JPS63256129A - Method for controlling vehicular mixture and vehicular structure - Google Patents

Abstract

PURPOSE:To optically control the solubility of a polyamino acid in a bimolecular film by introducing a photosensitive group into the side chain of the polyamino acid, and utilizing a change in the polarity due to the resulting photoisomeriza tion reaction. CONSTITUTION:The solubility of a membrane protein substance in a lipid bimolecular film is controlled by changing the bipolarity with the photoisome rization reaction of the polyamino acid wherein azobenzene as the photosensitive group is introduced into the side chain. In addition, a polyamino acid wherein a photosensitive group, the hydrophilic head group of a lipid bimolecular film forming substance, and an oppositely chargeable functional group are introduced into the side chain is used to change the bipolarity with the photoisomerization reaction of the photosensitive group, and the solubility of the polyamino acid in the lipid bimolecular film is changed. The interionic action between the functional group of the polyamino acid and the hydrophilic head group of the lipid bimolecular film is controlled, and the vehicular structure is controlled in an aggregated and dispersed state.

Description

[Detailed Description of the Invention] [Summary] The present invention uses a low degree of polymerization polyamino acid with a molecular length comparable to the thickness of a bilayer membrane as a membrane protein model substance, and a cationic double-stranded polyamino acid as a bilayer membrane forming substance. This invention relates to a method of controlling the interaction between membrane proteins and lipid molecules using surfactants, particularly the solubility of membrane proteins in a lipid 9-mask phase layer due to differences in polarity.

Furthermore, the present invention introduces azobenzene, which is a photosensitive group, into the side chain of a polyamino acid, and utilizes the change in polarity caused by the photoisomerization reaction to photocontrol the solubility of the polyamino acid in a bilayer membrane. Regarding the method.

The present invention also provides a lipid bilayer-forming substance having a hydrophobic long-chain alkyl group and a hydrophilic head group, which takes a gel state at a temperature lower than the Tc temperature and a liquid crystal state at a temperature higher than the Tc temperature, and , change its polarity from the first state to the second state
A polyamino acid having a photosensitive group that reversibly changes its polarity from the second state to the first state upon irradiation with a second excitation light, and a hydrophilic head group and an oppositely charged functional group introduced into the side chain. A mixture containing vesicles dispersed in fine particle form in water or an aqueous solution is heated to a temperature lower than the Tc temperature, the lipid bilayer membrane-forming substance of the vesicles is brought into a gel state, and the first excitation light is irradiated. , change the polarity of the photosensitive group to the second state, change the solubility of the polyamino acid in the lipid bilayer forming substance, elute the polyamino acid from the bilayer membrane, and improve the hydrophilicity of the lipid bilayer forming substance. The ionic interaction between the head group and the oppositely charged functional group of the eluted polyamino acid causes the vesicles to form an aggregated structure, and the mixture in which the vesicles are in the aggregated structure is irradiated with a second excitation light to release the photosensitive groups. heating the mixture in which the vesicles are in an aggregated structure above the Tc temperature to bring the lipid bilayer membrane-forming substance of the vesicles into a liquid crystal state, and redissolve the polyamino acid in the bilayer membrane; According to
This invention relates to a method for controlling the vesicle structure by releasing ionic interactions and making the vesicle a decomposed structure.

(Industrial fields of application and conventional technology) Development of functional membranes related to energy conversion functions such as high-sensitivity membrane sensors with gradient H'' concentration regarding active transport phenomena unique to biological membranes, and consideration of application fields of biological membrane function expression. Purposeful extraction of the functions put into the system has begun.

These membrane functions are not always expressed in biological systems, but concentrate and separate specific molecules and produce energy when and as needed. We cannot afford to ignore the mechanism of membrane function control that biological membranes have. These control mechanisms often involve structural changes in membrane materials. For example, when a KCI aqueous solution is separated by a phospholipid bilayer membrane containing colicin K and a potential difference is applied to both sides of the membrane, an all or none voltage-current characteristic is exhibited after 45 mV.

A system in which permeability is controlled by inducing structural changes using Na'' concentration gradients or light as acting factors is also known. When lectins act on lymphocytes, Ca'' permeation is controlled. This is thought to be due to structural changes in the membrane caused by the binding of lectins to membrane proteins, resulting in changes in the transport of Ca. Neuronal membrane excitation coupled to the control of monovalent cation permeability. The effect of calcium ions on membranes is an example of the relationship between structural changes and permeability that has been known for a long time.

However, it is strongly desired to elucidate and apply a method for controlling the permeability by conjugating such changes in membrane structure and permeability. (Journal of the Chemical Society of Japan, 868.19
83) [Means and effects for solving the problems] The present invention improves the solubility of membrane protein substances in lipid bilayer membranes by using a pre-containing reaction of polyamino acids with photosensitive groups introduced into their side chains. It is controlled by changing the dipole mo-ment.

