Quantifying molecular partition into model systems of biomembranes: an emphasis on optical spectroscopic methods

Optical spectroscopies have been intensively used to determine partition coefficients by a plethora of methodologies. The present review is intended to give detailed and useful information for the determination of partition coefficients and addresses several relevant aspects, namely: (i) definition and calculation of the partition coefficient between aqueous and lipidic phases; (ii) partition coefficients vs. “binding” formalisms; (iii) advantages of spectroscopic methodologies over separation techniques; (iv) formalisms for various experimental approaches based on UV-Vis absorption or fluorescence parameters (fluorescence intensity, lifetime, anisotropy and quenching); (v) experimental hints, artifacts and model limitations; and (vi) a brief survey of nonoptical techniques.     

Interaction of peptides with binary phospholipid membranes: application of fluorescence methodologies

The application of fluorescence methodologies to obtain information about the extent, dynamics and topology of peptide interaction with binary phospholipid (mainly zwitterionic/anionic) mixtures is reviewed. First, general approaches based on peptide (tryptophan residues) fluorescence properties that give information about its partition, location and dynamics will be presented. Then, methodologies based on membrane probes fluorescence that report the influence of peptide binding and/or incorporation on the lateral organization (phase separation) of membrane phospholipids will be described. Specific examples taken from the literature that illustrate both situations are presented as well as formalisms for data analysis. It is shown that steady-state and time-resolved fluorescence data (particularly important in the case of fluorescence resonance energy transfer studies) give complementary information, allowing a molecular picture of peptide interaction with biphasic systems to be drawn.     

Binding of a fluorescent dansylcadaverine-substance P analogue to negatively charged phospholipid membranes

We have investigated the binding of a new dansylcadaverine derivative of substance P (DNC-SP) with negatively charged small unilamellar vesicles composed of a mixture of phosphatidylcholine (PC) and either phosphatidylglycerol (PG) or phosphatidylserine (PS) using fluorescence spectroscopic techniques. The changes in fluorescence properties were used to obtain association isotherms at variable membrane negative charges and at different ionic strengths. The experimental association isotherms were analyzed using two binding approaches: (i) the Langmuir adsorption isotherm and the partition equilibrium model, that neglect the activity coefficients; and (ii) the partition equilibrium model combined with the Gouy-Chapman formalism that considers electrostatic effects. A consistent quantitative analysis of each DNC-SP binding curve at different lipid composition was achieved by means of the Gouy-Chapman approach using a peptide effective interfacial charge (v) value of (0.95 +/- 0.02), which is lower than the physical charge of the peptide. For PC/PG membranes, the partition equilibrium constant were 7.8 x 10(3) M(-1) (9/1, mol/mol) and 6.9 x 10(3) M(-1) (7/3, mol/mol), whereas for PC/PS membranes an average value of 6.8 x 10(3) M(-1) was estimated. These partition equilibrium constants were similar to those obtained for the interaction of DNC-SP with neutral PC membranes (4.9 x 10(3) M(-1)), as theoretically expected. We demonstrate that the v parameter is a determinant factor to obtain a unique value of the binding constant independently of the surface charge density of the vesicles. Also, the potential of fluorescent dansylated SP analogue in studies involving interactions with cell membranes is discussed.     

Quenching of fluorescence in membrane protein by hypocrellin B

The hypocrellin B (HB) was used as a fluorescence quencher to study the basic physical characteris-tics of HB in membrane systems, including the diffusion speed of quencher from aqueous phase into membrane phase, the partition coefficient (P) of quencher between membrane and water, and the fluorescence quenching constant of protein (Ksv; Kq). The experimental results show that the quenching of fluorescence in membrane protein by HB can be determined by the principle of dynamic quenching. The experimental process of fluorescence quenching was ob-served in detail by using the ESR technique. The signal of HB" was found to arise from an electron transfer from ex-cited trytophan to HB.     

