JPH1135603A - New heparin, heparin-bonded carrier and separation of fructose-1,6-bisphosphate aldolase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject carrier having affinity for an enzyme, useful for diagnosing progressive muscular dystrophy, acute hepatitis, etc., by bonding a heparin in which the glucosamine residue contains a specific group to a carrier. SOLUTION: A heparin in which >=50% of the glucosamine residue contains 6-O-sulfate group, preferably >=50% of the glucosamine residue contains 6-O- sulfate and the amount of the residue containing N-sulfate group is 10% at most is bonded to an insoluble carrier or a water-soluble polymer carrier (preferably aminoagarose) to give the objective carrier. The heparin is obtained by desulfating a heparin on the market, acetylating the amino group of glucosamine and selectively sulfating a hydroxyl group at the 6-position of the glucosamine. An aldehyde group at the reduction terminal part in the heparin and the amino group of an amino agarose gel are bonded, for example, by forming a Schiff base and treating the base with a reducing agent [e.g. NaB(CN)H3 ] to form an alkylamino crosslinking.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a carrier to which heparin is bound, and to fructose-1,6- by affinity chromatography using the heparin-bound carrier.
The present invention relates to a method for separating the enzyme from the content of bisphosphate aldolase, and further relates to heparin having a specific affinity for the enzyme.

[0002]

2. Description of the Related Art Heparin has a heparin skeleton composed of a series of disaccharide units consisting of a uronic acid residue and a glucosamine residue, and has a hydroxyl group at the 2-position of the uronic acid residue, an amino group at the 2-position of the glucosamine residue and a 6-amino acid group. It is a type of glycosaminoglycan in which the hydroxyl group is sulfated to various degrees. This substance generally has an antithrombin III binding site (FEB
SLett. (1980) 117, 203-206), which inhibits thrombin activity as a result of binding to ATIII and develops anticoagulant activity. Has been used. Heparin also interacts with lipoprotein lipase (J. Biol. C
hem. (1981) 256, 12893-12898), having an affinity for basic fibroblast growth factor (J. Cell Biol. (1990) 111,
1651-1659) and other bioactive factors.

[0003] As described above, since heparin has affinity for various substances, attention has been paid to elucidation of the structure involved in the binding between heparin and a physiologically active substance, and the anticoagulant action derived from the ATIII binding site has been attracting attention. For the purpose of enhancing the interaction with a bioactive factor after reducing it, chemical modification centering on desulfation of heparin polymer has been actively performed (J. Carbohydr. Chem. (1993) 12 , 507-52
1; Carbohydr. Res., 193, 165-172 (1989); Carbohydr.
Res. 46, 87-95 (1976); Can. J. Chem., 67, 1449 (1989);
U.S. Pat. No. 5,296,471). Heparin derivatives have been created by modifying heparin using techniques such as sulfation and desulfation, and some of the heparin derivatives have an affinity for a physiologically active substance. Heparin derivatives that have an affinity for a substance or can control the activity of a physiologically active substance are being searched for.

On the other hand, fructose-1,6-bisphosphate aldolase (EC 4.1.2.1), which is a useful enzyme for the diagnosis of progressive muscular dystrophy, acute hepatitis, myocardial infarction and malignant tumors
3) is based on the use of crude extracts obtained from muscle, liver, heart, kidney, brain, etc., using methods such as ammonium sulfate precipitation, cellulose phosphate column chromatography, DEAE cellulose column chromatography, and crystallization. Isolated and purified by complex combination (Methods
Enzymol. (1975) 42, 240-249).

[0005]

SUMMARY OF THE INVENTION Fructose-1,6
-Bisphosphate aldolase is useful for diagnosis of the above-mentioned various diseases, and although it is required in an isolated state, a means for obtaining the enzyme required a complicated column work. However, the amount of the enzyme obtained by the column work is very small, and a simplified and efficient purification method is required to obtain a large amount of the enzyme. The present invention provides a method for separating and purifying the enzyme by a very simple technique by affinity chromatography using a specific carrier, and further comprises heparin having a specific structure having affinity for the enzyme and a heparin-binding carrier for the enzyme. To provide.

[0006]

Means for Solving the Problems The present inventors have noticed that glycosaminoglycans having a heparin skeleton (repeated structure of uronic acid and glucosamine) have an affinity for various physiologically active substances, and have studied fructose- 1,6-bisphosphate aldolase has a high affinity for heparin having a specific structure, and affinity chromatography using a carrier to which the heparin is bound allows high-purity separation / purification of only the enzyme from the contents of the enzyme. The present inventors have found that the present invention can be performed and completed the present invention. That is, 50% or more of heparin in which the 6-position hydroxyl group of glucosamine is sulfated has a high affinity for the above enzyme. Therefore, using such a heparin-bound heparin binding carrier, If affinity column chromatography is performed in the process of isolating and purifying the enzyme from the contents, the enzyme can be efficiently separated and purified with high purity. As the heparin bound to the affinity carrier, glucosamine in which the hydroxyl group at position 6 is sulfated is 50%.
More than 10% of glucosamine residues in which a sulfate group is bonded to the amino group at the 2-position, and more than 50% of uronic acid residues in heparin have no 2-O-sulfate group Makes it possible to separate and purify the enzyme most efficiently.

A first gist of the present invention is a heparin-binding carrier in which heparin having at least 50% of glucosamine residues in the heparin skeleton has a 6-O-sulfate group is bound to the carrier. Is a method for separating the enzyme containing fructose-1,6-bisphosphate aldolase from the content of fructose-1,6-bisphosphate aldolase by affinity chromatography using the heparin-binding carrier of the present invention. More than 50% of the groups have 6-O-sulphate groups and at most 10 residues with N-sulphate groups
% Heparin.

