Patent Publication Number: US-7719273-B2

Title: Apparatus of nuclear magnetic resonance measurement by using circulation flow for sample condition control

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application is a continuation application of U.S. application Ser. No. 11/940,398, filed Nov. 15, 2007now U.S. Pat. No. 7,492,157, the contents of which are incorporated herein by reference. 
    
    
     CLAIM OF PRIORITY 
     The present application claims priority from Japanese application JP 2006-311380, filed on Nov. 17, 2006, the content of which is hereby incorporated by reference into this application. 
     BACKGROUND OF THE INVENTION 
     (1) Field of the Invention 
     The present invention relates to an NMR (nuclear magnetic resonance) apparatus, in particular, to an apparatus of nuclear magnetic resonance measurement by using circulation flow for sample condition control which apparatus is for carrying out measurement repeatedly while changing the measuring conditions of a sample in the NMR apparatus. 
     (2) Description of the Related Arts 
     Molecules such as proteins having certain functions within a living body have a larger molecular weight than that of compounds used for medicines or the like and have properties as a large molecule. 
     Large molecules typified by proteins have a molecular function in a solution thereof and this function is inhibited or accelerated when they bind to a certain small molecule. There are many methods for detecting binding of a large molecule to a certain small molecule or interaction therebetween. In particular, in measurement (which will hereinafter be called “NMR measurement”) utilizing nuclear magnetic resonance permitting direct observation of the information of a molecular structure of the large molecule or small molecule, it is possible to analyze, from a change in the measurement spectrum of the concentration of the molecule, not only a dissociation constant between the large molecule and low molecule or reaction rate of them but also interaction of them based on the structure of the molecules. Refer to, for example, the description in International Publication No. WO01/023889 (Japanese Patent Application No. 2001-527224) with regard to a method of measuring the interaction between a protein and a low molecule by means of NMR. 
     Of large molecules to be used for NMR measurement, proteins are produced, for example, by extraction from naturally occurring living organisms, extraction from an abundant expression system making use of  Escherichia coli  or the like having, incorporated therein, a gene related to the formation of a protein, or by a method utilizing a non-cell expression system causing abundant expression of a protein without using a living cell. Labeling of hydrogen, carbon and nitrogen, which are main elements constituting a protein, with a radioisotope is sometimes carried out. This labeling includes labeling of a combination of three elements, that is, hydrogen, carbon and nitrogen constituting a protein, comprehensive labeling method for labeling all them, and selective labeling method of labeling only atoms belonging to a specific amino acid residue. In any method, a labeling cost is high. 
     A nuclear magnetic resonance spectrometer (which will hereinafter be called NMR apparatus) has typically a magnet for causing a magnetostatic field B 0  and a nuclear magnetic resonance probe located at a bore portion inside of this magnet. This nuclear magnetic resonance probe includes at least one coil for applying an RF magnetic field B 1  to a sample of interest and detecting reaction (response) of the sample to the magnetic field thus applied. 
     Conventional nuclear magnetic resonance probes include probes for the static setting sample and flow through probes. In probes for the static setting sample, the sample is put in a glass tube or ampoule (which will hereinafter be called “sample tube”) and measurement is started after setting this sample tube at a predetermined position of the NMR apparatus. 
     In conventional probes for the static setting sample, a change in the NMR spectrum due to an increase in the concentration can be observed by carrying out NMR measurement while titrating a small molecule in a sample tube having an opening portion. 
     Conventional flow through probes each has a sample inlet, a sample outlet, and an internal tubular portion extending between the inlet and outlet. The internal tubular portion includes a cell for retaining a sample therein. The sample is put in this probe from the sample inlet, flows in the internal tubular portion, and enters the cell. After measurement, it flows in the tubular portion and is discharged out of the probe. 
     A combination of the conventional flow through probe and a robot-using sample transfer system has been used. Various sample transfer systems are employed for this combination and one of them is commercially available from Gilson, Inc. In such a system, samples adjusted for a plurality of measurement conditions must be prepared and put in a plurality of containers in advance, respectively. The samples are each fed to a predetermined flow through probe after passing through an apparatus capable of taking it out from the container. Each sample is discharged out of the probe after completion of the NMR measurement. Refer to International Publication No. WO2003/007009 (Japanese Unexamined Patent Application Publication No. 2004-534958) with regard to a preferred conventional system for transferring the sample to the probe. 
     SUMMARY OF THE INVENTION 
     The conventional probe for the static setting sample however has a difficulty in carrying out NMR measurement of a sample solution, which contains a small molecule at a concentration increased to a certain level, under the condition of a concentration less than the certain level because the sample solution is a mixture of a large molecule, a small molecule for evaluating its effect as a drug, and other reagents. 
     Titration of a small molecule in a sample tube typically used for NMR measurement causes an increase in the volume of the whole sample solution, a change in the concentration of a large molecule sample and a change in the liquid level of the sample solution. The titration volume relative to the volume of the sample solution must therefore be decreased as much as possible in order to suppress these changes. 
     The concentration of a small molecule to be titrated must be decreased in order to reduce the titration volume. The upper limit of the concentration of the solution to be titrated is determined, depending on the solubility of the small molecule. The solubility of a substance typically differs greatly with a kind or temperature of a solvent. Accordingly, in the NMR measurement in which the concentration of a small molecule is changed by titration, a difference in the kind of the solvent or temperature has an influence on the stability of the concentration of a large molecule. 
     In the combination of the conventional flow through probe and sample transfer system, samples different in concentrations must be prepared in advance. Large molecule solutions adjusted to respective predetermined concentrations must be prepared as many as the number of measurement conditions so that preparation of samples costs much. 
     Large molecules or small molecules whose measurement condition range is unknown also pose the problem that measurement for functional evaluation must be carried out in repetition until a preferred measurement condition range or degree of change in condition is established. 
     An object of the present invention is therefore to provide an apparatus and a method for carrying out nuclear magnetic resonance measurement using a solution sample containing a large molecule such as protein and a small molecule while efficiently exchange the large molecule; and an apparatus for carrying out measurement in repetition while stably controlling the concentration condition of the sample and changing the concentration of the small molecule. 
     The present invention makes it possible to measure a plurality of samples under a certain concentration condition of a small molecule by circularly transferring a small molecule solution from a small molecule concentration control portion  30  which injects, discharges or feeds the small molecule solution, a mixing filter portion  32  for mixing a solution (sample) containing a large molecule sample with the small molecule solution, a container  10  set in a nuclear magnetic resonance probe  24 , a separating filter portion  34  for separation for separating the mixture into the sample and the small molecule solution and then the control portion  30  again; injecting the sample from the mixing filter portion  32 , retaining the sample in a measurement portion of the container  10 , and discharging the sample from the separating filter portion  34  after completion of the NMR measurement. 
