JPH03227999A - Method for cutting saccharide chain out of glycoprotein - Google Patents
Method for cutting saccharide chain out of glycoproteinInfo
- Publication number
- JPH03227999A JPH03227999A JP25456389A JP25456389A JPH03227999A JP H03227999 A JPH03227999 A JP H03227999A JP 25456389 A JP25456389 A JP 25456389A JP 25456389 A JP25456389 A JP 25456389A JP H03227999 A JPH03227999 A JP H03227999A
- Authority
- JP
- Japan
- Prior art keywords
- glycoprotein
- hydrazine
- reaction
- reaction vessel
- sugar chains
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102000003886 Glycoproteins Human genes 0.000 title claims abstract description 27
- 108090000288 Glycoproteins Proteins 0.000 title claims abstract description 27
- 238000005520 cutting process Methods 0.000 title claims abstract description 10
- 238000000034 method Methods 0.000 title claims description 19
- 125000000837 carbohydrate group Chemical group 0.000 title abstract 3
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 claims abstract description 56
- 238000006243 chemical reaction Methods 0.000 abstract description 27
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 abstract description 12
- 230000035484 reaction time Effects 0.000 abstract description 4
- 229940039227 diagnostic agent Drugs 0.000 abstract description 3
- 239000000032 diagnostic agent Substances 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 3
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 abstract 1
- 239000011369 resultant mixture Substances 0.000 abstract 1
- 238000007738 vacuum evaporation Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 15
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 12
- 238000000354 decomposition reaction Methods 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 8
- 238000007796 conventional method Methods 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 108010058846 Ovalbumin Proteins 0.000 description 5
- 229940092253 ovalbumin Drugs 0.000 description 5
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 3
- 238000006640 acetylation reaction Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- 102000000447 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Human genes 0.000 description 2
- 108010055817 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Proteins 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000003456 ion exchange resin Substances 0.000 description 2
- 229920003303 ion-exchange polymer Polymers 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 235000011437 Amygdalus communis Nutrition 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000220304 Prunus dulcis Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- YZCKVEUIGOORGS-NJFSPNSNSA-N Tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 1
- 235000020224 almond Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000029983 protein stabilization Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、糖タンパク質の糖鎖分析を行うに際して、前
処理としての糖タンパク質からの糖鎖の切り出し法に関
する。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for cutting out sugar chains from glycoproteins as a pretreatment when performing sugar chain analysis of glycoproteins.
（従来の技術）
近年、生体由来の生理活性を有する糖タンパク質を医薬
、あるいは診断薬として利用する試みがなされている。(Prior Art) In recent years, attempts have been made to utilize physiologically active glycoproteins derived from living organisms as medicines or diagnostic agents.
この対象となる糖タンパク質を生産する手段としては組
織からの抽出、細胞の大量培養、遺伝子操作による組み
替えタンパク質の大量生産などが挙げられる。これまで
、得られた糖タンパク質の生理活性の同一性として、ア
ミノ酸組成分析に代表されるように、そのポリペプチド
部分のみによって特徴付けることが一般に行われてきた
。しかし、最近、糖タンパク質の生理的発現において糖
鎖が重要な役割を果たしていることが明らかにされつつ
ある。Means for producing the target glycoprotein include extraction from tissues, mass culture of cells, and mass production of recombinant proteins through genetic manipulation. Until now, the identity of the physiological activity of the obtained glycoprotein has generally been characterized only by its polypeptide portion, as typified by amino acid composition analysis. However, recently it has been revealed that sugar chains play an important role in the physiological expression of glycoproteins.
例えば、タンパク質の安定化、代謝におけるシグナル作
用、細胞内局所のシグナル作用、及びレセプターや標的
細胞に対する認識としてのシグナル作用などである。Examples include protein stabilization, signal action in metabolism, intracellular local signal action, and signal action as recognition of receptors and target cells.
このように、糖タンパク質を医薬或いは診断薬として有
効且つ特異的に利用するためには糖鎖構造を目的に沿っ
て規定することが重要となってくる。そのためには生産
された糖タンパク質糖鎖の構造の迅速な分析法が必要と
なってくる。Thus, in order to effectively and specifically utilize glycoproteins as medicines or diagnostic agents, it is important to define the sugar chain structure according to the purpose. To this end, a rapid method for analyzing the structure of produced glycoprotein sugar chains is required.
