WO2004005899A1

WO2004005899A1 - Method and device for detecting protein association by dispersion constant variation

Info

Publication number: WO2004005899A1
Application number: PCT/JP2003/008442
Authority: WO
Inventors: Masahide Terajima
Original assignee: Kansai Technology Licensing Organization Co., Ltd.; Shimadzu Corporation
Priority date: 2002-07-05
Filing date: 2003-07-02
Publication date: 2004-01-15
Also published as: AU2003281304A8; JP2004085528A; AU2003281304A1

Abstract

A method for detecting protein association by dispersion constant variation. A specimen such as a protein is added to a protein-containing liquid where a target protein is dispersed to produce a particle mixture system (13). A pair of excitation pulse laser beams (14) of the same wavelength are applied to the particle mixture system (13) in such ways that the laser beams intersect in the particle mixture system to form a transient diffraction grating. A probing laser beam (15) of different wavelength is applied to the transient diffraction grating at a different angle. The intensity of the transient diffracted wave of the laser beam is measured to determine the variation of the dispersion constant.

Description

Satsuki Itoda β Method and apparatus for detecting protein association by change in diffusion constant

The present invention relates to a novel protein association detection method and apparatus, and more particularly to an association detection method and apparatus using a change in diffusion constant.

Background art

In the medical and pharmaceutical fields, it is extremely important to determine whether a protein and a substance such as another protein have a specific interacting relationship, but its detection involves considerable difficulty. is there. One method is to fractionate the cell lysate with an ion-exchange column, flow each onto a column immobilized on the partner protein, and then conduct an association experiment with a specific protein. The equipment configuration is complicated, and the operation is cumbersome and time-consuming. Another method is spectroscopic analysis involving weight analysis using surface plasmons. For example, a test solution is attached to the surface of a sensor chip (a glass substrate with a deposited film of gold, etc.). Such as troublesome pre-processing and careful operation when applying this to the prism (for example,

Patent Document 1: Japanese Patent Application Laid-Open No. 7-159319 (refer to the “Summary” column)).

Furthermore, a fluorescence polarization measurement system is also used to measure and analyze the interaction between biological substances such as proteins and nucleic acids, but there is a problem that the configuration of the optical system is extremely complicated and large. Therefore, development of a method for simply and accurately measuring the degree of association between substances without using these conventional methods is awaited.

One object of the present invention is to provide a method and an apparatus for simply and accurately measuring the degree of protein association by measuring the change in the diffusion constant of a mixed system of a target protein and a test protein. is there.

A second object of the present invention is to provide a method for determining the diffusion of a mixture of a target protein and a test protein. It is an object of the present invention to provide a novel method and apparatus suitable for measuring a change in number in a very short time.

Disclosure of the invention

In order to solve the above-mentioned problems, the present invention firstly comprises adding a test protein to a protein-containing solution in which evening protein is dispersed, and detecting a change in the diffusion constant of the particle-mixing system caused thereby. And a method for measuring the degree of association between the two proteins.

Second, the present invention secondly causes a pair of excitation pulse lasers having the same wavelength to enter the particle mixing system so as to cross each other in the mixing system to generate a transient diffraction grating. The method consists of irradiating the probe laser beam at another angle and measuring the transient diffracted wave intensity of the laser beam to measure the change in the diffusion constant.

Thirdly, in the present invention, in measuring the diffusion constant change by the transient diffraction grating method, a dye capable of photochemically reacting at an excitation wavelength and binding to a protein is added to the particle mixture system, It constitutes a method of performing laser excitation and probe irradiation. This dye uses a dye that is photoreactive to most of the excitation wavelengths, regardless of whether the protein is photoreactive or not, eliminating the hassle of adjusting the wavelength of the laser. In addition, the use of laser light that matches the light absorption of the dye facilitates wavelength adjustment in the measurement of various proteins that originally differ in light absorption and photoreactivity.

Fourth, the present invention provides a light source of an excitation pulse laser, a light source of a probe laser light having a wavelength different from that of the excitation pulse laser, and the excitation pulse laser and one of the probe lasers overlapped on the same optical path. An optical path means, a transmission type diffraction grating for making the superposed laser incident from the end of the optical path means, a lens means for focusing a diffraction line of the laser emitted from the transmission type diffraction grating, and A measurement cell for containing a protein-containing liquid in which evening protein is dispersed and comprising a container of a permeable material and for adding a test protein, One of which is arranged to be irradiated with one of the excitation pulse lasers divided by diffraction and at least one probe laser, and the diffraction of the probe laser emitted from the measurement cell. A protein association measurement device includes a photoelectric detector for receiving light and converting the light into an electric signal.

