CC BY
16
UDC 577.1.04; 577.15.03
O. M. Alekseeva, A. V. Krementsova, Yu. A. Kim, A. V. Krivandin, O. V. Shatalova, G. E. Zaikov
MELAFEN EFFECTS TO STRUCTURAL PARAMETERS OF DMPC AND EGG PHOPHOLIPIDS MEMBRANES
Keywords: melafen, dimyristoilphosphatidylcholine, egg lecithin, multilammelar liposome, adiabatic differential microcalorymetry,
small-angle X-ray scattering.
The main goal of this study was the examining of action mechanism of Melafen - melamine salt of bis (oximethyl) phosphinic acid, applauded as regulator of plant development, on experimental objects of animal origin. With aid of differential scanning microcalorymetry (DSC) and small-angle X-ray scattering (SAXS) we tested the thermodynamic and structural properties of phospholipid membranes formed from individual synthetic dimyristoilphosphatidylcholine (DMPC) or natural phospholipid mixture - egg lecithin. The microdomain structural organization of DMPC membranes was altered by Melafen aqueous solutions. The integral structural changes did not were found in membranes of egg lecithin liposomes and period and degree of order under the Melafen aqueous solutions action.
Ключевые слова: мелафен, димиристоилфосфатидилхолин, яичный лецитин, мультиламмелярные липосомы, адиабатная дифференциальная микрокалориметрия, малоугловое дифракционное рентгеновское рассеяние.
Основная цель данного исследования - изучение механизма действия регулятора роста растений - мелафена (меламиновой соли бис оксиметилфосфиновой кислоты) на экспериментальные объекты животного происхождения. С помощью методов дифференциальной сканирующей микрокалориметрии (ДСК) и малоуглового рентгеновского рассеяния были изучены термодинамические и структурные свойства фосфолипидных мембран. Мембраны были сформированы из индивидуального синтетического фосфолипида димиристоилфосфатидилхолина (ДМФХ) или из смеси природных фосфолипидов яичного лецитина. Было обнаружено, что микродоменная организация ДМФХ мембран изменяется под действием водных растворов мелафена. Но интегральные структурные изменения под действием мелафена в липосомах, сформированных из яичного лецитина, не были выявлены.
In most case the primary targets for biologically active substance (BAS) are the cell membranes. In connection with this fact, the actions of Melafen aqueous solutions to the structural components of membranes have been examined. Considering that Melafen is used as the plant growth regulator (it inhibits the development of seeds when large concentrations and activates when low and ultra small) we held the complex examining with applying the aqueous solutions of Melafen over a wide range of concentrations from 10-21 M up to 10-3 M.
It is known that the main structural bases of membranes are the lipids. In our examining for formation of model experimental objects the lipids of one of the 8 most important classes of lipids of living organisms the glycerophospholipids (the phospholipids) [1] were used. Most of phospholipids molecules are consisted of hydrophilic polar head with charged (or neutral) phosphate group, and hydrophobe non polar part. Thus the molecule may be neutral or be negative charged summary. The hydrophobe part consists of fatty acids residues. Such architecture of phospholipid molecules permits to phospholipids to form or bilayer or the hexagonal education in water media. This fact is as the subject to stereo specific parameters and size relation of polar head and non-polar fatty acids residues. Bilayer or the hexagonal lipid structures are typical for cellular compartment, and were being discovered in vivo.
Different biologically active substances may exert the significant influence on the lipid bilayer. The structure of lipid bilayer is possible to study, watching changes of phase transitions of lipids and so to gain information about place when the material localizes in
bilayer, and about subtle mechanisms cooperation with lipids. The phase's transitions into lipid membranes have been studied in sufficient detail. And it is available to discover the reliable information on how the molecules localize in lipid bilayer. Localization was mirrored in lipids thermodynamic characteristics.
The lipid bilayer can be existed in two main phase states - crystal (gel L£> - phase) and liquid-crystal (La - phase) in temperature dependence. The bilayer transition from crystal into liquid crystalline state (and back) occurs when strictly specified temperature, characteristic data for lipid (7m). Differential scanning calorimetric (DSC) study determines the heat capacity dependence of lipids or membranes in suspension from temperature when constant pressure.
The low molecular weight compounds may shift the phase-transition temperature. Any causes of that phenomenon are the preferential interaction of these materials with solid or liquid states of membrane. The most of learned materials, (having, for example, anesthetic activity), decrease the temperature of membrane-phase main transition. These substances connect with outer layer on the surface of membrane, primarily with liquid phase. These relationships stabilize any areas of liquid phase, and the temperature of membrane melting decreases. BAS, which are able to shift the temperature of phase-transition, increase the transition duration that are mirrored as a width of endothermic peak in the thermograms curve.
