JPH037067B2 - - Google Patents

Description

Detailed Description of the Invention: Among molecules that interact with each other (referred to as A and B), one molecule B is fixed to a stationary phase by adsorption or chemical bonding, and the other molecule A is flowed together with a mobile phase. Chromatography that utilizes the interaction that occurs when a layer of molecule A contacts molecule B is usually called affinity chromatography, and is used to separate solute molecules and study interactions, such as between enzymes and substrates, and between antigens. It is used for separation and research on specific interactions using specific interactions, such as between antibodies or between proteins and acids.

In conventional affinity chromatography, one molecule B is immobilized on a stationary phase, but immobilization may be difficult, or it may gradually come off and flow out, or the nature of the interaction may change due to immobilization. There were some drawbacks. In order to solve the above-mentioned drawbacks, the present invention is designed to flow the molecule B in a layered manner without immobilizing it.

That is, the present invention is characterized in that, when introducing a sample into a chromatograph, groups of solute molecules that interact and have different development rates are introduced at different times or at different positions so that they intersect during development of the chromatograph. This is a column chromatography method.

Column chromatography development is typified by liquid chromatography development, but is not limited thereto.

Hereinafter, it will be explained in detail using the drawings.

Although FIG. 1 shows a block diagram of the apparatus used to carry out the present invention, a conventional liquid chromatography apparatus or high-performance liquid chromatography apparatus can be used, and no special apparatus is required. It is preferable that the pump 2 has little pulsation and excellent flow stability. Although the sample introduction method is not particularly limited, a sampling valve 3 was used in this example. It is also possible to use multiple sampling valves or automatic valves. The separation column 4 may be any type of packing material as long as it has different retention powers for either one of the two molecules and the conjugate of the two molecules, such as normal phase packing, reversed phase packing, etc. Although the elution position is mainly determined by the size of the molecule, the gel filtration packing material allows for a wide selection of salt concentration, pH, and solvent composition. Liked to be used. The detector 5 is selected from among detectors typified by absorbance detectors and fluorescence detectors in consideration of detection sensitivity and selectivity. In this device, solute molecules with a longer retention time are injected first, and solute molecules with a shorter retention time are subsequently injected within a time shorter than the difference in retention time, allowing them to cross within the column. As an embodiment of the present invention, single-stranded deoxyribonucleic acid (hereinafter abbreviated as DNA) and ribonuclease (hereinafter referred to as
The interaction with RNase (abbreviated as RNase) was measured using an aqueous GPC column (TSKGEL G2000SW).
Since RNase has a size smaller than the pore size, it is eluted later than the exclusion volume. On the other hand, DNA
and DNA and RNase conjugates are larger than the pore size and are therefore eluted in the excluded volume. In this example, fluorescence was detected using 230 nm excitation light and a 290 nm cut-off filter, and it was found that it was sensitive only to RNase and did not detect DNA. Figure 2 shows the dependence of the elution pattern of RNase on DNA concentration. RNase concentration was 2.5 μM, DNA
The concentration is the monomer concentration from the left: 26×10 ^-4 M, 10.8×
10 ^-4 M, 5.2 × 10 ^-4 M, 2.6 × 10 ^-4 M, 0 M,
Moreover, 40mM phosphate buffer (PH7) was used as the eluent. Figure 3 shows the dependence of RNase peak shift on DNA concentration. Based on this slope, the binding constants of DNA and RNase were determined by computer simulation at salt concentrations of 64mM and 48mM, respectively.
They were determined to be 1.26±0.03×10 ⁴ M ⁻¹ and 3.92±0.05×10 ⁴ M ⁻¹ . This value showed good agreement with the value obtained by the boundary sedimentation method.

According to the present invention, it has become possible to separate multiple molecular species based on the strength of their interaction with a certain molecule, and it has also become possible to determine interaction parameters between solute molecules from the elution pattern. . Since the present invention allows solute molecules to flow in a narrow layer, it can be applied to valuable samples and samples that are difficult to immobilize. The present invention is a rapid interaction analysis tool that replaces the boundary precipitation method and dialysis equilibrium method, and is widely used in biochemistry, medicine, and pharmacy.

[Brief explanation of the drawing]

Figure 1 is a block diagram of the apparatus used to carry out the present invention, Figure 2 is an example of measurement showing the dependence of RNase elution pattern on DNA concentration, and Figure 3 is
FIG. 3 is a diagram showing the dependence of the peak shift amount of RNase on DNA concentration. 1... Eluent, 2... Liquid pump, 3... Sampling valve, 4... Separation column, 5... Detector, 6, 7, 8, 9, 10... Each DNA monomer concentration is 26 x 10 ^-4 , 10.8× ^10-4 , 5.2× ^10-4 ,
2.6×10 ^-4 , RNase elution profile at 0M, 11, 12...DNA of RNase peak shift amount at salt concentration of 48mM and 64mM, respectively
Monomer concentration dependence.

Claims (1)

[Claims] 1 Solute molecules interact with each other,
and a plurality of samples having different chromatographic development rates and containing at least one type of solute molecule are introduced into the chromatograph at different times or positions so that the chromatographic development bands of the solute molecules intersect. Column chromatography featuring: