Dielectric properties of synaptosomes isolated from rat brain cortex.

Dielectric measurements were performed on the suspensions of synaptosomes isolated from rat brain cortex. The synaptosomes in buffered salt media showed typical dielectric dispersions caused by the presence of a thin limiting membrane of sufficiently low conductivity. An analysis of the dielectric data revealed that the electric conductivity of the synaptosome interior was about 37% of the external medium conductivity under isotonic conditions and that the dielectric constant for the interior phase was about 35. The membrane capacitance (0.7 muF cm-2) remained constant irrespective of nature and concentration of the univalents salts examined. Significant reduction in both the conductivity and the dielectric constant of the internal phase can be explained theoretically proveded that some intra-synaptosomal structural (synaptic vesicles and/or small mitochondria) of non-conducting nature occupies about 50% of the particulate volume, the remainder being in equilibrium with the external salt medium.     

Dielectric analysis of mitochondria isolated from rat liver I. Swollen mitoplasts as simulated by a single-shell model

A re-evaluation of the dielectric studies on isolated mitochondria (Pauly, H., Packer L., and Schwan, H.P. (1960) J. Biophys. Biochem. Cytol. 7, 589–601, and ibid. 7, 603–612) is presented. The suspensions of ‘mitoplasts’ prepared from rat liver mitochondria by a hyposmotic (10 mM KCI) treatment showed a dielectric dispersion with its characteristic frequency lying in the 1–100 MHz range. In the analysis of data special emphasis was put on the choice of the theoretical models to employ after serutiny of their applicability to the suspensions tested. As such we adopted the theory of Hanai et al. (Hanai, T., Asami, K., and Koizumi, N. (1979) Bull. Inst. Chem. Res., Kyoto Univ. 57, 297–305) that was advanced to include concentrated suspensions of shelled spheres. Curve fittings based on that theory resulted in a better agreement with experiment than the fittings based on a conventional theory for dilute suspensions. Major findings from our analyses on the swollen mitoplasts are that: (i) the limiting membrane of the mitoplasts has a specific electrical capacity of 1 μF/cm², (ii) the ratio of permittivity (or dielectric constant) for the mitoplast interior and permittivity for the external medium is 0.6–0.7, and (iii) the conductivity ratio between the interior phase and the medium is approx. 0.6. Reasons for discrepancy between the results of Pauly et al. and ours are discussed.     

Dielectric properties of yeast cells

Summary Dielectric measurements were made on suspensions of intact yeast cells over a frequency range of 10 kHz to 100 MHz. The suspensions showed typical dielectric dispersions, which are considered to be caused by the presence of cytoplasmic membranes with sufficiently low conductivity. Since the conductivity of the cell wall was found to be of nearly the same value as that of the suspending medium, composed of KCl solutions in a range from 10 to 80mm, the cell wall may be ignored in establishing an electrical model of the cells suspended in such media. An analysis of the dielectric data was carried out by use of Pauly and Schwan's theory. The membrane capacitance was estimated to be 1.1±0.1 F/cm², which is compared with values reported so far for most biological membranes. The conductivity of the cell interior was almost unchanged with varying KCl concentrations and showed low values owing to the presence of less conducting particles, presumably intracellular organelles. The relatively low dielectric constant of about 50 obtained for the cell interior, in comparison with values of aqueous solutions, may be attributed also to the presence of intracellular organelles and proteins.     

The Relationship of Internal Conductance and Membrane Capacity to Mitochondrial Volume

A study was made of the effect of mitochondrial size on the electrical properties of the membrane and the internal conductivity of mitochondria. The dielectric constant and electrical conductivity of suspensions of guinea pig heart mitochondria were examined in the frequency range 5 x 10⁵ to 2.5 x 10⁸ C.P.S. Membrane capacity was calculated to be 1.1 to 1.3 µf./cm.² and was virtually the same in mitochondria whose surface area was made to vary by a factor of 4 by osmotic means. This finding suggested that some mechanism must exist for the transfer of mitochondrial material into membrane structure during fluctuations in mitochondrial size. The electrical capacity of the membrane was unaffected by a 33-fold change in potassium chloride concentration. The internal conductance of swollen mitochondria was 2 to 3 times lower than that of the external medium. In shrunken mitochondria the internal conductance was virtually independent of the conductivity of the external medium. These results were discussed in relation to current concepts of mitochondrial structure.     