With engagement of model experimental objects it has managed to define any targets for influence of Melafen aqueous solution on cell membranes structure.
In our experiments the multilayered liposomes, formed from phospholipids, were used as model membranes. Thermodynamic parameters of lipids melting were determined by the DSC method in the presence of biologically active substances tested.
The method is based on the measurement of power, which was brought to cells. Cells that contained control and experimental objects are warmed up with strictly equal speed. It makes it possible to estimate the thermodynamic parameters of explored systems melting: enthalpy, temperature and half of width of main phase transition. But in diluted solutions the thermal effects, conditioned by macromolecules, are small extremely (the biopolymers heat capacity in 0,3% solution is only thousandth quota from common heat capacity of dissolvent). And only measurements by means of very sensitive differential adiabatic scanning microcalorimeter (DASM-4) [2] allowed estimating of melting parameters. The melting parameters are correlated with the structural changes in organization of lipid microdomains in bilayer in the presence of BAS.
Performance characteristics of DASM-4 [2] are: temperature range: 0-100° C, warm-up speed - 0,22,0 grad/min, workers volume of cells - 1,0 ml, sensibility on heat capacity - 1,6 x 10-5 J grad-1 ml-1, determination accuracy of relative heat capacity - 8 x 10-5 J grad-1 ml-1, record accuracy of temperature- 0,1 grad.
The sensibility of DASM-4 is determined by three construction principles of device: differential measurement circuit, continual warm-up with exactly designated speed and complete adiabatic of measuring cells. When feeding the electric current in heaters of microcalorymetry cells, these cells rise in temperature. Simultaneously, proportionally-integrated controllers provide automatically with a high degree of accuracy the temperature equalization of thermal shields with cells temperature. The cameras happen to be in conditions, close to adiabatic, when the camera heat-exchange with environment is virtually absent. These conditions are implemented in wide temperature range when cells were heated with a constant rate [2].
The synthetic individual glycerophospholipid -dimyristoilphosphatidylcholine (DMPC), was chosen in our work as of material for formation of liposome. As hydrocarbon chains are two residues of saturated myristic acid. The molecules of DMPC have the shape as cylinder. Because the size of phosphate head is small; this is why from such phospholipid it is possible to form of bilayer liposomes with short crook of membrane [3].
When liposomes were melted, 2 thermally induced endothermic transitions were discovered: first -pre transition and second - main transition (fig 1 (1)).
The interpretation of calorimetric curve that describes the main transition for individual substance is simple most. The peak, which non clear expressed in the area 14-15°C, reflects the pre transition, when the restructuring ranked from order phospholipids packing (3) as "gel-phase"(6) to "ripple-phase" (4), (7). Then the main endothermic transition occurs into less rank order condition "liquid crystal" (5), (8). The "ripple-phase" presence was observed for certain phosphatidylcholines-membrane by electron microscopy method. In
particular, it was seen that the corrugation period is different and typical for each kind of lipids. The corrugation periodicity and it the quantities were changed by many factors affecting to the bilayer structure [4].
For exploration of membrane structure of bilayer membrane, composed of DMPC, the multulammelar liposomes were used fig 1 (2). Such liposomes were formed when thin films were acted by hydration in phosphate buffer when neutral pH and temperature above of phase transition. Subtle films on the walls of flask were acquired when DMPC was dried under vacuum. These multilayer films were acted by hydration consistently as - the stratum for stratum. Thus such method allowed to education of multilayer vesicular structures, composed of bilayer membranes. Obtained liposomes have the multulammelar structure by the size up to 2000A from data of electron microscopy [5].
Fig. 1 - Modified scheme [4] of thermally induced endothermic phase transitions of DMPC in bilayer of multulammelar liposomes when melting by the DSC method. (1) - thermograms of endothermic phase transitions that were registered on DASM-4 when thermally induced phase transitions of DMPC: Pre transition and main transition;(2) - electron microscopic photo of multulammelar liposomes, obtained by Y.S. Tarahovsky by means of crio-transmission microscopy [4]; (3, 4, 5) - diagrams of phospholipid DMPC molecules locations when three phase states: "gel-phase" (3), "ripple-phase" (4), "liquid crystal phase" (5); (6, 7, 8) - electron micrographs when freeze spelling of phospholipid bilayer is able to "gel-phase" (6), "ripple-phase" (7), "liquid-crystal phase" (8) [4]
The proposed approach with aid of DSC method for inspection of properties of membranes is fairly correct. Since thermally induced phase main transition, i. e. the transition from solid - gel state in rank order to the liquid state - liquid crystal, play a major role in membranes of living organism. When liquid phase all conformational rearrangements of proteins occur easily. The oligomers formation, and the lipids flip-flop moving, lateral shifting are occurred easily too. In solid environment in membranes all these species of biological mobility have been hampered.
