JPH0431546B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for separating glycoalbumin using a crosslinked copolymer having a dihydroxyboronyl group.

In diseases such as diabetes, where blood sugar levels are high for a long period of time, various proteins in living organisms undergo non-enzymatic glycation reactions. Serum albumin is one of those proteins.

Conventionally, measurements of blood sugar, urine sugar, insulin, glucagon, oral glucose tolerance tests, etc. have been used to diagnose diabetes, but the results are easily affected by physiological conditions or the measurement method, and they are burdensome for the test subject. There are other problems. In contrast, the concentration of glycated proteins in living organisms has attracted attention in recent years because it is less affected by temporary physiological conditions and serves as an index of the average blood sugar concentration over the past few weeks to several months.

For example, glycated hemoglobin is an indicator of the average blood sugar level over the past 1 to 3 months and has a positive correlation with the severity of diabetes, so it has already been clinically applied as a new diagnostic method for diabetes and an indicator of blood sugar control. There is. In particular, it is said that the ratio of glycated hemoglobin to total hemoglobin correlates well with the fasting blood sugar level three months ago.

However, in clinical practice, information on blood sugar status from several weeks to a month ago is useful, and glycoalbumin has a half-life of 17 days, so it has recently attracted attention from various quarters as an indicator that can best meet this demand. is bathed in That is, compared to glycated hemoglobin, glycated albumin responds more quickly to blood sugar conditions, and unlike blood sugar levels, it does not fluctuate greatly due to the influence of temporary physiological conditions.

For example, the usefulness of glycoalbumin has been suggested, such as that it is effective in determining at an early stage whether a current or newly changed dietary therapy is appropriate for the patient. [KFMcfarland et al,
DIABETES, 28, 1011, 1979), C.E.
Gaslow, Proc Natal Akad Sai (CE
Guthrow, et al, Proc. Natl. Acad. Sci. USA,
76, No. 9, 4258, 1979)] (Prior art) Conventionally, as a method for separating and detecting glycoalbumin, ion exchange chromatography using carboxymethyl cellulose or the like as a stationary phase [G.F.D.・James F.Day et al, J.Bio.Chem., 254 , No.
3, 595, 1979)], affinity chromatography in which dihydroxy introduces a boronyl group into a soft gel such as cellulose, and utilizes the interaction between this functional group and glucose [A.K. Maria, Analytical Letters ( AKMallia et al.
Analytical Letters, 14(B8), 649-661,
1989)], a method using a color reaction represented by the thiobarbituric acid method [Kay F. McFarland et al.
Diabetes, 28 , 1011, 1979)] have been attempted.

(Problems to be Solved by the Invention) Among the above-mentioned conventional techniques, ion exchange chromatography is a method that utilizes the difference in isoelectric points of samples, but in the case of glycoalbumin and non-glycoalbumin, the difference between The difference in electric point is extremely small,
Separation is poor. Therefore, a very long time is required to achieve a satisfactory separation.

On the other hand, the method of coloring glucose bound to proteins, typified by the thiobarbituric acid method, first removes free glucose, then adds acid and hydrolyzes it at 100°C for 5 hours, followed by protein removal. ,
Add thiobarbituric acid to the supernatant and incubate at 40°C for 40 min.
A complicated operation of reacting for minutes and measuring absorbance is required.

On this point, the method using affinity chromatography utilizes the specific reaction between the dihydroxyboronyl group introduced into the stationary phase and 1,2-cis diol, as shown below, so can effectively separate glycoalbumin bound to non-glycoalbumin,
Its operation is also less complicated than the thiobarbituric acid method.

However, conventionally, the above-mentioned dihydroxyboronyl groups have been introduced into soft gels such as cellulose and agarose, which have extremely low mechanical strength and may break during handling or move when packed in columns. It has drawbacks such as the inability to flow the phase at a high flow rate.

In fact, most columns packed with these gels cannot be reused, even with extremely low mobile phase flow rates. In addition, the inability to pass a mobile phase at a high flow rate not only precludes high-speed analysis, but also means that it cannot be incorporated into a so-called high-performance liquid chromatography (HPLC) system, and operability, which is most required in clinical testing. , leading to the major drawback that reproducibility and even automation cannot be expected.