Phloem mobility of xenobiotics: tabular review of physicochemical properties governing the output of the Kleier model

The Kleier model of phloem-mobility of xenobiotics combines the intermediate permeability hypothesis with the acid trap mechanism for weak acids. The output of the model is dependent on the lipophilicity of a compound, for which octanol/water partition coefficients (log K_ow) have been used as a measure. The membrane permeability of xenobiotics is predicted from these partition coefficients, and the nature of the sieve tube membranes has been modelled using regressions derived from Nitella or potato permeability data. A wide range of log K_ow values for herbicides, fungicides, insecticides and experimental compounds (400) have been tabulated along with the model output for various membrane parameters. The application of the model is in broad agreement with literature and experimental observations on many of the known phloem mobile herbicides and predicts low phloem mobility for the fungicides and insecticides considered here, again in agreement with the literature. The behaviour of herbicides representative of the main chemical families and modes of action are reviewed, along with examples of the few phloem-mobile fungicides and insecticides identified.Abbreviations K_ow octanol-water partition coefficient - pK_a –log₁₀ acid dissociation constant - C_f Concentration factor - P membrane permeability     

Partition coefficients of beta-blockers in bile salt/lecithin micelles as a tool to assess the role of mixed micelles in gastrointestinal absorption

The objective of this study was to develop non-invasive spectroscopic methods to quantify the partition coefficients of two beta-blockers, atenolol and nadolol, in aqueous solutions of bile salt micelles and to assess the effect of lecithin on the partition coefficients of amphiphilic drugs in mixed bile salt/lecithin micelles, which were used as a simple model for the naturally occurring mixed micelles in the gastrointestinal tract. The partition coefficients (Kp) at 25.0 +/- 0.1degreesC and at 0.1 M NaCl ionic strength were determined by spectrofluorimetry and by derivative spectrophotometry, by fitting equations that relate molar extinction coefficients and relative fluorescence intensities to the partition constant Kp. Drug partition was controlled by the: (i) drug properties, with the more soluble drug in water (atenolol) exhibiting smaller values of Kp, and with both drugs interacting more extensively in the protonated form; and by (ii) the bile salt monomers, with the dihydroxylic salts producing larger values of Kp for the beta-blockers, and with glycine conjugation of the bile acid increasing the values of Kp for the beta-blockers. Addition of lecithin to bile salt micelles decreases the values of Kp of the beta-blockers. Mixed micelles incorporate hydrophobic compounds due to their large size and the fluidity of their core, but amphiphilic drugs, for which the interactions are predominantly polar/electrostatic, are poorly incorporated in mixed micelles of bile salts/lecithin.     

The application of aqueous two-phase systems to the purification of pharmaceutical proteins from transgenic sheep milk

Transgenic sheep milk containing the protein human₁-Antitrypsin (AAT) was partitioned in Poly(ethyleneglycol) (PEG)-Sulphate and PEG-Phosphate biphasic systems. Individual partition coefficients for AAT and some of the milk proteins were determined in these systems. The effects of PEG molecular weight, pH and the inclusion of NaCl on the partitioning of the proteins were also studied. It was found that increasing the concentration of NaCl and decreasing the molecular weight of the PEG resulted in an increase of the partition coefficients of the proteins to the upper (PEG) phase. This partitioning effect was greater for the more hydrophobic proteins and particularly in systems having a pH close to the isoelectric point of the protein. Solubilities of the proteins in increasing concentrations of ammonium sulphate were measured in order to investigate the effects of hydrophobic and electrostatic interactions on the partitioning of these proteins in aqueous two-phase systems. Those proteins that precipitated at low levels of ammonium sulphate showed an increase in partition coefficient at low concentrations of NaCl, or they were precipitated at the interface of the phases at low concentrations of NaCl. Proteins that had low salting out constants in ammonium sulphate solutions were relatively unaffected by NaCl in ATPS. It is probable however that conformational changes and the state of aggregation of proteins are also important and should be invoked in describing the partitioning behavior observed for -Lg for example. Comparison of theoretical and experimental values for AAT yield and purity showed clearly that partition coefficients are influenced by the degree of purity and values obtained with purified standards are not necessarily the same as for the same protein present in a complex mixture. Under the most favourable conditions using a 4% w/w loading of transgenic ovine milk, we obtained a 91% yield of AAT in the PEG phase with a purity of 73%.This revised version was published online in October 2005 with corrections to the Cover Date.     

