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E Nachliel

Publications and source records attributed to E Nachliel.

33 records · Page 2Linked to original sources
PubMed1993

Gaugement of the inner space of the apomyoglobin's heme binding site by a single free diffusing proton. II. Interaction with a bulk proton.

The reaction mechanism and the dynamic aspects of protonation of a defined moiety located inside an aqueous cavity in a protein were monitored by time resolved spectroscopy using the pyranine apomyoglobin complex as a model (Shimoni, Tsfadia, Nachliel, and Gutman, 1993, Biophys. J. 64:472-479). The reaction was synchronized by a short laser pulse and the reprotonation of the ground state pyranine anion (phi O-) was monitored, in the microsecond time scale, by its transient absorption at 457 nm. The observed signal was reconstructed by a numeric solution of nonlinear, coupled differential equations which account for the direct reaction of phi O- with bulk proton and by proton transfer from the nearby amino acids: His 64, Asp 44, Asp 60, and Glu 59. A unique combination of rate constant was obtained which quantitates the contribution of each pathway to the overall relaxation process. In the first phase of the dynamics phi O- abstracts a proton from the nearby protonated histidine. The bulk proton interacts preferentially with the cluster of three carboxylates and immediately shuttled to the deprotonated histidine. The high proximity of the reactive groups and the strong electrostatic forces operating inside the heme binding cavity render the rate of proton transfer in the site ultrafast.

Animals↗
PubMed1992

Quantitation of physical-chemical properties of the aqueous phase inside the phoE ionic channel.

The anion-specific channel of the phoE porine is a miniature body of water surrounded by peptide walls. The physical and chemical properties of the water in such a microscopic space were measured by monitoring the dynamics of a well-studied reaction--the protolytic dissociation of a strong acid. To attain this purpose, we allowed pyranine (8-hydroxypyrene-1,3,6-trisulfonate) to bind to the anion-specific channel. The dye is bound, with a 1:1 stoichiometry, with a delta G = -9.5 kcal/mol. Photoexcitation of the dye, to its first electronic singlet state (phi OH*), renders it very acidic and the hydroxyl proton dissociates to H+ and excited anion (phi O*-). We employed single photon-counting time-resolved fluorimetry, to monitor the reversible dissociation of pyranine as it proceeds within the channel and reconstructed the observed signal by a numerical integration of the differential diffusion equation pertinent for a proton within the channel. The most characteristic feature of the water-filled channel, is the intensified electrostatic interactions attained by the low dielectric constant of the diffusion space, epsilon eff = 24. For this reason, the electric field of a few positive charges is sufficient to ensure that an anion entering the channel will be effectively sucked in. The interaction of the water molecules with the peptide structure forming the channel affects the physical properties of the water. Their capacity to conduct proton, quantitated by the protons diffusion coefficient (4.5.10(-5) cm2/s), is reduced by 50% with respect to that of bulk water. The activity of the water in the channel is reduced to alpha H2O = 0.966. These observation are in accord with our previous studies of water in small defined cavities in proteins.

Algorithms↗
PubMed1991

Proton transfer dynamics in the nonhomogeneous electric field of a protein.

By adsorption of pyranine (8 hydroxypyrene 1, 3, 6 trisulfonate) to lysozyme we create on the positively charged protein a fluorophoric site with a total charge of -3. Photo dissociation of the dye's hydroxyl proton changes its absorption and fluorescence spectrum, permitting a continuous monitoring of the reprotonation dynamics. Absorbance measurements in the microsecond time scale monitor how the bulk protons penetrate the Coulomb cage of the bound dye. Time-resolved fluorescence monitors how the proton is escaping out of the Coulomb cage of the bound dye. These probe reactions were studied with a series of dye-enzyme complexes where the number of free carboxylate was reduced by amidation, increasing the total charge of the complex from +5 to +12.6. The time-resolved measurements demonstrate the complexity of the electric field in the immediate vicinity of the dye. It is consistent with a negative potential wall (of the anion) surrounded by a positive potential wall of proteinaceous moieties.

Adsorption↗
PubMed1990

Proton dissociation dynamics in the aqueous layer of multilamellar phospholipid vesicles.

