JPH0221260A - Quantitative determination of amino group reaction - Google Patents
Quantitative determination of amino group reactionInfo
- Publication number
- JPH0221260A JPH0221260A JP1599588A JP1599588A JPH0221260A JP H0221260 A JPH0221260 A JP H0221260A JP 1599588 A JP1599588 A JP 1599588A JP 1599588 A JP1599588 A JP 1599588A JP H0221260 A JPH0221260 A JP H0221260A
- Authority
- JP
- Japan
- Prior art keywords
- group
- amino group
- carrier
- substance
- reaction
- 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.)
- Pending
Links
- 125000003277 amino group Chemical group 0.000 title claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 title abstract description 10
- 239000000126 substance Substances 0.000 claims abstract description 16
- HAXFWIACAGNFHA-UHFFFAOYSA-N aldrithiol Chemical compound C=1C=CC=NC=1SSC1=CC=CC=N1 HAXFWIACAGNFHA-UHFFFAOYSA-N 0.000 claims abstract description 11
- WHMDPDGBKYUEMW-UHFFFAOYSA-N pyridine-2-thiol Chemical compound SC1=CC=CC=N1 WHMDPDGBKYUEMW-UHFFFAOYSA-N 0.000 claims abstract description 11
- 125000002228 disulfide group Chemical group 0.000 claims abstract description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 10
- 238000002835 absorbance Methods 0.000 abstract description 3
- 150000001875 compounds Chemical class 0.000 abstract description 2
- 238000003776 cleavage reaction Methods 0.000 abstract 2
- 230000007017 scission Effects 0.000 abstract 2
- AQYNZOSCOWGGTP-UHFFFAOYSA-N 2-pyridin-2-ylsulfanylpyridine Chemical group C=1C=CC=NC=1SC1=CC=CC=N1 AQYNZOSCOWGGTP-UHFFFAOYSA-N 0.000 abstract 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical group OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 abstract 1
- 230000008878 coupling Effects 0.000 abstract 1
- 238000010168 coupling process Methods 0.000 abstract 1
- 238000005859 coupling reaction Methods 0.000 abstract 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 abstract 1
- 125000004076 pyridyl group Chemical group 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- SEKLFMRSNLFPRB-UHFFFAOYSA-N 2-(pyridin-2-yldisulfanyl)ethanamine;hydrochloride Chemical compound Cl.NCCSSC1=CC=CC=N1 SEKLFMRSNLFPRB-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical compound C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- -1 ethylene, propylene Chemical group 0.000 description 2
- ZTVZLYBCZNMWCF-UHFFFAOYSA-N homocystine Chemical compound [O-]C(=O)C([NH3+])CCSSCCC([NH3+])C([O-])=O ZTVZLYBCZNMWCF-UHFFFAOYSA-N 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 description 1
- IZFHEQBZOYJLPK-SSDOTTSWSA-N (R)-dihydrolipoic acid Chemical compound OC(=O)CCCC[C@@H](S)CCS IZFHEQBZOYJLPK-SSDOTTSWSA-N 0.000 description 1
- UHBAPGWWRFVTFS-UHFFFAOYSA-N 4,4'-dipyridyl disulfide Chemical group C=1C=NC=CC=1SSC1=CC=NC=C1 UHBAPGWWRFVTFS-UHFFFAOYSA-N 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229960001305 cysteine hydrochloride Drugs 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
アフィニティークロマトグラフィーや免疫測定法等の生
化学的分野において、蛋白質を結合させた担体が利用さ
れている。このような蛋白質結合担体を調製するには蛋
白質のアミノ基を利用した化学的結合法を用いるのが一
般的であるが、担体の蛋白質結合量、即ち担体の有する
アミノ基反応基を定量することが得られた結果の解析等
に必要である。この作業はまた、蛋白質結合担体を調製
するにあたって好ましい蛋白質結合容量を有する担体の
選定のためにも必要である。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) In biochemical fields such as affinity chromatography and immunoassay, carriers to which proteins are bound are used. In order to prepare such protein-bound carriers, it is common to use a chemical bonding method that utilizes the amino groups of proteins. is necessary for analyzing the obtained results. This operation is also necessary for selecting a carrier having a preferable protein-binding capacity in preparing a protein-binding carrier.