And, respectively, all structural and functional activities are inhibited. The membrane structures were remained, when liquid crystal state of membrane exists. And the braking of all conformation processes is lacking [4]. This is why examining of BAS action on thermally induced transitions of lipid phase is the most important link in circuit test of biological effects of BAS.
Melafen is material, applied in agriculture, as of plant growth regulator. Subject to concentrations it can operate, as the stimulator (10-18 M - 10-13 M), or as the inhibitor (10-9 M - 10-3 M) of the developments of plant seeds and body. When studying its effects on the experimental objects we used the aqueous solutions of Melafen from large concentrations up to ultra small ones (10-3 M - 10-21 M).
Taking into account the close interdependence of vegetation and animal bodies in nature, it was necessary to study the action of plant growth regulator to objects of animal origin. As of simple model of animal cellular biomembranes, being by the primary target for biologically active substances, the phospholipid multulammelar liposomes, formed from DMPC, were used.
For second part of this work the method of small-angle X-ray scattering (SAXS) was used. As experimental object the egg lecithin liposomes were formed.
These two methods, applauded for testing of Melafen-lipids relationships with two membranes models, formed from synthetic individual phospholipid and natural mixture of egg lecithin, were used for solving of primary aim of these investigations -evaluations of the impact of plant growth regulator on membranes of animal origin.
Results
At fist part of this work we investigated the action of Melafen aqueous solution under the wide range of concentrations (10-17 M - 10-3 M) to the phase state of model phospholipid membranes. Data that obtained by the DSC method, are submitted: thermodynamic parameters of liposomes DMPC melting in the presence of aqueous solutions of Melafen over a wide range of concentrations are indicated in table. 1. The results were obtained by means of program "Microcal Origin 5.0" [5].
The temperature, in which maximum heat capacity increasing of DMPC is seen for main endothermic phase transition (Tmax) occurs when 24,1 -24,3° C. The Melafen aqueous solutions were added to liposomes right before melting endoterm registration. Solutions were used over a wide range of Melafen concentrations (10-17 M - 10-3 M). Thermograms of endothermic thermally induced main transition of DMPC in the presence of aqueous solutions of Melafen when large concentrations are submitted at fig. 2. On the assumption of data, that were shown at table 1, one can infer that Melafen in aqueous solutions when low and ultra small concentrations affected the fine structure of membranes in liposomes from DMPC. Melafen at concentrations above 10-17 M enhanced the enthalpy by 1.6-8%. The cooperatives of thermally induced transitions was subtracted, as demonstrated by half
width increasing on half altitude transition, up to 17%. And insignificantly (in limits of accuracy of method) Tmax was shifted apart to higher temperatures on 0,1° C.
The standard DSC conditions of liposomes melting had not shown any important changes for DMPC liposomes, when Melafen everyone aqueous solution concentrations were added. Tmax of main endothermic peak, which were corresponded of thermally induced transition, insignificantly were changed.
23
Fig. 2 - The Melafen influencing to DMPC liposomes melting. The peaks of main endothermic transitions were shown. Thermograms of melting of multulammelar liposomes, formed from DMPC, and melting in the presence of large concentrations of aqueous solution of Melafen. (1) - control 0,15 mg/ml DMPC; (2) - DMPC + 10-5 M Melafen; (3) - DMPC + 10-3 M Melafen; (4) - DMPC + 10-2 M Melafen
Table 1 - Effect of Melafen aqueous solutions under various concentrations on parameters of thermally induced endothermic DMPC main phase transition
Melafen concentration in DMPC suspension (M) Tmax (Co) Cooperativity of main transition (arb.un.) Enthalpy of main transition (arb.un.)
Without Melafen 24.1 0.6 358.1
10-21 24.1 0.7 351.3
10-19 24.1 0.7 347.1
10-17 24.2 0.7 363.9
10-15 24.2 0.6 379.5
10-13 24.2 0.6 370.1
10-11 24.2 0.7 375.2
10-7 24.2 0.7 366.4
10-5 24.2 0.7 372.6
10-3 24.2 0.8 385.4
And the enthalpy and the width of transitions (the reciprocal value of the transition cooperatives) polymodal have undergone of changing.
As a next step of investigation of Melafen influencing to DMPC liposomes we complicated of experimental model study by the special modification. In order to educe the Melafen solution actions to the phospholipid membrane structures the specific approach of different speed of heat supplied (1 degree/min has been used; 0,5 degree/min; 0,25 degree/min; 0,125 degree/min) to cells with control and experimental samples was used by us. Such approach use in order to educe some eventual restructuring of additive cooperative units in membrane microdomains.
