JP2000221136A - Method and apparatus for tracking changes with time passage - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure a stopped-flow infrared absorption, even when light does not pass a normal flow-through cell by adopting attenuated total reflectance(ATR) method for measuring infrared rays, and obtaining an infrared absorption signal without measuring the intensity of passing light. SOLUTION: A buffer solution, which includes a protein to be measured and a modifier solution, is prepared respectively to different solutions and stored in a sample reservoir of a stopped-flow apparatus 3. The sample solutions are sent by a constant flow rate to an ATR flow-through cell 2, mixed by a mixer in the cell 2 and supplied to a cell chamber to measure an infrared absorption. The protein of the sample is modified from the moment when it is mixed with the modifier, starting a change in secondary structure. Since the cell chamber is located immediately behind the mixer, an infrared absorption signal corresponding to the structure at a time point after a considerably short time has passed can be detected. Thereafter, the supply of the solutions by the stopped-flow apparatus is stopped, and in synchronism the infrared absorption signal is started to be obtained. An elapsed time of the secondary structure change subsequent to the modification is captured as an infrared absorption time change of an amide group.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a technique for tracking the course of chemical and structural changes of sample molecules in a solution over time.

[0002]

2. Description of the Related Art An appropriate reaction reagent is added to a solution containing a sample to be studied to cause a reaction, and a change in a chemical structure or a change in a three-dimensional structure caused by the reaction is measured over time. It is performed as a means to analyze the process.

When the speed of this reaction is on the order of milliseconds to sub-milliseconds, a technique called stopped flow measurement is used. This involves preparing samples, reaction reagents, etc. in separate solutions, sending them at a constant flow rate, mixing them immediately before supplying them to the flow-through cell provided in the measuring means, and supplying the mixed solution. Is stopped suddenly, and the subsequent change of the measurement signal is recorded with time in synchronization with the stop.

[0004] Measurement means include ultraviolet / visible absorption, fluorescence intensity,
Circular dichroism is widely used. However, these measurement means basically reflect the electronic state of the sample substance / molecule, and although they are universal to the sample substance / molecule, their specificity to the structure is small, that is, a structural change occurs. Can be detected, but there is a weak point that there is little information to know what it is.
When trying to see what kind of structural change is occurring,
It is advantageous to employ a measuring means that directly reflects the molecular structure, and the simplest such measuring means is infrared absorption.

However, ordinary solvents used in such a measurement system generally show infrared absorption by themselves, and solvents that do not show infrared absorption are extremely limited. There is almost no reaction system constituted only by such a solvent. On the other hand, a commonly used solvent, especially water, has a very strong infrared absorption.In the flow-through cell combined with the stopped flow method, the infrared light is almost completely absorbed and is not transmitted at all, Measurement is not possible. In order to allow the measurement infrared light to pass through as it is and to be able to perform infrared absorption measurement, there are ways to reduce the cell thickness and appropriately shorten the optical path length. In order to ensure the light transmittance required for measurement, it is essential that the optical path length be several tens of microns or less. It is not possible to flow at the possible flow rate, and the stopped flow measurement cannot be performed this time. Under such circumstances, it has been considered that the configuration of a measurement system called stopped-flow infrared absorption is difficult to realize.

[0006]

A system that combines stopped flow with infrared absorption and that is suitable for analyzing structural changes has not been practically realized because most of the solvents usually used for constructing a reaction system are used. This is because flow-through cells such as those used for stopped flow do not transmit light and do not perform measurement because they exhibit strong infrared absorption.

The present invention combines a stopped-flow method with an appropriate infrared measurement method, so that even in a system in which infrared absorption is strong and light cannot pass through a normal flow-through cell and cannot be measured,
To provide a system that enables the measurement of stopped-flow infrared absorption by obtaining a signal corresponding to infrared absorption, tracks structural changes over time with the reaction, and enables its analysis. is there.

[0008]

In order to solve the above-mentioned problems, the present invention employs an ATR method (attenuated total reflection method) for infrared measurement, and obtains an infrared absorption signal not based on transmitted light intensity measurement. Get it.

As is known in static state measurement, in the infrared ATR method, an optical system is configured such that measurement light is totally reflected inside the interface of the prism, but the wavelength is also outside the prism. It exudes to the extent of a minute and is attenuated by absorption by substances present there. When the absorbance is calculated by regarding this attenuation as the attenuation of transmitted light, it is possible to obtain the absorbance of the sample corresponding to the case where a measurement cell having an optical path length of approximately a wavelength level is used. On the other hand, a portion separated from the prism surface by about a wavelength or more does not affect the intensity of the total reflected light.Therefore, if a portion where the solution flows when the mixed sample solution is supplied is secured here, the stopped flow can be performed. It is possible to secure a sufficient permeation cross-sectional area, that is, a cell cross-sectional area necessary for sending the solution at an appropriate flow rate.