     To enable circulation transfer of the small molecule solution through the control portion  30 , mixing filter portion  32 , container  10  and separating filter portion  34 , an injection port  14  of the container  10  set in the nuclear magnetic resonance probe  24  is connected to a first sample transfer tubing  16   1 , a discharge port  12  of the container is connected to a second sample transfer tubing  16   2 , the first sample transfer tubing  16   1  is connected to a discharge port  50  of the mixing filter portion  32  (equipped with a small molecule injection port  51 , a sample solution injection port  52  and the discharge port  50 ), a first small molecule solution transfer tubing  18   1  is connected to the small molecule injection port  51  of the mixing filter portion  32 , an injection port  60  of the separating filter portion  34  (equipped with a small molecule solution discharge port  61 , a sample discharge port  62 , and the injection port  60 ) is connected to the second sample transfer tubing  16   2 , a second small molecule solution transfer tubing  18   2  is connected to the small molecule discharge port  61  of the separating filter portion  34 , the second small molecule solution transfer tubing  18   2  is connected to an injection port  40  of the small molecule concentration control portion  30  (having the injection port  40  and a discharge port  42  of the small molecule solution), and the first small molecule solution transfer tubing  18   1  is connected to the discharge port  42 . 
     In the small molecule concentration control portion  30 , the injection port  40  of the small molecule concentration control portion is connected to a small molecule solution injection portion  70 , discharge valve  44 , injection valve  46 , feed pump  48 , pressure gauge  49  and discharge port  42  of the small molecule concentration control portion. The small molecule solution injection portion  70  is composed of one or more syringe pumps and an electronic apparatus for controlling it (them). The feed pump  48  is composed of a pump for feeding the solution under pressure and an electronic apparatus for controlling the feed pump. 
     The feed pump continuously pushes out the solution in an amount corresponding to a total volume of the sample passing regions of the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1  and  18   2 , filter portions  32  and  34  and control portion  30  so that the solution passes through the container  10  set in the probe  24  and reaches the input side of the feed pump  48 . This enables circulation transfer of the solution. After the solution is filled in the container  10  set in the probe  24  and reaches the input side of the feed pump  48 , a constant volume of the solution always fills the container therewith. 
     (Injection of Buffer Solution) 
     The operation of the feed pump  48  is started and a buffer solution is introduced from the injection valve  46 . The buffer solution is fed to the mixing filter portion  32  after passing through the first small molecule solution transfer tubing  18   1 . In the mixing filter portion  32 , the buffer solution passes through portions  55   1  and  55   2  through which only a small molecule solution passes and hollow filters  54   1  and  54   2 , reaches a portion  56  through which a mixed solution passes, is discharged from the discharge port  50 , and reaches the container  10  after passing through the first sample transfer tubing  16   1 . Then, the buffer solution passes through the second sample transfer tubing  16   2 , separating filter portion  34 , and second small molecule solution transfer tubing  18   2  and reaches the input side of the feed pump  48  of the control portion  30 . 
     (Injection of Small Molecule) 
     At the time of the injection of the small molecule, a flow of a solution is formed in a transfer tubing by circulating a solution by means of the feed pump  48 . A small molecule solution for injection is set in the small molecule solution injection portion  70  in advance and the solution is injected in an amount determined by a measurer. The solution thus injected reaches the feed pump  48  with the flow and fed to the mixing filter portion  32  via the first small molecule solution transfer tubing  18   1 . 
     In the mixing filter portion  32 , the solution is fed from the small molecule solution injection port to the portions  55   1  and  55   2  through which only a small molecule solution passes, passes through the hollow filters  54   1  and  54   2 , and reaches the portion  56  through which a mixed solution passes. When only the small molecule solution is injected, only the small molecule solution is present in the portion  56  through which a mixed solution passes. The solution discharged from the discharge port  50  reaches the container  10  after passing through the first sample transfer tubing  16   1 . 
     Simultaneously with the injection, an excess solution appears. The discharge valve  44  is opened and the excess solution is discharged therefrom. With the completion of the discharge of the excess solution, the pressure indicated by a pressure gauge returns to that at the time of circulation feeding. After returning of the pressure is confirmed, the discharge valve  44  is closed, followed by steady circulation feeding. 
     At this time, the small molecule has an increased concentration in the sample passing regions of the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1  and  18   2 , filter portions  32  and  34  and control portion  30 . 
     The concentration of the small molecule in the sample solution to be used for the NMR measurement can be increased by performing the injection operation of the small molecule in plural times. Supposing that a total volume of the sample passing regions of the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1  and  18   2 , filter portions  32  and  34  and control portion  30  is V, a concentration of the small molecule present in the volume V before all the injection operations is α, a concentration of the small molecule to be injected is β, a volume per single injection operation is v, an amount of the small molecule discharged from the filter portion by the i-th injection operation is E(i), an amount of the small molecule which has remained after the injection operation is M(i), and an average concentration is δ(i), a discharged amount E( 1 ) of the small molecule, a remaining amount M( 1 ) of the small molecule and an average concentration δ( 1 ) after the first injection operation can be determined based on the following equations, respectively. 
                     [     Equation   ⁢           ⁢   1     ]     ⁢                                     E   ⁡     (   1   )       =     α   ×   v             (   1   )                 [     Equation   ⁢           ⁢   2     ]     ⁢                                     M   ⁡     (   1   )       =       α   ×     (     V   -   v     )       +     β   ×   v               (   2   )                 [     Equation   ⁢           ⁢   3     ]     ⁢                                     δ   ⁡     (   1   )       =       M   ⁡     (   1   )       V             (   3   )               
After the injection operation of the small molecule is performed i times, an amount E(i) of the small molecule, a remaining amount M(i) of the small molecule and an average concentration δ(i) can be determined based on the following equations, respectively.
 
                     [     Equation   ⁢           ⁢   4     ]     ⁢                                     E   ⁡     (   i   )       =       δ   ⁡     (     i   -   1     )       ×   v             (   4   )                 [     Equation   ⁢           ⁢   5     ]     ⁢                                     M   ⁡     (   i   )       =         δ   ⁡     (     i   -   1     )       ×     (     V   -   v     )       +     β   ×   v               (   5   )                 [     Equation   ⁢           ⁢   6     ]     ⁢                                     δ   ⁡     (   i   )       =       M   ⁡     (   i   )       V             (   6   )               
(Dilution of Small Molecule)
 
     The small molecule is diluted using, as a solution to be injected, a small-molecule-free solvent in a similar manner to that employed for the injection of the small molecule. At the time of injecting the small-molecule-free solution, a solution flow is formed in the transfer tubing by the circulation of the solution by means of the feed pump  48 . The small-molecule-free solution for injection is set in advance in the small molecule solution injection portion  70  and the solution is injected in only a volume predetermined by a measurer. The solution thus injected reaches the feed pump  48 , passes through the first small molecule solution transfer tubing  18   1  and is fed to the mixing filter portion  32 . 