From data of thermograms registered when different melting rates of membranes, the concentration dependences of melting enthalpy have been built (the data are introduced on fig. 3). Tmax and the half widths on half altitude of main transition under the wide range of the Melafen concentration strongly marked the extreme, which have been observed in the area of Melafen concentrations 10-14 M - 10-10 M, when all examined melting rates. The extremes and dose-depending of Tmax and half width on half altitude of transition (the quantity reciprocally proportional the cooperativity) were appeared in the same Melafen concentration range, when such method was applauded for DMPC liposomes melting.
It is known that the deceleration of heat supplied to cells with control and experimental samples much changes parameter of thermally induced transitions. Evidently, the relaxation processes in bilayer of phospholipid membranes were so much differently, when such approach was occurred. Indeed, in control samples the changes have been noted too. The deceleration of heat supplied to cells considerably reduces the main phase-transition Tmax and the enthalpy, but the cooperatives were changed so small. The complex polymodal picture of action of Melafen solutions, when large and medium concentrations, was found, when addition of Melafen aqueous solutions in wide concentrations range was produced. It should be noted that enthalpy changes were great in control samples. It was great when ultra small 10-17 M, 10-16 M and large 10-6 M, 10-5 M of Melafen concentrations. Melafen concentration 10-13 M, 10-12 M "hindered" of cooperativity changing. And for changing of Tmax and for width of transitions the same regularity was observed.
So, it has been ascertained that additions of Melafen aqueous solutions to DMPC liposomes exerted the significant complex dose-dependence effects on lipid bilayer structure. It should be noted that Melafen -the hydrophilic substance, deprived of any possibility to be incorporated into lipid bilayer. However, it is known that when bilayer melting the growth of membrane permeability was observed. This phenomenon may be linked with the processes of short-lived nana-size pours forming [6]. The bilayer melting was held when physiologically appropriate temperatures.
As considering that the multulammelar liposomes have as defects of bilayer, and some pores , which are permeable for water [6], it can be assumed that Melafen aqueous solutions operates to all liposomes in multilayer. However all of Melafen effects may to take place only on the surface of each bilayer.
Evidently, Melafen may act or as transducer of water structures near the surface of bilayer, or it directly influences on DMPC phospholipid heads. It was disclosed that in concentration range 10-14 M - 10-10 M Melafen exerts the maximum impact on thermally induced transitions parameters: temperature, in which maximum of heat capacity increasing is seen (Tmax), enthalpy and cooperatives. Obtained data by model membranes and appears to and on biomembranes, once again indicated that are likely, affect may be determined by formation in water media the supramolecular complexes Melafen -aqueous in vitro [7].
Fig. 3 - The of Melafen aqueous solutions with various concentrations effect to cooperativity of main thermally induced DMPC transition when different speeds of heat supplied to DSC cells with control and experimental samples. Different speed of heat supplied (1 degree/min has been used; 0,5 degree/min; 0,25 degree/min; 0,125 degree/min) to cells with control and experimental samples. (Melafen concentration was given in logarithmic scale. The point "0" was conditional)
As was sounded out above, low and ultra small Melafen concentrations do not exert destructive action on model membranes that formed from individual neutral phospholipid. But restructuring of lipid microdomains in bilayer occurred. Large, middle and ultra small Melafen concentration changed the intra membranous organization - microdomains structures of lipids in liposomes formed from individual neutral phospholipid. The polymodal changes of dose-dependent parameters of thermally induced transition may to take place due to certain relaxation conditions of structural elements of bilayer by means of certain changing of melting rates. The experimental model object that were formed from individual neutral phospholipid DMPC, is likely, more vulnerable for Melafen actions compared to liposomes that were composed of mixture neutral and charged phospholipids. Mixtures fairly close simulate the nature stable membranes. Precisely equilibrium distribution of phospholipids provides the bilayer stability. For example, it is known that the destruction of plasma membrane of cells when programmed cell death -apoptosis is associated with the phospholipids redistribution. So when apoptosis, in starting equable on charges and topography of native membrane was
occurred, the phosphatidylserine moving to the exterior leaf of bilayer was occurred too. These phosphatidylserine moving causes the charge destabilization and leads to destroying of the packing regularity in bilayer. The structure breaks down, that was followed by membrane fragmentation, and the cell death comes [8].
These are why the following part of our work was the study on the effect of Melafen aqueous solutions over a wide range of concentrations to the structural organization of lipids in membranes that were composed from mixture of nature phospholipids. The multulammelar liposomes formed from egg lecithin were used as of experimental object. Such multulammelar liposomes formed from egg lecithin are the convenient model, which may be approximated compositionally and structure to nature membranes.
The locations of membranes change in the cell interior due to cellular life activity. Some membranes are coming together. The structures of most membrane are sequenced or become disordered, or removed. The biologically active substances actions may be lacking of certain target of its effects. However, so much of BAS touch the whole processes. It may influence to the structural orders of different organizational levels from the regularity degree of membranes and bilayer thickness up to reciprocal locations of bilayer.
