JP3216436B2 - Basic protein adsorbent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an adsorbent which specifically adsorbs only basic proteins when separating and purifying or separating and removing proteins in the biochemical or medical fields.

[0002]

2. Description of the Related Art As a method for separating and purifying proteins,
Conventionally, liquid chromatography has been used. In the liquid chromatography, a column is filled with an adsorbent, a protein solution is flowed into the column, the adsorbent is adsorbed on the column, and then the adsorbed protein is eluted by flowing a specific solvent, whereby separation and purification can be performed. . In such liquid chromatography, protein acidity,
As a method for separating and purifying proteins using basicity, ion exchange chromatography is known. The ion exchange chromatography uses an anion exchange resin as an adsorbent, an ion exchange resin such as a cation exchange resin, depending on the ion exchange resin used at that time,
An acidic protein or a basic protein can be specifically adsorbed, respectively. However, these ion exchange resins have a drawback that when adsorbing a physiologically active substance such as an enzyme, a part thereof is denatured and irreversible binding may occur, so that the recovery rate is reduced. Further, when a high-concentration salt solution must be used as a solvent for eluting the adsorbed protein, there is a disadvantage that the activity of the protein is reduced.

[0003] Therefore, recently, it has been proposed to use hydroxyapatite as an adsorbent capable of recovering proteins efficiently and without deactivation. Hydroxyapatite chromatography utilizes ionic interactions, and its separation mechanism is similar to that of affinity chromatography, and is a method that can easily adsorb and elute proteins. However, hydroxyapatite, which has been used as an adsorbent, is produced at a low sintering temperature, has a small negative zeta potential, and adsorbs both acidic and basic proteins. It is impossible to specifically adsorb one of the proteins.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide an adsorbent capable of specifically adsorbing and separating only basic proteins from a serum or protein mixture.

[0005]

According to the present invention, Ca /
A hydroxyapatite having a P molar ratio of 1.62 to 1.72 and calcined at 1100 ° C. or higher, and a specific surface area of
A basic protein adsorbent characterized by comprising hydroxyapatite having a value of 1.0 m2 / g or less . Further, according to the present invention, the hydroxyapat
Is fired at 1100 ° C or higher and 1250 ° C or lower.
Negative surface zeta potential due to droxyapatite
Wherein the basic protein adsorbent is
Provided. Furthermore, according to the invention, the Ca / P molar ratio
From 1.62 to 1.72, 11
When the temperature is between 00 ° C and 1250 ° C, the surface zeta potential
Baking so that the specific surface area is 1.0 m2 / g or less.
A method for producing the basic protein adsorbent, comprising:
A law is provided.

Hereinafter, the present invention will be described in more detail.

The basic protein adsorbent of the present invention has a specific Ca / P molar ratio and comprises hydroxyapatite fired at a specific firing temperature as a constituent material. By making such a specific hydroxyapatite a constituent material,
The specific adsorption of only the basic protein is considered to be due to the following adsorption mechanism.

The zeta potential of hydroxyapatite varies depending on the firing temperature when distilled water is used as a fluid. For example, the zeta potential at a firing temperature of 600 ° C. or less is −
Constant at 1.5 mV, but -1.6 mV at 700 ° C.
-1.7 mV at 800 ° C, -2.0 mV at 900 ° C, 1
-2.3 mV at 000 ° C, -2.7 mV at 1100 ° C,
The temperature increases to −3.1 mV at 1200 ° C. as the firing temperature increases. On the molecular surface of the hydroxyapatite, as a protein adsorption site, a Ca site where a negatively charged acidic protein adsorbs to calcium having a positive potential, and a phosphoric acid where a positively charged basic protein has a negative potential And a P site adsorbed on the surface. Since the above-mentioned zeta potential indicates the potential of the entire surface, the zeta potential of the hydroxyapatite is the sum of the positive potential of the Ca site and the negative potential of the P site. On the other hand, even if the firing temperature of the hydroxyapatite is changed, the composition ratio and the state of the Ca site and the P site on the hydroxyapatite surface do not change, so that the change in the zeta potential due to the firing temperature of the hydroxyapatite is different from that of the Ca site. It is believed that both potentials at the P site change in the same manner. Therefore, the Ca site potential of the hydroxyapatite calcined at 1200 ° C. is set to 0 mV, and the amount of change in the zeta potential according to the calcining temperature is divided equally between the Ca site and the P site, and the zeta potential is divided between the Ca site and the P site. Decomposed into the following Table 1 (for example, 1
At 200 ° C., the zeta potential of the hydroxyapatite is −3.1 mV, and the zeta potential at 1100 ° C. is −2.
7 mV and the difference between these zeta potentials is +0.4 mV
Becomes If this is halved, it becomes +0.2 mV, so that the potential of the Ca site at 1100 ° C. is 0 mV + 0.2 m
V = 0.2 mV, and the potential at the P site is −3.1 mV
+0.2 mV = −2.9 mV. ).