Fifthly, the present invention is a measuring cell for containing a dispersion medium in which a target substance is dispersed and comprising a container made of a light-transmitting material and for adding a test substance, the measurement cell being along the same optical axis When placed so as to be irradiated with an excitation pulse laser and a probe laser for exciting a mixed system of the target substance and the test substance.

A particle-mixed transient diffraction grating detection device in which a grating pattern that causes diffraction of both the excitation pulse laser and the probe laser is printed or engraved on one surface of a front or back wall to be irradiated. It is what constituted.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram schematically showing a relationship (A) between a target protein and a test protein (drug) facing each other before or without association, and an associated state (B).

Figure 2 schematically shows the state before diffusion of target particles and test particles in a particle mixture system (A), the state where diffusion is progressing (B), and the state where diffusion is slow due to the presence of an inter-particle association phase (C). FIG.

FIG. 3 is a schematic diagram showing a state in which a sample including a particle mixing system generates a transient diffraction grating by an excitation pulse laser, and a probe laser emits a transient diffraction light signal. FIG. 4 is a schematic diagram showing a state in which rhodopsin is activated by photoexcitation and associates with G-protein.

FIG. 5 is a graph showing changes in the intensity of the transient diffracted light signal with and without protein association.

FIG. 6 is a schematic diagram showing a basic device configuration for performing a diffusion constant change measurement by the transient diffraction grating method of the present invention.

Fig. 7 shows the configuration of the device shown in Fig. 6 with the addition of a separation column and a sample injection unit, and a detector 5 is a schematic view showing a configuration in which an oscilloscope is connected to the oscilloscope.

FIG. 8 is a perspective view and a partial view of an optical system showing a simplified transient diffraction grating measurement cell in which the sample cell and the transmission diffraction grating in the device configuration shown in FIGS. 6 and 7 are combined.

BEST MODE FOR CARRYING OUT THE INVENTION

Generally, a state in which a protein and a protein or other substance are associated means that a relatively weak binding force such as a hydrogen bond, a charge transfer bond, or a hydrophobic bond acts between the same molecules in the two molecules, and the two molecules or the two molecules have a relatively small binding force. It means that the above molecules combine to form an aggregate with relatively regularity. Fig. 1 is a schematic representation of a simple example of the binding between a certain protein 1 and a drug 2. From the state before the association (A) to the state after the association (B) (aggregate) 3, Since the structures of protein 1 and drug 2 are essentially the same, they are designated as compositions 1 'and 2' after association. If such an association does not occur between the two substances (ie, if they remain in state A), they will diffuse rapidly in the medium.

Figure 2 shows that the test protein 12 was added to a protein-containing liquid (usually an aqueous solution) in which the evening protein 11 was dispersed, and the diffusion state of the resulting particle mixture and the presence or absence of association were determined. Unevenly distributed state without diffusion (A), state where diffusion progressed in about 0.1 second after addition due to no association (B), and even about 0.1 second after addition due to association This is shown as a state where diffusion has not progressed (C). In other words, the presence and degree of association are closely related to the diffusion speed (diffusion constant) of the particle-mixing system, and the inventor measures the diffusion constant of either one of the particles to obtain a mixture of particles such as protein. It was found that the presence / absence and degree of association in the system could be detected.

In this case, effective methods for measuring the diffusion state of the mixed particle system include (1) the light scattering method, (2) the tailoring method for observing the distribution state in a capillary, the dispersion method, and (3) the electric method. There are electrophoresis method and (4) transient diffraction grating method. Here, an example of measurement using the transient diffraction grating method is described. In the transient diffraction grating method, either the target protein or the test protein must have light absorbency. When neither absorbs light, it can be carried out by attaching PYP or another photoreceptor to one of the proteins or the like.

FIG. 3 is a schematic diagram illustrating the principle of the transient diffraction grating method. In Fig. 3, sample 13 in the permeation type cell contains a protein-containing liquid (aqueous solution) and is in a state immediately after addition of the test protein. Here, the axis z passing through sample 13 is the center axis. A pair of the same wavelengths; a pulse laser 14 for excitation by I (for example, 450 nm) is made to enter the sample 13 so as to intersect at an angle of 0. If protein association occurs in sample 13 and diffusion is not progressing, this excitation causes transient formation of interference fringes with lattice spacing d, where 2 d = A / s i η (Θ / 2). When this transient interference fringe (transient diffraction grating) formation state is irradiated with a probe laser beam 15 (for example, at a wavelength of 633 nm), the diffracted light until the transient diffraction grating disappears due to pulse excitation. The signal 16 is detected, and the relaxation curve of the sample, especially the relaxation phenomenon due to diffusion, is detected from the attenuation curve, and the diffusion constant of the excited species is determined.