In addition, the present invention uses a polyamino acid into which a photosensitive group, a hydrophilic head group of a lipid bilayer membrane-forming substance, and an oppositely charged functional group are introduced into the side chain, and a dipolar by changing the solubility of polyamino acids in lipid bilayer membranes, controlling the ionic interaction between the functional groups of polyamino acids and the hydrophilic head groups of lipid bilayer membranes, and changing the vesicle structure into agglomerated or dispersed states. control to the state.

〔Example〕

Examples of the present invention will be described in detail below.

[1] Experiment, low-density copoly(γ-methyl-L-glutamate/L-glutamic acid), a membrane protein model substance
(MG/GA), and poly(L-glutamic acid)
(PGA) (degree of polymerization 72) was used. Distearyldimethylammonium chloride (DSACL) was used as a bilayer membrane forming substance.

The molecular structures of PIIIG, MG/GA, PGA, and DSACL are as follows.

4NH-CH-CO) 4NH-CH-Co valve-IH-C
I-Co) (Nl(-C8-COarrow PMG
MG/GA PGA Also, the samples used for fluorescence measurements were term, ANS-PMG, in which the PMG terminal was labeled with ANS, 5ide, ANe-PGA, 5ide, and ANS-MG in which the side chain was labeled with ANS.
/GA, and 5ide, whose side chain was labeled with pyrene,
Pyrene-(87/13 MG/GA) was used.

Furthermore, zao-MG/GA containing 13% azobenzene in its side chain was used as a photoresponsive polyamino acid.

For fluorescence spectrophotometry, RF-54ot manufactured by Shimabara Manufacturing Co., Ltd.
Fluorescence spectra were measured using (DSACL) = 8.5 XIOM-''.
Using ION Giken FS-501A, (DSACL)
= 8.5 Xl0M −'<7) 2.2
OM-' was added and measured.

For ion permeation experiments in vesicle systems, a Tris-HEPES buffer containing 0.1M sodium gluconate was used.
After gel filtration of the vesicles adjusted to H6,8, the same T
0 adjusted to pH 6.8 with ris-HEPES buffer.
In addition to 1M potassium gluconate aqueous solution, changes in the sodium ion concentration of the aqueous solution outside the vesicles were measured using Horiba Seisakusho N.
Measurements were made using a -7ion H-type ion meter. Furthermore, optical stimulation (bovine No. 500W, D-33S or Shi-
No. 39 was used as a filter. ) by 13-12/
Changes in the solubility of 75azo-MG/GA in bilayer membranes using changes in polarity were measured using a fluorescence method, and morphological changes in vesicles were also observed using a phase contrast microscope (Oli≠Bass B).
HR).

[2] As a result, Figure 1 shows the relationship between long-chain alkyl chain moieties of vesicles not containing polyamino acids and vesicles containing polyamino acids measured by fluorescence depolarization method using diphenyl-1,3,5-hexatriene DPH. The results of measuring fluidity are shown. In both systems, the fluidity changes significantly at Tc (40°C), and by introducing polyamino acids, the fluidity of the system is increased in both the gel state and liquid crystal state. The trend was more pronounced in PGA. The dissolution of polyamino acids into bilayer membranes was measured using samples in which polyamino acid side chains were modified with ANS, which shifts the maximum fluorescence wavelength depending on the polarity of the medium. (Figure 2). As a result, term, A
PM
The G molecule dissolves in the rearrangement and exists as if penetrating the bilayer membrane, while 5ide, ANS-PGA's λ, and n+ax exist on the hydrophilic side, so the PGA molecule exists near the bilayer membrane head group. It is possible that From the above results, PMG
The molecule interacts hydrophobically with the long chain alkyl group of the DSACL molecule in the bilayer membrane, disrupting its backing structure and increasing its fluidity, and the PGA molecule interacts with the side chain carboxylic group and D S
It is thought that this bilayer membrane structure is disturbed by the ionic interaction of ACL with quaternary ammonium.

5ide, ANS-(88/12 MG/GA), 5
Similar measurements were made for IDE and ANS- (80/20 MG/GA), and as a result, there were two peaks, one on the high wavelength side and one on the low wavelength side, instead of just a - peak as shown in Figure 3. The emission on the wavelength side is assumed to be the emission in the bilayer membrane, and the emission on the high wavelength side is assumed to be the emission near the head group, and from this intensity ratio, the equilibrium constant of the equilibrium reaction as shown below was determined and a thermodynamic consideration was attempted.