[0009]

[Table 1]

On the other hand, when the protein is adsorbed on the hydroxyapatite, the sum of the surface potential of the adsorption site and the surface potential of the protein is a positive value including zero. Is assumed to be the case where the sum of と and the surface potential of the protein is negative. For example, assuming that the surface potential of bovine serum albumin (hereinafter referred to as BSA), which is an acidic protein, is -0.4 mV, the C of hydroxyapatite calcined at 1000 ° C.
Since the a-site potential is +0.4 mV, -0.4 mV +
0.4mV = 0mV, becomes 0, and the BSA is 1000
Adsorbs on the hydroxyapatite calcined at ℃. On the other hand, since the Ca site potential of hydroxyapatite calcined at 1100 ° C. is +0.2 mV, it is −0.4 mV.
V + 0.2 mV = -0.2 mV becomes negative, and the BSA does not adsorb to the hydroxyapatite calcined at 1100 ° C. On the other hand, assuming that the surface potential of the basic protein cytochrome C is +3.5 mV, even the hydroxyapatite calcined at 1200 ° C. where the negative value of the P-site potential is the largest is −3.1 mV.
The value becomes positive at 5 mV-3.1 mV = + 0.4 mV, and the cytochrome C is adsorbed to hydroxyapatite calcined at any temperature. Experimentally, the surface potential of an acidic protein is considered to be -0.3 mV to -1.6 mV, and the surface potential of a basic protein is considered to be +2.9 mV to +3.5 mV. Although adsorbed on apatite, the basic protein also adsorbs on the hydroxyapatite at 1100 ° C. or higher. Therefore, the adsorbent of the present invention finds such an adsorption mechanism, and uses hydroxyapatite fired at 1100 ° C. as a constituent material to specifically adsorb only basic proteins without adsorbing acidic proteins. That is, the calcination temperature is set to 1100 ° C. or higher, preferably 1150 ° C. to 1250 ° C. in order to prevent the negative value of the zeta potential of the adsorbent from becoming small and preventing both the acidic protein and the basic protein from being adsorbed.

The hydroxyapatite constituting the basic protein adsorbent of the present invention has been calcined at the above-mentioned specific calcination temperature, and has a Ca / P molar ratio of 1.62 or more.
It must be 1.72. The lower limit of the Ca / P molar ratio of the hydroxyapatite is that the crystal structure changes,
The value is 1.62, preferably 1.65, in order to prevent the basic protein from being absorbed. On the other hand, the upper limit is 1.72, preferably 1.65, in order to prevent the adsorbent from becoming alkaline and denaturing the adsorbed protein.

Further, the specific surface area of the hydroxyapatite is preferably 1.0 m ² / g or less in order to make the specific surface area free of pores, since the protein may not be eluted if the protein enters the pores on the surface. 0.3-0.01
m ² / g is desirable.

In order to synthesize the hydroxyapatite, for example, 5 mol of calcium hydroxide is suspended in water,
Preferably, 3 mol of phosphoric acid is gradually dropped and reacted,
It can be obtained by a known method such as a wet method of drying after curing, or a method of combining 3 mol of calcium pyrophosphate and 4 mol of calcium carbonate and synthesizing by a dry method.

To prepare the basic protein adsorbent of the present invention, the hydroxyapatite synthesized by the above method is calcined at 1100 ° C. or higher, and then pulverized by a known method.
After the pulverization, it can be obtained by a method of baking at 1100 ° C. or more, and classifying to 5 to 500 μm.

