JPH0472559A - Method and kit for analyzing alpha-acyl amino acid - Google Patents
Method and kit for analyzing alpha-acyl amino acidInfo
- Publication number
- JPH0472559A JPH0472559A JP18416090A JP18416090A JPH0472559A JP H0472559 A JPH0472559 A JP H0472559A JP 18416090 A JP18416090 A JP 18416090A JP 18416090 A JP18416090 A JP 18416090A JP H0472559 A JPH0472559 A JP H0472559A
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- alpha
- acyl amino
- kit
- analysis
- 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
- 238000000034 method Methods 0.000 title claims description 14
- 239000000126 substance Substances 0.000 claims abstract description 14
- 238000004458 analytical method Methods 0.000 claims abstract description 12
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 6
- 238000007385 chemical modification Methods 0.000 claims description 7
- 239000003607 modifier Substances 0.000 claims description 6
- 125000001165 hydrophobic group Chemical group 0.000 claims description 5
- 125000004442 acylamino group Chemical group 0.000 claims description 2
- 239000000243 solution Substances 0.000 abstract description 6
- 238000010828 elution Methods 0.000 abstract description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract description 4
- 150000001413 amino acids Chemical class 0.000 abstract description 4
- 239000007864 aqueous solution Substances 0.000 abstract description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 abstract 6
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 abstract 1
- 239000012295 chemical reaction liquid Substances 0.000 abstract 1
- 230000002209 hydrophobic effect Effects 0.000 abstract 1
- OVARTBFNCCXQKS-UHFFFAOYSA-N propan-2-one;hydrate Chemical compound O.CC(C)=O OVARTBFNCCXQKS-UHFFFAOYSA-N 0.000 abstract 1
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 102100032488 Acylamino-acid-releasing enzyme Human genes 0.000 description 5
- 108010061216 Acylaminoacyl-peptidase Proteins 0.000 description 5
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 5
- LIGACIXOYTUXAW-UHFFFAOYSA-N phenacyl bromide Chemical compound BrCC(=O)C1=CC=CC=C1 LIGACIXOYTUXAW-UHFFFAOYSA-N 0.000 description 5
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 102000018832 Cytochromes Human genes 0.000 description 3
- 108010052832 Cytochromes Proteins 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- XAKBSHICSHRJCL-UHFFFAOYSA-N [CH2]C(=O)C1=CC=CC=C1 Chemical class [CH2]C(=O)C1=CC=CC=C1 XAKBSHICSHRJCL-UHFFFAOYSA-N 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 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 1
- FKJSFKCZZIXQIP-UHFFFAOYSA-N 2-bromo-1-(4-bromophenyl)ethanone Chemical compound BrCC(=O)C1=CC=C(Br)C=C1 FKJSFKCZZIXQIP-UHFFFAOYSA-N 0.000 description 1
- CTENSLORRMFPDH-UHFFFAOYSA-N 4-(bromomethyl)-7-methoxychromen-2-one Chemical compound BrCC1=CC(=O)OC2=CC(OC)=CC=C21 CTENSLORRMFPDH-UHFFFAOYSA-N 0.000 description 1
- HVCNXQOWACZAFN-UHFFFAOYSA-N 4-ethylmorpholine Chemical compound CCN1CCOCC1 HVCNXQOWACZAFN-UHFFFAOYSA-N 0.000 description 1
- -1 9-anthrylcyatmethane Chemical compound 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- OKJIRPAQVSHGFK-UHFFFAOYSA-N N-acetylglycine Chemical compound CC(=O)NCC(O)=O OKJIRPAQVSHGFK-UHFFFAOYSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 238000003277 amino acid sequence analysis Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 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α−アシルアミノ酸の簡便、高感度な分析方法
に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a simple and highly sensitive method for analyzing α-acylamino acids.
本発明はまた、このような分析のための分析用キットに
関する。The invention also relates to an analytical kit for such an analysis.