[0008]

Embodiments of the present invention will be described below in detail. The heparin-binding carrier of the present invention is heparin in which 50% or more of glucosamine residues are sulfated at the 6-position, particularly 5%.
At least 6% of glucosamine residues in 0% or more of glucosamine residues are sulfated, and further, at most 10% of glucosamine residues having an N-sulfate group in position 2 are those in which heparin is bound to the carrier. The carrier in the binding carrier of the present invention is not particularly limited, and an insoluble carrier and a water-soluble polymer carrier are used. As the insoluble carrier, a carrier such as an insoluble natural polymer and its activated product used as a carrier for chromatography is preferable. Examples thereof include amino agarose, aminohexyl agarose, cyanogen bromide-activated agarose, and the like. Amino agarose is most preferred, but is not particularly limited. These carriers can be appropriately selected from those commercially available under trade names such as Sepharose and Biogel. As the water-soluble polymer carrier, polyethylene glycol or the like is used.

In the present invention, the average molecular weight of heparin bound to the carrier is not particularly limited, but is usually 8,000 to 13,000 Da, preferably 9,000 Da.
0 to 11,000 Da. The preferred structure is that the hydroxyl group at the 6-position of 50% or more of the glucosamine residue in the heparin skeleton is sulfated, and the N-position is at the 2-position.
The glucosamine residue having a sulfate group is characterized by being at most 10%. Such a heparin satisfies the requirement of (a) in a disaccharide composition represented by the following structural formula obtained by disaccharide analysis obtained by combining analysis by glycosaminoglycan degrading enzyme and analysis by high performance liquid chromatography. And heparin characterized by having the physical properties defined in (b). (A) ΔDiHS-tri (U, 6, N) S, ΔDiH
S-di (6, N) S and ΔDiHS-di (U, N)
S mol% is not more than 3% and ΔDiH
The mol% of S-6S is 50% or more. (However, ΔDi
HS-tri (U, 6, N) S is represented by R ¹ ,
R ^2, R ³ is SO ₃ ^- means it is, ΔDiHS-d
i (6, N) S means that R ¹ and R ² are SO ₃ ⁻ and R ₃ is H in the formula, and ΔDiHS-di
(U, N) S means that R ¹ is H, R ² and R ³ are SO ₃ ⁻ in the formula, and ΔDiHS-6
S means that R ¹ is SO ₃ ^— and R ² and R ³ are H in the formula. (B) The average molecular weight is 9,000 to 11,000 Da.

[0010]

Embedded image

More preferred heparin is the above (a) and
In addition to (b), ΔDiHS in the disaccharide composition
And satisfying that the mol% of NS is 3% or less.
(Where ΔDiHS-NS is R in the above equation)¹
And R^ThreeIs H and R^TwoIs SO_Three ^-Means
You. ). As the heparin-binding carrier of the present invention, these
Optimal heparin bound to amino agarose
Is also preferred. Heparin having the above specific structure according to the present invention
Is converted to fructose-1,6-bisphosphate aldolase
It has an affinity for. Here, the affinity is
Fructose-1,6-bisphosphine shown in Test Example 4 below
The specific activity is measured by the acid aldolase activity measurement method.
Is at least 1.0.

In the present invention, the disaccharide composition of heparin is calculated from the value measured by the disaccharide analysis method described in Examples described later, and the molecular weight is measured by the molecular weight measurement method described in Examples. It is. Test method 1 for disaccharide composition
The amount of unsaturated disaccharide having a specifiable structure obtained by disaccharide analysis by enzymatic digestion described in [ΔDiHS-0S,
ΔDiHS-6S, ΔDiHS-US, ΔDiHS-d
i (6, N) S, ΔDiHS−di (U, N) S, ΔD
iHS-di (U, 6) S and ΔDiHS-tri
(U, 6, N) S mol% total] as 100%
It shows the ratio of the disaccharide having the above specific structure,
This value reflects the position of sulfation in the constituent sugars of heparin before enzyme digestion. In addition, in this specification, the description of a disaccharide composition means the following.

[0013]

TABLE 1 TABLE - 1 substituent unsaturated disaccharide structure ^{R 1 R 2 R 3 ΔDiHS-} OS H COCH 3 H ΔDiHS-6S SO 3 - COCH 3 H ΔDiHS-NS H SO 3 - H ΔDiHS-US H ^{_{COCH 3 SO 3 - ΔDiHS-di}} (6, N) S SO 3 - SO 3 - H ΔDiHS-di (U, N) S H SO 3 - SO 3 - ΔDiHS-di (U, 6) S SO 3 - COCH ^{_{3 SO 3 - ΔDiHS-tri (}} U, 6, N) S SO 3 - SO 3 - SO 3 -

The structures indicated by the abbreviations in this specification may be described as follows. ΔDiHS-0S: ΔHexA1 → 4GlcNAc, Δ
DiHS-6S: ΔHexA1 → 4GlcNAc (6
S), ΔDiHS-NS: ΔHexA1 → 4GlcNA
c (NS), ΔDiHS-US: ΔHexA1 (US)
→ 4GlcNAc, ΔDiHS-di (6, N) S: Δ
HexA1 → 4GlcNAc (6, N-di) S, ΔD
iHS-di (U, N) S: ΔHexA1 (2S) → 4
GlcNAc (NS), ΔDiHS-di (U, 6)
S: ΔHexA1 (2S) → 4GlcNAc (6S),
ΔDiHS-tri (U, 6, N) S: ΔHexA1
(2S) → 4GlcNAc (6, N-diS) In the above formula, ΔHexA is an unsaturated uronic acid, GlcNAc
Indicates N-acetyl-D-glucosamine, and the numbers in parentheses indicate the bonding positions of sulfate groups.

The method for producing heparin bound to the heparin-binding carrier of the present invention can be obtained by sulfating the hydroxyl group at the 6-position of glucosamine in commercially available heparin. After commercially available heparin is desulfated by a known complete desulfation method, the amino group of glucosamine is acetylated, and then the glucosamine 6 is prepared by the method described in JP-B-6-99485.
It can be obtained by selectively sulphating the hydroxyl group at the position. Heparin in which the hydroxyl group at position 6 of glucosamine is selectively sulfated is hereinafter referred to as selective 6-O-sulfated heparin.