Dielectric analysis of mitochondria isolated from rat liver. I. Swollen mitoplasts as simulated by a single-shell model

A re-evaluation of the dielectric studies on isolated mitochondria (Pauly, H., Packer, L. and Schwan, H.P. (1960) J. Biophys. Biochem. Cytol. 7, 589-601, and ibid. 7, 603-612) is presented. The suspensions of 'mitoplasts' prepared from rat liver mitochondria by a hyposmotic (10 mM KCl) treatment showed a dielectric dispersion with its characteristic frequency lying in the 1-100 MHz range. In the analysis of data special emphasis was put on the choice of the theoretical models to employ after scrutiny of their applicability to the suspensions tested. As such we adopted the theory of Hanai et al. (Hanai, T., Asami, K., and Koizumi, N. (1979) Bull. Inst. Chem. Res., Kyoto Univ. 57, 297-305) that was advanced to include concentrated suspensions of shelled spheres. Curve fittings based on that theory resulted in a better agreement with experiment than the fittings based on a conventional theory for dilute suspensions. Major findings from our analyses on the swollen mitoplasts are that: (i) the limiting membrane of the mitoplasts has a specific electrical capacity of 1 microF/cm2, (ii) the ratio of permittivity (or dielectric constant) for the mitoplast interior and permittivity for the external medium is 0.6-0.7, and (iii) the conductivity ratio between the interior phase and the medium is approx. 0.6. Reasons for discrepancy between the results of Pauly et al. and ours are discussed.     

Synaptosomes and synaptosome membrane vesicles from the brain of Mamestra configurata: Application to voltage-dependent and ATP-dependent Ca2+ ion transport studies

High yields of relatively pure, morphologically well-preserved, functionally competent synaptosomes were prepared from brains of moths of Mamestra configurata using a modified microscale Ficoll flotation technique. Typical preparations yielded 10 mg of synaptosomal protein per gram of moth brains. The moth brain synaptosomes were virtually free of endoplasmic reticulum and mitochondrial contaminants as judged from marker enzyme studies and electron microscopy.Voltage-dependent Ca²⁺ ion transport was studied using the moth brain synaptosome preparations. Synaptosomes took up radioactive ⁴⁵Ca²⁺ from the incubation medium. The rate of uptake was increased up to three-fold when the synaptosomes were incubated in a depolarizing, high [K⁺] medium. Time course studies indicated that voltage-dependent Ca²⁺ uptake was composed of an early (<2 sec) fast phase and a late (>10 sec) slow phase.ATP-dependent Ca²⁺ ion transport was studied in moth brain synaptosome membrane vesicles prepared from synaptosomes by osmotic shock and purified on a second Ficoll gradient. The inside-out synaptosome membrane vesicles contained an ATP-dependent calcium ion pump which transported ⁴⁵Ca²⁺ from the incuation medium into the interior of the vesicle in the presence of ATP. The calcium ionophore A23187 rapidly released accumulated ⁴⁵Ca²⁺ from the vesicles. The maximal rate of ATP-dependent Ca²⁺ transport occurred at a [Ca²⁺ free] of 0.1 to 0.2 nM, indicating that the transport process has a very high affinity for Ca²⁺ ions.     