     In the mixing filter portion  32 , the solution is fed from the small molecule solution injection port to the portions  55   1  and  55   2  through which only a small molecule solution passes and after passing through the hollow filters  54   1  and  54   2 , it reaches the portion  56  through which a mixed solution passes. The solution discharged from the discharge port  50  passes through the first sample transfer tubing  16   1  and reaches the container  10 . 
     Appearance of an excess solution, which has occurred simultaneously with the injection, raises the pressure in the whole sample passing region. A pressure increase which has started from the circulation feeding of the solution by injection is confirmed by the pressure gauge  49  and the excess solution is discharged from the discharge valve  44  opened. When the discharge of the excess solution is completed, the pressure indicated by the pressure gauge returns to that at the time of circulation feeding. After confirmation of the returning of the pressure, the discharge valve  44  is closed to start steady circulation feeding again. 
     At this time, in the sample passing regions of the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1  and  18   2 , filter portions  32  and  34 , and control portion  30 , the small molecule has a reduced concentration. The concentration of the small molecule in the sample solution to be used for NMR can be reduced by repetition of the above-described injection operations. 
     Supposing that a total volume of the sample passing regions of the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1  and  18   2 , filter portions  32  and  34  and control portion  30  is V; a concentration of the small molecule before dilution is δ( 0 ), a volume of the small-molecule-free solution per single injection operation is v, an amount of the small molecule discharged from the filter portion by the i-th injection operation is E(i), an amount of the small molecule which has remained after the injection operation is M(i), and an average concentration is δ(i), an amount E( 1 ) of the small molecule discharged, a remaining amount M( 1 ) of the small molecule and an average concentration δ( 1 ) after first injection operation are determined based on the following equations, respectively. 
     
       
         
           
             
               
                 
                   
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     By using the above-described procedures in combination, it is possible to increase or decrease the concentration of the small molecule in the container  10  and to change the concentration of the small molecule again after NMR measurement. In addition, repetition of them makes it possible to carry out a series of NMR measurements while changing the concentration of the small molecule. 
     (Injection of Sample Solution) 
     By starting the operation of the feed pump  48 , a fixed flow is generated throughout the circulation routes of the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1  and  18   2 , filter portions  32  and  34 , and control portion  30 . A sample solution obtained by incorporating a large molecule in a solvent suited for the large molecule is injected from a sample solution injection portion  90 . In the portion  56  through which a mixed solution passes, the small molecule solution which has already been present there is mixed with the large molecule solution. The injection raises the pressure of the solution, but the hollow filters  54   1  and  54   2  do not pass the large molecule therethrough so that the solution containing the large molecule transfers, with the fixed flow, to the container  10  set in the probe. Since the container  10  has an inner diameter larger than that of the sample transfer tubings  16   1  and  16   2 , the solution which has passed the sample transfer tubing  16   2  is mixed further in the container  10 . 
     (Holding of Sample Solution) 
     When the mixed solution containing the large molecule reaches the container  10  and fills it therewith, the feed pump  48  stops its operation. By the termination of the pump, the pressure acting on the solution decreases and the circulation flow of the solution stops. The container  10  has an inner diameter larger than that of the sample transfer tubings  16   1  and  16   2 , which disturbs smooth flow of the solution through the sample transfer tubings  16   1  and  16   2 . The solution in the container  10  is therefore retained therein after the termination of the pump. 
     (NMR Measurement) 
     After the sample solution containing the large molecule is filled in the container set in the probe, NMR measurement of the sample is performed while applying a uniform magnetostatic field to the sample from the magnet. 
     (Discharge of Sample Solution) 
     By starting the operation of the feed pump  48 , a fixed flow is generated throughout the circulation routes of the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1  and  18   2 , filter portions  32  and  34 , and the control portion  30 . By the fixed flow thus formed, the mixed solution retained in the container  10  passes through the sample transfer tubing  16   2  and reaches the filter portion  34 . The small molecule solution which has passed through the portion  66  through which a mixed solution passes and then hollow filters  64   1  and  64   2 , passes through the  61 , and is discharged to the  18   2 . The solution containing the large molecule transfers from the sample discharge port  62  to a sample discharge syringe  91 . 
     The present invention makes it possible to efficiently carry out NMR measurement of a plurality of large molecules by using a small molecule having a predetermined concentration and a large molecule. 
     The present invention also makes it possible to carry out selection, from a group of large molecules, of a specific large molecule which binds to a small molecule more efficiently by NMR measurement. 
     The present invention also makes it possible to efficiently determine, based on NMR measurement, the conditions of a small molecule solution causing a change in the properties of a large molecule in a mixed solution. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and other features, objects and advantages of the present invention will become more apparent from the following description when taken in conjunction with the accompanying drawings wherein: 
         FIG. 1  is a schematic view of a circulation flow NMR apparatus according to a preferred embodiment of the present invention; 
         FIG. 2  is a schematic view of a connection between a sample holding vessel and a sample transfer tubing; 
         FIG. 3  is a flow chart of the measurement procedures according to the present invention; 
         FIG. 4  is a graph showing a change in the concentration of a small molecule in a sample solution caused by plural injection operations of the small molecule according to the present invention; 
         FIG. 5  is a graph showing a change in the concentration of a small molecule in a sample solution caused by plural dilution operations of the small molecule according to the present invention; 
         FIG. 6  is a schematic view of a circulation flow type NMR apparatus according to a preferred embodiment of the present invention which has a similar constitution to that of  FIG. 1  except that the sample transfer tubing is inserted into the magnet in parallel with the probe; 
         FIG. 7  is a schematic view of a circulation flow type NMR apparatus according to another preferred embodiment of the present invention which has a similar constitution to that of  FIG. 1  except that the sample transfer tubing is placed inside the probe; 
         FIG. 8  is a schematic view of a circulation flow type NMR apparatus according to a further preferred embodiment of the present invention in which the sample transfer tubing on one side of a vertical magnet is placed inside the probe; 
         FIG. 9  is a schematic view of a circulation flow type NMR apparatus according to a still further preferred embodiment of the present invention which has a similar constitution to that of  FIG. 8  except that the sample transfer tubing is placed inside the probe; and 
         FIG. 10  is a schematic view of a circulation flow type NMR apparatus according to a still further preferred embodiment of the present invention which has a similar constitution to that of  FIG. 8  except that the sample transfer tubing is placed so as to penetrate the magnet from the upper portion to the lower portion thereof. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Examples of the present invention will hereinafter be described specifically based on accompanying drawings. 
     EXAMPLE 1 
     The circulation flow constitution of a sample solution in NMR measurement according to the present invention and preferred embodiments of its method will next be described referring to some drawings. 