PKQuest: volatile solutes - application to enflurane,nitrous oxide,halothane, methoxyflurane and toluene pharmacokinetics

BACKGROUND: The application of physiologically based pharmacokinetic models (PBPK) to human studies has been limited by the lack of the detailed organ information that is required for this analysis. PKQuest is a new generic PBPK that is designed to avoid this problem by using a set of "standard human" default parameters that are applicable to most solutes. RESULTS: PKQuest is used to model the human pharmacokinetics of the volatile solutes. A "standard human" value for the lipid content of the blood and each organ (klip) was chosen. This set of klip and the oil/water partition coefficient then specifies the organ/blood partition for each organ. Using this approach, the pharmacokinetics of inert volatile solute is completely specified by just 2 parameters: the water/air and oil/water partition coefficients. The model predictions of PKQuest were in good agreement with the experimental data for the inert solutes enflurane and nitrous oxide and the metabolized solutes halothane and toluene. METHODS: The experimental data that was modeled was taken from previous publications. CONCLUSIONS: This approach greatly increases the predictive power of the PBPK. For inert volatile solutes the pharmacokinetics are determined just from the water/air and oil/water partition coefficient. Methoxyflurane cannot be modeled by this PBPK because the arterial and end tidal partial pressures are not equal (as assumed in the PBPK). This inequality results from the "washin-washout" artifact in the large airways that is established for solutes with large water/air partition coefficients.PKQuest and the worked examples are available on the web www.pkquest.com.     

FRET Detects the Size of Nanodomains for Coexisting Liquid-Disordered and Liquid-Ordered Phases

Biomembranes with as few as three lipid components can form coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. In the coexistence region of Ld and Lo phases, the lipid mixtures 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/chol or brain sphingomyelin (bSM)/DOPC/chol form micron-scale domains that are easily visualized with light microscopy. Although large domains are not observed in the mixtures DSPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/chol and bSM/POPC/chol, lateral heterogeneity is nevertheless detected using techniques with nanometer-scale spatial resolution. We propose a simple and accessible method to measure domain sizes below optical resolution (～200 nm). We measured nanodomain size for the latter two mixtures by combining experimental Förster resonance energy transfer data with a Monte-Carlo-based analysis. We found a domain radius of 7.5?10 nm for DSPC/POPC/chol, similar to values obtained previously by neutron scattering, and ～5 nm for bSM/POPC/chol, slightly smaller than measurable by neutron scattering. These analyses also detect the domain-size transition that is observed by fluorescence microscopy in the four-component lipid mixture bSM/DOPC/POPC/chol. Accurate measurements of fluorescent-probe partition coefficients are especially important for the analysis; therefore, we exploit three different methods to measure the partition coefficient of fluorescent molecules between Ld and Lo phases.     

Partition coefficients of fluorescent probes with phospholipid membranes

A method for determination of membrane partition coefficients of five fluorescent membrane probes, 1,6-diphenyl-1,3,5-hexatriene (DPH), p-((6-phenyl)-1,3,5-hexatrienyl) benzoic acid (DPH carboxylic acid), 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH propionic acid), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and N-4-(4-didecylaminostyryl)-N-methylpyridinium iodide (4-di-10-ASP), was developed utilizing the fluorescence enhancement of a constant probe concentration by titration with excess phospholipid liposomes. The partition coefficients of DPH, DPH carboxylic acid, DPH propionic acid, TMA-DPH and 4-di-10-ASP into dipalmitoylphosphatidylcholine membranes were determined to be 1.3.10(6), 1.0.10(6), 6.5.10(5), 2.4.10(5) and 2.8.10(6) respectively. Knowledge of the partition coefficients may help select a lipid concentration for membrane studies that necessitate a probe's dominant incorporation into membranes.     