糖タンパク質糖鎖を分析するためには、まず糖鎖を糖タ
ンパク質から切り出すことが必要である。糖鎖の切り出
し方法としては、ヒドラジン分解法、［純生化学実験講
座４、複合糖質研究法■、１４１頁、東京化学同人］、
及びグリコペプチダーゼ（アーモンド由来） ［Ｂｉ
ｏｃｈｅｍ、Ｂｉｏｐｈｙｓ 、 Ｒｅｓ 、 Ｃｏｍ
ｍｕｎ 、 、第７６巻、１１９４頁（１９７７） ］
［Ｊ、Ｂｉｏｃｈｅｍ、、第８４巻、１４７６頁（１９
７８） ］、或いはＮ−グリカナーゼ［Ｊ、Ｂｉｏｌ、
Ｃｈｅｍ、、第２５９巻、１０７００頁（１９８４）
］などの酵素による切断法がある。切り出された糖鎖は
通常、トリチウム標識化や蛍光標識化されて分析に供せ
られる。In order to analyze glycoprotein sugar chains, it is first necessary to cut out the sugar chains from the glycoprotein. Methods for cutting out sugar chains include the hydrazine decomposition method, [Pure Biochemistry Experiment Course 4, Complex Carbohydrate Research Methods ■, p. 141, Tokyo Kagaku Doujin],
and glycopeptidase (derived from almond) [Bi
ochem, Biophys, Res, Com
mun, vol. 76, p. 1194 (1977)]
[J, Biochem, vol. 84, p. 1476 (19
78) ], or N-glycanase [J, Biol.
Chem, vol. 259, p. 10700 (1984)
] There are enzyme-based cleavage methods such as The excised sugar chains are usually labeled with tritium or fluorescent and subjected to analysis.
（発明が解決しようとする課題）
しかしながら、従来の酵素法は反応時間が１０時間以上
と時間がかかり、更に糖鎖の構造、タンパク質の構造に
よって、切り出し効率が著しく影響を受けるなどの問題
点があった。又従来のヒドラジン分解法（無水ヒドラジ
ンを試料に直接添加、１００°Ｃで反応）は反応時間が
約１０時間であり、反応後に過剰なヒドラジンを除くた
めに更に数時間を要する。(Problem to be solved by the invention) However, the conventional enzymatic method takes a long reaction time of 10 hours or more, and further has problems such as the excision efficiency is significantly affected by the structure of the sugar chain and the structure of the protein. there were. Furthermore, the conventional hydrazine decomposition method (directly adding anhydrous hydrazine to a sample and reacting at 100°C) requires a reaction time of about 10 hours, and several hours are additionally required to remove excess hydrazine after the reaction.
そこで本発明は、従来のヒドラジン分解を改良すること
により、より迅速で簡便な糖タンパク質からの糖鎖の切
り出し法を提供することにある。Therefore, the present invention aims to provide a faster and simpler method for cutting out sugar chains from glycoproteins by improving conventional hydrazine degradation.
（課題を解決する為の手段）
本発明が特徴とするところは、糖タンパク質試料をヒド
ラジン蒸気と接触させ分解反応を行うことにある。(Means for Solving the Problems) The present invention is characterized by bringing a glycoprotein sample into contact with hydrazine vapor to carry out a decomposition reaction.
従来、ヒドラジン蒸気と接触させる分解反応により糖鎖
を糖タンパク質から切り出すという方法は検詞されてい
ない。Until now, there has been no method of cutting out sugar chains from glycoproteins by a decomposition reaction in which they are brought into contact with hydrazine vapor.
以下、本発明の詳細な説明する。The present invention will be explained in detail below.
本発明者らは鋭意検討の結果、糖タンパク質から糖鎖を
切り出すに際し、公知のヒドラジン分解法に変えて、ヒ
ドラジン蒸気による分解によれば、迅速で良い結果が得
られることを見出した。As a result of extensive studies, the present inventors have found that when cutting out sugar chains from glycoproteins, rapid and good results can be obtained by decomposition using hydrazine vapor instead of the known hydrazine decomposition method.
