant pK io of 3.64 ± 0.46. Ion exchange and Donnan equilibrium models adequately predicted the multi-component equilibrium data for competitive adsorption of H+, Na+, and Ca+2 onto bleached kraft wood pulp fibers. The ion exchange model was fully predictive, whereas the Donnan model required that the solution pH be known. At pH 2.7–6, the Donnan model predicted the adsorption of Na+ and Ca+2 onto both bleached and unbleached wood pulp fibers better than the ion exchange model. The ion exchange model assumed that residual carboxylate in the pulp served as the only site for the competitive binding of hydrogen and metal ions. In contrast, the Donnan model assumed a non site-specific distribution of metal ions between charged fiber and external solution phases and a carboxylate site specific adsorption of hydrogen ions. Above pH 6, both models failed to predict that the calcium adsorption on unbleached brownstock pulp increased beyond the carboxylate site capacity, suggesting that other functional groups within the brownstock pulp with intrinsic dissociation constant values higher than carboxylate were providing additional binding sites for calcium.

The Human EMAP-like Protein-70 (ELP70) Is a Microtubule Destabilizer That Localizes to the Mitotic Apparatus

In this report, we show that the echinoderm microtubule (MT)-associated protein (EMAP) and related EMAP-like proteins (ELPs) share a similar domain organization with a highly conserved hydrophobic ELP (HELP) domain and a large tryptophan-aspartic acid (WD) repeat domain. To determine the function of mammalian ELPs, we generated antibodies against a 70-kDa human ELP and showed that ELP70 coassembled with MTs in HeLa cell extracts and colocalized with MTs in the mitotic apparatus. To determine whether ELP70 bound to MTs directly, human ELP70 was expressed and purified to homogeneity from baculovirus-infected Sf9 cells. Purified ELP70 bound to purified MTs with a stoichiometry of 0.40 +/- 0.04 mol of ELP70/mol of tubulin dimer and with an intrinsic dissociation constant of 0.44 +/- 0.13 microm. Using a nucleated assembly assay and video-enhanced differential interference contrast microscopy, we demonstrated that ELP70 reduced seeded nucleation, reduced the growth rate, and promoted MT catastrophes in a concentration-dependent manner. As a result, ELP70-containing MTs were significantly shorter than MTs assembled from tubulin alone. These data indicate that ELP70 is a novel MT destabilizer. A lateral destabilization model is presented to describe ELP70's effects on microtubules.

Hydrogen Ion Titration of Long Alkyl Chain Amine Oxide Micelles

The hydrogen ion titration of long alkyl chain amine oxide micelles was carried out at various salt concentrations, Cs, surfactant concentrations, CD, and temperatures T. The surfactants studied were alkyldimethylamine oxides (CnDAO) with hydrocarbon chain lengths of 10–16 (n=10, 12, 14, and 16), and N, N-bis(2-hydroxyethyl)alkylamine oxide (CnDHEAO) with hydrocarbon chain lengths of 10–14 (n=10, 12, and 14). The intrinsic dissociation constant exponents, pKM, of the micelle were greater than those of the monomer, pK1, for all systems examined. Thus, the introduction of electric charges is more favored on the micelle surface than to the monomer in solution. The values of pKM of CnDAO at 25°C in 0.1 M NaCl were dependent on the hydrocarbon chain length in the following order: C10 (5.5)<C12 (5.9)<C14 (6.3)≈C16 (45°C; 6.2). This order can be interpreted reasonably in terms of different curvatures of the micelles that are determined by the intrinsic packing parameter value modified by the proposed hydrogen bond between protonated and unprotonated species on the micelle surface. For CnDHEAO, we observed practically identical pKM values for different chain lengths. Comparing CnDAO with CnDHEAO with the same hydrocarbon chain length at the same Cs and CD, pKM values for CnAO were greater than those for CnDHEAO. Values of pKM were independent of Cs and CD examined for both CnDAO and CnDHEAO. Anomalous titration behavior in the low charge density region was attributed to inaccurate estimation for the blank titrations. The monomer concentrations in a range higher than critical micelle concentration (cmc) were evaluated from the hydrogen ion titration with the assumed Gibbs–Duhem relation. These values coincided with the cmc values for C10DAO and C12DHEAO.

Biochemical analyses of the interactions between human immunodeficiency virus type 1 Vpr and p6(Gag).

The nonstructural human immunodeficiency virus type 1 Vpr protein is packaged into progeny virions at significant levels (approximately 200 copies/virion). Genetic analyses have demonstrated that efficient Vpr packaging is dependent upon a leucine-X-X-leucine-phenylalanine (LXXLF) motif located in the p6(Gag) domain of the structural Gag polyprotein. Recombinant proteins spanning full-length Vpr (Vpr(1-97)) or the amino-terminal 71 amino acids (Vpr(1-71)) formed specific complexes with recombinant p6 proteins in vitro. Complex formation required an intact LXXLF motif and exhibited an intrinsic dissociation constant of approximately 75 microM. Gel filtration and cross-linking analyses further revealed that Vpr(1-71) self-associated in solution. Our experiments demonstrate that Vpr can bind directly and specifically to p6 and suggest that oligomerization of both Vpr and Gag may serve to increase the avidity and longevity of Vpr-Gag complexes, thereby ensuring efficient Vpr packaging.

Folding units in calcium vector protein of amphioxus: Structural and functional properties of its amino- and carboxy-terminal halves.

