JPH08183798A - Ovoglycoprotein, chromatographic separation agent containing the compound and separation method - Google Patents

Abstract

PURPOSE: To provide the subject new protein having respective specific molecular weight, N-terminal amino acid sequence and sugar content, free from trypsin- inhibiting activity, producible at a low cost, resistant to the denaturation with organic solvent, exhibiting excellent liquid permeability and optical recognition capability and useful e.g. as a separation agent for optical isomer. CONSTITUTION: This new ovoglycoprotein has a molecular weight of about 30,000 determined by a matrix-supported laser-desorption ionized time-of-flight mass spectrometer, an amino acid sequence of T-E-S-P-X-S-A-P-L-V-P-A-D-M-D (X is cysteine or an amino acid bonded with sugar) as the 15 amino acid residues from the N-terminal and a sugar content of about 25%. It is free from trypsin inhibiting activity and useful e.g. as a separation agent for optical isomer by bonding to a carrier such as silica gel. The protein can be produced by adding 0.5M trichloroacetic acid/acetone to albumen, centrifuging the mixture to remove the precipitate, adding cold acetone to the supernatant, separating the produced precipitate by centrifugal separation, dissolving the separated precipitate and purifying by ion-exchange chromatography and reversed phase liquid chromatography.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel ovoglycoprotein, a chromatographic separating agent containing the ovoglycoprotein, and a separation method. More specifically, the ovoglycoprotein having optical recognition ability or a part of the molecular structure is modified. Separation agent for chromatography using ovoglycoprotein and a stationary phase in which it is bound to a carrier, and affinity electrokinetics obtained by adding ovoglycoprotein or ovoglycoprotein partially modified in molecular structure to a separation solution as a chiral selector. The present invention relates to a method for separating optical isomers by chromatography.

[0002]

2. Description of the Related Art For chiral chemical substances containing asymmetric carbon atoms, it is strongly required to separate the optical isomers, especially in the field of pharmaceuticals. That is, one of a plurality of optical isomers constituting one racemate shows a particularly remarkable medicinal usefulness, for example, a remarkable pharmacological action, a remarkable bioavailability, or conversely, a remarkable effect. Toxicities are generally shown to be clear, and therefore, it is more rational to administer as separated optical isomers as a drug than to administer as racemate, resulting in enhanced therapeutic effect. This is because there are cases. Although many laboratory methods have been reported for the separation of optical isomers, there are few that can be carried out on an industrial scale, and it has been considered that this is a very difficult technical problem. However, with the progress of column chromatography, methods for separating optical isomers by liquid chromatography have become generally known, and methods such as those shown in the following Documents 1) to 7) have been proposed.

1) Jergen Hermannson: Journal of Chromatography, 325 (1985).
Pages 379-384 (Joergen Hermansson: Journal of
Chromatography, 325 (1985) 379-384) 2) S. Allenmark et al: Journal of Chromatography, 264 (1983) 63-68.
264 (1983) 63-68) 3) S. Arenmark et al., Journal of Chromatography, 237 (1982) pp. 473-477.
Page (S. Allenmark et al: Journal of Chromatography
, 237 (1982) 473-477) 4) Japanese Patent Laid-Open No. 60-41619 5) Toshin Shin Miwa et al .: Chemical and Pharmaceutical Bulletin, Vol. 35 (1987) page 682-6
Page 86 (T. Miwa et al: Chemical and Pharmaceutical
Bulletin, Vol 35 (1987) 682-686) 6) Atsushi Haginaka et al .: Chromatographer, Vol. 29 (19
90) pp. 587-592 (J. Haginaka et al: Chroma
tographia, Vol 29 (1990) 587-592) 7) JP-A-64-3129