The election of egg lecithin for exploration of Melafen action on membranes, which are composed of nature phospholipids, had been conditioned by the following reasons. The egg lecithin primarily contains phosphatidylcholine and phosphatidylethanolamine, but so less it contains phosphatidylinositol and sfingocholine. To the composition of these phospholipids molecules the polar groups are entering: choline, ethanolamine, and inositol. And fatty acids residues of different quantity and degree of saturation are contained in these molecules. These phospholipids are kept in being in all cell membranes of animal origin. It composes the basis of structural ensuring of membranes integrity. And phospholipids as well as support the membrane components functional activity. For active works of integrated and associated membrane proteins (receptors, channels, ionic exchangers, pumps and enzymes) the certain environment compositions near these proteins are needed. For maintaining of integrated and associated membrane proteins activity the certain types of annular lipids are so important. The proteins primarily interact with zwitterionic lipid -phosphatidylcholine. It molecule in general is electro neutral, since it carries and positive, and negative charges.
The fluidity of membrane lipid phase can control of protein conformation fluctuations, which are required for maintaining of corresponding protein activity. Membrane thickness was conditioned by the length of fatty acids residues and sizes of phospholipid heads in phospholipids molecules. Membrane thickness permits to control of intensity of function of proteins that are built into membrane. For example, the large integral protein - Ca2+-ATPase (from family of SERCA2) changes it affinity for ligands in dependence of membrane thickness. So, the operating of depth or
outcropping of membranous loops with ligands-binding centers regulates the work activity of this Ca2+-pump. Ca2+-ATPase of sarcoplasmic reticulum (the main Ca2+-store in muscle cells) is surrounded by the phosphatidylethanolamine, witch has the cone molecular shape. This type of molecular structure allows of lipids molecules to form the hexagonal phase. For example, associated proteins - G-proteins, which are linked and implanted in membrane, are surrounded by phosphatidylethanolamine too [9]. G-proteins conduct the signals from receptors located in the outer surface of plasmalemma to cytoplasm enzymes. Their activities are in great dependence in lipid environment.
In connection with enumerated arguments, we separated the phospholipid models for test action of biologically active substance as experimental models, which are fairly actuating for many cell structural and functional parameters.
Above it was seen that DSC method, which was based on different heating rate of cell with experimental sample of liposomes DMPC, brought to light to the significant influences of Melafen aqueous solution (over a wide range of concentrations) on organization of lipid microdomains in bilayer. The 3 parameters of thermally induced endothermic main transitions of DMPC were registered by us. It was testified that the microdomains organization of phospholipid in bilayer was in subject to Melafen concentration.
It was known that Melafen is the hydrophilic molecule. But we noted that it action extends to all thickness of multulammelar liposomes. Since the peak of main transition remain clearly discovered. In visible, Melafen enters via defects of bilayer. It is possible of education of nana-pores during phase transitions also. Melafen action shall be exercised on hydrophilic surface of each bilayer. It can be assumed that Melafen changes the water medium around liposomes. It either forms the supramolecular complexes of Melafen-aqua, or it changes the gas solubility in the water solution.
However in liposomes formed from the egg lecithin, i.e. from the natural phospholipids mixture, we could not measure the parameters of microdomain organization of lipid bilayer by the DSC method correctly because the heat capacity peaks for each phospholipid in such mixture were overlapped. Due to this fact we did not get any clear picture suitable for quantitative parameter estimation of thermally induced transitions in the egg lecithin membranes. This is why we investigated the structural changes of lipid membranes, composed of natural phospholipids mixture, under the action of Melafen with the aid of another method, namely by the small-angle X-ray scattering technique. Thus we have studied the next more complicated level of membrane structural organization in multilammelar liposomes.
At the second part of this work the action of Melafen on the structure of phospholipid membranes in multilayer liposomes was studied by one of the methods of X-ray diffraction analysis, namely by small-angle X-ray scattering (SAXS) [10]. This method permits one on the basis of the analysis of the measured SAXS intensity to get information about low-resolution structure of
lipid membranes and to characterize membrane stacking parameters in liposomes.
The SAXS study of liposomes was carried out with an automated small-angle X-ray diffractometer assembled in the Institute of Biochemical Physics RAS. This diffractometer was developed on the basis of the construction of the small-angle X-ray diffractometer AMUR-K elaborated and exploited earlier in the Institute of Crystallography RAS.
Because the diffractometer used in this study is a custom-built device a general description of its construction is given hereinafter.