(Means for Solving the Problems) As a result of intensive research to overcome the above problems, the present inventors have found that the present inventors have found that the present inventors have developed a system with high speed, operability, and reproducibility.
Furthermore, they discovered a method for separating glycoalbumin that is easy to automate, leading to the completion of the present invention.

That is, the present invention provides a solution containing at least albumin with alcoholic hydroxyl groups of 1.0 to 14.0 meq/g, dihydroxyboronyl groups of 0.05 to 5.0 meq/g, and a specific surface area of 5 to 1000 per weight of crosslinked copolymer.
A method for separating glycoalbumin, which comprises contacting a crosslinked copolymer having a water retention capacity of ^0.5 to 6.0 g/g and separating albumin into glycoalbumin and non-glycoalbumin. Regarding.

In the present invention, glycoalbumin refers to albumin to which glucose is covalently bonded, and non-glycoalbumin refers to albumin to which glucose is not bonded. Moreover, albumin as used in the present invention refers to both glycoalbumin and non-glycoalbumin.

In order to achieve a method for separating glycoalbumin that is rapid, easy to operate, highly reproducible, and easy to automate,
The following three conditions are required for a carrier containing a dihydroxyboronyl group.

(1) Low non-specific adsorption (2) High mechanical strength (3) High chemical stability The crosslinked copolymer used in the present invention has 1.0% per crosslinked copolymer weight in addition to the dihydroxyboronyl group.
It has ~14.0 meq/g alcoholic hydroxyl groups.
When the amount of hydroxyl groups is within this range, non-specific adsorption of albumin (mainly derived from hydrophobic interactions) can be suppressed.

If the amount of hydroxyl groups exceeds 14.0 meq/g, the strength will decrease, and if it is less than 1.0 meq/g, non-specific adsorption of albumin due to hydrophobic interactions may occur.

An example of a copolymer having alcoholic hydroxyl groups is a copolymer consisting of vinyl alcohol units. The amount of hydroxyl groups can be determined, for example, by the method described in JP-A-57-30945.

The specific surface area of the copolymer used in the present invention is 5 to
It is in the range of 1000m ² /g. Soft gels such as Cephadex shrink when drying and lose their pores.
Although it exhibits a specific surface area of less than m ² /g, the copolymer of the present invention is hard and has permanent pores, so it maintains almost its swollen state even when dry.
If the specific surface area is less than 5 m ² /g, the contact efficiency will be poor and separation of glycoalbumin and non-glycoalbumin will be poor. Moreover, if it exceeds 1000m ² /g, the resin becomes excessively porous and brittle.
Not suitable for column chromatography. The specific surface area can be determined, for example, by the method described in JP-A-57-30945.

The water retention amount (hereinafter referred to as W _R ) of the copolymer of the present invention is suitably in the range of 0.5 to 6.0 g/g, preferably in the range of 1.0 to 5.0 g/g.
W _R is the amount of water that a copolymer can contain when the copolymer is brought into equilibrium with water, expressed as a value per dry weight of the copolymer. That is, W _R is a measure of the amount of pores within the copolymer. As W _R increases, the weight of the part forming the skeleton per unit volume of the copolymer in water, that is, the weight of the copolymer itself, decreases relatively. Therefore
When W _R exceeds 6.0 g/g, the mechanical strength of the copolymer decreases in water. When W _R becomes less than 0.5 g/g, the effective pore volume for separation decreases.
Separation ability decreases. Therefore, it is preferable that _WR be within an appropriate range. W _R can be determined, for example, by the method described in JP-A-57-30945.

Regarding the proportion of crosslinkable monomer units in all monomer units forming the copolymer used in the present invention, it is preferable that X in the following formula () is in the range of 0.05≦X≦0.4.

X=nb/a+nb () (where a is the mole fraction of monomer units excluding crosslinkable monomer units in the copolymer, b is the mole fraction of crosslinkable monomer units, and n is (Represents the number of functional groups such as polymerizable vinyl groups that one molecule of the crosslinkable monomer has.) When X is within the above range, high mechanical strength can be exhibited. When separating glycoalbumin and non-glycoalbumin by high performance liquid chromatography, it is preferable to use the above-mentioned crosslinked copolymer in the range of 0.2≦X≦0.4.