Among the above documents, 1) discloses a technique using a chiral α ₁ -acid glycoprotein. 2) and 3) disclose a method for separating bovine serum albumin using a stationary phase bound to silica and agarose, respectively. 4) discloses a method for separation using orosomucoid, its functional analogue, and the like. 5) and 6) disclose a method of separating ovomucoid using a stationary phase bound to a carrier. 7) discloses a method of separating avidin using a stationary phase having a support bound to it. However, the materials used in the techniques 1) to 4) and 7) are generally expensive. In addition, since the separation method in these techniques is mainly performed by liquid chromatography using a large amount of organic solvent, the materials used must be stable against denaturation by an organic solvent.For example, albumin and orosomucoid must meet this condition sufficiently. Can't be satisfied with. In addition, the sample loading of the separating agent for optical isomers on which a protein is immobilized is small. An HPLC chiral stationary phase column using a protein as a ligand shows optical recognition for many kinds of racemates, and is currently most widely used. However, the mechanism of optical recognition is complicated and has not been clarified, and several kinds of expensive columns must be prepared in order to know the suitability of optical separation. On the other hand, affinity electrokinetic chromatography
It has an advantage that the suitability of optical separation can be easily known with a small amount of protein and high resolution can be obtained. However, at present, only a few examples using albumin are shown, and it cannot be said that separation equivalent to liquid chromatography is obtained.

[0005]

Therefore, the object of the present invention is to provide a novel protein useful for the separation of optical isomers, and to be cheaper and stable to denaturation by an organic solvent,
The present invention is to provide a separating agent for chromatography, which has excellent optical recognition ability and high sample loading and can efficiently separate optical isomers, and further a novel protein useful for separating optical isomers. The present invention provides an optical separation method by affinity electrokinetic chromatography, in which is added to a separation solution.

[0006]

Means for Solving the Problems The present inventor has conducted various studies in order to achieve the above object. As a result, they have found that ovoglycoprotein, which can be easily obtained from egg white, has excellent optical recognition ability, has a high sample loading amount, and is stable against denaturation by an organic solvent, and completes the present invention. Came to. That is, the present invention is
Matrix-assisted laser desorption / ionization time-of-flight mass spectrometer has a molecular weight of about 30,000 and 15 from the N-terminus
The amino acid sequence of the residue is TESPXSAPLVPADM-
D (where X represents an amino acid to which cysteine or a sugar chain is bound), which provides an ovoglycoprotein having a sugar content of about 25% and no trypsin inhibitory activity. The present invention also provides a stationary phase in which the above ovoglycoprotein is bound to a carrier, a stationary phase in which a part of the molecular structure of ovoglycoprotein immobilized in a carrier is modified, or an ovoglycoprotein in which a part of the molecule is modified. The present invention provides a separating agent for chromatography, which comprises a stationary phase in which is bound to a carrier. The present invention further provides an optical separation method by affinity electrokinetic chromatography, which is characterized in that the ovoglycoprotein or a part of the molecular structure thereof is modified and added to the separation solution as a chiral selector. is there.

Hereinafter, the present invention will be described in detail. The ovoglycoprotein according to the present invention can be prepared by the following method. Preparation of ovoglycoprotein 1) Separation of acetone-precipitated powder from egg white 900 ml of 0.5M trichloroacetic acid / acetone (1: 2, v / v) (pH 3.5) was added to 450 ml of egg white at 4 ° C.
Stir for 4 hours. Centrifuge at 5000 rpm for 30 minutes at 4 ° C. to remove the precipitate. Add 2 volumes of cold acetone to the supernatant and stir for 15 minutes. 5000 rp at 4 ° C
Centrifuge at m for 1 hour and remove the supernatant. The precipitate is dissolved in demineralized distilled water, dialyzed overnight and freeze-dried. The obtained acetone-precipitated powder is used for the following isolation.

2) Isolation of Ovoglycoprotein from Acetone Precipitated Powder About 3 g of acetone precipitated powder is dissolved in 10 mM CH ₃ COONH ₄ (pH 4.6) and filtered. This is separated by ion exchange chromatography to obtain fractions SP-1, SP-2 and SP-3. The chromatogram of the obtained fraction is shown in FIG. After desalting each component, it is freeze-dried. The conditions in ion exchange chromatography are shown below. Column: SP-Sepharose (length 6 cm, inner diameter 5 cm), Eluent: Eluent A 10 mM CH ₃ COONH ₄ (pH 4.6) 250 ml Eluent B 500 mM CH ₃ COONH ₄ (pH 4.6) 250 ml Eluent C 700 mM CH ₃ COONH ₄ (pH 4.6) (eluent A and eluent B were run in a linear gradient for 5 hours and then eluent C was run) Flow rate: 100 ml / h Detection: 280 nm