As a specific method for producing optimal heparin, for example, the following method can be mentioned. After dissolving 500 mg of heparin (derived from pig small intestine: manufactured by SPL) in 10 ml of distilled water, Amberlite IR-120 equilibrated with distilled water.
The mixture was adsorbed on a B column (φ2 x 15 cm), the acidic fraction of the eluate was collected, and pyridine was added to adjust the pH to 5.0 to 7.5.
Preferably, it is adjusted to 6.0 to 7.0. The solution is freeze-dried to obtain a heparin pyridinium salt.
To 500 mg of the obtained heparin pyridinium salt, for example, 5 ml of methanol, 30 ml of dimethyl sulfoxide (DMSO) and 15 ml of 1,4-dioxane were added to completely dissolve the heparin pyridinium salt, and this solution was added to Ogamo et al. Method (Carbohydr. Res., 193, 165-17)
2 (1989)) at 80 to 95 ° C for 20 hours or more,
The complete desulfation of heparin is performed by heating for preferably 40 hours or more, more preferably 60 to 75 hours.
After completion of the desulfurization reaction, 100 to 200 ml of ice-cold distilled water is added, and the pH of the reaction mixture is adjusted to 8 to 11, preferably 9 to 10 by adding NaOH. Then
After dialyzing the reaction mixture against distilled water for 20 hours or more, the inner solution of the dialysate is subjected to freeze-drying treatment, and completely desulfated heparin can be recovered as a yellow powder.

Next, the method of Danishefsky et al. (Arch. Bio
According to Chem. Biophys., 90, 114-121 (1960), N-acetylation of glucosamine of fully desulfated heparin is performed. For example, 400 mg of fully desulfated heparin is added to 60
After dissolving in 10 ml of 10% methanol containing 50 mM sodium carbonate, the pH of the solution is adjusted to 6 or more, preferably 7 to 8 by adding an appropriate amount of acetic acid. Then, the reaction mixture is stirred well while cooling on ice. 3 during the next hour
By adding 00 to 500 μl of acetic anhydride 5 to 8 times, and decreasing the pH of the reaction mixture each time by adding an appropriate amount of 10% methanol containing saturated sodium carbonate,
Adjust between 7 and 8. After the reaction, 100-150m
One liter of distilled water is added, and dialyzed against distilled water for 48 hours or more, preferably for about 72 hours. By freeze-drying the inner dialysis solution, completely desulfated / N-acetylated heparin can be obtained.

Next, 400 mg of completely desulfated / N-
After dissolving acetylated heparin in about 10 ml of distilled water, Amberlite IR-120B equilibrated with distilled water.
The solution is passed through a column (φ2 × 15 cm), the acidic fraction of the eluate is collected, and a 10% tributylamine ethanol solution is added to adjust the pH to 4.5 to 7.0, preferably 5.5 to 6.
Adjust to 5. The completely desulfated / N-acetylated heparin tributylammonium salt obtained by freeze-drying this solution was used according to the method of Takano et al. (Biosci. Biotech.
Biochem., 56, 1413-1416 (1992)).
After dissolving in dimethylformamide (DMF) and cooling with ice, 10 to 15 ml of 4.6 ml of DMF containing dichlorohexylcarbodiimide (DCC) is added. After further stirring, D containing 4-5 ml of 1.2 mmol sulfuric acid
MF is added, and stirring is continued under ice-cooling conditions for 10 to 20 minutes to advance the reaction. After completion of the reaction, the total volume of the reaction mixture
Pour into 0-100 g of ice and immediately neutralize with sodium bicarbonate to adjust the pH to 6.5-7.5. Next, the reaction mixture is dialyzed against distilled water, passed through a glass fiber filter paper and a membrane filter (4.5 μm) in that order to remove salts of dicyclohexylurea, and then subjected to a freeze-drying treatment to be selectively dried. -O-sulfated heparin can be synthesized.

In the present invention, the method of bonding heparin to the carrier is not particularly limited, but covalent bonding is preferred, and the covalent bond between the aldehyde group which may be present at the reducing end of heparin and the carrier has a strong binding force. Most preferred as an affinity carrier. Selective 6 obtained as above
The heparin-binding carrier of the present invention can be obtained by binding -O-sulfated heparin to the carrier. Selective 6-O
-Various functional groups of sulfated heparin and carriers for activated chromatography (derivatives with functional groups such as amino groups introduced)
6-O using coupling reaction with a functional group of
The immobilization of sulfated heparin on a carrier can be performed. In general, it is preferable to use an aldehyde group, which may be present at the reducing end in the selective 6-O-sulfated heparin molecule, for the coupling reaction with the carrier. In this case, an insoluble carrier having an amino group as the carrier (Eg, aminohexyl agarose or amino agarose) is used. When a carboxyl group in a selective 6-O-sulfated heparin molecule is used for a coupling reaction with a carrier, a carrier having an amino group similar to the above is used as the carrier, and the selective 6-O- When a free amino group in a sulfated heparin molecule is used for a coupling reaction with a carrier, a carrier of cyanogen bromide-activated agarose is used. Amino agarose carriers are preferred because they most easily bind to heparin.

The method for binding the selective 6-O-sulfated heparin to the carrier includes the following methods. As a method of binding an aldehyde group which may be present at the reducing end in selective 6-O-sulfated heparin to an amino group of an amino agarose gel, a Schiff base is formed, and then NaB (CN) H
A reducing agent such as 3 is allowed to act to form an alkylamino bridge. As this method, for example, a known general method can be used, and the method of Sakai et al. (J. Chromatog
r., 400, 123 (1987)). Selective 6
As a method for bonding a carboxyl group in a uronic acid residue in -O-sulfated heparin to an amino group in an amino agarose gel, the carboxyl group is converted to 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) Activated by a carbodiimide reagent such as an acid amide bond (-CO
This is a method for generating crosslinks of NH-). However, selective 6
Due to the three-dimensional structure of -O-sulfated heparin, the former binding method, which is considered to have stronger binding power as a carrier, is preferable.