The water layers interspacing between the phospholipid membranes of a multilamellar vesicle are 3-10 water layers across and their width is adjusted by osmotic pressure (Parsegian, V.A., et al., 1986. Methods Enzymol. 127:400-416). In these thin water layers we dissolved pyranine (8 hydroxypyrene 1,3,6 trisulfonate), a compound which, upon photo excitation, ejects it hydroxy proton with time constant of 100 psec. (Gutman, M. 1986. Methods Enzymol. 127:522-538). In the present study we investigated how the width of the aqueous layer, the density of phosphomoieties on the membrane's surface and the activity of water in the layer affect the capacity of protons to diffuse out from the electrostatic cage of the excited anion before it decays to the ground state. Using a combination of steady-state and subnanosecond time-resolved fluorescence measurements we determined the average number of proton excited-anion recombinations before the proton escapes from the Coulomb cage. The probability of recombination in thin water layer is significantly higher than in bulk. The factor contributing most to enhancement of recombination is the diminished water activity of the thin aqueous layer. The time frame for proton escape from an electrostatic trap as big as a membrane-bound protein is 3 orders of magnitude shorter than turnover time of membrane-bound enzymes. Thus the effects of local forces on proton diffusion, at the time scale of physiological processes, is negligible.

Arylsulfonates↗
PubMed1989

Dynamics of proton diffusion within the hydration layer of phospholipid membrane.

The diffusion of protons at the immediate vicinity of (less than 10 A from) a phospholipid membrane is studied by the application of the laser-induced proton pulse. A light-sensitive proton emitter (8-hydroxypyrene-1,3,6-trisulfonate) was trapped exclusively in the hydration layers of multilamellar vesicles made of egg phosphatidylcholine, and the protons were dissociated by a synchronizing laser pulse. The recombination of the proton with pyranin anion was monitored by time-resolved spectroscopy and analyzed by a diffusion-controlled formalism. The measured diffusion coefficient is only slightly smaller than the diffusion coefficient of proton in bulk water. Modulating the width of the hydration layer by external pressure had a direct effect on the diffusibility of the proton: the narrower the hydration layer, the slower is the diffusion of protons.

Arylsulfonates↗
PubMed1987

Time-resolved protonation dynamics of a black lipid membrane monitored by capacitative currents.

The laser-induced proton pulse (Gutman, M. (1986) Methods Enzymol. 127, 522-538) was used for transient protonation of one side of a black lipid membrane. The charging of the membrane drives an electric (voltage or current) signal selectively representing the fast proton exchange at the membrane/electrolyte interface. The sensitivity of the electric signal to the presence of buffer indicates that proton transfer is measured, not some dyes or membrane photoelectric artifact. The same event can be visualized in an analogous system consisting of a pH indicator adsorbed to neutral detergent-phospholipid mixed micelles. The time-resolved light absorption transient is equivalent to the electrically determined transient charging of the membrane surface. The sensitivity of the current measurement exceeds the spectrophotometric method by 6-8 orders of magnitudes. As little as 10(-18) mol of H+ reacting with 0.75 mm2 of the membrane surface can be monitored in a time-resolved observation. Both types of observed transients were accurately reconstructed by the numerical solution of coupled, non-linear, differential equations describing the system. The rate constants of the various proton transfer reactions were calculated and found to be of diffusion controlled reactions. There is no evidence for any barrier at the interface which either prevents protons from reaching the membrane, or keeps proton on the interface. The electric measurements can be applied for monitoring proton transfer kinetics of complex biomembrane preparations.

Hydrogen-Ion Concentration↗
PubMed1985

Effect of buffer on kinetics of proton equilibration with a protonable group.

The laser-induced proton pulse generates a massive, brief, proton pulse capable of perturbing biochemical equilibria. The time resolution of the monitoring system can follow the diffusion-controlled protonation of specific sites on macromolecular bodies [Gutman, M. (1984) Methods Biochem. Anal. 30, 1-103]. In order to apply this method in enzymology, one must first evaluate how the buffer capacity of biochemical systems (substrates and proteins) will affect the observed dynamics. Unlike equilibrium measurements, where buffer is an inert component, in kinetic studies buffer modulates the observed dynamics. In this paper we analyze the effect of buffer on the dynamics of protonation in a model system. We describe the experimental technique and introduce the mathematical formalism that determines the various rate constants involved in the reaction. The analysis of the experiments indicates that in buffered solution proton flux is carried by two mechanisms: (A) proton dissociation followed by free proton diffusion; (B) collisional proton transfer between small diffusing solutes. We demonstrate how to evaluate the contribution of each pathway to the overall proton flux.

Arylsulfonates↗
PubMed1985

Kinetic analysis of protonation of a specific site on a buffered surface of a macromolecular body.