3) Reverse-phase liquid chromatography of ovoglycoprotein (FIG. 2) A is a chromatogram of SP-1, B is a SP-2, and C is a chromatogram of SP-3. SP-1 contained ovomucoid and an unidentified substance, SP-2 was ovomucoid, and SP-3 was determined to be ovoglycoprotein from the results of 4). The reverse phase liquid chromatography conditions are shown below. Column: Cosmosil 5C18-AR (length 250 mm, inner diameter 4.6 mm), eluent: Eluent A H ₂ O / CH ₃ CN (80/20, v / v) (containing 0.1% trifluoroacetic acid) Eluent B H ₂ O / CH ₃ CN (20/80, v / v) (containing 0.1% trifluoroacetic acid) 0% eluent B to 90 minutes 100% eluent B with a linear gradient flow did. Flow rate: 1.0 ml / min Detection: 280 nm 4) Characteristics of SP-3 a. Molecular weight of SP-3 Matrix-assisted laser desorption / ionization time-of-flight type (MA
It was determined to be about 30,000 by the LDI-TOF) mass spectrometer (Figure 3). B. Sugar content of SP-3 SP-3 is 15.1% glucosamine, 7.3% hexose,
It contains 2.8% sialic acid and has a sugar content of about 25%. C. Enzyme inhibitory activity of SP-3 SP-3 has no trypsin inhibitory activity. D. N-terminal amino acid sequence of SP-3 The amino acid sequence of 15 residues from the N-terminal is TESPXSAP-
It was LVPADMD (where X represents an amino acid to which cysteine or sugar chain is bound). NBRF-P
IR Database (National Biomedical Research Founda
As a result of the search using the Protein Protein Information Resource, the above amino acid sequence was different from the N-terminal amino acid sequence of the known ovomucoid, and no identical or similar amino acid sequence was found. Determined to be protein.

The carrier used in the present invention may be any one that can form a stationary phase by binding to ovoglycoprotein or ovoglycoprotein obtained by modifying a part of its molecular structure. The chromatographic separating agent of the present invention is mainly used for liquid chromatography, and examples of the carrier for use in such an application include silica gel, glass, cellulose, carbon and synthetic polymers. it can. The method of binding the ovoglycoprotein to the carrier may be carried out according to the method usually used for forming a stationary phase. Therefore, for example, aminopropyl silica gel or a synthetic polymer having an amino group bonded thereto is used as a carrier, ovoglycoprotein is bonded with N, N'-disuccinimidyl carbonate as a cross-linking agent, or glass is used as a carrier, and 3-glycid Xypropyltrimethoxysilane is used as a cross-linking agent to bind ovoglycoprotein, or cellulose is used as a carrier,
A method of activating with bromocyan and then binding ovoglycoprotein to it can be considered.

Generally, a method for modifying a protein molecule includes
There are chemical methods, enzymatic methods, physical methods and the like. That is,
Focusing on amino groups, imidazole groups, and carboxyl groups in protein molecules, reacting these with aldehydes, acid anhydrides, and alcohols produces Schiff bases, N-substituted imidazole groups, and esters for chemical modification. If the various actions of the enzyme are used, modification of the functional group,
Reactions such as oxidation and reduction of molecules and removal of a part of molecules can be performed under mild conditions. For example, an ovoglycoprotein obtained by glutarizing a part of the ovoglycoprotein can be obtained as follows. Add ovoglycoprotein and glutaraldehyde to phosphate buffer pH 6.8,
After stirring at 0 ° C for 15 hours, the resulting glutarized ovoglycoprotein (non-reduced form) or sodium borohydride was used at 4 ° C in a phosphate buffer of pH 6.8.
After stirring for 12 hours and reducing, the resulting glutarized ovoglycoprotein (reduced form) can be purified and obtained.

The method for purifying the glutarized ovoglycoprotein is not particularly limited and may be a commonly used method. For example, glutarized ovoglycoprotein can be purified by removing unreacted glutaraldehyde and sodium borohydride from the above reaction solution using Sephadex G25 column chromatography. In addition, to obtain ovoglycoprotein in which a part of ovoglycoprotein is diol,
It can be obtained by adding ovoglycoprotein and 2,3-epoxypropanol to a phosphate buffer of pH 8.0, stirring the mixture at room temperature for 24 hours, and purifying the reaction product. Furthermore, to obtain an ovoglycoprotein in which a part of the ovoglycoprotein is acylated, ovoglycoprotein and a corresponding acid anhydride, for example, acetic anhydride are added at pH 8.
It can be obtained by purifying the produced acylated ovoglycoprotein after adding it to the borate buffer solution of 5 and stirring at 25 ° C. for 30 to 60 minutes.