The X-ray source in this diffractometer is an X-ray generator IRIS-M (Nauch-Pribor, Orel, Russia) with a fine focus X-ray tube with a copper anode BSV29Cu (Svetlana-Roentgen, St. Petersburg, Russia). The X-ray beam passes through a Ni p-filter, and then is focused with a glass mirror and collimated with tantalum slits. A sample for SAXS study (liposome suspension) is placed in a thin-walled (wall thickness ~10 ^m) glass capillary of ~1 mm outer diameter (sample volume ~15 ^l).
SAXS patterns are recorded with the gas-filled (85% Xe, 15% Me, 4 atm) linear position-sensitive detector constructed in the Joint Institute for Nuclear Research (Dubna, Russia) [11]. The detector has 1024 channels of discretization, the width of each channel is 97 micrometers. For CuKa X-ray radiation (Aavr=0.1542 nm) the detector has the special resolution of 150-200 micrometers and the efficiency of registration about 75%. SAXS patterns recorded with the detector are transferred to a personal computer for storage and processing.
For an X-ray tube with a copper anode and sample-to-detector distances from 130 to 425 mm accessible in the diffractometer it is possible to record the intensity of X-ray scattering in the range of the values of diffraction vector module S = (2sin0)/A from ~0.015 nm to ~3.5 nm-1 (A is the wavelength of X-radiation, 0 is a half of a scattering angle). This range of S values corresponds to the range of Wolf-Bragg distances D = (S)-1 from ~67 nm to ~0.286 nm.
Due to the X-ray beam focusing with a mirror and simultaneous registration of X-ray diffraction pattern with a position-sensitive detector a high intensity of X-ray scattering and high sensitivity of registration are achieved in such a diffractometer. This is very important for efficient SAXS study of liposomes and other biologic objects which have labile structure and scatter X-rays rather poorly.
Multilayered liposomes for SAXS study were formed by the method of hydration of lipid films. At first the egg lecithin (Sigma, USA) was dissolved in chloroform in a glass flask. Then chloroform was evaporated from this solution under argon gas flow and a thin lipid film was formed on the walls of the flask. For a complete removal of chloroform from a lipid film it was kept under vacuum not less than 12 hours. After this a small amount of buffer solution (50 mM phosphate buffer, pH 7.5, 200 mM of NaCl) was poured in the flask with a lipid film, the flask was filled with argon gas, heated up to ~45° C and then was vigorously shaken with an electric shaker until a lipid film was totally dispersed in the buffer solution. The lipid
concentration in liposome suspensions used in SAXS study was about 10%. Melafen was dissolved in the same buffer solution and was added to liposome suspensions at ~25° C in quantities required to achieve Melafen concentration in these suspensions equal to 1021 M, 10-18 M, 10-2 M and 10-6 M. Prepared liposome suspensions with Melafen were stored in closed vials under argon gas at a temperature of ~5°C before being used in the SAXS study.
The SAXS study of liposome suspensions was done at the ambient room temperature (~22° C). The duration of the SAXS intensity measurement for each liposome specimen was 30-50 min.
For background scattering correction the SAXS intensity of the capillary with buffer solution was measured and then subtracted from the SAXS intensity measured for each liposome sample. To take account of lipid concentration variation in liposome samples arising from addition of Melafen solutions in liposome dispersion the SAXS intensity for all samples was normalized to have the equal peak intensity at S ~ 0.14 nM-1. SAXS patterns smoothing and desmearing with method [12] were done with PRO software developed in the Institute of Crystallography RAS (SAXS patterns smearing is intrinsic for diffractometers with slit collimation systems).
SAXS patterns for all liposome samples studied (liposome suspensions containing 10-21 M, 10-18 M, 10-12 M, 10-6 M of Melafen and without Melafen) disclose two diffraction peaks (reflections) with maxima at S ~ 0.14 nm-1 and S ~ 0.29 nm-1. As an example, two experimental SAXS patterns of liposome suspensions containing 10-6 mM of Melafen and without Melafen are shown in fig. 4a. In these patterns (fig. 4a) background scattering was subtracted and intensities were normalized. The same SAXS patterns after smoothing and collimation correction are submitted in fig. 4b.
The X-ray diffraction peaks on the SAXS patterns for all studied liposome suspensions were equidistant (the abscissa of the second peak is approximately twice the abscissa of the first peak). Consequently, these diffraction peaks can be considered as the first and the second orders of reflection for membrane multilayers in liposomes (lipid lamellar phase).
The normalized SAXS intensities for all liposome samples studied (liposome suspensions containing Melafen and control suspensions without Melafen) were very similar, as illustrated for two samples of liposome suspensions containing 10-6 mM of Melafen and without Melafen in figs. 4a and 4b.
This indicates that the structure of lipid membranes and their stacking parameters in liposomes are not noticeably altered by Melafen addition to liposome suspension.