Functional membrane vesicles from the nervous system of insects: I. Sodium- and chloride-dependent γ-aminobutyric acid transport

(1) A synaptosomal fraction obtained from locust nervous tissue has been shown to possess an active γ-aminobutyric acid transport mechanism. This activity is preserved and even enriched by the membrane vesicles derived from osmotically shocked synaptosomes. (2) Electron-microscopy examination indicates that the above membrane vesicles are derived predominantly from the neuronal plasma membrane and are devoid of any internal cellular organelles and components. Active transport of γ-aminobutyric acid into these vesicles has been demonstrated with artificially imposed ion gradients as the sole energy source. (3) γ-Aminobutyric acid transport can be driven by an Na⁺ gradient (out>in) and /or by a gradient of Cl^? (out>in). This process is absolutely dependent on the simultaneous presence of both types of ion in the external medium. The stimulation of the process by valinomycin indicates that γ-aminobutyric acid transport is an electrogenic process which is stimulated by a membrane potential (interior negative).     

Dielectric Properties and Ion Mobility in Erythrocytes

The impedance of erythrocytes of man, cattle, sheep, dog, cat, rabbit, and chicken was measured in the range from 0.5 to 250 Mc. The dielectric constant of the red cell interior is 50 at 250 Mc, varies but little with species, and can readily be accounted for by the cells' hemoglobin content. The electrical conductivity of the red cell interior was determined between 70 and 100 Mc. The values differ from species to species within the rather limited range from 4.4 to 5.3 mmho/cm. Removal of the cell membranes does not affect the conductivity. Hence, the cell interior behaves, from an electrical point of view, like a highly concentrated hemoglobin solution. A theoretical value for the electrical conductivity of erythrocyte interiors, which is calculated on the basis of the salt content of the cell, ion mobility, and the volume concentration of the hemoglobin, is roughly twice as large as the measured value. This discrepancy is typical not only of the red blood cell. Pertinent measurements show that it is probably caused by hydrodynamic and possibly by electrostatic effects also, which lower the mobility of the ions. From the lower electrical mobility it appears that a lowered diffusion constant of the electrolytes and nonelectrolytes within the cell is indicated.     

Met-enkephalin receptor-mediated increase of membrane fluidity modulates nitric oxide (NO) and cGMP production in rat brain synaptosomes

The association of [³H]-Met-enkephalin with synaptosomes isolated from rat brain cortex, when incubated for 30 min at 25°C follows a sigmoid path with a Hill coefficient h=1.25±0.04. Binding of Met-enkephalin into synaptosomes was saturable, with an apparent binding constant of 8.33±0.48 nM. At saturation, Met-enkephalin specific receptors corresponded to 65.5±7.2 nmol/mg synaptosomal protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of Met-enkephalin into synaptosomes of at least one class of high affinity specific receptors. Met-enkephalin increased the lipid fluidity of synaptosomal membranes labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r_o/r)–1]^–1. Arthenius-type plots of [(r_o/r)–1]^–1 indicated that the lipid separation of the synaptosomal membranes at 23.4±1.2°C was perturbed by Met-enkephalin such that the temperature was reduced to 15.8±0.8°C. Naloxone reversed the fluidizing effect of Met-enkephalin, consistent with the receptor-mediated modulation of membrane fluidity. Naloxone alone had no effect on membrane fluidity. NO release and cGMP production by NO-synthase (NOS) and soluble guanylate cyclase (sGC), both located in the soluble fraction of synaptosomes (synaptosol) were decreased by 82% and 80% respectively, after treatment of synaptosomes with Met-enkephalin (10^–10–10^–4 M). These effects were reversed by naloxone (10^–4 M) which alone was ineffective in changing NO and cGMP production. We propose that Met-enkephalin achieved these effects through receptor mediated perturbations of membrane lipid structure and that inhibition of the L-Arg/NO/cGMP pathway in the brain may result in the antinociceptive effects of Met-enkephalin.     