In order to obtain a stationary phase to which an ovoglycoprotein having a part of its molecular structure modified, a ovoglycoprotein having a part of its molecular structure modified by a covalent bond or an ionic bond is used. And a method in which the stationary phase to which the ovoglycoprotein has been bonded is modified by the method described above to obtain the desired stationary phase. The method of binding the ovoglycoprotein having a part of the molecule modified to the carrier may be carried out according to the method usually used for forming a stationary phase. Therefore, for example, aminopropyl silica gel or a synthetic polymer having an amino group bonded thereto is used as a carrier, and glutaraldehyde or N, N'-disuccinimidyl carbonate is used as a cross-linking agent to bond a partially modified ovoglycoprotein. Or, silica gel, glass or carbon as a carrier and 3-glycidoxypropyltrimethoxysilane as a cross-linking agent to bind ovoglycoprotein with a part of the molecule modified, or cellulose as a carrier and activated with bromocyan Therefore, a method of binding an ovoglycoprotein whose molecule is partially modified to these is conceivable. To bind the glutarized ovoglycoprotein to aminopropyl silica gel, specifically, the following may be performed.

The glutarized ovoglycoprotein is added to the pH
Dissolve in the sodium bicarbonate buffer of 6.8. Separately, aminopropyl silica gel and N, N'-disuccinimidyl carbonate were dissolved and suspended in a sodium hydrogen carbonate buffer solution having a pH of 6.8, and the mixture was stirred overnight and washed with preparative water to obtain an activated aminopropyl silica gel suspension. obtain. The previously prepared solution of glutarized ovoglycoprotein was added to the activated aminopropyl silica gel suspension, stirred and washed with water, and a separating agent for optical isomers in which glutarized ovoglycoprotein was bound to silica gel via a crosslinking agent was added. Obtainable. Further, the chemical modification of the ovoglycoprotein on the stationary phase to which the ovoglycoprotein is bound may be carried out as follows. For example, a carrier obtained by introducing a polyamine such as pentaethylhexamine into a hydrophilic synthetic polymer (polyvinyl alcohol copolymer) and N, N'-disuccinimidyl carbonate are dissolved and suspended in a sodium hydrogen carbonate buffer solution having a pH of 6.8. The mixture is turbid, stirred overnight, and washed with preparative water to obtain a suspension of activated synthetic polymer.

Separately, a solution prepared by dissolving ovoglycoprotein in a sodium hydrogencarbonate buffer solution having a pH of 6.8 is prepared and added to the activated synthetic polymer suspension to obtain a polymer separating agent having ovoglycoprotein bound thereto. The separating agent and glutaraldehyde were placed in a phosphate buffer of pH 6.8 and
After stirring at 0 ° C for 15 hours, the resulting glutarized ovoglycoprotein (non-reduced form) or sodium borohydride was used at 4 ° C in a phosphate buffer of pH 6.8.
After stirring for 12 hours and reducing, the resulting glutarized ovoglycoprotein (reduced form) is bound to the synthetic polymer via the amide bond and the cross-linking agent to obtain a separating agent for optical isomers. By chemically modifying the ovoglycoprotein in this way, the column life (number of times of sample injection) as a separation column for optical isomers can be significantly extended.

The present invention is characterized by using an ovoglycoprotein or an ovoglycoprotein in which a part of the molecular structure thereof is modified for the separation of optical isomers. The type of carrier, ovoglycoprotein or It is not particularly limited by the method for binding the ovoglycoprotein having a part of its molecular structure modified to the carrier, the method for modifying the ovoglycoprotein, or the like. Therefore, the stationary phase is contained in the separating agent of the present invention as an essential constituent component, but at the same time, other components such as silica gel, glass, cellulose, carbon and polymer are arbitrarily selected and added to the separating agent. It is free and these conventional separating agents can be appropriately used in combination with the separating agent of the present invention for improving the separation.