本発明では、糖タンパク質は小チューブの中で予め良く
乾燥させておく。この糖タンパク質試料の入ったチュー
ブを密閉可能な反応容器内に設置する。それから、反応
容器内を真空ポンプによって減圧する。その際、窒素、
アルゴンなとの不活性な気体で反応容器内を置換してか
ら減圧するとより望ましい。ヒドラジンは減圧前に、反
応容器内に加えておいても良いが、望ましくは、反応容
器内を減圧してから、注入するのが良い。In the present invention, the glycoprotein is thoroughly dried in a small tube in advance. The tube containing this glycoprotein sample is placed in a sealable reaction container. Then, the pressure inside the reaction vessel is reduced using a vacuum pump. At that time, nitrogen,
It is more preferable to purge the inside of the reaction vessel with an inert gas such as argon and then reduce the pressure. Although hydrazine may be added to the reaction vessel before the pressure is reduced, it is preferable to inject it after the pressure inside the reaction vessel is reduced.
本発明でのヒドラジン分解の際の標準的な反応条件は減
圧下、１００〜１５０°０１１５分から２時間で、好ま
しくは、１３０℃、３０分から１時間である。反応後、
試料チューブを反応容器より取り出し、トルエンを加え
て減圧乾固する。場合によっては、この操作をもう一度
繰り返すことにより糖タンパク質から糖鎖を切り出すこ
とが出来る。次に公知の方法により、Ｎ−アセチル化を
行ない、さらにピリジルアミノ化やＡＢＥＥ（パラアミ
ノ安息香酸）化による標識化を行った後にＨＰ Ｌ Ｃ
分析に供する。The standard reaction conditions for hydrazine decomposition in the present invention are under reduced pressure from 100 to 150° for 15 minutes to 2 hours, preferably at 130° C. for 30 minutes to 1 hour. After the reaction,
Take out the sample tube from the reaction container, add toluene and dry it under reduced pressure. In some cases, sugar chains can be excised from glycoproteins by repeating this operation once more. Next, by a known method, N-acetylation is performed, and further labeling is performed by pyridylamination and ABEE (para-aminobenzoic acid), followed by HPLC.
Provide for analysis.
従来法では無水ヒドラジンによる分解反応に約１０時間
要していたが、本発明の方法によると３０分から １時
間で反応を終了することが可能となった。更に、糖タン
パク質試料にはヒドラジン蒸気し２か接触しないため、
反応後に除去すべきヒドラジン量か従来法に比べて著し
く少量になり、糖タンパク質試料からのヒドラジンの減
圧による除去時間を著しく短縮することも可能となった
。In the conventional method, the decomposition reaction using anhydrous hydrazine required about 10 hours, but according to the method of the present invention, it became possible to complete the reaction in 30 minutes to 1 hour. Furthermore, since only hydrazine vapor comes into contact with the glycoprotein sample,
The amount of hydrazine that needs to be removed after the reaction is significantly smaller than in conventional methods, and it is also possible to significantly shorten the time required to remove hydrazine from a glycoprotein sample under reduced pressure.
以下に、本発明の実施例を示し、本発明を具体的に説明
するが、本発明はこれらの実施例に限定されるものでは
ない。EXAMPLES Below, the present invention will be specifically explained by showing examples of the present invention, but the present invention is not limited to these examples.
（実施例）
実施例１ オブアルブミン糖鎖の分析■ オブアルブ
ミン糖鎖の切り出し
オブアルブミン（シグマ社）２００μｇを試料チューブ
の中でよく乾燥させた後、密閉可能な反応容器に設置し
た。反応容器内を減圧にした後、ヒドラジン５００μｍ
を反応容器内に注入した。反応容器内を１３０°Ｃにし
、４５分反応させた。反応後、容器内を冷却し、常圧に
してから試料チューブを取り出し、別の密閉可能な容器
内に移し、２００μｍのトルエンを加え５分間減圧乾固
する操作を２回繰り返し糖鎖の切り出しを行った。(Example) Example 1 Analysis of ovalbumin sugar chain ■ Excision of ovalbumin sugar chain After thoroughly drying 200 μg of ovalbumin (Sigma) in a sample tube, it was placed in a sealable reaction vessel. After reducing the pressure inside the reaction vessel, 500 μm of hydrazine was added.
was injected into the reaction vessel. The inside of the reaction vessel was heated to 130°C, and the reaction was carried out for 45 minutes. After the reaction, the inside of the container was cooled down to normal pressure, the sample tube was taken out, transferred to another sealable container, 200 μm of toluene was added, and the tube was dried under reduced pressure for 5 minutes, which was repeated twice to cut out the sugar chains. went.