Muscle of amphioxus contains large amounts of a four EF-hand Ca2+-binding protein, CaVP, and its target, CaVPT. To study the domain structure of CaVP and assess the structurally important determinants for its interaction with CaVPT, we expressed CaVP and its amino (N-CaVP) and carboxy-terminal halves (C-CaVP). The interactive properties of recombinant and wild-type CaVP are very similar, despite three post-translational modifications in the wild-type protein. N-CaVP does not bind Ca2+, shows a well-formed hydrophobic core, and melts at 44 degrees C. C-CaVP binds two Ca2+ with intrinsic dissociation constants of 0.22 and 140 microM (i.e., very similar to the entire CaVP). The metal-free domain in CaVP and C-CaVP shows no distinct melting transition, whereas its 1Ca2+ and 2Ca2+) forms melt in the 111 degrees -123 degrees C range, suggesting that C-CaVP and the carboxy- domain of CaVP are natively unfolded in the metal-free state and progressively gain structure upon binding of 1Ca2+ and 2Ca2+. Thermal denaturation studies provide evidence for interdomain interaction: the apo, 1Ca2+ and 2Ca2+ states of the carboxy-domain destabilize to different degrees the amino-domain. Only C-CaVP forms a Ca2+-dependent 1:1 complex with CaVPT. Our results suggest that the carboxy-terminal domain of CaVP interacts with CaVPT and that the amino-terminal lobe modulates this interaction.

Potentiometric titration and enthalpy evaluation of horseradish peroxidase in the presence of n-dodecyl trimethylammonium bromide

The reversible proton dissociation equilibria of horseradish peroxidase (HRP) is investigated in 100 mM NaCl solution at 25°C and over a pH range of 3.0–11.0 in the absence and presence of n-dodecyl trimethylammonium bromide (DTAB). Intrinsic dissociation constants for the carboxyl groups of acidic residues and also for the imidazole side chains of histidine residues in HRP are estimated using the Tanford expression. From the obtained titration curve the intrinsic pK a (pK a,int) of titratable groups, the electrostatic free energy ( W el), the enthalpy of unfolding (ΔH u) and especially the enthalpy of ionisation (ΔH ion) for the enzyme molecule are estimated. Also the state of ionisation is discussed in terms of ionisation enthalpy changes. Results indicate that 79% of the total carboxyl groups are present in the form of buried or masked groups, which are not exposed to titration. While in the presence of DTAB, 65% of these groups remain buried and the isoelectric pH varies from 8.8 (in the absence of DTAB) to 5.5 (in the presence of DTAB). In the absence of surfactant no conformational changes are observed in the whole pH region of the titration experiment.

The Size and Curvature of Anionic Covesicle Substrate Affects the Catalytic Action of Cytosolic Phospholipase A2

Cytosolic phospholipase A2(cPLA2) is normally located in the cytosol, but in response to cellular activation the enzyme binds to the membrane at the lipid/water interface where it catalyzes the hydrolysis of thesn-2 ester of arachidonate-containing phospholipids. Synthetic phospholipid vesicle systems have been used in kinetic and mechanistic analyses of cPLA2, but these systems result in a rapid loss of enzyme activity. In the present research, covesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) containing ≤10 mol% 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) as substrate were used to show that this premature cessation of enzyme-catalyzed hydrolysis is dependent on vesicle size with 25-nm-diameter vesicles supporting little activity as compared to 100-, 200-, and 400-nm vesicles. This suggests that the curvature of the vesicle may shift a conformational equilibrium toward an enzyme state which does not support activity. Interestingly, the presence of 30% (v/v) glycerol greatly enhanced the activity of the enzyme, although vesicle size-dependent premature cessation of hydrolysis was still observed. While the premature cessation of hydrolysis in the absence of glycerol is accompanied by enzyme inactivation, little inactivation occured in the presence of glycerol, indicating that premature cessation and inactivation are not absolutely coupled. When using this covesicle substrate system under conditions (6–10 mM CaCl2) where the vesicles are fusing, no premature cessation of hydrolysis has been observed. This is despite a mean vesicle diameter of 400–450 nm under vesicle-fusing conditions, which is comparable to the largest vesicles used under nonfusing conditions (0.5 mM CaCl2) where considerable premature cessation of hydrolysis was observed. Since DMPM has an intrinsic active site dissociation constant at least 330 times larger than that of PAPC, the optimum conditions for conducting kinetic and mechanistic analyses of cPLA2with this covesicle substrate is one in which cPLA2is assayed in the presence of glycerol and with fusion-inducing concentrations of calcium. The use of 1,2-dioleoyl-sn-glycero-3-phosphomethanol (DOPM) instead of DMPM in this system supports much less activity and adds the complication of a strong affinity of DOPM for the active site.

Behavior of Nonalternating Maleic Acid Copolymers in Aqueous Solution

A nonalternating 1:3 maleic acid−methyl methacrylate copolymer was synthesized and characterized, and its polyelectrolyte behavior was studied by potentiometric and conductometric titrations. Two intrinsic dissociation constants were found equal to 3.5 and 7.6, and only a slight influence of the nature of the counterions and of the presence of added salt (0.1 M) was observed. The transport coefficients, determined from conductometry, for mono- and divalent cations were in relatively good agreement with the values calculated from Manning theory, especially at low polymer concentrations. A conformational change around αN ∼ 0.25 was confirmed by means of viscometric and fluorescence measurements.