In the present specification, the term "optical isomer" means
It refers to a chiral compound having an asymmetric carbon atom in the molecule, and an example can be found in many pharmaceuticals. Examples thereof include chlorpheniramine, chlorprenaline, pindolol, verapamil, propranolol, dimethindene, ethiazide, oxazepam, flurbiprofen and the like. In these compounds, a plurality of optical isomers having a mirror image relationship to each other exist, and they together form a racemate. The separating agent of the present invention is particularly effective for separating these racemates and separating the optical isomers constituting them. The separating agent of the present invention is mainly used in liquid chromatography. Therefore, the method of use may be carried out by a usual operation in liquid chromatography, for example, packing the separating agent of the present invention in a column,
Charge the racemate of the optical isomer, and then pass through a phosphate buffer or a mobile phase of a buffer solution containing ethanol, isopropanol, etc., depending on the retention time,
The required optical isomers may be separated. When the ovoglycoprotein of the present invention or the ovoglycoprotein partially modified in its molecular structure is used as an additive for affinity electrokinetic chromatography, it may be carried out by a usual operation in affinity electrokinetic chromatography, and a separation solution is used. Ovoglycoprotein or ovoglycoprotein partially modified in its molecular structure is dissolved in it, and this is filled in a capillary for affinity electrokinetic chromatography, and the racemate of the optical isomer is injected, and the difference in electrophoretic mobility is used. The required optical isomers may be separated.

[0018]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Comparative Example 1 3 g of aminopropyl silica gel and 2 g of N, N'-disuccinimidyl carbonate were placed in 100 ml of 0.1 M sodium hydrogen carbonate buffer (pH 6.8), stirred overnight, taken on a glass filter and washed with water to activate. A suspension of modified aminopropyl silica gel was prepared. Separately, add 2g of SP-1 to 0.
A solution dissolved in 30 ml of 1M sodium hydrogen carbonate buffer (pH 6.8) was prepared and added to the above suspension to obtain an SP-1 immobilized separating agent consisting of SP-1-bonded silica gel. It was packed in a steel column and used as a column for separating optical isomers. Comparative Example 2 3 g of aminopropyl silica gel and 2 g of N, N'-disuccinimidyl carbonate were placed in 100 ml of 0.1 M sodium hydrogen carbonate buffer (pH 6.8), stirred overnight, taken on a glass filter and washed with water to activate. A suspension of modified aminopropyl silica gel was prepared. Separately, add 2g of SP-2 to 0.
A solution dissolved in 30 ml of 1M sodium hydrogen carbonate buffer (pH 6.8) was prepared and added to the suspension to obtain an SP-2 immobilized separating agent composed of SP-2 bonded silica gel. It was packed in a steel column and used as a column for separating optical isomers. Comparative Example 3 3 g of aminopropyl silica gel and 2 g of N, N'-disuccinimidyl carbonate were added to 100 ml of 0.1 M sodium hydrogen carbonate buffer (pH 6.8), stirred overnight, taken on a glass filter and washed with water to activate. A suspension of modified aminopropyl silica gel was prepared. Separately, 2 g of commercially available ovomucoid was added to 0.1 M sodium hydrogen carbonate buffer (pH 6.
8) A solution dissolved in 30 ml was prepared and added to the suspension to obtain an ovomucoid-immobilized separating agent consisting of ovomucoid-bonded silica gel. It was packed in a steel column and used as a column for separating optical isomers. Example 1 3 g of aminopropyl silica gel and 2 g of N, N'-disuccinimidyl carbonate were added to 100 ml of 0.1 M sodium hydrogen carbonate buffer (pH 6.8), stirred overnight, taken on a glass filter and washed with water to activate. A suspension of modified aminopropyl silica gel was prepared. Separately, prepare a solution prepared by dissolving 2 g of ovoglycoprotein (SP-3) in 30 ml of 0.1 M sodium hydrogen carbonate buffer (pH 6.8), add it to the suspension, and add a solution consisting of ovoglycoprotein-bonded silica gel. An ovoglycoprotein-immobilized separating agent of the invention was obtained. It was packed in a steel column and used as a column for separating optical isomers.