     As illustrated in  FIG. 1 , a nuclear magnetic resonance probe  24  is fixed in bores  22   1  and  22   2  placed inside of magnets  20   1  and  20   2  for applying a magnetic field to a sample. A container  10  for retaining a certain amount of the sample is set in the nuclear magnetic resonance probe  24  in the optimum positional relationship with a probe coil  28  for detecting a nuclear magnetic resonance signal. In general, the container  10  is preferably made of a glass. The container  10  is connected to a second sample transfer tubing  16   2  via a discharge port  12 . The container  10  is connected to a first sample transfer tubing  16   1  via an injection port  14 . 
     In this Example, the container  10 , discharge port  12 , first sample transfer tubing  16   1 , injection port  14  and second sample transfer tubing  16   2  are connected on the same axis inside the bores  22   1  and  22   2 . 
     The second sample transfer tubing  16   2  is connected to a separating filter portion  34 . The separating filter portion  34  has an injection port  60 , small molecule solution discharge port  61 , sample discharge port  62 , hollow filter substances  64   1  and  64   2 , portions  65   1  and  65   2  through which only a small molecule solution passes, and a portion  66  through which a mixed solution passes. 
     A solution injected from the injection port  60  is brought into contact with the hollow filter substances  64   1  and  64   2  at the portion  66  through which a mixed solution passes. The hollow filter substances disturb the passage of a large molecule so that no large molecule is present in the portions  65   1  and  65   2 . A solution containing the large molecule can be discharged from the sample discharge port  62  connected to the portion  66 . A solution containing the small molecule is discharged from the small molecule solution discharge port  61  connected to the portions  65   1  and  65   2 . 
     Although no particular limitation is imposed on the hollow filter substances  64   1  and  64   2  insofar as they can separate large molecules such as protein from the other substances, use of a hollow substance with pores having a diameter small enough to inhibit the passage of the protein but large enough to permit passage of the other components including small molecules is preferred. 
     The small molecule solution discharge port  61  of the separating filter portion is connected to an injection port  40  of a control portion  30  via a second small molecule solution transfer tubing  18   2 . 
     In the control portion  30 , the injection port  40  is connected to a small molecule solution injection portion  70 , discharge valve  44 , injection valve  46 , feed pump  48 , pressure gauge  49  and discharge port  42  of the small molecule concentration control portion; 
     the small molecule solution injection portion  70  has one or more syringe pumps and an electronic appliance for controlling the syringe pump(s); and 
     the feed pump  48  has a pump of feeding a solution by application of a pressure and an electronic appliance for controlling the feed pump. 
     In the small molecule solution injection portion  70 , one or more pressurized syringe pumps are preferably controlled electronically. For example, use of a syringe pump “KDS200”, product of Kd Scientific, makes it possible to carry out an injection operation of a solution into a circulation route while precisely controlling the syringe containing the solution. 
     As the feed pump  48 , those typically used in high performance liquid chromatography (which will hereinafter be abbreviated as HPLC) are preferred. A feed pump which drives a plunger to feed a liquid under a constant pressure by means of an electronically controllable stepping motor or the like is desirable. 
     The discharge port  42  of the control portion  30  is connected to a small molecule solution injection port  51  of a mixing filter portion  32  via a first small molecule solution transfer tubing  18   1 . 
     The mixing filter portion  32  has a discharge port  50 , small molecule solution injection port  51 , sample solution injection port  52 , hollow filters  54   1  and  54   2 , portions  55   1  and  55   2  through which only a small molecule solution passes, and a portion  56  through which a mixed solution passes. 
     A solution injected from the small molecule solution injection port  51  is brought into contact with the hollow filters  54   1  and  54   2  at the portions  55   1  and  55   2 . A sample solution containing the large molecule is injected from the sample solution injection port  52 . The hollow filter substances hinder the passage of the large molecule so that the large molecule is not transferred to the side of the portions  55   1  and  55   2 . The solution containing the large molecule is mixed with the small molecule solution and discharged from the discharge port  50  connected to the portion  56 . 
     Although no particular limitation is imposed on the hollow filters  54   1  and  54   2  insofar as they can separate a large molecule such as protein from the other components, a hollow substance with pores having a diameter small enough to prevent the passage of protein but large enough to permit passage of the other components including a small molecule. 
     The material of the sample transfer tubing  16   1 ,  16   2 ,  18   1 , and  18   2  must be selected depending on the properties of the sample solution. Polyethylene ethylene ketone (PEEK), Tefzel, Kel-F, fused silica and the like are often employed in the measurement of a bio-related large molecule such as protein. The sample transfer tubing has an inner diameter of preferably from 0.5 mm to 0.065 mm. 
     The container  10  must reserve therein a sufficient amount of a sample for NMR measurement. When protein is employed as the large molecule, the amount of the sample is from 400 μL to 100 μL. 
     When the probe has improved detection sensitivity and magnetic field can be kept uniform, even 100 μL or less is sufficient for measurement. 
     The container  10  and sample transfer tubing  16   2  are connected at a connection portion  12 , while the container  10  and the sample transfer tubing  16   1  are connected at a connection portion  14 . They are connected by utilizing a pushing pressure to keep the hermetic sealing of the container. One example of the connection form is illustrated in  FIG. 2 . The container  10  has a narrow tube  220  and this narrow tube  220  is connected to the sample transfer tubing  16   1  and  16   2  at the connection portions  12  and  14 . The connection portions  12  and  14  are each made of a connector  200  and fitting  210 . Connection tools typically employed in HPLC are preferred. The fitting  200  has two types, that is, integrated one and that separated into nut and ferrule, but either one is suited for connection. The connector  200  and fitting  210  are made of preferably PEEK, PTEF, Kel-F, Tefzel or other materials known in the field of HPLC. The narrow tube  220  of the container  10  has preferably a similar outer diameter to that of the sample transfer tubing and desirably falls within a range of from 1.57 mm to 0.36 mm. 
     A preferred embodiment of NMR measurement carried out by adjusting the conditions of a small molecule solution and injecting a large molecule solution under predetermined conditions will next be described in accordance with  FIG. 3 . 
     After starting  100  of measurement, degassing  102  of pure water and buffer solution to be injected is carried out. Degassing of the water or solution may be carried out by installing a degasser at the injection valve  46  or feed pump  48  and removing dissolved oxygen from the water or solution passing through the degasser, or by preliminary reducing the pressure of the water or solution to be injected and causing generation of air bubbles therefrom. For degassing by pressure reduction, it is recommended to connect an aspirator, for example, that manufactured by Tokyo Rikakikai to a pressure vessel such as an eggplant flask having the water or solution charged therein, dip the flask in a ultrasonic bath “B-220H” manufactured by Branson, and expose it to ultrasonic waves for 60 minutes. 
     Injection  104  of the pure water into a circulation route is performed in the following manner. 