When unreacted (free) amino groups remain in the heparin-bound carrier to which the selective 6-O-sulfated heparin is bound, non-specific interaction is caused, and it is used especially as a carrier for affinity chromatography. In this case, specific binding does not occur and unspecified substances are bound, which is not preferable. Therefore, it is preferable that the unreacted residual amino group in the heparin-bound carrier is acetylated and inactivated by the following method. That is, for example, the above-prepared heparin-bound carrier is suspended in 5 ml of a 0.2 M sodium acetate solution, 2.5 ml of acetic anhydride is added, and the mixture is added under ice-cooling conditions for 20 to 40 minutes and then at room temperature for 20 to 40 minutes. By reacting for ４０40 minutes, it is possible to acetylate and inactivate the amino group. A heparin-binding carrier on which heparin has been immobilized (possibly after further acetylation reaction)
It is preferred to wash with water and desalinate completely. Also, Bitter
Carboxyl groups are quantified according to the method of & Muir (Anal. Biochem., 4, 330-334 (1962)), and the weight of the selective 6-O-sulfated heparin immobilized on the agarose gel per unit weight is estimated. This makes it possible to confirm the amount of immobilized heparin.

The bound carrier in which heparin is bound to the insoluble carrier of the present invention is packed in a column and brought into contact with an enzyme-containing substance.
It can be used to separate it from the non-adsorbed fraction, or it can be separated by filtration, centrifugation or the like after contact with the enzyme-containing substance by a batch method. Further, the binding carrier of the water-soluble polymer carrier and heparin can be separated by a method such as molecular sieve after contacting with the enzyme-containing substance.

The method of separating and purifying fructose-1,6-bisphosphate aldolase of the present invention comprises the steps of: separating the fructose-1,6-bisphosphate aldolase from the fructose-1,6-bisphosphate aldolase by affinity chromatography using the above-mentioned heparin-binding carrier. This is a method for separating and purifying the enzyme. In the method of the present invention, if a method using affinity chromatography using the heparin-binding carrier of the present invention is used, a preparatory step performed in accordance with a conventional method to separate and purify fructose-1,6-bisphosphate aldolase is used. Other processing operations such as processing are not particularly limited. As a preferred embodiment of the present invention, for example, a liquid obtained by homogenizing the raw material of the enzyme is fractionated by performing affinity chromatography immobilized on chondroitin polysulfate, and the effluent of the adsorbed fraction is purified by the heparin of the present invention. There is a method in which fractionation is carried out by affinity chromatography using a binding carrier, and the above enzyme is separated and obtained from the adsorbed fraction.

The affinity chromatography in the method of the present invention uses a column packed with the heparin-binding carrier of the present invention, and the other operations are performed according to a conventional method, and the specific adsorption fraction is eluted with an eluent (eg, NaC).
1, an aqueous solution of an alkali metal salt such as KCl) to obtain an eluate containing the enzyme.
Further, the eluate can be subjected to molecular sieving such as gel filtration chromatography and ultrafiltration to obtain a higher purity enzyme.

In the method of the present invention, fructose-1,6
The raw material for separating and purifying bisphosphate aldolase is not limited as long as it is a tissue in which the enzyme is expressed, but a tissue derived from a mammal is preferable. The tissue preferably includes muscle, liver, heart, kidney, brain and the like. Among them, brain is preferable, and cerebrum is particularly preferable. These tissues are usually homogenized in a buffer and centrifuged to use as an enzyme source.

[0026]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples unless it exceeds the gist thereof. In addition, each test method in an Example is as follows.

Test Method 1 [Disaccharide Analysis by Enzymatic Digestion] The position of the sulfate group of heparin was analyzed as follows. That is, each sulfated polysaccharide is enzymatically digested, and the resulting unsaturated disaccharide (the above structural formula: Chemical Formula 1) is subjected to high performance liquid chromatography (HPL).
C) (Shinsei Kagaku Kenkyusho 3, Carbohydrate II (Tokyo Kagaku Dojin, 1991), p.
Structural analysis combining glycosaminoglycan degrading enzyme and HPLC ”). The peak area for each unsaturated disaccharide was calculated and the peak area relative to the total area was expressed as a percentage.

(1) Complete desulfation of heparin Digestion of heparin produced by 6-O-sulfation treatment with a degrading enzyme Heparin 1.0 mg was obtained according to the method described in Shinsei Kagaku Jikken Kozai 3, Carbohydrate II, pp. 54-59. 20 mM sodium acetate (pH 7.0) solution containing 2 mM calcium acetate 22
Dissolve in 0 μl and add 20 mU of heparitinase,
U heparitinase I and II were added and reacted at 37 ° C. for 2 hours.

(2) Analysis by HPLC 20 μl of solution after digestion of heparin with a digestive enzyme
1 was analyzed using HPLC (Medicalization, model 852). Ion exchange column (Dionex, Carb
oPac PA-1 column φ4.0mm × 250m
m) was used to measure the absorbance at 232 nm. At a flow rate of 1 ml / min, the method was based on a method using a gradient system (50 mM → 2.5 M) using lithium chloride (Kari).
ya, et al., Comp. Biochem. Physiol., 103B, 473, (199
2)).