For example, when rhodopsin is used as the target protein and the process of G-protein binding is detected, as shown in Figure 4, (a) when light h is applied to rhodopsin, (b) activated rhodopsin is obtained. It binds to (c) G-protein. (D) The bound G-protein (GTP) is converted to GDP, resulting in (e) G-protein separation. The graph drawn with a solid line in FIG. 5 clearly shows that the above-mentioned association between proteins occurred from the peak height and the sustained state of the diffracted light intensity signal by the probe laser. In this case, the peak appeared within about 0.2 seconds, and the diffusion advanced by about 0.8 seconds. The graph drawn by the dashed line is for a sample in which there was no association between proteins, and indicates that diffusion was almost completed in an instant of 0.1 to 0.2 seconds. The sudden fall before the peak in the solid line graph is caused by heat based on photoexcitation.

Referring again to FIG. 2, a strong transient diffraction signal is detected in the skewed distribution state (A) without diffusion, and the transient diffraction signal is detected in the state (B) where diffusion has progressed because there is no protein association. In the state (C) in which diffusion has not progressed even after about 0.1 second from the addition due to association, a strong transient diffraction signal is detected, and the phase 11 of the target protein and the target protein are not detected. It is well understood that the larger the associating phases 1 1 ′ and 1 2 ′ intervening between the phases 1 and 2 of the test protein, the slower the diffusion, and therefore the longer the lifetime of the excited state (transient diffraction grating). Will be.

As described above, in the protein association detection method of the present invention performed by the transient diffraction grating method, both the target and the test protein are prepared in an aqueous solution, and they do not require complicated operations such as attaching to the measurement substrate. The measurement can be performed in the form of a solution, and the measurement can be performed in a short time within 1 second after adding the test protein to the sample cell. In addition, the sample is required to have light absorbency, but does not require a luminescent label from a molecule as in fluorescence analysis.

Furthermore, in the measurement of the diffusion constant change by the transient diffraction grating method, the particle mixing system is used not only in the case where neither one of the sample and the test protein reacts with light, but also in the case where any of the proteins reacts with light. As described above, a specific effect can be obtained by adding a dye that binds to these proteins by performing a photochemical reaction at the excitation wavelength and then performing laser excitation and probe irradiation. In this case, an effective additive dye is, for example, 2-nitrobenzaldehyde. The dye is photoresponsive to most of the excitation wavelengths, eliminating the hassle of tuning the laser wavelength.

Example

Hereinafter, a configuration example of the transient diffraction grating measurement device according to the present invention will be described. FIG. 6 shows an example of a basic apparatus configuration, in which reference numerals in the figure, reference numeral 20 denotes a light source of an excitation pulse laser having a wavelength of 450 nm, for example, and reference numeral 21 denotes another different wavelength such as 6 33 The probe is a light source that emits 33 nm of laser light for the probe.The lasers emitted from these are projected from both sides of the half mirror 22 to the beginning of the optical path means 24 consisting of a single optical fiber, and have the same optical path. Layered on. At the end of the optical path means 24, a convex lens 25 is arranged, and the synthetic laser light focused (collimated) by this lens is a transmission type grating. A pair of excitation pulse lasers 27 incident on the wing 26 and diffracted and emitted at an appropriate opening angle, and a pair of probe lasers 28 diffracted and emitted at a larger opening angle, are terminated. The laser beam incident on the step-convex lens 29 and coming out of the transmission grating 26 immediately before is focused so that the pair of excitation pulse lasers 27 intersect at an appropriate angle 0. In addition, the outer pair of probe lasers 28 are focused so as to intersect at a larger angle.

These focused laser beams are incident on the measuring cell 30. The measurement cell 30 is composed of a container made of a light-transmitting material and accommodates a protein-containing liquid in which evening get protein is dispersed, and is used for adding a test substance such as another protein during measurement. It is. In this case, the two excitation pulse lasers 27 are made to cross inside the measurement cell 30, and at least one of the probe lasers 28 is also made to enter the sample cell 30. To be. Further, behind the measurement cell 30, a photoelectric detector 31 for receiving the diffracted light of the probe laser emitted from the cell 30 and converting it into an electric signal is arranged.

With the above basic device configuration, it is apparent that the detector 31 emits a signal output indicating the presence / absence and degree of the protein association described above. In this configuration, since the sample cell 30 is a single container type, when measuring another sample, the sample solution must be replaced or the sample cell 30 itself must be replaced. Therefore, the apparatus configuration shown in Fig. 7 is provided with an additional configuration for the measurement of various types of samples, particularly, multiple components that are sequentially eluted from the column.

