M. Gutman

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.

THE DYNAMIC FEATURE OF THE PROTON COLLECTING ANTENNA OF A PROTEIN SURFACE

The surface of a protein is a condense matrix of proton binding sites having wide range of pK values. In domains where proton uptake is a part of the catalytic cycle, the surface sites endow the region with special kinetic features which represents the ensemble properties of the proton binding sites. Low pK carboxylate can merge their Coulomb cages to form an extended proton trap, where the binding of a proton to one is rapidly followed by shuttling to another. Neutral pK moieties can act as a temporary proton reservoir which delay the proton at the site, enhancing the probability that upon dissociation it will be taken up by the other elements of the active site. These features had been experimentally identified in small model molecules, where detailed kinetic analysis was carried out. On the base of these measurements the dynamics of protonation of the proton entry sites of bacteriorhodopsin and cytochrome oxidase were investigated.

Dynamic studies of proton diffusion in mesoscopic heterogeneous matrix: II. The interbilayer space between phospholipid membranes.

The thin water layer, as found in chloroplast or mitochondria, is confined between low dielectric amphypathic surfaces a few nm apart.The physical properties of this mesoscopic space, and how its dimensions affect the rate of chemical reactions proceeding in it, is the subject for this study.The method selected for this purpose is time resolved fluorometry which can monitor the reversible dissociation of a proton from excited molecule of pyranine (8 hydroxy pyrene 1,3,6 tri sulfonate) trapped in thin water layers of a multilamellar vesicle made of neutral or slightly charged phospholipids.The results were analyzed by a computer program of N. Agmon (Pines, E., D. Huppert, and N. Agmon. 1988. J. Am. Chem. Soc. 88:5620-5630) that simulates the diffusion of a proton, subjected to electrostatic attraction, in a thin water layer enclosed between low affinity, proton binding surfaces. The analysis determines the diffusion coefficient of the proton, the effective dielectric constant of the water and the water accessibility of the phosphomoieties of the lipids.These parameters were measured for various lipids [egg-phosphatidylcholine (egg PC), dipalmitoyl phosphatidylcholine (DPPC), cholesterol + DPPC (1:1) and egg PC plus phosphatidyl serine (9:1)] and under varying osmotic pressure which reduces the width of the water layer down to approximately 10 approximately across.WE FOUND THAT: (a) The effective dielectric constant of the aqueous layer, depending on the lipid composition, is approximately 40. (b) The diffusion coefficient of the proton in the thin layer (30-10 approximately across) is that measured in bulk water D = 9.3 10(-5) cm(2)/s, indicating that the water retains its normal liquid state even on contact with the membrane. (c) The reactivity of the phosphomoiety, quantitated by rate of its reaction with proton, diminishes under lateral pressure which reduces the surface area per lipid.We find no evidence for abnormal dynamics of proton transfer at the lipid water interface which, by any mechanism, accelerates its diffusion.

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 dyes 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.

The mechanism of monensin-mediated cation exchange based on real time measurements.

Monensin is an ionophore that supports an electroneutral ion exchange across the lipid bilayer. Because of this, under steady-state conditions, no electric signals accompany its reactions. Using the Laser Induced Proton Pulse as a synchronizing event we selectively acidify one face of a black lipid membrane impregnated by monensin. The short perturbation temporarily upsets the acid-base equilibrium on one face of the membrane, causing a transient cycle of ion exchange. Under such conditions the molecular events could be discerned as a transient electric polarization of the membrane lasting approx. 200 microseconds. The proton-driven chemical reactions that lead to the electric signals had been reconstructed by numeric integration of differential rate equations which constitute a maximalistic description of the multi equilibria nature of the system (Gutman, M. and Nachliel, E. (1989) Electrochim. Acta 34, 1801-1806). The analysis of the reactions reveals that the ionic selectivity of the monensin (H+ > Na+ > K+) is due to more than one term. Besides the well established different affinity for the various cations, the selectivity is also derived from a large difference in the rates of cross membranal diffusivities (MoH > MoNa > MoK), which have never been detected before. (v) Quantitative analysis of the membranes crossing rates of the three neutral complexes reveals a major role of the membranal dipolar field in regulating ion transport. The diffusion of MoH, which has no dipole moment, is hindered only by the viscose drag. On the other hand, the dipolar complexes (MoNa and MoK) are delayed by dipole-dipole interaction with the membrane. (vi) Comparison of the calculated dipoles with those estimated for the crystalline conformation of the [MoNa(H2O)2] and [MoK(H2O)2] complexes reveals that the MoNa may exist in the membrane at its crystal configuration, while the MoK definitely attains a structure having a dipole moment larger than in the crystal.