Test Method 2 [Measurement of molecular weight] 10 μl of a 3% solution of heparin was subjected to HPLC.
Was analyzed by gel filtration. The column is TSKgel-
(G4000 + G3000 + G2500) Using PWX (Tosoh, 7.8 mm × 30 cm), 0.2 M as eluent
It was developed using sodium chloride at a flow rate of 1.0 ml / min. For the detection of heparin, a differential refractometer (Shimadzu Corporation, AID-2A) was used. The average molecular weight was determined with reference to a heparin molecular weight standard (Kaneda et al., Bioc.
hem. Biophys. Res. Comm., 220, 108-112 (1996)). The measurement of the molecular weight of the standard heparin was performed using the light scattering method (Nagasawa et al., J. Biochem., 81, 989-993 (197
7)).

Test Example 3 [NMR Analysis] For ¹³ C-NMR, using a QE300 type NMR spectrometer manufactured by GE Co., Ltd., a 10% D ₂ O solution was prepared for each sample subjected to 100% D ₂ O substitution. , TPS is 0 ppm, the chemical shift is 5
The measurement was performed under the conditions of 1.66 ppm methanol as an internal standard, a measurement temperature of 80 ° C., a pulse width of 60 °, and a cumulative number of 90,000.

Test Example 4 [Measurement of fructose-1,6-bisphosphate aldolase activity] The activity was determined as follows according to the method of Herbert G. Lebherz and William J. Rutter (Methods Enzymol., 42, 249-258 (1975)). A measurement was made. That is, the following five kinds of reagents were prepared. 0.1 M glycylglycine buffer (pH 7.
5), a 50 mM sodium fructose-1,6-bisphosphate solution, a 0.1 M fructose-1-dimonocyclohexylammonium phosphate solution, a glycerol-3-phosphate dehydrogenase / triose phosphate isomerase complex solution having a concentration of 10 mg / ml, NADH.

4 mg of NADH, 50 μl of glycerol-3-phosphate dehydrogenase / triose phosphate isomerase complex solution, 1 ml of fructose 1,6-
0.1 M glycylglycine buffer (pH 7.5) containing sodium bisphosphate solution and 1 ml fructose-1-dimonocyclohexylammonium phosphate solution
5 to 50 μl of fructose-1 for 20 ml of
The reaction was started by adding a measurement sample containing 6-bisphosphate aldolase. 340 per unit time
The decrease in the absorbance in nm was measured under a temperature condition of 25 ° C. Since the molecular extinction coefficient of NADH at 340 nm is 6.22 × 10 ⁶ cm ² · mol, when the change in absorbance is a decrease from 12.44 to 6.22, 1 μmol of fructose-1,6-bisphosphate is used. Alternatively, it was assumed that 1 μmol of fructose-1-phosphate was cleaved, and based on this, the amount of cleavage in various samples was calculated. The unit (activity; unit) of fructose-1,6-bisphosphate aldolase was defined as 1 unit as the amount of an enzyme capable of cleaving 1 μmol of a substrate in 1 minute at 25 ° C. in the above reaction system. Specific activity was defined as the number of units per mg of protein.

Example 1 Synthesis of selective 6-O-sulfated heparin 500 mg of heparin (derived from porcine small intestine; manufactured by SPL) was dissolved in 10 ml of distilled water, and then Amberlite IR-120B equilibrated with distilled water. Column (φ2 × 15cm)
And the eluate was fractionated every 3 ml. After measuring the pH of each of the fifty finally obtained fractions, the acidic fractions were collected and pyridine was added to adjust the pH to 6.5. The solution was freeze-dried to obtain about 510 mg of heparin pyridinium salt. To perform a complete desulfation reaction, 5 ml of methanol, 30 ml of dimethylsulfoxide (DMSO), and 15 ml of 1,4-dioxane were added to 500 mg of this to completely dissolve the heparin pyridinium salt. Ogamo et al.'S method (Carbo
hydr. Res., 193, 165-172 (1989)) and the reaction mixture was heated at 90 ° C. for 72 hours. After the desulfation reaction, 150 ml of ice-cold distilled water was added, and the pH of the reaction mixture was adjusted.
By adding 1N sodium hydroxide to 9.
Adjusted to 5. Then, the reaction mixture was added to distilled water for 24 hours.
After dialysis for an hour, the dialysis solution was subjected to a freeze-drying treatment. As a result, 430 mg of fully desulfated heparin was recovered as a yellow powder.

Next, the method of Danishefsky et al. (Arch. Bio
N-acetylation reaction was performed according to Chem. Biophys., 90, 114-121 (1960)). That is, 400 mg of completely desulfated heparin was dissolved in 60 ml of 10% methanol containing 50 mM sodium carbonate, and the pH of the solution was adjusted to 7 to 8 by adding an appropriate amount of acetic acid. Then, stirring was continued well while cooling the reaction mixture in an ice-water bath. During the next hour, 400 μl of acetic anhydride was added 6-7 times, and each time the pH of the reaction mixture was lowered by adding an appropriate amount of 10% methanol containing saturated sodium carbonate to 7-8. Adjusted in between. After the reaction,
120 ml of distilled water was added and dialyzed against distilled water for 72 hours. 410m by freeze-drying the dialysis solution
g of fully desulfated / N-acetylated heparin was obtained.

Then, 400 mg of completely desulfated / N-
After dissolving the acetylated heparin in 10 ml of distilled water,
The solution was passed through an Amberlite IR-120B column (φ2 × 15 cm) equilibrated with distilled water, and the eluate was fractionated every 3 ml. After measuring the pH of each of the 50 fractions finally obtained, the acidic fractions were collected and the pH was adjusted to 5.5 by adding a 10% ethanol solution of tributylamine. This solution was freeze-dried to obtain 430 mg of completely desulfated / N-acetylated heparin tributylammonium salt. This was obtained using the method of Takano et al. (Biosci. Biotech. Bi
ochem., 56, 1413-1416 (1992)), dissolved in 8 ml of dimethylformamide (DMF) and cooled to 0 ° C.
4.6 ml of dichlorohexylcarbodiimide (DCC)
And 12 ml of DMF was added. After further stirring, 4.4 ml of DMF containing 1.2 mmol sulfuric acid was added, and stirring was continued at 0 ° C. for 15 minutes to advance the reaction.