本発明は前記の様な担体の有するアミノ基反応基の定量
方法に関するものである。The present invention relates to a method for quantifying the amino reactive group contained in a carrier as described above.
（従来の技術）
従来、担体間のアミノ基反応基数を比較する方法として
、担体にこのアミノ基を介して蛋白質を結合させ、この
蛋白質を生理活性を測定する方法か知られている。(Prior Art) Conventionally, as a method for comparing the number of amino group-reactive groups between carriers, a method is known in which a protein is bound to a carrier via the amino group, and the physiological activity of this protein is measured.
（従来技術の問題点）
しかしながら、従来知られた方法では、結合させる蛋白
質として例えば酵素、抗体等の特殊な性質を有する蛋白
質を用いなければならず、例えば醇索を用いた場合には
、その酵素の触媒作用を受ける基質を添加して酵素活性
を測定した後、得られた値とその酵素量と活性を関連ず
ける標準曲線とを比較して、また例えば抗体を結合させ
た場合には、その抗体と抗原抗体を行う標識された抗原
等を添加した後、標識抗原を測定して行うが、この様な
測定操作は複雑であり、誤差を生じ易いと言う問題点が
ある。しかもこれらの方法では、担体のアミノ菖反応基
を定量することが出来ないと言う大きな問題点がある。(Problems with the prior art) However, in the conventionally known methods, it is necessary to use a protein with special properties, such as an enzyme or an antibody, as the protein to be bound. After measuring the enzyme activity by adding a substrate that is catalyzed by the enzyme, the values obtained are compared with a standard curve that correlates the amount of enzyme and the activity; The labeled antigen is measured after adding a labeled antigen, etc. that performs an antigen-antibody reaction with the antibody, but such a measurement operation is complicated and has the problem of being prone to errors. Moreover, these methods have a major problem in that they cannot quantify the amino acid reactive groups of the carrier.
本発明者はこれら従来技術に見られる問題点を解決すべ
く鋭意研究を行った結果本発明を完成させた。即ち本発
明は、アミノ基反応基を有する担体にアミノ基を介して
ピリジルジスルフィド基を有する物質を結合させ、該物
質中のジスルフィド基を開裂させて得られる遊離メルカ
プトピリジンを測定することを特徴とするアミノ基反応
基の定量方法を提供するものであり、以下詳細に説明す
る。The present inventor completed the present invention as a result of intensive research to solve the problems seen in these conventional techniques. That is, the present invention is characterized in that a substance having a pyridyl disulfide group is bonded to a carrier having an amino reactive group via an amino group, and the free mercaptopyridine obtained by cleaving the disulfide group in the substance is measured. The present invention provides a method for quantifying amino group-reactive groups, which will be described in detail below.
（問題点を解決するための手段）
本発明でアミノ基反応基とは、例えばＮ、Ｎ’−カルボ
ニルジイミダゾールによって活性化された０ＨＣＣＨ２
ＣＨ２ＣＨ２ＣＨ−Ｎ−等アミノ基と化学結合可能な官
能基を意味する。担体とは、シリカ、ガラス等の無機物
質等、エチレン、プロピレン、イソプロピレン、ブタジ
ェン、塩化ビニル等の重合体あるいはこれらの共重合し
た樹脂等を基材とした担体で、蛋白質をそのアミノ基を
利用して化学的に結合させるために例えば、Ｎ、Ｎ−カ
ルボニルジイミダゾール等により活性化される一級水酸
基、グルタルアルデヒド等により活性化されるアミノ基
等を導入されたものであれば良い。(Means for solving the problem) In the present invention, the amino group-reactive group refers to 0HCCH2 activated by, for example, N,N'-carbonyldiimidazole.