     A vessel containing the degassed pure water is connected to the injection valve  46 . By means of the feed pump  48 , it is sucked, discharged from the discharge port  42 , and fed to the transfer tubing  18   1 . During feeding, the discharge valve  44  is opened, while the injection port  52  and discharge port  62  for the large molecule solution is closed. The pure water is fed to the mixing filter portion  32  via the first small molecule solution transfer tubing  18   1 . At the mixing filter portion  32 , it passes through the portions  55   1  and  55   2  through which only a small molecule solution passes and the hollow filters  54   1  and  54   2 , reaches the portion  56  through which a mixed solution passes, is discharged from the discharge port  50 , passes through the first sample transfer tubing  16   1 , and reaches the container  10 . Then, the pure water passes through the second sample transfer tubing  16   2 , separating filter portion  34 , and second small molecule solution transfer tubing  18   2 , reaches the discharge valve  44  of the control portion  30 , and is discharged outside. Until mixing of air bubbles in the pure water discharged from the discharge valve  44  stops, supply of the pure water is continued. When the discharge valve  44  is closed without stopping the injection of the pure water from the injection valve  46 , the pure water reaches the input side of the feed pump  48 , leading to a pressure rise. When the pump  48  is thereafter once stopped and the injection valve  46  is closed, the whole of the sample passage regions in the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1 , and  18   2 , filter portions  32  and  34 , and the control portion  30  is filled with the pure water. 
     Exchange  106  of the pure water with the buffer solution is performed in the following manner. 
     A vessel containing the buffer solution which has completed the degassing treatment  102  is connected to the injection valve  46 , followed by similar operations to those employed in the injection  104  of pure water, whereby the whole of the sample passing regions in the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1 , and  18   2 , filter portions  32  and  34 , and the control portion  30  is filled with the buffer solution. 
     Injection and concentration control  108  of small molecule solution is performed in the following manner. 
     First, solution circulation is performed by means of the feed pump  48  to form a flow of the solution in a transfer tubing. A small molecule solution for injection is set in the small molecule solution injection portion  70  and it is injected in an amount determined by a measurer. During injection, the discharge valve  44  is opened. Use of a syringe pump for injection enables precise injection of the solution in an injection amount at an injection rate, each determined by the measurer. The solution thus injected reaches the feed pump  48  with the flow, passes through the first small molecule solution transfer tubing  18   1  and is fed to the mixing filter portion  32 . In the mixing filter portion  32 , the small molecule solution is fed from the small molecule solution injection port to the portions  55   1  and  55   2  through which only a small molecule solution passes, passes through the hollow filters  54   1  and  54   2 , and reaches the portion  56  through which a mixed solution passes. During the injection of only the small molecule solution, only the small molecule solution is present in the portion  56  through which a mixed solution passes. The solution discharged from the discharge port  50  reaches the container  10  after passing through the first sample transfer tubing  16   1 . An excess solution which has appeared simultaneously with the injection is discharged from the discharge valve  44 . After completion of the discharge of the excess solution, the pressure indicated by a pressure gauge returns to that during the circulation feeding. After confirmation of the returning of this pressure, the discharge valve  44  is closed, followed by steady circulation feeding. The concentration of the small molecule has increased in the sample passing regions in the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1 , and  18   2 , filter portions  32  and  34 , and control portion  30 . The inner concentration becomes substantially uniform by carrying out steady circulation feeding about five cycles. 
     An increase in the concentration of the small molecule in the sample solution to be used for the NMR measurement can be controlled by conducting the injection operation of the small molecule plural times. Variations in the concentration of the small molecule in the container  10  as a function of injection frequency occur in accordance with the equations (4), (5) and (6).  FIG. 4  shows the concentration of the small molecule in the container  10  when the total volume of the sample passing regions in the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1  and  18   2 , filter portions  32  and  34  and control portion  30  is 1000 μL, the initial concentration of the small molecule is 0 mol/L, the concentration of the small molecule to be injected by every injection operation is 0.01 mol/L, and the volume of the solution injected from the solution injection portion  70  by single injection operation is 10 μL. With an increase in the injection frequency, the concentration of the small molecule in the container  10  rises. Accordingly, the desired small molecule concentration can be realized by designating the injection concentration, volume and frequency and the NMR measurement is performed at this concentration. 
     The concentration of a target small molecule in the sample solution used for the NMR measurement can be reduced by repeating the injection operation of, instead of the solution containing a small molecule, only a buffer or a solution not containing a specific small molecule. The concentration of the small molecule in the container  10  varies as a function of injection frequency in accordance with the equations (10), (11) and (12). 
       FIG. 5  shows the concentration of the small molecule in the container  10  when the total volume of the sample passing regions in the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1  and  18   2 , filter portions  32  and  34 , and control portion  30  is 1000 μL, the initial concentration of the small molecule is 0.0019 mol/L, the concentration of the small molecule to be injected by every injection operation is 0.00 mol/L, and the volume of the solution injected from the solution injection portion  70  by single injection operation is 100 μL. With an increase in the injection frequency, the concentration of the small molecule in the container  10  decreases. Accordingly, a desired small molecule concentration is realized by designating the volume and operation frequency and the NMR measurement is carried out at this concentration. 
     By using the operation for increasing the concentration of the small molecule and that for decreasing it in combination, the concentration of the small molecule can be controlled, that is, increased or decreased freely. This suggests that a measurement condition once employed can be used again. 
     In the injection operation of the solution containing or not containing the small molecule, addition of a lock solvent having a certain concentration to a solution to be injected makes it possible to keep its concentration in the container  10  constant. 
     The equalization  110  of the concentration  110  is conducted in the following manner. 
     The concentration can be made almost uniform by carrying out steady circulation feeding about five cycles, but an increase in the circulation frequency enables further equalization of the concentration. 
     Adjustment of magnetic field homogeneity  112  is conducted in the following manner. 
     When a lock solvent is added to the small molecule solution in advance, adjustment of magnetic field homogeneity (shimming) can be carried out utilizing a lock signal in the NMR measurement. Shimming can also be performed not using the lock signal but using a signal derived from the small molecule. 
     The injection and concentration control  120  of the small molecule solution) are carried out in the following manner. 
     When the concentration of the small molecule is different from the predetermined concentration based on the NMR signal derived from the small compound or measurement of spectrum, the concentration can be controlled by carrying out injection and concentration control  108  of a small molecule solution and equalization  110  of the concentration. 
     Injection  122  of large molecule solution is performed in the following manner. 
     The operation of the feed pump  48  is started to generate a certain flow in all the circulation routes in the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1  and  18   2 , filter portions  32  and  34  and control portion  30 . From the sample solution injection portion  90 , the solution containing the large molecule is injected in an amount designated by the measurer. In the portion  56  through which a mixed solution passes, the low molecule solution which has already been present is mixed with the large molecule solution. The hollow filters  54   1  and  54   2  do not allow the passage of the large molecule so that the solution containing the large molecule transfers, along the certain flow, to the container  10  set in the probe. Since the container  10  has a larger inner diameter than that of the sample transfer tubings  16   1  and  16   2 , further mixing of the solution which has passed through the sample transfer tubing  16   1  occurs in this container  10 . 