After the completion of the reaction, the whole reaction mixture was poured into 80 g of ice, and immediately neutralized with sodium hydrogen carbonate to adjust the pH to 7.0. Next, the reaction mixture was dialyzed against distilled water, and then passed through a glass fiber filter paper and a membrane filter (4.5 μm) in this order to remove dicyclohexyl urea salts. Finally, a freeze-drying treatment was performed to synthesize 450 mg of selective 6-O-sulfated heparin. When the molecular weight of this selective 6-O-sulfated heparin was measured by a light scattering method, it was 1.37 × 10 ⁴ D of untreated heparin.
a, 0.97 × selective 6-O-sulfated heparin
It was 10 ⁴ Da. The elution pattern of these gel filtration HPLCs is as shown in FIG. 1. The elution time of untreated heparin is 33.5 minutes (a), and the elution time of selective 6-O-sulfated heparin is 35.1 minutes. (B). In addition, disaccharide analysis of these substances was performed (FIG. 2). The disaccharide composition was calculated from this elution pattern (Table 2).

[0038]

[Table 2] Table 2 ΔDiHS- OS NS 6S US (6, N) S (U, N) S (U, 6) S (U, 6, N) S Hep 4 2 4 2 12 7 1 68 6- OS Hep 10 0 57 23 0 0 10 0 Note Hep: untreated heparin 6-OS Hep: selective 6-O-sulfated heparin

In addition, the selective 6-O-sulfated heparin is converted to ¹³
It was analyzed by C-NMR (FIG. 3). As a result, as with the disaccharide composition, selective 6-O-sulfated heparin could not be obtained by conventional heparin. Heparin having no N-sulfate group in glucosamine residue and having a large amount of 6-O-sulfate It became clear that it was. When the heparin is bound to a carrier for affinity chromatography, heparin having a specific binding is ΔDiHS-tri
(U, 6, N), ΔDiHS-di (U, N) S and Δ
The mol% of DiHS-di (6, N) S was 3
% Is preferable.

Example 2 Preparation of Heparin-Binding Carrier Using Selective 6-O-Sulfated Heparin as Ligand Selective 6-O-sulfated heparin was prepared according to the method of Sakai et al. (J. Chromatogr., 400, 123 (1987)). 5 mg of sulfated heparin 200 mg
In 2M phosphate buffer (pH 7.2). Then, 5 g of amino agarose gel powder was suspended, and 15 m
g of NaB (CN) H ₃ was added and the reaction was allowed to proceed at room temperature for 24 hours with sufficient stirring. After completion of the reaction, the gel containing the coupling product was filtered. Furthermore, desalination was performed completely by performing a water washing process two or three times.

When unreacted (free) amino groups remain,
The remaining amino groups were acetylated and inactivated because they could cause non-specific interactions. That is, the prepared selective 6-O-sulfated heparin-immobilized agarose gel was suspended in 5 ml of a 0.2 M sodium acetate solution, and 2.5 ml of acetic anhydride was added thereto. For 30 minutes. After completion of the reaction, the generated gel was washed with water and completely desalted. Bitter
According to the method of & Muir (Anal. Biochem., 4, 330-334 (1962)), the amount of the carboxyl group was determined, whereby the selective 6-O immobilized on an agarose gel per unit weight was determined.
-The weight of sulfated heparin was estimated. As a result, the wet weight of the gel was 1 g, which was comparable to the maximum binding amount reported hitherto.
In this case, it was confirmed that about 10 mg of selective 6-O-sulfated heparin had been immobilized, and thus no free amino group was present.

Example 3 Purification of Fructose-1,6-bisphosphate Aldolase Using a Selective 6-O-Sulfated Heparin-Binding Carrier For 9 g of bovine cerebrum, 80 ml of 1 m cooled to 0 ° C.
10 mM Tris-HCl containing M EDTA (pH
(7.4 buffer) was added to cool the cerebral tissue. Then, after removing the cerebral tissue and shredding using a razor,
The buffer solution cooled again to 0 ° C. was added twice (V / W) and homogenized in a glass homogenizer. The homogenate prepared in this manner is put into an appropriate centrifuge tube, and
Centrifugation was performed at 100,000 × g at 60 ° C. for 60 minutes. After completion of the centrifugation, the supernatant (crude extract) was used for further purification.

The above crude extract was subjected to chondroitin polysulfate-immobilized agarose affinity column (φ2 × 20 c
m) in 10 mM Tris-H containing 1 mM EDTA
Equilibrated with Cl (pH 7.4) buffer. After applying the whole amount of the crude extract to this affinity column, 6
10 mM Tris- containing 0 ml of 1 mM EDTA
HCl (pH 7.4) buffer, 1 mM ED
10 mM Tris-HCl containing TA, 0.5 mM mercaptoethanol and 10% glycerol (pH
7.4) The adsorbed fraction was eluted with 100 ml of the buffer solution. This adsorbed fraction was subjected to SDS-PAGE (Laemmli, U.S.A.).
K., J. Biol. Chem., 252, 1102-1106 (1977)).

Next, a column (φ1 × 10 cm) packed with the heparin-binding carrier prepared in Example 2 was applied to 1 mM
EDTA, 0.5 mM mercaptoethanol, 10
% Glycerol in 10 mM Tris-HCl (p
H 7.4) Equilibrated with buffer. After applying 40 ml of the adsorbed fraction of the agarose affinity column chromatography immobilized with chondroitin polysulfate to this affinity column, 1 mM EDTA, 0.5 mM
The plate was washed with 80 ml of 10 mM Tris-HCl (pH 7.4) buffer containing mM mercaptoethanol and 10% glycerol. 1 mM per specific adsorption fraction
EDTA, 0.5 mM mercaptoethanol and 10
% Glycerol in 10 mM Tris-HCl (p
H 7.4) 10 mM Tr with 15 ml buffer and 1.5 M sodium chloride, 1 mM EDTA, 0.5 mM mercaptoethanol and 10% glycerol
Linear concentration gradient of sodium chloride (0 to 1. 0) with two kinds of eluates of 15 ml of is-HCl (pH 7.4) buffer.
5M). The detection was performed based on the absorbance at 220 nm, and the elution pattern is shown in FIG. Since a single peak appeared in the linear concentration gradient of sodium chloride, this fraction was collected and subjected to SDS-PAGE.
As a result, a single band having a molecular weight of about 40 kDa was observed. In addition, this fraction was fructose-1, 6
-Bisphosphate aldolase activity.