Particularly preferable skeletons of the copolymers used in the present invention include those in which vinyl alcohol units are crosslinked with triallyl isocyanurate units.

The crosslinked copolymer forming the skeleton of the copolymer used in the present invention can be obtained, for example, by the method described in JP-A-57-30945.

The crosslinked copolymer used in the present invention has 0.05 to 5.0 meq/g of dihydroxyboronyl groups per weight. When the amount of dihydroxybonyl groups is within this range, glycoalbumin and non-glycoalbumin can be efficiently separated.
If the amount of dihydroxybonyl groups is less than 0.05 meq/g, the retention of glycoalbumin will be insufficient and separation from non-glycoalbumin will not be possible. On the other hand, even if the amount of dihydroxyboronyl groups increases, glycoalbumin and non-glycoalbumin can be separated satisfactorily;
5.0meq/g is sufficient, and there is no practical point in increasing it more than this.

The method for introducing dihydroxyboronyl group is, for example,
Introducing carboxymethyl groups into the hydroxyl groups of the crosslinked copolymer having hydroxyl groups as described above by a known method,
Then, after converting the carboxymethyl group into azide, it can be obtained by reacting it with meta-aminophenylboronic acid [H.L. Weiss et al., Biochemistry (HL.
Weith, et al, Biochemistry), 9 , 4396 ('
70)]. It can also be obtained by reacting the hydroxyl group with bromocyanide, then reacting it with oligoptide, and then reacting it with meta-aminophenylboronic acid in the presence of carbodiimide [USP 3912595]. Furthermore, it can also be obtained by introducing epihalohydrin, bisepoxide, etc. into the hydroxyl group by a known method, and then reacting it with etaminophenylboronic acid.

The dihydroxyboronyl group can be quantitatively determined by, for example, coexisting a polymer having a dihydroxyboronyl group and sorbitol in an aqueous solvent and titrating with an alkali (New Experimental Chemistry Course, Analytical Chemistry, P77, Maruzen). In addition, a polymer having a dihydroxyboronyl group is treated with hydrogen peroxide,
It can also be obtained by determining the amount of liberated boron using the aforementioned titration method, atomic absorption method, luminescence method, or the like.

The copolymer of the present invention may be in any shape such as spherical, lumpy, or film-like, but when packed in a column and used, it is preferably spherical, and in that case, the particle size is 3 to 3.
Preferably it is in the range of 1000 μm.

As mentioned earlier, the principle of separation of glycoalbumin and non-glycoalbumin in the present invention is based on the interaction between the dihydroxyboronyl group of the crosslinked copolymer and the cis diol of chain glucose bonded to albumin. Based. More specifically, a dehydration reaction occurs between the dihydroxyboronyl group and the 1,2-cis diol of the chain glucose bonded to albumin to form a covalent bond. This reaction is inhibited on an acidic side or in a system where 1,2-cis diol such as sugar coexists. Therefore, by bonding glycoalbumin to a crosslinked copolymer having a dihydroxyboronyl group in a system in which 1,2-cis diol does not coexist on the alkali side (hereinafter referred to as an adsorption solution), non-glycoalbumin can be obtained. separation is achieved.

In addition, glycoalbumin can be recovered by placing the crosslinked copolymer to which glycoalbumin is bound in an acidic side or in a system containing a substance containing 1,2-cis diol (hereinafter referred to as an eluent). You can also do it.

The adsorption liquid does not contain a substance having 1,2-cis diol and preferably has a pH of 7.5 to 9.5. On the other hand, the eluent preferably has a pH of 2 to 6.5, and even if the pH is 7.5 or higher, the pH is 1 or 2.
- It is sufficient if it contains a substance having cis diol. The concentration of 1,2-cis diol is 50~
500mM is preferred. Salt concentration is 10mM for both solutions.
1M is preferable, and the type of buffer is not particularly limited.

In the present invention, when separating glycoalbumin and non-glycoalbumin, from the viewpoint of rapidity, operability, reproducibility, and ease of automation, the crosslinked copolymer is packed in a column and the mobile phase is passed through it. Preferably, so-called chromatography is used. At that time, before and during the introduction of the components to be separated into the column, the adsorption solution is passed through the column, and then,
Possible methods include a method of switching to an eluent (so-called stepwise method), or a method of continuously converting from adsorbent to eluent using a concentration gradient forming device (gradient mixer) (so-called gradient method).