     Holding  124  of the sample in the NMR measurement portion is conducted in the following manner. 
     Since the volume and flow rate of the large molecule solution to be injected, and the volumes of the region  56 , transfer tubing  161  and container  10  are already known, the time necessary for filling the container  10  with the mixed solution can be determined from the time that has elapsed after injection. At the time when the container  10  is filled, the feed pump  48  is terminated. By the termination of the pump, pressure acting on the solution decreases and the circulation flow of the solution stops. Since the container  10  has a larger inner diameter than that of the sample transfer tubings  16   1  and  16   2 , a difference in the resistance to the solution between when it passes through the container  10  and when it passes through sample transfer tubings  16   1  and  16   2  enables holding of the solution in the container  10 . 
     NMR measurement  126  is conducted in the following manner. 
     After the container set in the probe is filled with the sample solution containing the large molecule, NMR measurement of the sample is performed while applying a uniform magnetostatic field to the sample from the magnet. 
     The discharge  128  of the large molecule solution is performed in the following manner. 
     The operation of the feed pump  48  is started and a certain flow is generated in all the circulation routes in the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1  and  18   2 , filter portions  32  and  34 , and control portion  30 . With the certain flow thus generated, the mixed solution retained in the container  10  passes through the sample transport tube  16   2  and reaches the filter  34 . The small molecule solution which has passed through the portion  66  through which a mixed solution passes and hollow filters  64   1  and  64   2  passes through the  61  and is discharged to the  18   2 . When a sample discharging syringe  91  is connected to the sample discharge port  62  and the sample discharge port  62  is opened, the solution containing the large molecule transfers from the sample discharge port  62  to the sample discharging syringe  91  and the large molecule solution is discharged. 
     Judgment  130  on whether the repetition of the exchange of the large molecule solution is completed or not is made in the following manner. 
     It is judged whether measurement is conducted under all the conditions under which the large molecule solution is to be measured or not. One of the judging methods is to record, prior to the measurement, the number of the conditions under which the large molecule solution is to be measured and each time, compare the number of the conditions under which the measurement is performed in practice with the recorded number of the measurement conditions of the large molecule solution. When the measurement has been made under all the measurement conditions of the large molecule solution in practice as a result of judgment, the next procedure, that is, judgment  140  on whether the repetition of a concentration change or exchange of the small molecule solution is completed or not is made. If measurement is not performed under all the conditions, the injection of the large molecule solution and NMR measurement are carried out again, after returning to the concentration control  120  of small molecule solution. When the measurer judges that the repetition is completed, the repetition is terminated. 
     Judgment  140  on whether the repetition of a concentration change or exchange of the small molecule solution is completed or not is performed in the following manner. 
     It is judged whether measurement is conducted under all the conditions under which the small molecule solution is to be measured or not. One of the judging methods is to record, prior to the measurement, the number of the conditions (such as frequency of concentration change and kind of the small molecule) under which the small molecule solution is to be measured and each time, compare the number of the conditions under which the measurement is performed in practice with the recorded number of the measurement conditions of the small molecule solution. When measurement has been made under all the measurement conditions of the small molecule solution in practice as a result of judgment, the next procedure, that is, discharge  150  of the small molecule solution is made. If not so, the concentration control of the small molecule is carried out again after returning to the injection  108  of the small molecule and the measurement is carried out in repetition. When the measurer judges that the repetition is completed, the repetition is terminated. 
     The discharge  150  of the small molecule solution is carried out in the following manner. 
     The vessel of the buffer solution treated in the degassing procedure  102  is connected to the injection valve  46  and by similar treatment to that employed in the injection  104  of the pure water, the whole of the sample passing regions in the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1 , and  18   2 , filter portions  32  and  34 , and control portion  30  is filled with the buffer solution. The small molecule solution present in the circulation portions is discharged from the discharge valve  44 . 
     Exchange  152  with pure water is carried out in the following manner. 
     The vessel of the pure water treated in the degassing treatment  102  is connected to the injection valve  46  and by similar treatment to that employed in the injection  104  of the pure water, the whole of the sample passing regions in the container  10  set in the probe  24 , sample transfer tubings  16   1 ,  16   2 ,  18   1 , and  18   2 , filter portions  32  and  34 , and control portion  30  which are circulation portions is filled with the pure water. The buffer solution is discharged from the discharge valve  44 . 
     Some of the samples need mixing of a lock solvent therein at the time of NMR measurement. When a large portion of the solvent is light water such as a phosphate buffer employed frequently for the NMR measurement of a protein, use of deuterated water as a lock solvent is preferred. A preferred concentration of deuterated water is from 5 to 10%. 
     Measurement using a preferred solvent such as buffer solvent or lock solvent selected depending on the large molecule to be measured is also within the scope of the Example of the present invention. 
     A magnetic field B 0  is applied from the magnet  20  to a lock-solvent-containing sample solution in the container  10  to effect magnetic field locking. The uniformity of the magnetic field B 0  for enabling the NMR measurement can be maintained by utilizing the magnetic field locking for controlling the uniformity of the magnetic field B 0  generated from the magnet  20 . 
     It is known that proteins, among large molecules, have a property of undergoing an enzymatic reaction. This enzymatic reaction is a reaction of changing a substrate into a product with a protein as a catalyst and the protein reacts only with a specific substrate. This reaction is inhibited by a compound called “inhibitor”. Inhibitors are compounds controlling the enzymatic reaction of proteins and search of them is industrially useful. 
     In the research and development of inhibitors, it is necessary to study the interaction between a protein and a small molecule, elucidate their binding state, and develop a small molecule having a stronger binding performance. 
     In order to study the interaction between a protein and a small molecule, it is useful to study not the interaction with one protein but the interaction between a plurality of proteins, each obtained by modifying a specific residue of the protein whose active site (binding site to the compound, substrate) has been subjected to activation/deactivation control, and a small molecule while varying the kind or concentration of the small molecule. 
     According to the present invention, the interaction with a plurality of proteins can be analyzed efficiently by means of NMR measurement. In accordance with the procedures as described above in  FIG. 3 , a specific small molecule solution is injected in the procedure  108 . After the solution conditions are fixed, a protein solution is injected in the procedure  122 , held in the container  10  in the procedure  124 , and then NMR measurement is performed. 
     A pulse sequence which is used for a small molecule solution or protein solution can also be applied to the NMR measurement. 