The period D of membrane stacking in liposomes was calculated for liposome samples after collimation correction according to the Wolf-Bragg formula as D=h(Sh)1. In this formula h is the order of reflection and Sh is the abscissa of this reflection determined as the value of S at which the first derivative of SAXS intensity goes to zero.
5 40
•t
c 1500
"a>
(A 1000
c
b
1-10 M of Melafen
2 — .vith -.v*. Melafen
,J La
C 0 1 0 2 03 OA 0.5 OS
S. nm~
Fig. 4 - SAXS patterns of liposome suspension containing 10-6 M of Melafen (1) and liposome suspension without Melafen (2): a - experimental SAXS patterns after subtraction of background scattering and intensity normalization, b - the same SAXS patterns after smoothing and collimation correction. S = (2sin0)A,
Results obtained showed that the values of period D of membrane stacking in liposomes at all studied Melafen concentrations and in liposomes without Melafen were the same with the accuracy ±0.01 nm (tab. 2). Such accuracy does not exceed the experimental error. So we can state that within experimental error of the SAXS method Melafen when concentrations 10-21 M, 10-18 M, 1012 M and 10-6 M has no influence on membrane stacking period in multilammelar liposomes formed from the egg lecithin.
Table 2 - Lipid membranes stacking period D in liposomes when various Melafen concentrations in buffer solution. The values of D were calculated after collimation correction of SAXS patterns and for each pattern were averaged on two orders of diffraction
Melafen concentrations D, nm
Without Melafen (control №1) 6.88
10-21 M 6.89
10-18 M 6.88
10-12 M 6.88
10-6 M 6.88
Without Melafen (control №2) 6.90
The widths of corresponding diffraction peaks were also one and the same for all liposome suspensions studied as illustrated for two samples in figs. 4a and 4b. This is the evidence for the same size of the regions of coherent scattering and the same degree of membrane stacking order in these regions for membrane multilayers in liposome suspensions containing 10-21 M, 10-18 M, 10-12 M, 10-6 M of Melafen and in liposome suspensions without Melafen.
For further analysis of Melafen action on lipid membrane structure in liposomes the electron density profiles of these membranes when various concentrations of Melafen in buffer solution were calculated on the basis of the SAXS data.
One-dimensional centrosymmetric electron density profiles p(x) of membrane multilayers in liposomes were calculated at the relative scale in the normal direction to membrane planes by means of Fourier transformation of collimation corrected SAXS patterns utilizing the formula:
p( x) = 2/D]TP(h)^A/1(h)cos(2^hx) , (1)
h=\ D In this formula D is a stacking period of membranes in liposomes, h is an order of reflection (in our case this is a number of a diffraction maximum), P(h) is a phase sign (+ or -) for the h order reflection, /(h) is the integrated intensity of the h order reflection, N is a maximum order of reflections (in our case N=2). The multiplier h in formula (1) is the intensity correction factor for membrane multilayers disorientation in liposome suspension (Lorenz factor).
The electron density profiles of lipid membranes calculated with formula (1) and utilizing SAXS patterns depicted in fig. 4b for liposome suspensions containing 10-6 M of Melafen and without Melafen are shown in fig. 5.
Fig. 5 - Electron density profiles of phospholipid membranes in multulammelar liposomes formed from egg lecithin: 1 - for liposome suspension containing 10-6 M of Melafen; 2 - for liposome suspension without Melafen (x is a distance in the direction normal to membrane planes)
The peaks of the electron density on these profiles at x ~ 1.4 nm and x ~ 5.4 nm can be associated with the position of lipid polar groups in a lipid bilayer,
and the electron density minimum at x ~ 3.4 nm corresponds to the central hydrophobic part of a lipid bilayer. The thickness of lipid membrane defined as the distance between the peaks of the electron density at x ~ 1.4 nm and x ~ 5.4 nm is about 4 nm. As is seen from fig 5, the electron density profiles of lipid membranes for liposome suspensions containing 10-6 M of Melafen and without Melafen are identical. The same electron density profiles of lipid membranes were obtained when Melafen concentrations in buffer solution equal to 10-21 M, 10-18 M and 10-2 M.
So, the results of the SAXS study of liposome suspensions show that Melafen, which presents in wide concentration range (from 10-21 M up to 10-6 M), has no visible effect on the structure and stacking parameters of phospholipid membranes in multilayered liposomes formed from egg lecithin. The absence of destructive changes of phospholipid membranes in the presence of Melafen in solution may be considered as a positive factor for practical applications of this biologically active substance.