Fluid-fluid membrane microheterogeneity: a fluorescence resonance energy transfer study

Large unilamellar vesicles of dimyristoylphosphatidylcholine/cholesterol mixtures were studied using fluorescence techniques (steady-state fluorescence intensity and anisotropy, fluorescence lifetime, and fluorescence resonance energy transfer (FRET)). Three compositions (cholesterol mole fraction 0.15, 0.20, and 0.25) and two temperatures (30 and 40 degrees C) inside the coexistence range of liquid-ordered (l(o)) and liquid-disordered (l(d)) phases were investigated. Two common membrane probes, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dimyristoylphosphatidylethanolamine (NBD-DMPE) and N-(lissamine(TM)-rhodamine B)-dimyristoylphosphatidylethanolamine (Rh-DMPE), which form a FRET pair, were used. The l(o)/l(d) partition coefficients of the probes were determined by individual photophysical measurements and global analysis of time-resolved FRET decays. Although the acceptor, Rh-DMPE, prefers the l(d) phase, the opposite is observed for the donor, NBD-DMPE. Accordingly, FRET efficiency decreases as a consequence of phase separation. Comparing the independent measurements of partition coefficient, it was possible to detect very small domains (<20 nm) of l(o) in the cholesterol-poor end of the phase coexistence range. In contrast, domains of l(d) in the cholesterol-rich end of the coexistence range have comparatively large size. These observations are probably related to different processes of phase separation, nucleation being preferred in formation of l(o) phase from initially pure l(d), and domain growth being faster in formation of l(d) phase from initially pure l(o).     

Partitioning of (+-)-5,6-dihydro-6-phenyl-2-n-alkyl-imidazo- [2,1-b]thiazoles into large unilamellar liposomes: a steady-state fluorescence quenching study.

The interaction of the tetramisole derivative (+-)-5,6-dihydro-6-phenyl-imidazo[2,1-b]thiazole and a number of its 2-n-alkyl homologues (-ethyl through -n-pentyl and -n-heptyl) with large unilamellar phosphatidylcholine/phosphatidylethanolamine/dipalmitoylphosphatidic acid (2:1:0.06, w/w) vesicles was studied by means of steady-state fluorescence quenching using 8-(2-anthryl)octanoic acid as membrane probe. Linear Stern-Volmer plots were obtained for each derivative, indicating dynamic quenching. The slopes of the plots decreased with increasing liposomal concentration. For four short-chain homologues (-H, -ethyl, -n-propyl and -n-butyl), the respective membrane partition coefficients Kp and bimolecular quenching rate constants kq were determined from the plots of the reciprocal of the apparent quenching rate constant (kappq)-1 against the lipid volume fraction alpha L of the liposomes. The partition coefficients increased with increasing chain-length of the tetramisoles. A linear relationship was found between the free energy of partitioning and the number of methylene units of the homologues (-delta G degrees per methylene group = 1.6 +/- 0.1 kJ mol-1). For the n-pentyl and n-heptyl derivatives, the fluorescence quenching technique did not allow one to determine their membrane partition coefficients. Analysis of the fluorescence intensity measurements with Scatchard plots gave further evidence for the partitioning nature of the tetramisole derivatives' association with the liposomal membranes.     

Thermodynamic modeling of the partitioning of biomolecules in aqueous two-phase systems using a modified Flory–Huggins equation

A modified Flory–Huggins equation accounting for the solvation of polymer molecules by water molecules was used to model the phase behavior of aqueous two-phase systems (ATPS) formed by poly(ethylene glycol) (PEG) and dextran. The parameters of the equation were obtained by fitting experimental equilibrium data either accounting for or disregarding dextran polidispersity. The modified equation was subsequently applied to calculate partition coefficients of biomolecules in these systems. It was found that accounting for polidispersity did not affect significantly the calculated phase equilibrium, but increased the agreement of calculated partition coefficients with experimental data. Further improvement was obtained by using a size dependent interaction parameter for dextran pseudo-components.     