     When a change in chemical shift value or line width of a peak derived from a small molecule is to be measured and a solvent contains a water molecule (H 2 O), proton one-dimensional measurement using WATERGATE, DPFGSE or Water-Flipback method capable of controlling a signal from the water molecule and efficiently detecting a signal derived from a target protein is suited. When the solvent does not contain a water molecule (H 2 O), on the other hand, use of WATERGATE, DPFGSE or Water-Flipback method is not required. 
     When correlation between peaks derived from a small molecule is measured and a solvent contains a water molecule (H 2 O), proton COSY, NOESY or TOCSY measurement using WATERGATE, DPFGSE or Water-Flipback method capable of controlling a signal from the water molecule and efficiently detecting a signal derived from a target protein is suited. When the solvent does not contain a water molecule (H 2 O), use of WATERGATE, DPFGSE or Water-Flipback method is not required. 
     When correlation between the peaks derived from an isotopically modified protein such as  13 C or  15 N and the solvent contains a water molecule (H 2 O), HSQC or HMQC measurement using WATERGATE, DPFGSE or Water-Flipback method capable of controlling a signal from the water molecule and efficiently detecting a signal derived from a target protein is suited. Use of a method of measuring heteronuclear correlation, as well as HSQC and HMQC measurements, is also embraced in the present invention. When the solvent does not contain a water molecule (H 2 O), use of WATERGATE, DPFGSE or Water-Flipback method is not required. 
     After the NMR measurement, the protein is discharged in the procedure  128 , preparation for injection of another protein is performed in the procedure  120 , and the protein is injected in the procedure  122 . 
     In the method of the present invention where proteins are exchanged successively in a small molecule solution under certain conditions, it is not necessary to fill the whole circulation route with a protein solution so that the protein amount to be measured can be limited to an amount necessary for the NMR measurement. The term “amount necessary for the NMR measurement” means an amount necessary for keeping the magnetic field of the solution in the container  10  uniform and being detected by a probe coil  28  placed in the NMR probe  24 . 
     In the present invention, the concentration of a small molecule can be increased or diluted so that a measurer can control the conditions of the solution during measurement and set them efficiently. Even if the concentration condition suited for the effect for a large molecule is unknown, the condition can be controlled while carrying out measurement. It is therefore possible to carry out measurement within a concentration range suited for measurement of correlation without preparing a plurality of samples corresponding to a wide range of concentration change. 
     EXAMPLE 2 
     A preferred example relating to the finding of association or aggregation conditions of a protein under various solution conditions will next be explained. 
     Protein is known to cause association, depending on the pH, temperature or concentration of a small molecule in the solution thereof. Association or aggregation of a protein causes a change in its protein function and sometimes leads to appearance of a new activity. A change in the protein due to aggregation can be detected as a spectrum change determined by the NMR measurement. 
     A concentration condition of a small molecule that causes association or aggregation of a protein, which occurs due to a difference in the concentration of a small molecule, can be found by using the system or procedure as described in Example 1. For example, lysozyme is known to cause aggregation in the presence of an amino acid such as arginine. In order to study the action of a specific small molecule against a single kind of a protein, injection of an amino acid such as arginine is carried out in the procedure of injection and concentration control  108  of a small molecule as described in  FIG. 3 . 
     Then, subsequent procedures for measurement are performed in accordance with  FIG. 3  and in the procedure  122  of injecting a large molecule solution, a target protein is injected. 
     In the NMR measurement  126  for determining the association or aggregation state, proton one-dimensional measurement using a WATERGATE, DPFGSE or Water-Flipback method capable of controlling a signal from a water molecule (H 2 O) of the solvent and efficiently detecting a signal derived from the target protein is suited. When the solvent does not contain a water molecule (H 2 O), use of the WATERGATE, DPFGSE or Water-Flipback method is not required. 
     The line width and height of the peak derived from the target protein on the NMR spectrum thus obtained are recorded for each condition of the small molecule solution. 
     When association or aggregation of proteins occurs, plural proteins which have so far moved in a solution as each molecule start moving in the bound form. This leads to an increase in the mass relative to the movement, slower random movement in the solution, increase in the line width of a peak derived from the protein, and reduction in the peak height. 
     It is judged based on the data recorded in advance whether an increase in the peak linewidth or decrease in the height occurs as a function of the concentration of the small molecule or not. The concentration of the small molecule at which a change starts is the solution condition for causing association or aggregation of the protein. 
     Measurement of not only a solution of a single protein but also a solution of plural proteins is embraced in this Example. 
     Measurement of plural protein solutions and plural small molecule solutions is also embraced in this Example. 
     EXAMPLE 3 
     In another embodiment of the present invention, as illustrated in  FIG. 6 , the injection port  14  and discharge port  12  are located outside the probe  24 , the first sample transfer tubing  16   1  and the second sample transfer tubing  16   2  are located in a direction perpendicular to the container  10 , the first sample transfer tubing  16   1  and the second sample transfer tubing  16   2  are located inside the bore  22   2  and along the nuclear magnetic resonance probe  24  and introduced outside of the magnet  20  from the probe insertion port of the bore  22   2 . In such an embodiment, it is possible to decrease the length of the first sample transfer tubing  16   1  and the second sample transfer tubing  16   2  and not to insert them in the probe having only a limited internal space. 
     EXAMPLE 4 
     In a further embodiment, as illustrated in  FIG. 7 , the injection port  14  and discharge port  12  are placed inside of the probe  24 , the first sample transfer tubing  16   1  and the second sample transfer tubing  16   2  are located in a direction perpendicular to the container  10  and the first sample transfer tubing  16   1  and the second sample transfer tubing  16   2  are introduced outside of the magnet  20  after allowing them to pass through the inside of the probe  24 . In such an embodiment, it is possible to decrease the length of the first sample transfer tubing  16   1  and the second sample transfer tubing  16   2  and integrate the probe with sample transfer tubings. 
     EXAMPLE 5 
     In a still further embodiment, as illustrated in  FIG. 8 , the second sample transfer tubing  16   2  is placed inside the bore  22  and the first sample transfer tubing  16   1  is placed inside of the probe  24  and the first sample transfer tubing  16   1  and the second sample transfer tubing  16   2  are connected to the container  10  at the injection port  14  and discharge port  12 . 
     EXAMPLE 6 
     In a still further embodiment, as illustrated in  FIG. 9 , the first sample transfer tubing  16   1  and the second sample transfer tubing  16   2  are placed inside the probe  24  and at the injection port  14  and discharge port  12 , the first sample transfer tubing  16   1  and the second sample transfer tubing  16   2  are connected to the container  10 . In such an embodiment, the probe can be integrated with the container  10  and sample transfer tubings. 
     EXAMPLE 7 
     In a still further embodiment, as illustrated in  FIG. 10 , the second sample transfer tubing  16   2  is inserted from the upper opening portion of the bore  22   1 , the first sample transfer tubing  16   1  is placed inside the probe  24 , and the first sample transfer tubing  16   1  and the second sample transfer tubing  16   2  are connected to the container  10  at the injection port  14  and discharge port  12 . 