Conclusion
These investigations are actual because the cell membranes are the primary targets for exogenous operative factors (biologically active substances at our case) entering into animal body by all ways. Lipids are the major and prevailing components of the animal cellular membranes. This is why phospholipids were chosen for our investigations, as the most widespread components of membrane lipid phase. For differential scanning calorimetric testing of membrane structural properties the synthetic individual neutral phospholipid DMPC was used. When melting, DMPC multilammelar liposomes have simple thermograms with two phase endothermic transitions in physiological temperature range. These thermograms are interpreted easily. Pretransition and the main phase transition in the presence of biologically active substances changed its parameters (enthalpy, Tmax and cooperativity of the main phase transition). Our formulation of experiment was complicated by the special modification. This modification consisted in four different speeds of heat supplied to DSC cells, which contained control and experimental samples. This type of model study is fairly physiological because restructurings of lipid phase in membranes take place in animal organism constantly. In certain cases (for instance, when hibernation) membrane restructurings are temperature-dependent. And the rates of temperature changing are different sometimes also.
For experiments with the aid of the small-angle X-ray scattering method the multulammelar liposomes formed from a natural phospholipid mixture were used. The multulammelar liposomes formed from egg lecithin are a difficult object for differential scanning calorimetric test. The phospholipid mixture gives the confluent peaks of heat capacity and the estimation of calorimetric parameters by deconvolution procedure does not allow correct deciphering of the biologically active substances effect on the certain lipid microdomains. This is why we studied by the SAXS technique the next organizational level of lipid membranes - the lamellar structure and mutual
arrangement of lipid membranes in multilayer liposomes.
We did not reveal by the SAXS method any noticeable structural changes of the egg lecithin membranes at Melafen concentrations used for a crop production. The SAXS method disclosed that the bilayer organization of multulammelar liposomes formed from egg lecithin was not changed in the presence of Melafen.
But on the base of differential scanning microcalorymetry data we discovered that the microdomain structure organization of DMPC liposome membrane was changed by Melafen aqueous solutions in the wide concentration range. The types of these variations were rather complex.
We concluded that Melafen changed the microdomain organization of structure in membrane formed from individual neutral phospholipid and did not affect the membrane structure at the next organizational level. Namely Melafen did not change the overall size and spacing of bilayers in multulammelar liposomes formed from natural phospholipid mixture. These results were reported shortly elsewhere [13].
However, we should indicate that the aqueous solutions of Melafen altered the structural properties of labile objects which can easily change their conformation. Such influence was shown for objects related not to plant life, but to animal life, namely for liposomes formed from individual phospholipid [13] and for the water soluble protein (bovine serum albumin) [14]. As membrane composition becomes more complicated, the membrane structure becomes more resistant to the bed environment (or biologically active substances as in our case). These phenomena in our works were shown for liposomes formed from a mixture of natural phospholipids and for erythrocyte ghost [15]. The aqueous solutions of Melafen did not influence on these objects. However, when experimental object acquired any functions, the structure of this object began to be exposed to Melafen influence. In this case the alteration of structural parameters of the model object under the action of biologically active substances may be mediated by the influence of these substances on some functions of this object.
As an example of this phenomenon one can mention the shape changing of erythrocytes [16]. The aqueous solutions of Melafen exert significant influence on cell function. The purine-dependent calcium signaling in insulated thymocytes, lymphocytes and cells of ascetic Ehrlich carcinoma decreased to the complete oppression when large concentrations of Melafen [17].
The main finding following from all our studies [13-17] is the necessity of very strict observance of concentration limitations of Melafen when it will use as the plant growth regulator. This is vitally important for the prevention of Melafen negative influence on bodies of animal origin.
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© O. M. Alekseeva - Ph.D., Senior Researcher, Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia, hvatovanatoliy@gmail.com; A. V. Krementsova - Ph.D., Researcher, Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia, Yu. A. Kim - Doctor of Physics and Mathematical Sciences, Leader Researcher, Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Russia, A. V. Krivandin - Ph.D., Senior Researcher, Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia, O. V. Shatalova - Researcher, Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia G. E. Zaikov - professor of plastics technology department of KNRTU, .
© О. М. Алексеева - кандидат биологических наук, старший научный сотрудник, Институт биохимической физики им. Н.М. Эмануэля РАН, Москва, Россия, hvatovanatoliy@gmail.com; А. В. Кременцова - кандидат физико-математических наук, научный сотрудник, Институт биохимической физики им. Н.М. Эмануэля РАН, Москва, Россия, Ю. А. Ким - доктор физико-математических наук, ведущий научный сотрудник, Институт биофизики клетки РАН, Пущино, Россия, А. В. Кривандин -кандидат физико-математических наук, старший научный сотрудник, Институт биохимической физики им. Н.М. Эмануэля РАН, Москва, Россия, О. В. Шаталова - научный сотрудник, Институт биохимической физики им. Н.М. Эмануэля РАН, Москва, Россия Г. Е. Заиков - профессор каф. ТПМ КНИТУ.