It means a functional group capable of chemically bonding with an amino group, such as CH2CH2CH-N-. A carrier is a carrier based on inorganic substances such as silica and glass, polymers such as ethylene, propylene, isopropylene, butadiene, and vinyl chloride, or resins made by copolymerizing these. For example, any material into which a primary hydroxyl group activated by N,N-carbonyldiimidazole or the like or an amino group activated by glutaraldehyde or the like may be introduced for chemical bonding.
本発明で使用するピリジルジスルフィド基を有する物質
としては、担体のアミノ基反応基と結合させるためのア
ミノ基を書９◆龜％１つ有し、かつ例えば２−ピリジル
ジスルフィド基、４−ピリジルジスルフィド基等を有す
る例えば２−アミノエチル−２−ピリジルジスルフィド
塩酸塩、２−アミノエチル−４−ピリジルジスルフィド
塩酸塩等が使用出来る。The substance having a pyridyl disulfide group used in the present invention has one amino group for bonding with the amino reactive group of the carrier, and includes, for example, a 2-pyridyl disulfide group, a 4-pyridyl disulfide group, and a pyridyl disulfide group. For example, 2-aminoethyl-2-pyridyldisulfide hydrochloride, 2-aminoethyl-4-pyridyldisulfide hydrochloride, etc. having a group such as 2-aminoethyl-2-pyridyldisulfide hydrochloride can be used.
前途の例えば−級水酸基、アミノ基等を活性化した担体
のアミノ基反応基とピリジルジスルフィド基を有する物
質のアミノ基反応基を結合させる操作に特別の制限はな
い。反応により生じた担体とピリジルジスルフィド基を
有する物質の結合体と担体に未結合の該物質を分離する
には、例えば遠心分離、′ａ過等の通常の生化学的手段
あるいは例えばバウンド／フリー洗浄分離等の免疫学的
洗浄分離法等を用いれば良い。There are no particular restrictions on the operation of bonding the amino group-reactive group of the carrier activated with, for example, a -class hydroxyl group or amino group, and the amino group-reactive group of the substance having a pyridyl disulfide group. To separate the conjugate of the carrier and a substance having a pyridyl disulfide group produced by the reaction from the substance unbound to the carrier, conventional biochemical means such as centrifugation, filtration, or bound/free washing can be used. An immunological washing separation method such as separation may be used.
以上の操作で得られる担体に結合したピリジルジスルフ
ィド基を有する物質中のジスルフィド基を開裂させるた
めには、該結合体に例えばシスティン、グルタチオン、
ジヒドロリポ酸、ホモシスティン等のチオール基を有す
る化合物を反応させれば良く、この操作を行う時の反応
温度、反応時間等の条件は適宜決定すれば良い。この操
作によりピリジルジスルフィド基は開裂され、メルカプ
トピリジンが遊離する。遊離したメルカプトピリジンは
、例えばバイオケミカル ジャーナル １５１巻、４１
７頁、１９７５年等に示された様に３４３ｎｍでの吸光
度をｎ１定する方法等で測定して定量すれば良い。In order to cleave the disulfide group in the substance having a pyridyl disulfide group bound to the carrier obtained by the above operation, the conjugate must be
A compound having a thiol group, such as dihydrolipoic acid or homocystine, may be reacted, and conditions such as reaction temperature and reaction time for performing this operation may be appropriately determined. This operation cleaves the pyridyl disulfide group and liberates mercaptopyridine. Free mercaptopyridine is described, for example, in Biochemical Journal, Vol. 151, 41.
7, 1975, etc., the absorbance at 343 nm may be determined by the method of determining n1 for quantitative determination.
（発明の効果）
本発明では、担体が有するアミノ基反応基を定量するこ
と、即ち担体中のアミノ基反応基数を知ることが可能で
ある。しかも本発明で、遊離されたメルカプトピリジン
量の測定は吸光度の測定等の比較的簡便な方法により可
能である。(Effects of the Invention) According to the present invention, it is possible to quantify the amino group-reactive groups that a carrier has, that is, to know the number of amino-reactive groups in the carrier. Furthermore, in the present invention, the amount of mercaptopyridine released can be measured by a relatively simple method such as absorbance measurement.