The preparation and properties of the membranal DPNH dehydrogenase from Escherichia coli

Abstract 1. The membrane bound DPNH oxidase of Escherichia coli can reduce the artificial electron acceptors: ferricyanide, dichlorophenolindophenol (DCIP) and menadione. All three are reduced by the flavoprotein of DPNH oxidase, but at different sites of the enzyme. 2. Freeze-drying of the bacterial membranes causes a selective detachment of DPNH dehydrogenase (DPNH: (acceptor) oxidoreductase, EC 1.6.99.3) from the membranes. This solubilization is accompanied by a decrease of K m (K 3 Fe(CN) 6 ) from 2.0 to 0.25 mM, while no change is detected in K m (DPNH). This enzyme is not the DPNH diaphorase found in the bacteria. 3. DPNH dehydrogenase of E. coli is a metalloflavoprotein, containing non-heme iron, labile sulfide, FMN and FAD. 4. Reduction of the enzyme with DPNH in the absence of electron acceptor (ferricyanide or DCIP) causes a rapid and irreversible change to a less active state, Form II. Form II is characterized by a higher K m (DPNH) and slower v max ., while the K m (K 3 Fe(CN) 6 ) remains unchanged. 5. The transformation of the enzyme to Form II is accompanied by the reduction of the non-heme iron component. The role of non-heme iron in the enzymic reaction is discussed.

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.

Studies on the activation of plasminogen I. Preparation and properties of an insoluble derivative of streptokinase

1. 1. An insoluble derivative of streptokinase was prepared by coupling streptokinase to a diazotized copolymer of p-aminophenylalanine and leucine. 2. 2. The product was shown to be functionally identical to soluble streptokinase. 3. 3. Some proteins, especially casein, were found to accelerate activation of plasminogen with bound streptokinase; other proteins, e.g. albumins, had no influence on the reaction, whereas tosyl-l-argininemethyl ester inhibited it. 4. 4. Using bound streptokinase, it was possible to separate the activation phase from the caseinolytic phase in the plasmin system.

Time-Resolved Study of the Inner Space of Lactose Permease

Pyranine (8-hydroxy pyrene-1,3,6-trisulfonate) is a commonly used photoacid that discharges a proton when excited to its first electronic singlet state. Follow-up of its dissociation kinetics reveals the physicochemical properties of its most immediate environment. At vanishing ionic strength the dye adsorbs to the Escherichia coli lactose permease with stoichiometry of 1:1 and an association constant of 2.5 x 10(5) M(-1). The reversal of the binding at high ionic strength and the lower pK value of the bound dye imply that positive charge(s) stabilize the dye in its site. The fluorescence decay curve of the bound dye was measured by time-correlated single photon counting and the measured transient was subjected to kinetic analysis based on the geminate recombination model. The analysis indicated that the binding domain is a cleft (between 9 and 17 A deep) characterized by low activity of water (a((water)) = 0.71), reduced diffusivity of protons, and enhanced electrostatic potential. The binding of pyranine and a substrate are not mutually exclusive; however, when the substrate is added, the dye-binding environment is better solvated. These properties, if attributed to the substrate-conducting pathway, may explain some of the forces operating on the substrate in the cavity. The reduced activities of the water strips the substrate from some of its solvation water molecules and replace them by direct interaction with the protein. In parallel, the lower dielectric constant enhances the binding of the proton to the protein, thus keeping a tight seal that prevents protons from diffusing.

Utilization of monensin for detection of microdomains in cholesterol containing membrane.

The effect of cholesterol on the monensin mediated proton-cation exchange reaction was measured in the time-resolved domain. The experimental system consisted of a black lipid membrane equilibrated with monensin (Nachliel, E., Finkelstein, Y. and Gutman, M. (1996) Biochim. Biophys. Acta 1285, 131-145). The membrane separated two compartments containing electrolyte solutions and pyranine (8-hydroxypyrene 1,3,6-trisulfonate) was added on to one side of the membrane. A short laser pulse was used to cause a brief transient acidification of the pyranine-containing solution and the resulting electric signal, derived from proton-cation exchange, was measured in the microsecond time domain. Incorporation of cholesterol had a clear effect on the electric transients as measured with Na+ or K+ as transportable cations. The measured transients were subjected to rigorous analysis based on numeric integration of coupled, non-linear, differential rate equations which correspond with the perturbed multi-equilibria state between all reactants present in the system. The various kinetic parameters of the reaction and their dependence on the cholesterol content had been determined. On the basis of these observations we can draw the following conclusions: (1) Cholesterol perturbed the homogeneity of the membrane and microdomains were formed, having a composition that differed from the average value. The ionophore was found in domains which were practically depleted of phosphatidylserine. (2) The diffusivity of the protonated monensin (MoH) was not affected by the presence of cholesterol, indicating that the viscosity of the central layer of the membrane was unaltered. (3) The diffusivity of the monensin metal complexes (MoNa and MoK) was significantly increased upon addition of cholesterol. As the viscosity along the cross membranal diffusion route is unchanged, the enhanced motion of the MoNa and MoK is attributed to variations of the electrostatic potential within the domains.