■ オブアルブミン糖鎖の分析
■で得られた試料に飽和炭酸水素ナトリウム水溶液を２
００μｍ加えてから、これに２０μｍの無水酢酸を５回
に分けて１５分間隔で攪拌しながら加えて最後に３０分
攪拌してＮ−アセチル化を行った。次に試料溶液をイオ
ン交換樹脂（Ｄｏｗｅｘ５０Ｗｘ２）のカラムに通して
脱塩し、カラムは更に純水で数回洗浄した。この素通り
画分と洗液とを合せ、減圧乾固した。試料残渣は常法通
りにピリジルアミノ化［純生化学実験講座４、複合糖質
研究法Ｉ、１４５〜１４９頁、東京化学同人］によって
蛍光標識してから５ｅｐｈａｄｅｘ Ｇ−１５で精製し
た後、ＨＰ Ｌ Ｃにて分析した。第１図にそのクロマ
トグラムを示す。カラムはＳｈｉｍａｐａｋ ＣＬＣ−
ＯＤＳ （内径０．６ｘ１５ｃｍ） （島津製作所）
を５５°Ｃの恒温槽中て用い、溶媒は１０ｍＭリン酸緩
衝液（ＰＨ３８）中、ｎ−ブタノールの０．１から０２
５％の直線濃度勾配を６０分間でかけ、流量を１．０ｍ
ｌ／ｍｉｎとした。■Analysis of ovalbumin sugar chains■ Add saturated aqueous sodium bicarbonate solution to the sample obtained in ■
After adding 00 μm of acetic anhydride, 20 μm of acetic anhydride was added in 5 portions with stirring at 15 minute intervals, and finally stirred for 30 minutes to perform N-acetylation. Next, the sample solution was desalted by passing through a column of ion exchange resin (Dowex 50W x 2), and the column was further washed several times with pure water. This pass-through fraction and the washing liquid were combined and dried under reduced pressure. The sample residue was fluorescently labeled by pyridylamination in a conventional manner [Pure Biochemistry Experiment Course 4, Complex Carbohydrate Research Methods I, pp. 145-149, Tokyo Kagaku Dojin], purified with 5ephadex G-15, and then purified with HPLC. It was analyzed in Figure 1 shows the chromatogram. The column is Shimapak CLC-
ODS (inner diameter 0.6x15cm) (Shimadzu)
was used in a constant temperature bath at 55°C, and the solvent was 0.1 to 0.02% of n-butanol in 10mM phosphate buffer (PH38).
A 5% linear concentration gradient was applied over 60 minutes with a flow rate of 1.0 m
It was set to 1/min.
なお、対照として従来法によるヒドラジン分解（無水ヒ
ドラジン添加、１００℃、１０時間）で切り出した糖鎖
のＨＰ Ｌ Ｃのクロマトグラムを第２図に示した。As a control, FIG. 2 shows an HPLC chromatogram of a sugar chain excised by conventional hydrazine decomposition (addition of anhydrous hydrazine, 100° C., 10 hours).
第１図、第２図に示したように、本発明による糖鎖の切
り出し法によっても従来法と全く同一のクロマトグラム
が得られた。As shown in FIGS. 1 and 2, the method for cutting out sugar chains according to the present invention yielded completely the same chromatogram as the conventional method.