Fragment based drug design and diversity-oriented synthesis of carboxylic acid isosteres

The medicinal chemist toolbox is plenty of (bio)isosteres when looking for a carboxylic acid replacement. However, systematic assessment of acid surrogates is often time consuming and expensive, while prediction of both physicochemical properties (logP and logD) as well as acidity would be desirable at early discovery stages for a better analog design. Herein in this work, to enable decision making on a project, we have synthesized by employing a Diversity-Oriented Synthetic (DOS) methodology, a small library of molecular fragments endowed with acidic properties. By combining in-silico and experimental methodologies these compounds were chemically characterized and, particularly, with the aim to know their physicochemical properties, the aqueous ionization constants (pKa), partition coefficients logD and logP of each fragment was firstly estimated by using molecular modeling studies and then validated by experimental determinations. A face to face comparison between data and the corresponding carboxylic acid might help medicinal chemists in finding the best replacement to be used. Finally, in the framework of Fragment Based Drug Design (FBDD) the small library of fragments obtained with our approach showed good versatility both in synthetic and physico-chemical properties.     

Octanol–water partition of nonzwitterionic peptides: Predictive power of a molecular size-based model

A remarkably simple, molecular size-based model developed to predict octanol–water partition coefficients for organic compounds is tested on a set of 188 neutral peptides with available experimental partition data. Despite using only two parameters, it gives a promising correlation (r² = 0.914; σ = 0.455, F = 1978.0), and predictions are in a realistic range even for larger peptides (cyclosporin, melanotan, sandostatin) where common, overparametrized fragment methods become quite unreliable. Ion-pair partitioning and the extraction constant formalism is briefly reviewed to describe the sigmoidal lipophilicity profile of ionizable, nonzwitterionic peptides. It seems possible to extend the present model to estimate apparent partition coefficients measured around neutral pH and physiological conditions for monoionic peptides; however, as no standard conditions are yet defined and only relatively small number of experimental data are available, the situation here is more complex. Proteins 30:86–99, 1998. © 1998 Wiley-Liss, Inc.     

Novel physical chemistry approaches in biophysical researches with advanced application of lasers: Detection and manipulation

Novel methodologies utilizing pulsed or intense CW irradiation obtained from lasers have a major impact on biological sciences. In this article, recent development in biophysical researches fully utilizing the laser irradiation is described for three topics, time-resolved fluorescence spectroscopy, time-resolved thermodynamics, and manipulation of the biological assemblies by intense laser irradiation. First, experimental techniques for time-resolved fluorescence spectroscopy are concisely explained in Section 2. As an example of the recent application of time-resolved fluorescence spectroscopy to biological systems, evaluation of the viscosity of lipid bilayer membranes is described. The results of the spectroscopic experiments strongly suggest the presence of heterogeneous membrane structure with two different viscosity values in liposomes formed by a single phospholipid. Section 3 covers the time-resolved thermodynamics. Thermodynamical properties are important to characterize biomolecules. However, measurement of these quantities for short-lived intermediate species has been impossible by traditional thermodynamical techniques. Recently, development of a spectroscopic method based on the transient grating method enables us to measure these quantities and also to elucidate reaction kinetics which cannot be detected by other spectroscopic methods. The principle of the measurements and applications to some protein reactions are reviewed. Manipulation and fabrication of supramolecues, amino acids, proteins, and living cells by intense laser irradiation are described in Section 4. Unconventional assembly, crystallization and growth, amyloid fibril formation, and living cell manipulation are achieved by CW laser trapping and femtosecond laser-induced cavitation bubbling. Their spatio-temporal controllability is opening a new avenue in the relevant molecular and bioscience research fields. This article is part of a Special Issue entitled “Biophysical Exploration of Dynamical Ordering of Biomolecular Systems” edited by Dr. Koichi Kato.     

Phospholipid lateral phase separation and the partition of cis-parinaric acid and trans-parinaric acid among aqueous, solid lipid, and fluid lipid phases.