At this time, since the fluorescence intensity of ANS changes depending on the polarity of the medium, correction was also made for this.

MG/GA □ MG/GA at mem
bran 5 surface in
membraneside, ANS-(80/20
MG/GA) (Fig. 4). This allows in
Since k is a positive value in the gel state and a negative value in the liquid crystal state, 80/20 MG/GA exists mainly near the bilayer membrane head group in the gel state and within the bilayer membrane in the liquid crystal state. It is thought that

Furthermore, the entropy change due to the incorporation of MG/GA into the bilayer membrane and the entropy change were determined using the following equation.

R, T R As a result, ΔH is 2.9 KJ/mo in gel and liquid crystal display states.
l.

is constant, but ΔS is 7.5 J/mol in gel state.
12J/mo1. in liquid crystal state. It can be said that the entropy term is mainly involved in the incorporation of the thick 80/20 MG/GA into the bilayer membrane. 88/12
A similar measurement was made for MG/GA, and it is thought that it increases with ΔH and the liquid crystal state. On the other hand, in the vesicle system Table, 1. Thermodynamfcpa
ramater ΔH1Δ(ΔS), for th
etransfer of MG/GA copoly
merfrom the membrane 5
surface to the interior.

For sodium ion permeation measurement of MG/GA12 MG/GA20, gluconate C6, which has a large molecular size, is used as an anion to prevent its leakage and human exposure.
HIZO? was used. In other words, the internal solution of the icicle has a pH of 6.
The osmotic pressure difference was brought to zero by using a 0.1 M aqueous solution of potassium gluconate at pH 6 and 8 as the external liquid of the vesicle.

88/12 MG/GA DMF solution (1mg/cc)
Figure 5 shows the results of measuring the sodium ion release rate before and after addition using an ion meter. In the gel state, the release rate after addition of 88/12 MG/GA is about 2.1 times that before addition, whereas in the liquid crystal state, the release rate after addition increases to about 3.0 times after addition. are doing. Also 5ide, Pyrene- (87/13 MG/GA)
The ratio of the excimer emission intensity to the monomer emission intensity based on the association in the vesicles was measured, and the ratio was larger in the liquid crystal state than in the gel state, so it was 87/13MG/
It is thought that GA has a stronger tendency to aggregate in the liquid crystal state (Figure 6). These results are 88/12 in liquid crystal state.
The increase in the ion release rate due to the addition of MG/GA is due to the increase in the solubility of 88/12 MG/GA in the bilayer membrane in the liquid crystal state. This suggests that a permeation path was formed. From the above results, it was quantitatively confirmed that the solubility in the bilayer membrane differs depending on the polarity of polyamino acids. We attempted to control the solubility of polyamino acids in bilayer membranes using light. azo-MG/GA in a bilayer film has an excitation wavelength of 290 nm and a maximum fluorescence wavelength of 445 nm;
Since m almost matches the excitation wavelength of ANS, 450 nm,
ANS was added to the vesicle solution, and the response of the fluorescence intensity of <470 nm to light and thermal stimulation was investigated based on the excitation of azo-MG-/GA (290 nm). Due to its presence near the bilayer membrane head group, azo-M can be stimulated by light or heat.
The solubility of G/GA in the bilayer membrane changes, and the solubility of azo-MG/GA in the bilayer membrane is increased by utilizing the fact that the fluorescence of azo-MG/GA existing near the ANS excites the ANS. The results are shown in Figure 7. 20°C
By irradiating UV in the gel state, azobenzene photoisomerizes from the trans form to the cis form, forming azo-MG/
As the polarity of GA increases, it is eluted from the bilayer membrane, and an increase in the fluorescence intensity of ANS is observed (FIG. 7B-+b). However, even if the trans isomer is restored by irradiation with visible light at this temperature, azo-MG/GA does not re-dissolve into the bilayer membrane in a gel state (Fig. 7b), but when the temperature is increased to 55°C, which is higher than Tc. When the temperature was increased, azo-MG/GA re-incorporated into the bilayer membrane and the fluorescence intensity of ANS decreased (Fig. 7C). Changes in vesicle morphology during this series of cycles of light and heat stimulation were observed using a phase contrast microscope. In the gel state below Tc, azo-MG/GA exists in the bilayer membrane and individual vesicles are dispersed;
Ki to take o-MG #Reihori = I. Even after irradiating this with visible light, no change was observed in this aggregated structure, but it returned to a structure higher than Tc). In other words, the 330 nm light stimulus is fixed in the form of intervesicle association, which is not disturbed by the 390 nm or higher light stimulus, but after that? It will be erased by increasing the temperature (Figure 3).