Further, in order to effectively perform the stopped flow measurement, it is necessary to efficiently mix the sample solution immediately before the cell chamber, which is a photometry site, and to minimize the time lapse from mixing to the cell chamber. It is necessary to shorten the length and to effectively maintain a constant temperature from the solution reservoir to the cell chamber via the flow path. The last of these items is that the rate of the reaction that causes a structural change depends on the temperature, and therefore it is necessary to perform the reaction at a constant temperature in order to perform measurement with good reproducibility.

[0011] Such a stopped flow-infrared ATR
In order to satisfy the requirements of the method measurement, the flow-through cell is composed of an ATR prism, spacer and cell body, mixes the sample solution efficiently, and has a built-in ATR flow with a built-in channel and mixer to reach the measuring section. Construct a through cell. The cell is provided with a function of controlling a constant temperature by providing a flow path for circulating constant-temperature water or the like, or by attaching a heating / cooling device using a Peltier element.

[0012]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described with reference to an example of a system shown in FIG. 1 and an application of the system to the analysis of a protein denaturation process.

The system comprises an infrared spectrophotometer 1 incorporating an infrared ATR flow-through cell 2 optimally configured for stopped flow measurement, a stopped flow device 3, and a data processing device 9. You. This infrared ATR flow-through cell 2 will be described later.

A buffer solution containing the protein of interest and a denaturing agent solution such as urea or guanidine which cause denaturation are prepared as separate solutions, and placed in a sample reservoir of a stopped flow apparatus. The sample reservoir is usually composed of a syringe. The stopped-flow apparatus sends these sample solutions at a constant flow rate to the ATR flow-through cell, where they are mixed by a mixer provided in the cell and supplied to the cell chamber, where the infrared absorption is converted to the infrared ATR. It is measured by the method.

From the viewpoint of the protein of the sample, when the mixture is mixed with the denaturing agent by the mixer, the denaturation starts from the moment, and the change of the secondary structure starts. The cell chamber is installed immediately after the mixer, and in the state where the solution is flowing, an infrared absorption signal corresponding to the structure at the time when a very short time elapses from the mixer to reach the cell chamber is detected. Will be. In this case, as the wave number for detecting the infrared absorption, attention is paid to the change in the secondary structure due to the metamorphism, and the vicinity of 1650 cm −1 of the amide group reflecting the change is selected.

Next, the liquid feeding by the stopped flow device is stopped. In synchronization with this, acquisition of an infrared absorption signal is started. Or at the same time as starting the acquisition of the infrared absorption signal,
The liquid feeding may be stopped. In this case, the time course of the change of the secondary structure due to the modification is regarded as the time change of the infrared absorption of the amide group.

The key to realizing the stopped flow-infrared ATR system is the ATR flow-through cell. FIG. 2 shows a configuration example for that purpose. FIG. 3 shows details of each part. The ATR flow-through cell includes an ATR prism 11, a spacer 12, and a cell substrate 13. For the ATR prism, germanium, zinc selenide, or the like, which is insoluble in a solvent used for the stopped flow measurement, has an infrared transmittance, and has a relatively high refractive index, is used. The ATR prism and the cell substrate constitute a cell with a spacer having a space for accommodating the measurement solution sample interposed therebetween. This space constitutes the cell cell chamber 19, and its size is determined in consideration of the size of incident light for infrared measurement and the flow rate of the stopped flow solution.

The cell substrate is provided with a sample introduction port 21 for receiving a sample sent from the stopped flow device, a mixer 18 for mixing them, and a sample discharge port 22 for discharging unnecessary sample solution. The number of sample introduction ports is the number of solutions to be used. The flow path from the sample introduction port 21 to the cell chamber via the mixer 18 in FIG. 2 through the mixer 18 and from there to the sample discharge port 22 is a concept. As an embodiment, the cell substrate top view 33 and the cell substrate front view 34 in FIG. As shown in the figure, the sample introduction port 2 for attaching a liquid feed pipe from the stopped flow device to the lower surface of the cell substrate
1 is provided, and a channel extending up and down through the cell substrate is used as a flow path to the upper surface of the cell substrate, and a thin groove is dug from the upper surface of the cell substrate to form a sample solution flow path 35 to the mixer 18. The mixer 18 was formed so as to intersect at 180 ° or less, and a groove was provided in the direction of a bisector of the intersection from the mixer 18 so as to provide a flow path to the cell chamber 19.