Example 4 Isoform of fructose-1,6-bisphosphate aldolase Next, 10 mM Tris-containing 1 mM EDTA
Mon fully equilibrated with HCl (pH 7.4) buffer
H equipped with oQ column (trade name: manufactured by Pharmacia)
A specific adsorption fraction of selective 6-O-sulfated heparin affinity chromatography was applied to PLC,
10 mM Tris containing 15 ml of 1 mM EDTA
Washed with -HCl (pH 7.4) buffer. 10 mM containing 7.5 ml of 1 mM EDTA per adsorbed fraction
6. Tris-HCl (pH 7.4) buffer and 7.
5 ml of 0.5 M sodium chloride and 1 mM EDT
10 mM Tris-HCl containing A (pH 7.4)
Elution was attempted using a linear concentration gradient of sodium chloride (0-0.5 M) with two buffer solutions. The detection was performed based on the absorbance at 220 nm, and the elution pattern is shown in FIG.

One broad peak immediately before the start of the linear concentration gradient of sodium chloride (Fr. 1), and four sharp peaks in the linear concentration gradient of sodium chloride (Fr. 1).
r.2 to Fr.5) appeared. Superd per 5 obtained fractions
When gel filtration analysis and fractionation were performed by HPLC equipped with ex G-200 to perform purity analysis and final purification, as shown in FIG. 6, a sharp peak was observed at a retention time of about 13 minutes for all fractions. It was detected (detection by absorbance at 220 nm), and it was found that all fractions were purified to an extremely high purity. The yield of Fr.1 to Fr.5 is respectively
2.7 mg, 16.5 mg, 7.1 mg, 5.4 mg,
It was 7.7 mg. As shown in Example 5, when the fructose-1,6-bisphosphate aldolase activities of these five fractions were measured, all the fractions had high activities.

The five fractions were further subjected to Native-PAGE according to the method of David (Ann. NY Acad. Sci., 121, 404 (1964)). As a result, Fr.1, F
Since the mobility increases in the order of r.2, Fr.3, Fr.4, and Fr.5, the negative charge is Fr.1 <Fr.2 <Fr.3 <Fr.4
<Fr.5 was found to increase in order. Therefore, Fr.
1 to Fr.5 are considered to be isoforms of fructose-1,6-bisphosphate aldolase. Fructose-1,6-bisphosphate aldolase present in the brain is a tetramer of two subunits, A having a high isoelectric point and C having a low isoelectric point, and five of A4, A3C, A2C2, AC3 and C4. It has been reported that species isoforms exist (Methods Enzymol., 42, 240-249 (1975)). A and C have almost the same molecular weight. Fr. 1, 2, 3, 4, and 5
Are considered to correspond to A4, A3C, A2C2, AC3, and C4, respectively.

Example 5 Measurement of the activity of fructose-1,6-bisphosphate aldolase Specific adsorbed fraction on the heparin-bound carrier bound with the selective 6-O-sulfated heparin obtained in Example 3, Example 4. F fractionated by ion exchange with MonoQ column in 4
The activity of fructose-1,6-bisphosphate aldolase of each of Fractions 1 to 5 and Fractions 1 to 5 after purification by Superdex G-200 was measured. As a result, selective 6-O-
The total activity and specific activity of the specific adsorption fraction of the sulfated heparin-binding affinity column were 142.65 and 1.
48. The activity and specific activity of Fr.1 fraction from the MonoQ column were 2.92 and 2.17, the activity and specific activity of Fr.2 were 32.42 and 5.0, and the activity and specific activity of Fr.3. Was 40.98 and 13.5, the activity and specific activity of Fr. 4 were 25.19 and 8.8, and the activity and specific activity of Fr. 5 were 16.11 and 4.6 (Table-
3). In addition, MonoQ column and Superdex in Table-3
The total activity and specific activity of the column represent the sum of the activities of Fr. 1 to 5, respectively, and the average of the activities of each Fr.

[0049]

[Table 3] Table 3 Purification process Total activity Total protein mass (mg) Specific activity Recovery (%) (unit / mg-protein) Crude extract 176.0 2670 0.07 100 6-OS Hep column 142.7 96.4 1.48 81 MonoQ column 118.1 17.3 6.81 67 Superdex colimn 96.3 11.5 8.38 55 Clude extract: Crude extract 6-OS Hep column: Column-specific adsorption fraction using heparin-binding carrier MonoQ column: MonoQ adsorption fraction Superdex column: Superdex G-200 Purification after HPLC Stuff

[0050]

Industrial Applicability According to the present invention, heparin having a specific structure having an affinity for fructose-1,6-bisphosphate aldolase useful for diagnosis of various diseases such as progressive muscular dystrophy, and a novel heparin to which heparin is bound. A heparin binding carrier is provided. In addition, by using affinity column chromatography using this heparin-binding carrier for the separation and purification of the fructose-1,6-bisphosphate aldolase-containing substance, advanced purification can be achieved with a simple operation. A method is provided for doing so.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the gel filtration HPLC elution patterns of (a) untreated heparin and (b) selective 6-O-sulfated heparin.

FIG. 2 (a) Unsaturated disaccharide preparation, (b) untreated heparin,
(c) The figure which shows the ion-exchange HPLC elution pattern of the enzyme digest of the selective 6-O-sulfated heparin.