A method for determining the dielectric constant and the conductivity of membrane-bounded particles of biological relevance

Numerical assessment is made regarding Pauly and Schwan's theory which describes the dielectric behaviour of a suspension of “shell spheres” as a model of biological membrane-bounded particles. The results indicate that approximate expressions of the theory may give rise to serious errors when applied to particles smaller than about 1 Μm in diameter. With a view to performing analysis according to a general expression of the theory, some of the characteristic responses of dielectric parameters upon changes in phase parameters are examined with particular reference to some numerical ranges of biological interest. On this basis a simplified and systematic procedure is proposed for estimating the phase parameters of particles whose shell phase can be regarded as non-conductive. As the application of the procedure proposed, a set of dielectric data of a synaptosome suspension is analyzed, so that the following three phase parameters are successfully determined: membrane capacitance (or shell phase dielectric constant), internal phase conductivity and internal phase dielectric constant. Some limitations of the procedure are discussed for the cases of conducting shells and small particles.     

Electrical Properties of Mitochondrial Membranes

The electrical capacity of the membrane of rat liver mitochondria is 0.5 to 0.6 µ./cm². This membrane capacity is obtained from the analysis of the frequency dependence of the admittance of a suspension of swollen mitochondria. In potassium chloride media the mitochondrial membrane capacity does not depend on the ion concentration. The internal conductance of the mitochondria was approximately one-half that of the external medium; the same applies if the mitochondria are equilibrated in a medium with a 10-fold difference in potassium chloride concentration. Hence the swollen mitochondria investigated here appear to be able to adjust their internal ion concentration in proportion with that of the external phase. The similarity of the membrane capacity of isolated mitochondria with the range of values known for other membranes suggests a common molecular structure. The analysis of experimental data suggests an anisotropic electrical behavior of the interior of mitochondria. This anisotropy is readily explained by the existence of internal membranes.     

Electrical Properties of Phospholipid Vesicles

The capacitance of the membrane of phospholipid vesicles and the electrical properties of the vesicle interior have been determined. To this end the electrical properties of phospholipid vesicles have been investigated over a frequency range extending from 1 kHz to 100 MHz. The dielectric behavior is characterized by two dispersions, one placed between 1 kHz and 1 MHz and the other between 1 and 100 MHz. The relaxational behavior at low frequencies is explained by counterion movement tangential to the vesicle surface and a reasonable value for the fixed charge of the vesicles is calculated from the dispersion magnitude. The relaxation at high frequencies is of the Maxwell-Wagner type and appears caused by the phospholipid bilayer bounding the interior phase of the vesicles. It is consistent with the existence of a closed bilayer with a capacitance of about 2 μF/cm² and an internal phase similar to the vesicle suspending medium. There is no indication of other than normally structured water inside the small vesicles.     

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.     

The effect of the lipid bilayer state on fluorescence intensity of fluorescein-PE in a saturated lipid bilayer

Fluorescein-PE is a fluorescence probe that is used as a membrane label or a sensor of surface associated processes. Fluorescein-PE fluorescence intensity depends not only on bulk pH, but also on the local electrostatic potential, which affects the local membrane interface proton concentration. The pH sensitivity and hydrophilic character of the fluorescein moiety was used to detect conformational changes at the lipid bilayer surface. When located in the dipalmitoylphosphatidylcholine (DPPC) bilayer, probe fluorescence depends on conformational changes that occur during phase transitions. Relative fluorescence intensity changes more at pretransition than at the main phase transition temperature, indicating that interface conformation affects the condition in the vicinity of the membrane. Local electrostatic potential depends on surface charge density, the local dielectric constant, salt concentration and water organisation. Initial increase in fluorescence intensity at temperatures preceding that of pretransition can be explained by the decreased value of the dielectric constant in the lipid polar headgroups region related in turn to decreased water organisation within the membrane interface. The abrupt decrease in fluorescence intensity at temperatures between 25 degrees C and 35 degrees C (DPPC pretransition) is likely to be caused by an increased value of the electrostatic potential, induced by an elevated value of the dielectric constant within the phosphate group region. Further increase in the fluorescence intensity at temperatures above that of the gel-liquid phase transition correlates with the calculated decreased surface electrostatic potential. Above the main phase transition temperature, fluorescence intensity increase at a salt concentration of 140 mM is larger than with 14 mM. This results from a sharp decline of the electrostatic potential induced by the phosphocholine dipole as a function of distance from the membrane surface.     