[0019]

According to the present invention, the stopped-flow measurement of various reaction processes can be performed by infrared absorption which directly reflects the structure of a molecule. In other words, the UV-visible absorption that has been used in combination with the stopped-flow device is to observe the change in absorbance associated with the change in the electronic state of the target chemical species, providing evidence that the chemical species has changed. However, it does not directly suggest what kind of chemical or structural change it is.
Stopped flow-fluorescence measurement has also been used to track protein denaturation, but it also observes changes in the state of fluorescent amino acid residues in proteins, that is, tryptophan, phenylalanine, and tyrosine. It does not indicate how it has changed. Circular dichroism (CD)
Even when the measurement is combined, the change in the electronic state of the target chemical species is observed as a steric asymmetry, and the change in the structure, particularly, the change in the chemical bond is not directly indicated. In contrast, infrared absorption reflects the chemical bond itself, so that its formation, extinction, and change can be directly observed, and it is now possible to track structural changes that are not estimated.

Further, when the stopped flow-circular dichroism (CD) measurement is applied to the tracking of the denaturation process of a protein, a certain protein aggregates and precipitates due to the denaturation and turbidity is generated. In the case of a sensitive CD measurement, there has been a problem that the measurement is disturbed. The infrared ATR method is not substantially affected by such scattering, so that accurate and highly reliable measurement is possible.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram of a system in which a stopped flow device and an infrared spectrophotometer incorporating an ATR device are combined.

FIG. 2 Infrared A configured for stopped flow measurement
It is a schematic diagram of a flow-through cell for TR.

FIG. 3 Infrared A configured for stopped flow measurement
FIG. 3 is an exploded view of an embodiment of a flow-through cell for TR.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 Infrared spectrophotometer 2 ATR flow-through cell 3 Stopped flow device 4 Liquid sending flow path and temperature control unit 5 Waste liquid reservoir 6 Stopped flow device I / F 7 Temperature controller 8 Infrared spectrophotometer I / F 11 ATR Prism 12 Spacer 13 Cell substrate 14 Measurement incident light 15 Measurement total reflection light 16 Sample solution introduction flow path 17 Sample discharge flow path 18 Mixer 19 Cell chamber 20 Cell constant temperature device 21 Sample introduction port 22 Sample discharge port 31 Spacer top view 32 Spacer front Figure 33 Top view of cell board 34 Front view of cell board 35 Front view of cell board

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G054 AA06 AB10 BB10 BB13 CB02 CD03 CE01 EA04 EA05 EB10 FA08 FA18 FA36 GA01 GB01 JA01 2G057 AA01 AB02 AB09 AC01 BA05 DA03 DB05 DC01 DC07 EA06 2G059 AA01 BB04 DD17EE12 DD13 FF03 HH01 JJ12 KK01 MM01

Claims (7)

[Claims] 1. A solution containing a sample whose change is to be measured is supplied to a flow-through cell of an infrared spectrophotometer while one or more solutions are mixed at a fixed ratio. By detecting the infrared absorption signal or infrared reflection signal of the sample, by measuring the change over time of the signal in synchronization with stopping the supply of the mixed solution,
A method to track processes such as chemical and structural changes caused by mixing mixed solutions. 2. A method according to claim 1, wherein an infrared absorption signal or an infrared reflection signal of the sample is detected by an ATR method. 3. A flow-through cell of the infrared spectrophotometer according to claim 1, wherein two or more sample solution introduction ports, a mixer for mixing the solution introduced therefrom to reach the measurement cell, and a cell unit. Is provided with a prism made of a material having a high refractive index and transmitting infrared light such as germanium, zinc selenide, etc., and measuring the infrared ATR method so that infrared light is totally reflected at a liquid contact surface of the prism. apparatus. 4. The AT of the infrared spectrophotometer according to claim 3.
By digging grooves in the cell substrate, the flow path of the solution after the sample solution introduction port, the mixer, the flow path for guiding the mixed solution to the cell part, the flow path for discharging the solution from the cell, etc. are formed in the R flow-through cell. Configured ATR flow-through cell. 5. The AT of the infrared spectrophotometer according to claim 3.
A constant temperature ATR flow-through cell with an R flow-through cell and a mechanism for keeping the cell section at a constant temperature. 6. A constant temperature ATR flow-through cell of the infrared spectrophotometer according to claim 5, wherein a flow path for circulating constant temperature water is separately provided in the cell substrate, and a constant temperature water or the like is circulated therein to form a cell portion. ATR flow-through cell maintained at a constant temperature. 7. A constant temperature ATR flow-through cell of the infrared spectrophotometer according to claim 5, wherein a temperature control mechanism using a Peltier element is attached to a cell substrate so that the cell portion is kept at a constant temperature. cell.