However, the separation method of the present invention is not limited to chromatography. For example, the crosslinked copolymer is immersed in an adsorption liquid, the components to be separated are added thereto, and the mixture is gently stirred and left to settle naturally, or the crosslinked copolymer is precipitated by centrifugation.
Non-glycoalbumin is obtained from the supernatant.

Furthermore, by immersing the precipitated copolymer in an eluent and stirring it, glycoalbumin can be obtained from the supernatant. This method is suitable for large-scale separation.

In the present invention, when separating glycoalbumin and non-glycoalbumin from a sample containing a large amount of components other than albumin, such as serum,
It is preferable to isolate albumin in advance by another method or to perform specific detection of albumin.

(Effect of the invention) Typical stationary phases for conventional liquid chromatography include gels of polysaccharides such as cellulose and agarose, silica gel, and styrene-divinylbenzene copolymers. It has extremely low strength, has low chemical stability (easily dissolves at pH 7.5 or higher), and has poor hydrophilicity, so it exhibits hydrophobic adsorption to albumin.

The crosslinked copolymer used in the separation of glycoalbumin of the present invention has alcoholic hydroxyl groups per weight.
1.0-14.0meq/g, dihydroxyboronyl group 0.05
~5.0meq/g, specific surface area 5~1000m ² /g, and the amount of water that can be retained is 0.5~6.0g/g, so it has high mechanical strength and its high hydrophilicity prevents nonspecific adsorption of albumin. It is also chemically stable (can be used at pH 2 to 12).

Therefore, the method for separating glycoalbumin of the present invention is superior to any conventional method in all aspects such as rapidity, operability, reproducibility, and ease of automation.

This method makes an extremely large contribution as a new method for early diagnosis and disease management of diabetes.

(Example) The present invention will be explained below using examples.

Example 1 Vinyl alcohol copolymer gel described in Example 1 of JP-A-57-30945 (X: 0.3, hydroxyl group content: 7.8 meq/g, W _R : 1.78 g/g, specific surface area: 78
m ² /g, particle size: 9.0 μm), 116.8 g of epichlorohydrin, 250 ml of dimethyl sulfoxide,
Add 12.5ml of 10N sodium hydroxide aqueous solution and 30℃
The reaction was carried out for 20 hours. The amount of epoxy group introduced is
It was 1.0meq/g. The epoxy group was determined by the method described in JP-A-57-190003.

Next, 13g of aminophenylboronic acid hemisulfate
was dissolved in 250 ml of water, adjusted to pH 11, and 20 g of the above epoxy group-introduced polymer was added, followed by a reaction at 60°C for 20 hours. Next, in order to protect the remaining epoxy groups, 25mM tris(hydroxymethyl)aminomethane was added and reaction was carried out at 50°C for 5 hours.

The amount of boric acid introduced was 0.28 meq/g. The determination of boric acid was carried out by the following method.

10 ml of 30% hydrogen peroxide solution was added to 1 g of the above polymer into which aminophenylboronic acid had been introduced, and the boron-carbon bond was severed by reacting for 5 hours. Next, the polymer was spun down and the supernatant was collected. After decarboxylation, a boric acid ester was formed by adding sugar, strongly oxidized, and boric acid was determined by titration with sodium hydroxide.

All instruments used in this analysis were made of quartz.

The copolymer into which aminophenylboronic acid was introduced had a hydroxyl group density of 9.2 meq/g (after cutting the dihydroxyboronyl group) and a W _R of 1.70 g/g. Also,
The particle size and specific surface area were the same as before introduction.

The above aminophenylboronic acid-introduced copolymer was packed into a stainless steel column with an inner diameter of 7.6 mm and a length of 100 mm to obtain a packed column for high performance liquid chromatography.

Using this column, glycoalbumin and non-glycoalbumin were separated by liquid chromatography under the conditions shown below.
A chromatogram as shown in the figure and FIG. 2 could be obtained.