Example 2 0.1 g of glutaraldehyde and 2 g of ovoglycoprotein were placed in 0.06M phosphate buffer (pH 6.8) and the mixture was heated to 30 ° C.
After stirring for 15 hours, glutarized ovoglycoprotein was synthesized. Unreacted glutaraldehyde was removed by Sephadex G25 column chromatography to isolate glutarized ovoglycoprotein (non-reduced form). Alternatively, it can be obtained by further stirring with sodium borohydride in a phosphate buffer of pH 6.8 at 4 ° C. for 12 hours for reduction, and then purifying the glutarized ovoglycoprotein (reduced form) produced. it can. Next, a hydrophilic polymer gel (polyvinyl alcohol copolymer) is added to the polyamine (for example,
Pentaethylhexamine) -introduced column separating agent (for example, Asahi Pack NH2P) (2 g) and N, N'-disuccinimidyl carbonate (2 g) were placed in 100 ml of 0.1 M sodium hydrogen carbonate buffer (pH 6.8) and stirred overnight. Then, it was taken on a glass filter and washed with water to prepare a suspension of the activated synthetic polymer gel. Separately, prepare a solution of 2 g of non-reduced or reduced glutarized ovoglycoprotein in 30 ml of 0.1 M sodium hydrogen carbonate buffer (pH 6.8), add it to the above suspension, and keep at 30 ° C. for 15 hours. After stirring, the mixture was placed on a glass filter and washed with water to obtain a separating agent of the present invention composed of a glutarized ovoglycoprotein-bonded hydrophilic polymer gel. The obtained separating agent was packed in a steel column to give a column for separating optical isomers.

Example 3 3 g of aminopropyl silica gel and 2 g of N, N'-disuccinimidyl carbonate were added to 100 ml of 0.1M sodium hydrogen carbonate buffer (pH 6.8), stirred overnight, taken on a glass filter and washed with water. To prepare a suspension of activated aminopropyl silica gel. Separately, 2 g of ovoglycoprotein was added to a 0.1 M sodium hydrogen carbonate buffer solution (pH 6.
8) A solution dissolved in 30 ml was prepared and added to the suspension to obtain an ovoglycoprotein-bonded silica gel separating agent. 2 g of this separating agent and 0.1 g of glutaraldehyde
In 30 ml of 0.06 M phosphate buffer (pH 6.8)
The mixture was stirred at ℃ for 15 hours to obtain the separating agent (non-reducing type) of the present invention. Add 0.2g of sodium borohydride and add 4 ℃
The mixture was stirred and reduced for 12 hours to obtain a separating agent of the present invention consisting of silica gel with glutarized ovoglycoprotein (reduced type). The obtained separating agent was packed in a steel column to give a column for separating optical isomers. Example 4 3 g of aminopropyl silica gel and 0.1 g of glutaraldehyde were added to 100 ml of 0.06M phosphate buffer (pH 6.8).
After stirring for 15 hours at 30 ° C., it was placed on a glass filter and washed with water. To this glutarized silica gel, 2 g of ovoglycoprotein was added to 0.1 M sodium hydrogen carbonate (pH 6.
8) It was dissolved in 30 ml and allowed to react, and glutarization of ovoglycoprotein was also performed to obtain a separating agent of the present invention consisting of glutarized ovoglycoprotein-bonded silica gel. The obtained separating agent was packed in a steel column to give a column for separating optical isomers.

Example 5A An ovoglycoprotein-bonded silica gel separating agent was obtained by using aminopropyl silica gel in the same manner as in Example 1. After drying this separating agent in a phosphorus pentoxide desiccator,
A separating agent of the present invention comprising a diolated ovoglycoprotein-bonded silica gel suspended in a 06 M phosphate buffer solution (pH 8.0), added with 0.5 ml of 2,3-epoxypropanol and stirred at room temperature for 24 hours. Got The obtained separating agent was packed in a steel column to give a column for separating optical isomers. Example 5B 2 g of ovoglycoprotein was suspended in 0.06 M phosphate buffer, 0.5 ml of 2,3-epoxypropanol was added, and the mixture was stirred at room temperature for 24 hours to obtain a diolified ovoglycoprotein. Then, 3 g of aminopropyl silica gel and 2 g of N, N'-disuccinimidyl carbonate were added to 0.2 g.
A suspension of activated aminopropyl silica gel was prepared by placing in 100 ml of 1M sodium hydrogen carbonate buffer (pH 6.8), stirring overnight, loading on a glass filter and washing with water.
Separately, prepare a solution prepared by dissolving 2 g of diolated ovoglycoprotein in 30 ml of 0.1M sodium hydrogencarbonate buffer (pH 6.8), add it to the above suspension, stir at 30 ° C. for 15 hours, and then on a glass filter. Then, the product was washed with water to obtain a separating agent of the present invention composed of diolated ovoglycoprotein-bonded silica gel. The obtained separating agent was packed in a steel column to give a column for separating optical isomers.