In FIG. 7, portions denoted by the same reference numerals as those in FIG. 6 have the same functions as the corresponding portions in FIG. 6, and further description will be omitted. The difference in this configuration is that the measuring cell 30 is of a flow type, and the beginning of the inlet channel 32 is connected to the outlet of the column 33. A test sample injection part 34 such as a microsyringe is connected to the middle of the flow path 32, and a test substance from the test sample injection part 34 is used for components eluted sequentially from the column 33. Each is added, and the transient diffraction grade is sequentially set in the measurement cell 30 *. Child measurements will be taken. Note that a drain tube hangs down and protrudes from the lower end of the measurement cell 30 ′, and faces the collection container 35. The output of the photoelectric detector 31 is connected to the illustrated oscilloscope 36 or a recorder or other data processing device, and an association detection signal corresponding to the component peak is displayed.

The structure shown in FIG. 8 shows a cell 37 with a diffraction grating in which a sample cell and a transmission diffraction grating, which are main parts in the measurement apparatus shown in FIGS. The cell 37 is made of a light-transmitting container or a solid, the front is located at the end of the combined optical path of the excitation pulse laser 127 and the probe laser 28, and the back is used for the excitation pulse laser and the probe. A grating pattern 38 that causes both laser diffractions is printed or engraved. The lattice pattern 38 may be formed on the front of the cell 37. The pitch of the grid is one to several wins.

In the above grating / cell combined structure, the incident excitation pulse laser 27 diffracts and disperses at an angle of 0 in the cell to generate a transient diffraction grating that accompanies the state of the test substance and target substance in the cell. The change in the diffusion constant, which is a relaxation phenomenon, is measured by the photoelectric detector 31 as the change in the intensity of the probe laser 28 that has also been diffracted. However, a slit plate 39 is provided to detect the probe laser 28 at a specific diffraction angle. In addition, this cell structure can be used not only for protein association but also for detection of various particle diffusion states as long as a transient diffraction grating is generated in the cell.

Industrial applicability

As described above, according to the present invention, the degree of protein association can be easily and accurately measured by measuring the change in the diffusion constant of a mixture of a target protein and a test protein. In particular, according to the apparatus configuration of the present invention, it is possible to measure the change in the diffusion constant of the mixed system of the target protein and the test protein in a very short time.

Therefore, the method and apparatus of the present invention can be used to search for proteins that bind to drugs, The present invention can be used for searching for a signal transduction system, an immune response, a receptor ligand assay, and the like, and enables easy and accurate detection of association of a protein or the like excellent in portability and mobility.

In addition, the use of a dye in the method of the present invention exerts an effect that all kinds of proteins can be easily detected. In other words, since the absorption wavelength and the reactivity of light differ depending on the protein, it is necessary to change the laser wavelength depending on the properties of the protein when the dye is not used. The complicated operation of adjusting is not required. In addition, the use of laser light in accordance with the light absorption of the dye makes it easy to measure various proteins that originally have different light absorption and photoreactivity (some of which do not react). This can be performed in wavelength adjustment.

Claims

The range of the expression. A test substance such as another protein is added to the protein-containing liquid in which the target protein is dispersed, and the resulting change in the diffusion constant of the particle-mixing system is detected. A method of measuring the degree of a meeting.

A pair of excitation pulse lasers having the same wavelength are incident on the particle mixing system so as to cross each other in the mixing system to generate a transient diffraction grating, which is then probed at another wavelength and another angle. 2. The method according to claim 1, wherein a change in the diffusion constant is measured by irradiating a laser beam for use and measuring a transient diffraction light intensity of the laser beam.

3. The method according to claim 2, wherein a dye capable of undergoing a photochemical reaction at an excitation wavelength and binding to a protein is added to the particle mixture system, and then laser excitation and probe irradiation are performed.

A light source for an excitation pulse laser, a light source for a probe laser light having a wavelength different from that of the excitation pulse laser, and an excitation pulse laser and a probe laser for overlapping the same optical path. Optical path means; a transmission type diffraction grating for inputting the superposed laser beam from the end of the optical path means; lens means for focusing a diffraction line of the laser emitted from the transmission type diffraction grating; A measuring cell for accommodating a protein-containing liquid in which a target protein is dispersed and containing a target protein therein, and for adding a test substance such as another protein, wherein the focused laser beam Of which are arranged so as to be irradiated with two excitation pulse lasers separated by diffraction and at least one probe laser, and a probe out of the measurement cell. Proteins associated measuring device characterized by comprising a photoelectric detector for converting into an electric signal by receiving the laser one diffracted light.

A measurement cell for storing a dispersion medium in which a target substance is dispersed and consisting of a container made of a light-transmitting material, and for adding a test substance, along the same optical axis. When placed so as to be irradiated with an excitation pulse laser and a probe laser to excite a mixed system of the target substance and the test substance, a wall body serving as a front or back surface to be irradiated. A grating pattern for generating diffraction of both the excitation pulse laser and the probe laser printed or engraved on one surface of the particle mixing system; .