     The constitutions of the present invention other than those described above will next be described. 
     (1) An apparatus for nuclear magnetic resonance measurement, which is equipped with a measurement portion having a magnet for applying a magnetic field to a sample, a bore placed inside the magnet, a nuclear magnetic resonance probe placed in the bore, and a container for retaining the sample therein, which container is installed in the nuclear magnetic resonance probe and has an injection port and a discharge port of the sample; a mixing filter portion for mixing a small molecule solution with a sample solution which has a first sample transfer tubing connected to the injection port of the container, a second sample transfer tubing connected to the discharge port of the container, an injection port of the small molecule solution, and an injection port and discharge port of the sample, the discharge port being connected to the first sample transfer tubing; a separating filter portion for separating the small molecule solution not containing the sample from the mixed solution of the small molecule and sample, which filter portion has a first small molecule solution transfer tubing connected to the small molecule solution injection port of the mixing filter portion, a small molecule discharge port, and a discharge port and injection port of the sample, the injection port of the sample being connected to the second sample transfer tubing; a small molecule concentration controlling portion for controlling the concentration of the small molecule and feeding the small molecule solution, which portion has a second small molecule solution transfer tubing connected to the small molecule discharge port of the separating filter portion and an injection port and discharge port of the small molecule solution, the injection port being connected to the second small molecule solution transfer tubing and the discharge port being connected to the first small molecule solution transfer tubing; a transmitter/receiver system for transmitting an electromagnetic wave to the nuclear magnetic resonance probe or receiving an electromagnetic wave from the nuclear magnetic resonance probe; a unit for carrying out circulation solution transfer among the small molecule concentration control portion, first small molecule solution transfer tubing, mixing filter portion, first sample transfer tubing, container, second sample transfer tubing, separating filter portion and second small molecule solution transfer tubing; a unit for injecting the small molecule solution; a unit for controlling the small molecule concentration; a unit for injecting the sample solution; a unit for holding the sample solution in the measurement portion; a unit for discharging the sample solution; and a unit for carrying out nuclear magnetic resonance measurement of the sample solution. 
     (2) An apparatus as described above in (1), wherein the container has an NMR measurement portion having a larger diameter than those of the first and second sample transfer tubings. 
     (3) An apparatus as described above in (1), wherein the mixing filter portion has a portion through which the mixed solution of the sample with the small molecule separated by a hollow filter substance through which only the small molecule solution can pass passes, a portion through which only the small molecule solution passes, the injection port of the small molecule solution, the injection port of the sample, and a discharge port for discharging the mixed solution; and the separating filter portion has a portion through which the mixed solution of the sample with the small molecule separated by a hollow filter substance through which only the small molecule solution can pass passes, a portion through which only the small molecule solution passes, an injection port of the mixed solution, the discharge port of the sample and the discharge port of the small molecule solution. 
     (4) An apparatus as described above in (1), wherein in the small molecule concentration control portion, the injection port of the small molecule concentration control portion is connected to the small molecule solution injection portion, discharge valve, injection valve, feed pump, pressure gauge and discharge port of the small molecule concentration control portion; the small molecule solution injection portion has one or more syringe pumps, and injection from the syringe pump is controlled by an electronic appliance for controlling the syringe pump; and the feed pump portion has an injection port to which a degasser can be attached, a pump for feeding the solution under pressure, and an electronic appliance for controlling the pump. 
     (5) An apparatus as described above in (1), wherein in the unit for carrying out circulation solution transfer, the solution pressurized at the feed pump portion is discharged from the discharge port of the small molecule concentration control portion, passes through the first small molecule solution transfer tubing, reaches the small molecule solution injection port of the mixing filter portion, passes through the hollow filter substance at the mixing filter portion, is discharged from the discharge port, passes through the first sample transfer tubing, reaches the container, passes through the second sample transfer tubing, passes through the injection port of the separating filter portion, the hollow filter substance at the separating filter portion, and the second small molecule solution transfer tubing, and reaches the injection port of the small molecule concentration control portion. 
     (6) An apparatus as described above in (1), wherein the unit for injecting the small molecule solution injects a solution containing the small molecule from the small molecule solution injection portion of the small molecule concentration control portion while carrying out circulation solution transfer and discharges the solution from the discharge valve until the value of the pressure gauge returns to that under a steady circulation state. 
     (7) An apparatus as described above in (1), wherein the unit for controlling the small molecule concentration is a unit for increasing the small molecule concentration in the container by injecting the small molecule solution from the small molecule solution injection portion of the small molecule concentration control portion and discharging an excess solution depending on the volume of the small molecule solution thus injected; or decreasing the small molecule concentration in the container by injecting a solution not containing the small molecule from the small molecule solution injection portion and discharging an excess solution depending on the injected volume. 
     (8) An apparatus as described above in (1), wherein the unit for injecting the sample solution feeds the sample solution to a portion of the mixing filter portion through which the mixed solution of the small molecule and sample passes by pressurizing with a syringe or the like attached to the sample injection port of the mixing filter portion while carrying out the circulation solution transfer and then feeds the sample to the container in accordance with a method of circulation transfer of a solution. 
     (9) An apparatus as described above in (1), wherein in the unit for holding the sample solution in the measurement portion, the sample solution is held in the NMR measurement portion of the container by terminating the pressurization of the feed pump when the sample reaches the NMR measurement portion of the container by circulation solution transfer. 
     (10) An apparatus as described above in (1), wherein in the unit for discharging the sample solution, the circulation solution transfer is caused by starting the feeding of the solution under pressure of the feed pump, the sample-containing mixed solution is separated at the separating filter portion into a sample-containing solution and a sample-free small molecule solution, and the sample-containing solution is taken out from the discharge port of the separating filter portion. 
     (11) An apparatus as described above in (1), wherein after controlling the small molecule concentration in the container by using the unit for injecting the small molecule solution and the unit for controlling the small molecule concentration, the unit for carrying out nuclear magnetic resonance measurement of the sample solution holds the sample in the NMR measurement portion by means of the unit for injecting the sample solution and the unit for holding the sample solution in the measurement portion, carries out nuclear magnetic resonance measurement, and after the measurement, discharges the sample by making use of the unit for discharging the sample solution. 
     Application of the present invention to large molecules, such as protein, having a function in the living body enables repetition of NMR measurement while maintaining a constant volume of the sample in the NMR measurement region and changing the solution conditions and large molecule efficiently. In the fields of life science, it contributes to the improvement in the efficiency of analysis of biochemical processes occurring in the living body, while in the medical or drug discovery fields, it contributes to the improvement in the efficiency of disease mechanism analysis or screening by measuring the binding strength with the disease-related protein. 
     Reference numerals used in the drawings of the present application will be explained below.