[Effects of the Invention] By introducing a photosensitive group into the side chain of a polyamino acid and utilizing the change in polarity caused by the tactile reaction, the solubility of the polyamino acid in a bilayer membrane can be optically controlled.

By including the above-mentioned polyamino acids in the bilayer membrane-forming substance, the vesicle structure can be optically controlled.

[Brief explanation of drawings]

Figure 1 shows the fluidity of the long alkyl chain portion of vesicles that do not contain polyamino acids and vesicles that contain polyamino acids. Figure 2 shows the maximum fluorescence wavelength (λmax) depending on the polarity of the medium.
Figure 3 shows the relationship between the Z value (polarity) and λmax measured using a sample in which ANS was modified to a polyamino acid side chain. Figure 4 shows the fluorescence spectrum of 80
/20 A diagram showing the temperature dependence of Ink for the transition from the film surface to the inside of the MG/GA copolymer film, Figure 5 is 88/1
Figure 6 shows the ion release rate of vesicles containing MG/GA copolymer in the gel and liquid crystal phase states.
Figure 7 shows the temperature dependence of the ratio of excimer emission intensity to monomer emission intensity based on the association of Azo-MG/GA) in vesicles. A diagram showing the dependence of the luminescence spectrum on the gel/liquid state of vesicles. The diagram shows the change in the morphology of vesicles according to the principles of the present invention during cycles of light and heat stimulation. o term AN3-psi/〆es/c/eb
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Claims (7)

[Claims] (1) A lipid bilayer-forming substance having a hydrophobic long-chain alkyl group and a hydrophilic head group, which assumes a gel state at a temperature lower than the Tc temperature and a liquid crystal state at a temperature higher than the Tc temperature, and the first excitation light irradiation. changing the polarity from the first state to the second state, and changing the polarity from the second state to the first state by irradiation with a second excitation light;
A mixture of a photosensitive group that undergoes a reversible change and a polyamino acid having a hydrophilic head group and an oppositely charged functional group introduced into its side chain contains vesicles dispersed in fine particles in water or an aqueous solution. A vesicle mixture characterized by: (2) A lipid bilayer-forming substance having a hydrophobic long-chain alkyl group and a hydrophilic head group that assumes a gel state at a temperature lower than the Tc temperature and a liquid crystal state at a temperature higher than the Tc temperature, and changing the polarity from the first state to the second state, and changing the polarity from the second state to the first state by irradiation with a second excitation light;
Water or an aqueous solution is added to an organic solution in which a photosensitive group that undergoes a reversible change and a polyamino acid in which a hydrophilic head group and an oppositely charged functional group are introduced into the side chain are dissolved, and the liquid-liquid phase is separated. A first step of forming vesicles in which the mixture containing the lipid bilayer membrane-forming substance and the polyamino acid is dispersed in water or an aqueous solution in the form of fine particles by irradiating ultrasonic waves; The temperature is lower than the Tc temperature,
a second step of bringing the lipid bilayer membrane-forming substance of the vesicle into a gel state; and irradiating the mixture containing the vesicles in which the lipid bilayer membrane-forming substance is in a gel state with the first excitation light. , changing the polarity of the photosensitive group to the second state, changing the solubility of the polyamino acid in the lipid bilayer membrane-forming substance, and eluting the polyamino acid from the bilayer membrane, and a third step of forming the vesicles into an aggregated structure through ionic interaction between the hydrophilic head group of the bilayer membrane-forming substance and the oppositely charged functional group of the eluted polyamino acid; A fourth method of irradiating the mixture with the second excitation light to bring the polarity of the photosensitive group into the first state.
heating the mixture in which the vesicles are in an aggregated structure above the Tc temperature to bring the lipid bilayer membrane-forming substance of the vesicles into a liquid crystal state, and redissolve the polyamino acid in the bilayer membrane; A method for controlling a vesicle structure, comprising a fifth step of releasing the ionic interaction and forming the vesicle into a dispersed structure. (3) The method according to item 2, wherein the lipid bilayer membrane-forming substance is a cationic double-stranded surfactant. (4) The method according to item 2, wherein the polyamino acid is a low polymerization degree polyamino acid having a molecular length comparable to the thickness of the bilayer membrane. (5) The method according to item 2, wherein the photosensitive group is azobenzene. (6) The method according to item 2, wherein the functional group is a carboxyl group. (7) The lipid bilayer membrane-forming substance is distearyldimethylammonium chloride, and the polyamino acid is a copoly(γ-methyl-L-glutamate/L-glutamic acid) having an azobenzene group in its side chain.
(azo-MG/GA), and the first excitation light is light with a wavelength of 330 nm or less, and the second excitation light is light with a wavelength longer than 390 nm. the method of.