タンパク質の中にはそのN末端アミノ酸がαアシル化さ
れているタンパク質(N−α−アシル化タンパク質)が
多く存在する。N−α−アシル化タンパク質のN末端ア
ミノ酸配列解析にはエドマン法を原理とする通常のタン
パク質−次構造解析方法をそのまま適用することはでき
ない。その分析方法としては、マススペクトル解析装置
を用いる方法〔フェブスレターズ(FFiBS Let
ters)第128巻、第37頁(1981)〕、アシ
ルアミノ酸遊離酵素を用いる方法(酵素法)〔ジャーナ
ル オブ バイオケミストリー(J、 Biochem
、 )第92巻、第607頁(1982−)]が知られ
ている。Among proteins, there are many proteins whose N-terminal amino acids are α-acylated (N-α-acylated proteins). Conventional protein-substructure analysis methods based on the Edman method cannot be directly applied to N-terminal amino acid sequence analysis of N-α-acylated proteins. The analysis method is a method using a mass spectrum analyzer [FFiBS Let
ters) Vol. 128, p. 37 (1981)], method using acyl amino acid releasing enzyme (enzymatic method) [Journal of Biochemistry (J, Biochem)
) Volume 92, Page 607 (1982-)] is known.
本発明者らも、既に該アシルアミノ酸遊離酵素を用いた
簡便なN−α−アシル化タンパク質のN末端アミノ酸配
列分析方法を提供している(特開昭63−305253
号)。すなわちN−α−アシル化タンパク質のN末端ア
ミノ酸であるα7アシルアミノ酸は、核酸をアシルアミ
ノ酸遊離酵素で遊離させ、次いでアミノ酸分析方法又は
逆相系カラムの高速液体クロマトグラフィーを行うこと
により同定される。The present inventors have already provided a simple method for analyzing the N-terminal amino acid sequence of N-α-acylated proteins using the acylamino acid releasing enzyme (Japanese Patent Laid-Open No. 63-305253
issue). That is, the α7 acylamino acid, which is the N-terminal amino acid of an N-α-acylated protein, is identified by releasing the nucleic acid with an acylamino acid releasing enzyme, and then performing an amino acid analysis method or high performance liquid chromatography using a reversed phase column. .
しかしながらN末端のα−アシルアミノ酸の同定におい
ては、アミノ酸の種類、アシル基の違いによるイオン交
換樹脂クロマトグラフィー逆相系カラムクロマトグラフ
ィー等における溶出位置の差が小さく、標準物質との比
較による明確な同定は困難であった。However, in the identification of N-terminal α-acyl amino acids, differences in elution position in ion-exchange resin chromatography, reversed-phase column chromatography, etc. due to differences in the type of amino acid and acyl group are small, and clear comparison with standard substances is difficult. Identification was difficult.
上記現状にかんがみ、本発明の目的は、αアシルアミノ
酸の高分離、高感度での分析方法及びそれに用いるキッ
トを提供することにある。In view of the above-mentioned current situation, an object of the present invention is to provide a method for highly separating and highly sensitive analysis of α-acyl amino acids, and a kit used therefor.
本発明を概説すれば、本発明の第1の発明は、α−アシ
ルアミノ酸の分析方法に関し、α−アシルアミノ酸のカ
ルボキシル基に疎水性基を含む化学修飾基を導入するこ
とを特徴とする。また本発明の第2の発明は、上記第1
の発明の方法を用いて分析を行うための分析用キットで
あって、α−アシルアミノ酸のカルボキシル基に疎水性
基を含む化学修飾基を導入することのできる化学修飾剤
を含有することを特徴とする。To summarize the present invention, the first aspect of the present invention relates to a method for analyzing α-acylamino acids, and is characterized by introducing a chemical modification group containing a hydrophobic group into the carboxyl group of α-acylamino acids. Further, the second invention of the present invention is the above-mentioned first invention.
An analytical kit for performing analysis using the method of the invention, characterized in that it contains a chemical modifier capable of introducing a chemical modification group containing a hydrophobic group into the carboxyl group of an α-acylamino acid. shall be.
本発明の方法に使用し得る化学修飾剤としては、化学修
飾基として疎水性基を含み、α−アシルアミノ酸のカル
ボキシル基に反応し共有結合を形成する化学修飾剤であ
ればよく、例えばフェナシルブロマイド、9−アンスリ
ルシアツメタン、パラブロモフェナシルブロマイド、l
。The chemical modifier that can be used in the method of the present invention may be any chemical modifier that contains a hydrophobic group as a chemical modification group and reacts with the carboxyl group of α-acylamino acid to form a covalent bond, such as phenacyl Bromide, 9-anthrylcyatmethane, parabromophenacyl bromide, l
.