Sample: () Human serum albumin (Fraction V) [manufactured by Miles] 4% solution (5 μ) () 25 mg in the above albumin solution (5 ml)
of glycol and under sterile conditions 37
Reacted for 14 days at °C (5μ) Mobile phase: (Solution A) 250mM ammonium acetate + 50mM magnesium chloride (PH8.5) (Solution B) 100mM Tris (hydroxymethyl) aminomethane + 200mM sorbitol + 50mM EDTA (PH8.5) ) Eight minutes after injecting the sample, the A solution was switched to the B solution.

Pump: JASCO Corporation product name (registered trademark name)
TRIROTAR-V (Flow rate: 0.5ml/mm) Detector: JASCO Corporation Product name Fluorescence detector
FP-210 (excitation wavelength: 280 nm, fluorescence wavelength: 340 nm) Temperature: 25°C (Results) As can be seen from Figures 1 and 2, the A peak decreased and the B peak increased due to the saccharification reaction.

When the A and B peaks of the chromatogram shown in Figure 2 were separated and collected and a thiobarbituric acid reaction was performed, the A peak was albumin with no sugar bound, and the B peak was albumin with sugar bound. I found out.

Example 2 Vinyl alcohol copolymer gel (X: 0.4, hydroxyl group content: 4.2 meq/g, WR: 2.48 g/g, specific surface area;
An epoxy group was introduced into 25 g (120 m ² /g, particle size: 9.6 μm) in the same manner as in Example 1. The amount of epoxy groups introduced was 0.95 meq/g.

Next, 20g of aminophenylboronic acid hemisulfate
was dissolved in 400 ml of water to adjust the pH to 11, and 20 g of the above epoxy group-introduced polymer was added, and a boronyl group was introduced in the same manner as in Example 1. Next, in the same manner as in Example 1, the remaining epoxy groups were protected. The amount of boric acid introduced was 0.45 meq/g. The copolymer into which aminophenylboronic acid was introduced had a hydroxyl group density of 4.8 meq/g and a WR of 2.42 g/g. Furthermore, the particle size and specific surface area were the same as before introduction.

The above aminophenylboronic acid-introduced copolymer was packed into a stainless steel column with an inner diameter of 6.0 mm and a length of 100 mm to make a column for high performance liquid chromatography. Using this column, glycoalbumin and non-glycoalbumin were separated by liquid chromatography under the conditions shown below.
A chromatogram as shown in the figure could be obtained.

Sample: () Human serum albumin [Midori Jujisha]
0.2% solution (10μ) Mobile phase: (Solution A) 50mM glycine + 150mM magnesium chloride (PH8.5) (Solution B) 100mM tris(hydroxymethyl)aminomethane + 100mM sorbitol + 50mM EDTA + 100mM NaCl (PH8.5) Inject the sample. After 3.5 minutes, remove liquid A from B.
Switched to liquid.

Pump: JASCO Corporation, product name 880-PU (2 units) (Flow rate: 2.0ml/min) Detector: JASCO Corporation, product name FP-210 (Excitation wavelength: 280nm Fluorescence wavelength: 340nm) Temperature: 35℃ (Results) As shown in Figure 3, two peaks, A peak and B peak,
Two peaks were obtained. When the A peak and the B peak were separately collected and subjected to a thiobarbituric acid reaction, it was found that the A peak was albumin with no sugar bound, and the B peak was albumin with sugar bound.

[Brief explanation of drawings]

Figure 1 shows the results before the saccharification reaction and the second stage in Example 1.
The figure is a chromatogram of albumin after the same saccharification reaction, and FIG. 3 is a chromatogram in Example 2.

Claims (1)

[Scope of Claims] 1. A solution containing at least albumin has alcoholic hydroxyl groups of 1.0 to 1.0 or more per weight of crosslinked copolymer.
14.0meq/g, dihydroxyboronyl group 0.05~
Separating albumin into glycoalbumin and non-glycoalbumin by contacting with a crosslinked copolymer having a specific surface area of 5.0 meq/g, a specific surface area of 5 to 1000 m ² /g, and a water retention capacity of 0.5 to 6.0 g/g. A method for separating glycoalbumin, characterized by: 2. The method for separating glycoalbumin according to claim 1, wherein the alcoholic hydroxyl group possessed by the crosslinked copolymer is derived from a vinyl alcohol unit.