In order to use the basic protein adsorbent of the present invention, a mixture containing a basic protein to be adsorbed may be brought into contact with the adsorbent. Specifically, for example, a conventional liquid chromatography column is used. It can be used by a method of filling and developing and eluting the basic protein adsorbed by a conventional method.

In the present invention, the basic proteins to be adsorbed and separated include lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen,
α-chymotrypsin, histone, protamine and the like can be preferably mentioned.

[0017]

The basic protein adsorbent of the present invention can specifically adsorb only basic proteins by using a specific hydroxyapatite as a constituent material. Furthermore, since the protein can be collected efficiently and without deactivation, a basic physiologically active substance or the like can be easily separated and purified from, for example, serum or crushed material of living tissue or cells or microorganisms. By passing blood through the adsorbent, basic harmful substances in blood can be removed.

[0018]

The present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to these examples.

[0019]

Example 1 The molar ratio of Ca / P was 1.67 and the firing temperature was 1
200 ° C hydroxyapatite (specific surface area 0.04m
² / g, particle size 50-150 μm) 0.9 cmφ × 15
The column was packed in a cmL column, equilibrated with distilled water, and then attached to a high performance liquid chromatograph. Then BSA
(Pl (equipotential): 4.7), fibrinogen (pl:
5.6), a protein mixed solution of lysozyme (pl: 10.8) and cytochrome C (pl: 10.6) is injected into the column, and after adsorption, the column is washed with distilled water,
0-0.35M calcium phosphate buffer (pH 7.
4) Development and dissolution test by linear gradient (flow rate:
0.3 ml / min, detection: UV (280 nm)). The results are shown in FIG. In addition, from FIG. 1, adsorption separation of albumin and fibrinogen which are acidic proteins was not recognized, and it was found that only basic protein was adsorbed and separated well.

[0020]

Example 2 Ca / P molar ratio was 1.67 and firing temperature was 1
100 ° C hydroxyapatite (particle size 50-150μ)
A protein mixed solution was injected in the same manner as in Example 1 except that m) was used, and a development and elution test was performed. The results are shown in FIG. From FIG. 2, it was found that adsorption separation of albumin and fibrinogen, which are acidic proteins, was not recognized, and only basic protein was adsorbed and separated favorably.

[0021]

Comparative Example 1 A Ca / P molar ratio of 1.67 and a firing temperature of 1
000 ° C. hydroxyapatite (particle size 50-150μ)
A protein mixed solution was injected in the same manner as in Example 1 except that m) was used, and a development and elution test was performed. The results are shown in FIG. In addition, from FIG. 3, albumin which is an acidic protein, fibrinogen and lysozyme which is a basic protein,
It was found that both cytochromes C were adsorbed and separated.

[Brief description of the drawings]

FIG. 1 is a chromatogram showing the results of a development and elution test performed in Example 1.

FIG. 2 is a chromatogram showing the results of a development and elution test performed in Example 2.

FIG. 3 is a chromatogram showing the results of a development and elution test performed in Comparative Example 1.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-8-71410 (JP, A) JP-A-7-88361 (JP, A) JP-A-3-218460 (JP, A) JP-A-4- 303766 (JP, A) (58) Field surveyed (Int. Cl. ⁷ , DB name) B01J 20/00-20/34

Claims (3)

(57) [Claims] 1. A hydroxyapatite having a Ca / P molar ratio of 1.62 to 1.72, calcined at 1100 ° C. or more, and having a specific surface area of 1.0 m 2 / g or less.
A basic protein adsorbent characterized by comprising roxyapatite . 2. The method according to claim 1, wherein the hydroxyapatite is 1100.
Hydroxyapatite calcined at a temperature between 1 and 250 ° C
2. The basic protein adsorbent according to claim 1 , wherein the surface zeta potential is negative . 3. The method according to claim 1, wherein the molar ratio of Ca / P is 1.62 to 1.72.
Hydroxyapatite, 1100 ° C or higher and 1250 ° C or lower
Below, the surface zeta potential is negative and the specific surface area is 1.0 m
Claims: Baking so as to be 2 / g or less.
2. The method for producing a basic protein adsorbent according to 1.