Molecular dynamics simulation of α-melanocyte stimulating hormone in a water-membrane model interface

The conformation of the tridecapeptide α-melanocyte stimulating hormone in the presence of a double water-membrane interface was studied by molecular dynamics simulation, using the computational package THOR. In this program the solvent is represented by a continuous medium with dielectric constant ɛ, and the interface between different media is simulated by a surface of discontinuity of the dielectric constant. The electrostatic image method was used to write down the terms, added to the force field, that describe the polarisation effects induced in the interface by the atomic charges. The program was further improved by the introduction of a second surface, parallel to the first one, to mimic the membrane. A conformational search using the software Prelude was employed to find an initial geometry for the peptide in water. The molecular dynamics simulation performed during 10 ns showed that the peptide structure is flexible in water, without stabilisation of any preferential conformation. In the presence of the model membrane, the peptide moved to the medium representing the interior of the membrane. Inside the low dielectric constant medium, the structure of the peptide showed a turn in the central sequence of amino acids and a packed conformation remained stabilised during more than 7.0 ns of simulation. Received: 27 November 1998 / Revised version: 11 March 1999 / Accepted: 8 April 1999     

Electrorotation of Erythrocytes Treated with Dipicrylamine: Mobile Charges within the Membrane Show their ``Signature' in Rotational Spectra

In this study, electrorotation spectra of individual cells (that is, frequency dependence of cell rotation speed) have been proved to yield information not only about the passive electric properties of cell constituents, but also about the presence of mobile charges within the plasma membrane being part of ion carrier transport systems. Experiments on human erythrocytes pretreated with the lipophilic anion dipicrylamine (DPA) gave convincing evidence that these artificial mobile charges adsorbed to the plasma membrane contributed significantly to the rotational spectrum at relatively low conductivity of the external medium (2–5 mS m⁻¹). Theoretical integration of the mobile charge concept into the single-shell model (viewing the cell as a homogenous sphere surrounded by a membrane) led to a set of equations which predicted electrorotational behavior of DPA-treated cells in dependence on medium conductivity. The quantitative data on the partition and the transmembrane translocation rate of the DPA anion extracted from the experimental rotational spectra agreed well with the corresponding literature values. Received: 14 February 1996/Revised: 29 May 1996     

Fluorescence changes of rhodamine 6G associated with changes in membrane potential in synaptosomes

The intensity of rhodamine 6G fluorescence was found to be a useful scale for measuring the membrane potential in synaptosomes. The fluorescence of rhodamine 6G in synaptosomal suspensions increases with depolarization in the synaptosomes induced by the replacement of cations in the medium or by the addition of agents known to depolarize the membrane potential. Considering the character of the dye, we have derived an equation which gives the relation between the fluorescence intensity of the dye and the membrane potential. The change in membrane potential (diffusion potential) of synaptosomes was calculated using the equation. The calculated membrane potential was proportional to the logarithm of the K⁺ concentration above 20 mM, and the slope of membrane potential against log[K⁺] was about 52 mV per decade of concentration. The permeability ratio ($\text{P}{}_{\mathrm{<mtext>X</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$; the ratio of the permeability constants of a given cation, X⁺, and K⁺) was estimated from the calculated membrane potential.     

Is the concentration of γ-aminobutyric acid in the nerve terminal regulated via product inhibition of glutamic acid decarboxylase?