実施例２ ＩｇＧ糖鎖の分析
■ ＩｇＧ糖鎖の切り出し
ＩｇＧ（シグマ社）２００μｇを試料チューブの中でよ
く乾燥させた後、密閉可能な反応容器に設置した。反応
容器内を減圧にした後、ヒドラジン５００μｍを反応容
器内に注入した。反応容器内を１３０℃にし、４５分反
応させた。反応後、容器内を冷却し、常圧にしてから試
料チューブを取り出し、別の密閉可能な容器内に移し２
００μｍのトルエンを加え５分間減圧乾固する操作を２
回繰り返し糖鎖の切り出しを行った。Example 2 Analysis of IgG sugar chains ■ Excision of IgG sugar chains After thoroughly drying 200 μg of IgG (Sigma) in a sample tube, it was placed in a sealable reaction container. After reducing the pressure inside the reaction vessel, 500 μm of hydrazine was injected into the reaction vessel. The inside of the reaction vessel was heated to 130°C, and the reaction was carried out for 45 minutes. After the reaction, cool the inside of the container and bring it to normal pressure, then take out the sample tube and transfer it to another sealable container.
Add 00 μm of toluene and dry under reduced pressure for 5 minutes.
The sugar chains were excised repeatedly.
■ ＩｇＧ糖鎖の分析
■で得られた試料に飽和炭酸水素ナトリウム水溶液を２
００μｍ加えてから、これに２０μｍの無水酢酸を５回
に分けて１５分間隔で攪拌しながら加えて最後に３０分
攪拌してＮ−アセチル化を行った。次に試料溶液をイオ
ン交換樹脂（ＤＯＷｅＸ５０Ｗｘ２）のカラムに通して
脱塩し、カラムは更に純水で数回洗浄した。この素通り
画分と洗液とを合せ、減圧乾固した。試料残渣は常法通
りにピリジルアミノ化によって蛍光標識してから５ｅｐ
ｈａｄｅｘ Ｇ−１５で精製した後、ＨＰ Ｌ Ｃにて
分析した。第３図にそのクロマトグラムを示した。カラ
ムはＳｈｉｍａｐａｋ Ｃ１、Ｃ−０ＤＳ （内径０．
６Ｘ１５ｃｍ）（島津製作所）を５５°Ｃの恒温槽中で
用い、溶媒は１０ｍＭリン酸緩衝液（ＰＨ３，８）中、
ｎ−ブタノールの０１から０．２５％の直線濃度勾配を
６０分間でかけ、流量を１．０ｍｌ／ｍｉｎとした。■Analysis of IgG sugar chains■ Add 2 saturated aqueous sodium bicarbonate solutions to the sample obtained in ■.
After adding 00 μm of acetic anhydride, 20 μm of acetic anhydride was added in 5 portions with stirring at 15 minute intervals, and finally stirred for 30 minutes to perform N-acetylation. Next, the sample solution was desalted by passing it through a column of ion exchange resin (DOWeX50Wx2), and the column was further washed several times with pure water. This pass-through fraction and the washing liquid were combined and dried under reduced pressure. The sample residue was fluorescently labeled by pyridylamination in a conventional manner, and then 5ep
After purification with hadex G-15, it was analyzed by HPLC. FIG. 3 shows the chromatogram. The column is Shimapak C1, C-0DS (inner diameter 0.
6 x 15 cm) (Shimadzu Corporation) in a constant temperature bath at 55 °C, the solvent was 10 mM phosphate buffer (PH 3, 8),
A linear concentration gradient of n-butanol from 0.01 to 0.25% was applied over 60 minutes, with a flow rate of 1.0 ml/min.
なお、対照として従来法によるヒドラジン分解で切り出
した糖鎖のトＩ Ｐ Ｌ Ｃのクロマトグラムを第４図
に示した。As a control, FIG. 4 shows an IPLC chromatogram of a sugar chain excised by hydrazine decomposition using a conventional method.
第３図、第４図に示すように、本発明による糖鎖の切り
出し法によっても従来法と全く同一のクロマトグラムが
得られた。As shown in FIGS. 3 and 4, the sugar chain excision method according to the present invention yielded completely the same chromatogram as the conventional method.
（発明の効果）
以」二、詳細に説明した通り、本発明によれば、糖タン
パク質より糖鎖を切り出す際の反応時間を著しく短縮す
ることが可能となり、更に反応後試料からのヒドラジン
の除去の操作時間を短縮することも可能となった。(Effects of the Invention) As explained in detail in Section 2, according to the present invention, it is possible to significantly shorten the reaction time when cutting out sugar chains from glycoproteins, and it is also possible to remove hydrazine from the sample after the reaction. It has also become possible to shorten the operation time.