Example 6A An ovoglycoprotein-bonded silica gel separating agent was obtained by using aminopropyl silica gel in the same manner as in Example 1. A solution prepared by dissolving 0.225 ml of acetic anhydride in 1.8 g of this separating agent and 1 ml of dioxane was used as a 0.1 M borate buffer solution (pH 8.
5) After adding to 50 ml, the mixture was stirred at 25 ° C. for 30 minutes, then placed on a glass filter and washed with water to obtain a separating agent comprising the acetylated ovoglycoprotein-bonded silica gel of the present invention.
The obtained separating agent was packed in a steel column to give a column for separating optical isomers. Example 6B 2 g of ovoglycoprotein in 0.2 ml of 1 ml dioxane.
A solution of 25 ml of acetic anhydride was added to a 0.1 M borate buffer solution (pH 8.5), and the mixture was stirred at 25 ° C. for 30 minutes to obtain acetylated ovoglycoprotein. Next, 3 g of aminopropyl silica gel and 2 g of N, N'-disuccinimidyl carbonate were added to 100 ml of 0.1 M sodium hydrogen carbonate buffer (pH 6.8), stirred overnight, taken on a glass filter, washed with water and activated. A suspension of modified aminopropyl silica gel was prepared. Separately, a solution prepared by dissolving 2 g of acetylated ovoglycoprotein in 30 ml of 0.1M sodium hydrogen carbonate buffer (pH 6.8) was prepared, added to the suspension, and the present invention consisting of acetylated ovoglycoprotein-bonded silica gel. Was obtained. The obtained separating agent was packed in a steel column to give a column for separating optical isomers. Example 7 The inner wall of the capillary was coated with linear polyacrylamide, inner diameter 77 μm, effective length 30 cm, total length 37 c.
m capillaries were prepared. After washing this capillary with water and a running solution of 50 mM phosphate buffer (pH 4.5), 50 mM containing ovoglycoprotein at 50 mM
A separation solution of a phosphate buffer (pH 4.5) was filled up to about 27 cm (before the detector) from the sample injection side. next,
After injecting a sample solution (chlorpheniramine: 250 μg / ml) at 0.035 kg / cm ² (0.5 psi) for 1 second, both ends of the capillary were in a 50 mM phosphate buffer (pH 4.
The sample was dipped in the electrophoresis solution of 5) and electrophoresed under conditions of an applied voltage of 12.0 kV, a capillary temperature of 23 ° C., and a detection of 254 nm. The results are shown in Fig. 8. As is clear from FIG. 8, there was no instability of the baseline due to the absorption of background, and a good separation result was shown. Example 8 The inner wall of the capillary was coated with linear polyacrylamide, inner diameter 77 μm, effective length 30 cm, total length 37 c.
m capillaries were prepared. After washing this capillary with water and an electrophoresis solution of 50 mM phosphate buffer (pH 5.0), 50 mM containing ovoglycoprotein of 50 mM
A separation solution of a phosphate buffer (pH 4.5) was filled up to about 27 cm (before the detector) from the sample injection side. next,
Sample solution (triperisone: 250 μg / ml) 0.0
After injecting at 35 kg / cm ² (0.5 psi) for 1 second,
Both ends of the capillary were immersed in an electrophoresis running solution of 50 mM phosphate buffer (pH 5.0), and electrophoresis was performed under conditions of an applied voltage of 12.0 kV, a capillary temperature of 23 ° C., and a detection of 254 nm. The results are shown in Fig. 9. As is clear from FIG. 9, there was no instability of the baseline due to the absorption of background, and a good separation result was shown.

The effects of the present invention will be shown below by experimental examples. Experimental Example 1 Using the columns for separating optical isomers prepared in Comparative Examples 1 and 2 and Example 1, benzoin, chlorophenylamine maleate and ketoprofen were separated. As a result, ovoglycoprotein (SP-3) was immobilized. The chromatography column showed good resolution (Figs. 4, 5 and 6). However, the SP-1 and SP-2 immobilized columns were not found to have optical recognition ability. The HPLC conditions are: column inner diameter 2.0 mm, length 100 mm,
Mobile phase 20 mM phosphate buffer (pH 5.1) / ethanol = 90/10 (V / V), column temperature 25 ° C., flow rate 0.1.
2 ml / min, detection 220 nm.