Fractions of synaptosomes were used to study the regulation of -aminobutyric acid (GABA) synthesis. The isolated synaptosomes were superfused in media of various compositions. [³H]GABA and GABA released into the medium or remaining in the synaptosomes were analyzed by liquid scintillation and HPLC techniques. Different conditions, designed to increase the GABA efflux rate were used: the rate of superfusion was varied and the concentrations of K⁺ and Ca²⁺ were altered. Stimulation of GABA efflux was paralleled with an increased synthesis of GABA, since, in spite of the increased GABA efflux, a relatively constant intraterminal level was found. The findings suggest that the intraterminal concentration of GABA and thus also its synthesis is regulated via product inhibition. In addition, [³H]GABA, exogenous, and GABA, endogenous, responded to external stimulae (Ca²⁺, veretradine, various GABA concentrations and the glutaminase inhibitor diazo-nor-leucine) in a way which was compatible with them being localized in and/or released from different compartments.     

Dielectric properties of mouse lymphocytes and erythrocytes

In order to study the effect of the nucleus on dielectric behavior of the whole cell, permittivity (dielectric constant) and conductivity of mouse lymphocytes and erythrocytes were measured over a frequency range from 0.1 to 250 MHz. Erythrocytes (spherocytes) showed a single dielectric dispersion, which was explained by a single-shell model that is a conducting sphere covered with a thin insulating shell. On the other hand, lymphocytes showed a broad dielectric dispersion curve which was composed of two subdispersions. The high-frequency subdispersion, which was not found for erythrocytes, was assigned to the Maxwell-Wagner dispersion of the nucleus occupying about 65% of the total cell volume. Analysis of the lymphocyte dispersion was carried out by a double-shell model, in which a shelled sphere, i.e., nucleus, is incorporated into the single-shell model. The following electrical parameters were consequently estimated; the capacitance of the plasma membrane, 0.86 microF.cm-2; the conductivity of the cytoplasm, 3.2 mS.cm-1; the capacitance and conductance of the nuclear envelope are, respectively, 0.62 microF.cm-2 and 15 S.cm-2, and the permittivity and conductivity of the nucleoplasm are 52 and 13.5 mS.cm-1.     

The redox properties of cytochromes b imposed by the membrane electrostatic environment.

The effect of the dipole potential field of extended membrane spanning alpha-helices on the redox potentials of b cytochromes in energy transducing membranes has been calculated in the context of a three phase model for the membrane. In this model, the membrane contains three dielectric layers; (i) a 40-A hydrophobic membrane bilayer, with dielectric constant em = 3-4, (ii) 10-20-A interfacial layers of intermediate polarity, ein = 12-20, that consist of lipid polar head groups and peripheral protein segments, and (iii) an external infinite water medium, ew = 80. The unusually positive midpoint potential, Em = +0.4 V, of the "high potential" cytochrome b-559 of oxygenic photosynthetic membranes, a previously enigmatic property of this cytochrome, can be explained by (i) the position of the heme in the positive dipole potential region near the NH2 termini of the two parallel helices that provide its histidine ligands, and (ii) the loss of solvation energy of the heme ion due to the low dielectric constant of its surroundings, leading to an estimate of +0.31 to +0.37 V for the cytochrome Em. The known tendency of this cytochrome to undergo a large -delta Em shift upon exposure of thylakoid membranes to proteases or damaging treatments is explained by disruption of the intermediate polarity (ein) surface dielectric layer and the resulting contact of the heme with the external water medium. Application of this model to the two hemes (bn and bp) of cytochrome b of the cytochrome bc1 complex, with the two hemes placed symmetrically in the low dielectric (em) membrane bilayer, results in Em values of hemes bn and bp that are, respectively, somewhat too negative (approximately -0.1 V), and much too positive (approximately +0.3 V), leading to a potential difference, Em(bp) - Em(bn), with the wrong sign and magnitude, +0.25 V instead of -0.10 to -0.15 V. The heme potentials can only be approximately reconciled with experiment, if it is assumed that the two hemes are in different dielectric environments, with that of heme bp being more polar.