The kinetics of protonation of a specific site on a macromolecular structure (micelle) in buffered solution was studied with the purpose of evaluating the effect of buffer on the observed dynamics. The experimental system consisted of the following elements: Brij 58 micelles serving as homogeneous uncharged macromolecular bodies, bromocresol green, a well-adsorbed proton detector, and 2-naphthol-3,6-disulfonate as a proton emitter in the bulk. Imidazole was the mobile buffer while neutral red, which has a high affinity for the micellar surface, served as the immobile buffer. An intensive laser pulse ejects a proton from the proton emitter, and the subsequent proton-transfer reactions are measured by fast spectrophotometric methods. The dynamics of proton pulse in buffered solution are characterized by a very rapid trapping of the discharged protons by the abundant buffer molecules. This event has a major effect on the kinetic regime of the reaction. During the first 200 ns the proton flux is rate limited by free-proton diffusion. After this period, when the free-proton concentration decayed to the equilibrium level, the relaxation of the system is carried out by the diffusion of buffer. Thus in the buffered biochemical system, at neutral pH, most of proton flux between active sites and bulk is carried out by buffer molecules--not by diffusion of free protons. Surface groups on a high molecular weight body exchange protons among them at a very fast rate. This reaction has a major role on proton transfer from a specific site to the bulk.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1984

Kinetic analysis of proton transfer between reactants adsorbed to the same micelle. The effect of proximity on the rate constants.

The dense packing of protogenic enzymes on the coupling membrane can furnish a route for a rapid proton flux which may avoid the adjacent bulk phase. In order to evaluate the role of proximity between reactants on the rate constant of proton transfer we generated a model system consisting of 2-naphthol and pH indicator (bromocresol green) both adsorbed on the same micelle of unchanged detergent. Excitation of the 2-naphthol by a short intensive laser pulse lowers its pK with subsequent synchronized proton ejection. The discharged protons are detected by their reaction with the indicator using a fast transient absorption technique. Evidence is produced that under certain conditions all of the observed proton flux represents proton transfer between 2-naphthol and indicator molecules sharing the same micelle. In this model system the entire proton flux proceeds through an aqueous phase fully accessible to phosphate ions. The high proximity of the reactants (the separation can not exceed approximately 6 nm) has a marked effect on the rate constant of the reaction k = 2.0 +/- 0.5 X 10(11) M-1 s-1. In spite of this extremely fast rate of reaction we observe unhindered competition, for the surface discharged proton, between the surface-bound reactants and phosphate ions in the bulk. Thus even in proton transfer between closely packed reactants on an interface, the diffusion of the proton is not limited to the interface. This finding implies that on bioenergetic surface the electrochemical potential of the proton on the surface will equal that of the bulk.

Adsorption↗
PubMed1983

Kinetic analysis of the protonation of a surface group of a macromolecule.

The dynamics of proton transfer between a surface-attached acidic moiety and the bulk of the solution was measured using the laser-induced proton pulse technique. [Gutman, M., Huppert, D. and Pines, E. (1981) J. Am. Chem. Soc. 103, 3709-3713]. The model system for this study consists of pH indicators (either neutral red or bromcresol green) adsorbed on Brij 58 micelles, as defined targets for protonation and a non-adsorbed proton emitter (2-naphthol-3,6-disulfonate) for generation of protons in bulk. The reaction was measured with 50-ns time resolution over a time period of about 200 microseconds. The results were analyzed by a numerical solution of the coupled nonlinear differential equation corresponding with the reaction system. [Gutman, M., et al. (1983) J. Am. Chem. Soc. 105, 2210-2216]. Quantitative analysis reveals two independent reactions which govern the observed dynamics: (a) a diffusion-controlled reaction between the proton and the surface targets; (b) translocation of the protonated target between the hydration layer of the interface and a more hydrophobic one. The contribution of the translocation reaction to the dynamics of surface protonation is more pronounced for compounds like carboxylates or phenolates which increase their hydrophobicity upon protonation. Amines and azoaromatic structures are more hydrophilic in their protonated states, the dynamics of their protonation is less affected by post-protonation distribution within the microenvironments of the interface. The interrelation between the partial rate constants and the macroscopic time constants and equilibrium parameters is analyzed.

Bromcresol Green↗
PubMed1982

Direct measurement of proton transfer as a probing reaction for the microenvironment of the apomyoglobin heme-binding site.