Experimental Example 2 In the same manner as in Experimental Example 1, a sample loading test was carried out when separating the enantiomers of benzoin. FIG. 7 shows the results. In FIG. 7, A is a commercially available ovomucoid-immobilized column, and B is the ovoglycoprotein-immobilized column of the present invention. The HPLC conditions are: column inner diameter 2.0 mm, length 100 mm, mobile phase 20 mM phosphate buffer (pH 5.1) / ethanol = 90/10 (V / V),
The column temperature was 25 ° C., the flow rate was 0.2 ml / min, and the detection was 254 nm. The injection amount is 2.5 nmol for A and 20 nmol for B
Met. From FIG. 7, ovomucoid-immobilized separating agent (A)
In comparison, the ovoglycoprotein-immobilized separating agent (B) of the present invention was found to exhibit a sample loading of 8 times or more.

INDUSTRIAL APPLICABILITY The separating agent for optical isomers of the present invention is inexpensive and stable against denaturation by organic solvents, has excellent liquid permeability, has excellent optical recognition ability, and has a high sample loading amount. The optical isomers can be efficiently separated. or,
In the optical separation by affinity electrokinetic chromatography, when it is used as an additive in a separation solution, it exhibits excellent optical recognition ability, less adsorption to a capillary column than ovomucoid, and the number of times of use is greatly extended.

[Brief description of drawings]

FIG. 1 is a chromatogram showing the isolation of ovoglycoprotein from acetone precipitated powder of egg white.

Fig. 2 Fraction SP isolated from acetone precipitated powder of egg white
-1 is a chromatogram of SP-2, SP-3 separation.

FIG. 3 is a chart showing the results of molecular weight analysis of ovoglycoprotein with a mass spectrometer.

[Fig. 4] Using an ovoglycoprotein-immobilized column,
It is a chromatogram which shows the result of the separation in the enantiomer of benzoin.

[FIG. 5] Using an ovoglycoprotein-immobilized column,
It is a chromatogram which shows the result of the separation in the enantiomer of chloropheniramine maleate.

FIG. 6 shows the case of using an ovoglycoprotein-immobilized column,
It is a chromatogram which shows the result of the separation in the enantiomer of ketoprofen.

FIG. 7 is a chromatogram showing the results of a sample loading test with separation in the enantiomers of benzoin.

FIG. 8 is a chromatogram showing the results of optical separation by affinity electrokinetic chromatography of the enantiomers of chlorpheniramine.

FIG. 9 is a chromatogram showing the results of optical separation by affinity electrokinetic chromatography of triperizone enantiomers.

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Claims (8)

[Claims] 1. A matrix-assisted laser desorption / ionization time-of-flight mass spectrometer has a molecular weight of about 30,000.
So, the amino acid sequence of 15 residues from the N-terminal is TESPXS-
APLVPADMD (where X is cysteine or an amino acid to which a sugar chain is bound), an ovoglycoprotein having a sugar content of about 25% and no trypsin inhibitory activity. 2. A chromatographic separating agent comprising a stationary phase in which ovoglycoprotein or ovoglycoprotein having a part of its molecular structure modified is bound to a carrier. 3. An ovoglycoprotein having a partly modified molecular structure is a glutarized ovoglycoprotein (non-reducing type), a glutarized ovoglycoprotein (reducing type), a diolated ovoglycoprotein, or an acylated ovoglycoprotein. The chromatographic separating agent according to claim 2, which is a protein. 4. The separating agent for chromatography according to claim 2 or 3, wherein the carrier is silica gel, glass, cellulose, carbon or a synthetic polymer. 5. The chromatographic separating agent according to claim 2, wherein the separating agent is a separating agent for optical isomers. 6. The separating agent for chromatography according to claim 3, wherein the separating agent is a separating agent for optical isomers. 7. The separating agent for chromatography according to claim 4, wherein the separating agent is a separating agent for optical isomers. 8. A method for separating optical isomers by affinity electrokinetic chromatography, which comprises adding ovoglycoprotein or ovoglycoprotein having a part of its molecular structure modified to a separation solution.