以上本発明により、担体の選定、あるいはアミノ基反応
基を介して蛋白質等を結合させた担体を用いて得られた
実験等の結果を容易に解析することが可能となる。As described above, according to the present invention, it becomes possible to select a carrier or easily analyze the results of experiments etc. obtained using a carrier to which a protein or the like is bound via an amino group reactive group.
（実施例）
以下本発明を更に詳細に説明するために実施例を示すが
本発明はこれら実施例に限定されるものではない。(Examples) Examples will be shown below to explain the present invention in more detail, but the present invention is not limited to these Examples.
実施例１
４種類の一級水酸基を有するエチレン製担体（直径１．
５ｍｍの球状）各１０００個に５０ｇＮ、Ｎ’−カルボ
ニルジイミダゾール及び３ｍｌの１００％アセトンを添
加し、２５Ｏｒｐｍで３０分間振盪した。担体をアセト
ン及び蒸溜水で洗浄した後、２５ｍｇの２−アミノエチ
ル−２−ピリジルジスルフィド塩酸塩と２．５ｍｌの５
０ｍＭホウ酸緩衡液（ｐＨ１３，５）を添加して、１２
Ｏｒｐｍで１８時間振盪して２−ピリジルジスルフィド
基を導入した。４種類の担体をそれぞれ１２個ずつ計り
取り、反応器に移し、０．５ｍＭシスティン塩酸塩を含
むＯ，ＬＭホウ酸緩衡液を加えて１２Ｏｒ ｐ ｍで２
時間振盪した後、３４３ｎｍでの吸光度を測定して遊離
２−メルカプトピリジンを定量した。２−メルカプトピ
リジン定量の結果と、その値より求めた担体１個当りの
アミノ基反応基数を表１に示す。尚、２−メルカプトピ
リジンのモル吸光係数は８．０８Ｘ１０３である。Example 1 Ethylene carrier having four types of primary hydroxyl groups (diameter 1.
50 g of N, N'-carbonyldiimidazole and 3 ml of 100% acetone were added to each of 1000 pieces (5 mm spheres), and the mixture was shaken at 25 Orpm for 30 minutes. After washing the carrier with acetone and distilled water, 25 mg of 2-aminoethyl-2-pyridyl disulfide hydrochloride and 2.5 ml of 5
Add 0mM boric acid buffer (pH 13,5) and
The 2-pyridyl disulfide group was introduced by shaking at Orpm for 18 hours. Weigh out 12 pieces of each of the four types of carriers, transfer them to a reactor, add O,LM boric acid buffer containing 0.5mM cysteine hydrochloride, and incubate at 12Orpm for 2 hours.
After shaking for an hour, free 2-mercaptopyridine was quantified by measuring absorbance at 343 nm. Table 1 shows the results of quantitative determination of 2-mercaptopyridine and the number of amino group-reactive groups per carrier determined from the results. Note that the molar extinction coefficient of 2-mercaptopyridine is 8.08×103.
表１Table 1
Claims (1)
ピリジルジスルフィド基を有する物質を結合させ、該物
質中のジスルフィド基を開裂させて得られる遊離メルカ
プトピリジンを測定することを特徴とするアミノ基反応
基の定量方法。(1) A substance having a pyridyl disulfide group is bonded to a carrier having an amino group-reactive group via an amino group, and the free mercaptopyridine obtained by cleaving the disulfide group in the substance is measured. Method for quantifying reactive groups.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1599588A JPH0221260A (en) | 1987-12-28 | 1988-01-28 | Quantitative determination of amino group reaction |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP32997787 | 1987-12-28 | ||
| JP62-329977 | 1987-12-28 | ||
| JP1599588A JPH0221260A (en) | 1987-12-28 | 1988-01-28 | Quantitative determination of amino group reaction |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0221260A true JPH0221260A (en) | 1990-01-24 |
Family
ID=26352233
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1599588A Pending JPH0221260A (en) | 1987-12-28 | 1988-01-28 | Quantitative determination of amino group reaction |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0221260A (en) |
-
1988
- 1988-01-28 JP JP1599588A patent/JPH0221260A/en active Pending
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