Aromatic alcohols fluoresce at different wavelengths in their neutral (phiOH*) and anionic (phiO-*) excited states. Consequently, time-resolved fluorescence measurements, at the respective wavelengths, can be used for measuring the rates of proton dissociation and recombination of the excited molecule. As the lifetime of the excited state is very short (a few nanoseconds), the measured reaction is that which takes place in a volume corresponding to the diffusion distance of the proton during the lifetime of the excited state. 8-Hydroxypyrene 1,3,6-trisulfonate (pK = 7.7, pK* = 0.5) is bound to apomyoglobin with a stoichiometry of 1:1. In the bound state its neutral form fluorescence increase 20-fold. The binding affinity is pH-dependent. Two protonatable groups, with pK = 6.5, participate in the stabilization of the negatively charged ligand in the binding site. The ligand is bound only to the apoprotein and is displaced from its site by hemin. Thus we suggest that the ligand is bound to the heme binding site of apomyoglobin. Time-resolved fluorescence of the bound ligand yields the rate constants of proton dissociation and recombination as taking place within the heme binding cavity of apomyoglobin. The rate of proton dissociation is slowed to 7% of the rate measured for the free ligand. Such a slow dissociation indicates a strong interaction of the water in the cavity with the walls [Gutman, M., Huppert, D., and Nachliel, E. (1982) Eur. J. Biochem. 121, 637-642]. The water activity in the site is equivalent to alpha (H2O) = 0.67.

Apoproteins↗
PubMed1982

Kinetic studies of proton transfer in the microenvironment of a binding site.

Excitation of 8-hydroxypyrene 1,3,6-trisulfonate to its first electronic singlet state converts the compound from weak base (pK degrees = 7.7) into a strong acid (pK* = 0.5). The dissociation of the proton in water or dilute salt solution is a very fast reaction, K12 = 1 X 10(10) S-1. In concentrated salt solutions the dissociation is slowed as an exponential function of the chemical activity of the water in the solution. This kinetic parameter has been used to gauge the properties of the microenvironment of the binding sites of bovine serum albumin at which this compound is bound. Time-resolved fluorometry reveals two distinct steps: a rapid dissociation of the proton with tau = 300 +/- 40 ps which lasts approximately 0.5 ns, followed by a slower reaction with tau = 3.3 ns. The first rapid phase represents proton dissociation taking place in the binding site. From the rate constant K = 3.3 X 10(9) s-1 we estimate that the ability of the water molecules in the site to hydrate the ejected proton is equivalent to a salt solution with water activity of 0.85. The slow phase represents the escape of the proton from the binding site. The rate of the escape, 1.4 X 10(8) s-1, is significantly slower than diffusion-controlled dissociation. It is concluded that the shape of the site or its lowered proton conductivity do not allow a rapid escape of the proton to the bulk. Still it should be remembered that the escape of the proton is 10(5)-10(6)-times faster than a typical turnover of an enzyme.U

Arylsulfonates↗
PubMed1981

Probing the micelle/water interface by a rapid laser-induced proton pulse.

The laser-induced pH jump (Gutman, M. and Huppert, D.J. (1979) Biochem. Biophys. Methods 1, 9-19) has a time resolution capable of measuring the diffusion-controlled rate constant of proton binding. In the present study we employed this technique for measuring the kinetics of protonation-deprotonation of surface groups of macromolecules. The heterogeneous surface of proteins excludes them from serving as a simple model, therefore we used micelles of a neutral detergent (Brij 58) as a high molecular weight structure. The charge was varied by the addition of a low concentration of sodium dodecyl sulfate and the surface group with which the protons react was an adsorbed pH indicator (bromocresol green or neutral red). The dissociation of a proton from adsorbed bromocresol green is slower than that from free indicator. This effect is attributed to the enhanced stabilization of the acid form of the indicator in the pallisade region of the micelle. The pK shift of bromocresol green adsorbed on neutral micelles is thus quantitatively accounted for by the decreased rate of proton dissociation. Indicators such as neutral red, which are more lipid soluble in their alkaline form, do not exhibit such decelerated proton dissociation in their adsorbed state nor a pK shift on adsorption to neutral micelles. The protonation of an indicator is a diffusion-controlled reaction, whether it is free in solution or adsorbed on micelles. By varying the electric charge of the micelle this rate can be accelerated or decelerated depending on the total charge of the micelle. The micellar charge calculated from this method was corroborated by other measurements which rely only on equilibrium parameters. The high time resolution of the pH jump is exemplified by the ability to estimate the diffusion coefficient of protons through the hydrated shell of the micelle.

Colloids↗