The partition of cis-parinaric acid (9,11,13,15-cis, trans, trans,cis-octadecatetraenoic acid, cis-PnA) and trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid, trans-PnA) among aqueous, solid lipid, and fluid lipid phases has been measured by three spectroscopic parameters: absorption spectral shifts, fluorescence quantum yield, and fluorescence polarization. The solid lipid was dipalmitoylphosphatidylcholine (DPPC); the fluid lipid was palmitoyldocosahexaenoylphosphatidylcholine (PDPC). Mole fraction partition coefficients between lipid and water were determined by absorption spectroscopy to be for ci--PnA, 5.3 X 10(5) with a solid lipid and 9 X 10(5) with fluid lipid and, for trans-PnA, 5 X 10(6) with solid lipid and 1.7 X 10(6) with fluid lipid. Ratios of the solid to the fluid partition coefficients (Kps/f) are 0.6 +/- 0.2 for cis-PnA and 3 +/- 1 for trans-PnA. A phase diagram for codispersions of DPPC and PDPC has been constructed from the measurements of the temperature dependence of the fluorescence quantum yield and polarization of cis-PnA and trans-PnA and their methyl ester derivatives. A simple analysis based on the phase diagram and fluorescence data allows additional calculations of Kps/f's which are determined to be 0.7 +/- 0.2 for the cis probes and 4 +/- 1 for the trans probes. The relative preference of trans-PnA for solid phase lipids and its enhanced quantum yield in solid phase lipids make it sensitive to a few percent solid. The trans probes provide evidence that structural order may persist in dispersions of these phospholipids 10 degrees C or more above their transition temperature. It is concluded that measurements of PnA fluorescence polarization vs. temperature are better suited than measurements of quantum yield vs. temperature for determining phospholipid phase separation.     

Fluorescence quenching in model membranes An analysis of the local phospholipid environments of diphenylhexatriene and gramicidin A′

Interactions between the fluorophors diphenylhexatriene or gramicidin A′ and lipids are examined using a spin-labeled phosphatidylcholine as a fluorescence quenching probe. It is found that in phospholipid vesicles of mixed lipid composition at temperatures where phospholipids are completely in the liquid crystal phase, several different species of phosphatidylcholines are randomly distributed around the fluorophors. In vesicles of mixed lipid composition which can undergo thermally induced phase separations, the fluorescence quenching observed at lower temperatures reflects a non-random distribution of lipids around each fluorophor. This observation is explained in terms of the partition of fluorophor between a spin-labeled lipid-rich liquid crystal phase, and a spin-labeled lipiddepleted gel phase. Gramicidin A′ strongly favors partition into the liquid crystal phase, whereas diphenylhexatriene partitions about equally between the two lipid phases. A method is described utilizing fluorescence quenching for the calculation of the partition coefficient for a fluorophor. The partition coefficients so calculated are shown to be in good agreement with previously reported values derived from other methods. It is also shown that Ca²⁺-induced lipid phase separations can be monitored by fluorescence quenching.     

Influence of different transition metals in phthalocyanines on their interaction energies with volatile organic compounds: an experimental and computational study

Experimental partition coefficients were determined for a series of volatile organic compounds (VOCs) (acetonitrile, n-butylamine, n-octane tetrachloroethene, and toluene) for the interaction with 2,3,9,10,16,17,23,24-octakis(octyloxy)-phthalocyaninato complexes, PcM(OR)₈, with varying central metal atoms [M=H₂ (metal-free), Ni, Pd, Cu, Zn]. Large partition coefficients for toluene were observed in the case of the nickel and palladium phthalocyanines, whereas for the corresponding zinc-containing compound, interaction with n-butylamine resulted in a high value for the partition coefficient. Interaction energies for model coordination complexes were obtained at the ab initio LMP2/ LACVP* level of theory. The interaction of various small volatiles with the various PcM(OR)₈ compounds was studied using the PM3 semiempirical Hamiltonian. Large values for interaction energies correspond to particularly strong partition coefficients, suggesting that coordination of the volatiles to the central metal dominates over the often discussed π-system stacking at the PcM(OR)₈’s. Figure: Chemical structure of the phthalocyanines investigated.