第１図は本発明による方法（ヒドラジン蒸気、１３０°
Ｃ１４５分）によって切り出したオブアルブミン糖鎖の
蛍光標識化物のＨＰ Ｌ Ｃのクロマトグラム、第２図
は従来法（ヒドラジン添加、１００°Ｃ１１０時間）で
切り出したオブアルブミン糖鎖の蛍光標識化物のＨＰ
Ｌ Ｃのクロマトグラムである。
第３図は本発明による方法（ヒドラジン蒸気、１３０℃
、４５分）によって切り出したＩｇＧ糖鎖の蛍光標識化
物のＨＰＬＣのクロマトグラム、第４図は従来法（ヒド
ラジン添加、１００°０１１０時間）で切り出したＩｇ
Ｇ糖鎖の蛍光標識化物のＨＰ Ｌ Ｃのクロマトグラム
である。
（以下余白）
０
第
１
薗
名
目
笛
回
葛４Figure 1 shows the method according to the invention (hydrazine vapor, 130°
Figure 2 shows the HPLC chromatogram of a fluorescently labeled ovalbumin sugar chain cut out using the conventional method (addition of hydrazine, 100°C for 110 hours).
It is a chromatogram of LC. Figure 3 shows the method according to the invention (hydrazine vapor, 130°C).
Figure 4 shows an HPLC chromatogram of a fluorescently labeled IgG sugar chain excised by the conventional method (addition of hydrazine, 100°C for 10 hours).
This is an HPLC chromatogram of a fluorescently labeled G-glycan. (Left below) 0 No. 1 Sononame Fue Round 4
Claims (1)
ことを特徴とする、糖タンパク質からの糖鎖の切り出し
法。(1) A method for cutting out sugar chains from glycoproteins, which is characterized by contacting glycoproteins with hydrazine vapor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25456389A JP2651941B2 (en) | 1989-09-29 | 1989-09-29 | Extraction method of sugar chain from glycoprotein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25456389A JP2651941B2 (en) | 1989-09-29 | 1989-09-29 | Extraction method of sugar chain from glycoprotein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03227999A true JPH03227999A (en) | 1991-10-08 |
| JP2651941B2 JP2651941B2 (en) | 1997-09-10 |
Family
ID=17266788
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP25456389A Expired - Lifetime JP2651941B2 (en) | 1989-09-29 | 1989-09-29 | Extraction method of sugar chain from glycoprotein |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2651941B2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5585473A (en) * | 1994-12-09 | 1996-12-17 | The Biomembrane Institute | Compounds and methods for monosaccharide analysis |
| JP2005308697A (en) * | 2004-04-26 | 2005-11-04 | Mitsubishi Chemicals Corp | Sugar chain separating method, specimen analyzing method, liquid chromatograph, sugar chain analyzing method, and sugar chain analyzing apparatus |
| JP2006329938A (en) * | 2005-05-30 | 2006-12-07 | Shimadzu Corp | Sugar chain cut-out device |
| CN111638291A (en) * | 2020-06-16 | 2020-09-08 | 江南大学 | Method for determining trace carbohydrate impurities in amino acid bulk drug |
-
1989
- 1989-09-29 JP JP25456389A patent/JP2651941B2/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5585473A (en) * | 1994-12-09 | 1996-12-17 | The Biomembrane Institute | Compounds and methods for monosaccharide analysis |
| JP2005308697A (en) * | 2004-04-26 | 2005-11-04 | Mitsubishi Chemicals Corp | Sugar chain separating method, specimen analyzing method, liquid chromatograph, sugar chain analyzing method, and sugar chain analyzing apparatus |
| JP2006329938A (en) * | 2005-05-30 | 2006-12-07 | Shimadzu Corp | Sugar chain cut-out device |
| CN111638291A (en) * | 2020-06-16 | 2020-09-08 | 江南大学 | Method for determining trace carbohydrate impurities in amino acid bulk drug |
| CN111638291B (en) * | 2020-06-16 | 2021-11-16 | 江南大学 | Method for determining trace carbohydrate impurities in amino acid bulk drug |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2651941B2 (en) | 1997-09-10 |
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