FIG. 3. ¹³ C-NM of selective 6-O-sulfated heparin
The figure which shows an R spectrum.

FIG. 4 is a view showing an elution pattern of affinity chromatography using a selective 6-O-sulfated heparin-binding affinity carrier.

FIG. 5 is a view showing an elution pattern of ion exchange chromatography in HPLC equipped with a MonoQ column.

FIG. 6: HPL equipped with Superdex column
The figure which shows the elution pattern of the gel filtration chromatography by C.

Claims (18)

[Claims] The present invention relates to a glucosamine residue in a heparin skeleton.
A heparin-binding carrier, wherein heparin having at least 0% having a 6-O-sulfate group is bound to the carrier. 2. The glucosamine residue 5 in the heparin skeleton.
2. The method according to claim 1, wherein at least 0% of the residues having 6-O-sulfate groups and at most 10% of the residues having N-sulfate groups are bound to the carrier. Heparin binding carrier. 3. The heparin having an average molecular weight of 9,000 to 1
3. The composition according to claim 1, wherein the density is 1,000 Da.
The heparin-binding carrier as described in the above. 4. A disaccharide composition represented by the following structural formula, which is obtained by disaccharide analysis combining analysis with glycosaminoglycan degrading enzyme and analysis by high performance liquid chromatography, and satisfies the definition of (a), and A heparin-binding carrier, wherein heparin having the physical properties defined in (b) is bound to the carrier. (A) ΔDiHS-tri (U, 6, N) S, ΔDiH
S-di (6, N) S and ΔDiHS-di (U, N)
S mol% is not more than 3% and ΔDiH
The mol% of S-6S is 50% or more. (However, ΔDi
HS-tri (U, 6, N) S is represented by R ¹ ,
R ^2, R ³ is SO ₃ ^- means it is, ΔDiHS-d
i (6, N) S means that R ¹ and R ² are SO ₃ ⁻ and R ³ is H in the formula, and ΔDiHS-di
(U, N) S means that R ¹ is H, R ² and R ³ are SO ₃ ⁻ in the formula, and ΔDiHS-6
S means that R ¹ is SO ₃ ^— and R ² and R ³ are H in the formula. (B) The average molecular weight is 9,000 to 11,000 Da. Embedded image 5. The heparin-bound carrier according to claim 1, wherein heparin is bound to the carrier via a covalent bond at its reducing end. 6. The method according to claim 1, wherein the heparin-binding carrier is a carrier for affinity chromatography.
A heparin-binding carrier according to any one of claims 1 to 5. 7. The heparin-binding carrier according to claim 1, wherein the carrier is a water-insoluble carrier. 8. A method for separating an enzyme containing fructose-1,6-bisphosphate aldolase from the substance containing the fructose-1,6-bisphosphate aldolase by affinity chromatography using the heparin-binding carrier according to any one of claims 1 to 7. For separating fructose-1,6-bisphosphate aldolase. 9. The fructose-1,6 according to claim 7, comprising the following steps (a) and (b).
-A method for separating bisphosphate aldolase. (1) fractionating the content of fructose-1,6-bisphosphate aldolase by performing affinity chromatography immobilized on chondroitin polysulfate; and (b) adsorbing the fraction obtained in step (a). ~
7. The enzyme is selectively adsorbed and eluted by affinity chromatography using the heparin-binding carrier according to any one of 7. 10. The method for separating fructose-1,6-bisphosphate aldolase according to claim 9, wherein the eluate obtained in step (b) is further purified by a molecular sieve. 11. The fructose-1,6-bisphosphate according to any one of claims 8 to 10, wherein the substance containing fructose-1,6-bisphosphate aldolase is obtained from a tissue of a mammal. A method for separating aldolase. 12. The separation method according to claim 11, wherein
A method for separating fructose-1,6-bisphosphate aldolase, wherein the mammalian tissue is one or more tissues selected from the group consisting of muscle, liver, heart, kidney and brain. 13. The glucosamine residue in the heparin skeleton has at least 50% a 6-O-sulfate group, and the residue having an N-sulfate group is at most 10%. Heparin. 14. The heparin according to claim 13, wherein
Furthermore, 50% or more of the uronic acid residues in the heparin skeleton are 2-
Heparin characterized by having no O-sulfate group. 15. An average molecular weight of 9,000 to 1,100.
The heparin according to claim 13 or 14, wherein the heparin is 0Da. 16. A disaccharide composition represented by the following structural formula, which is obtained by disaccharide analysis combining analysis with a glycosaminoglycan degrading enzyme and analysis by high performance liquid chromatography, and satisfies the definition of (a); Heparin having the physical properties as defined in (b). (A) ΔDiHS-tri (U, 6, N) S, ΔDiH
S-di (6, N) S and ΔDiHS-di (U, N)
S is not more than 3% for each mole%, and ΔD
The mol% of iHS-6S is 50% or more. (However, Δ
DiHS-tri (U, 6, N) S is represented by R
^1, R ^2, R ³ is SO ₃ ^- means it is, DerutaDiHS
-Di (6, N) S is that R ¹ and R ² are SO
₃ ^- means that R ³ is H, DerutaDiHS-
In di (U, N) S, R ¹ is H and R ²
And R ³ is SO ₃ ^- means it is further ΔDiHS
-6S is a compound of the formula wherein R ¹ is SO ₃ ^— , R ² and R
³ means H. (B) The average molecular weight is 9,000 to 11,000 Da. Embedded image 17. The heparin according to claim 16, wherein the molar percentage of ΔDiHS-NS is 3% or less in the disaccharide composition according to claim 16.
iHS-NS means that R ¹ and R ³ are H and R ² is SO ₃ ⁻ in the above formula. ). 18. The heparin according to any one of claims 14 to 17, wherein the heparin has an affinity for a fructose-1,6-bisphosphate aldolase enzyme.