2−ジアミノ−4,5−ジメトキシベンゼン、4−ブロ
モメチル−7〜メトキシクマリン等が挙げられる。Examples include 2-diamino-4,5-dimethoxybenzene and 4-bromomethyl-7-methoxycoumarin.
本発明においてはまず種々のα−アシルアミノ酸の標準
物質を化学修飾剤、例えばフェナシルブロマイドと反応
させてフェナシル誘導体とし、例えば逆相カラムを用い
る高速液体クロマトグラフィーに供試し、例えばフェナ
シル基の吸収極大がある248nmで該化学修飾α−ア
シルアミノ酸を検出すれば良い。次に同定したい物質を
同様に化学修飾し、同様に逆相カラムクロマトグラフィ
ーを行い、前述の化学修飾された標準物質との比較によ
り目的のα−アシルアミノ酸を同定する。同定限界感度
は10ピコモルである。In the present invention, first, standard substances of various α-acyl amino acids are reacted with a chemical modifier, such as phenacyl bromide, to form phenacyl derivatives, and the phenacyl derivatives are subjected to high performance liquid chromatography using, for example, a reversed phase column. The chemically modified α-acyl amino acid may be detected at 248 nm, which has a maximum wavelength. Next, the substance to be identified is chemically modified in the same manner, reversed phase column chromatography is performed in the same manner, and the target α-acylamino acid is identified by comparison with the chemically modified standard substance described above. The identification limit sensitivity is 10 pmol.
このα−アシルアミノ酸の化学修飾剤を試薬としキット
としておくことで、本発明のα−アシルアミノ酸の分析
を簡便に行うことができる。By preparing a kit using this chemical modifier for α-acylamino acids as a reagent, analysis of the α-acylamino acids of the present invention can be easily performed.
なお、化学修飾試薬は溶液状でも良いし、凍結乾燥品で
も良い。またキット中に、例えばアシルアミノ酸遊離酵
素、化学修飾されたα−アシルアミノ酸標準物質、緩衝
液、逆相ODSカラム等を入れておいても良い。Note that the chemical modification reagent may be in the form of a solution or may be a lyophilized product. Further, the kit may contain, for example, an acyl amino acid releasing enzyme, a chemically modified α-acyl amino acid standard substance, a buffer solution, a reversed phase ODS column, and the like.
以下、本発明を実施例により更に具体的に説明するが本
発明はこれに限定されない。EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.
実施例1
フェナシルブロマイドで修飾したα−ア七チルアミノ酸
の分析
各50 pmolのα−アセチルアミノ酸水溶液と20
nmolのフェナシルブロマイドのアセトン溶液を混
合し、5%N−エチルモルホリンのアセトニ) IJル
溶液を添加、50℃、2時間反応させることによりフェ
ナシル化α−アセチルアミノ酸を調製した。反応液をO
DSカラム(ワコーシル:和光純薬社製) (0,4
6X 25cm)に供試し、0.1%リン酸酸中上セト
ニ IJルの濃度勾配溶出により分離した。検出は25
4 nmで行った。第1図に分離したフェナシル化α−
アセチルアミノ酸のクロマトグラフを示す。第1図にお
いて、縦軸は吸光度(254nm)、横軸は時間(分)
を示す。また図中1はAsnの、2はGlnの、3はS
erの、4はGlyの、5はThrの、6は^laの、
7はHisの、8はProの、9はTyrの、10はV
alの、11はMetの、12は11eの、13はLe
uの、14は^spの、15はPheの、16はGlu
の各7エナシル化α−アセチルアミノ酸の溶出ピークを
示す。Example 1 Analysis of α-acetyl amino acid modified with phenacyl bromide 50 pmol each of α-acetyl amino acid aqueous solution and 20 pmol of α-acetyl amino acid aqueous solution
Phenacylated α-acetylamino acid was prepared by mixing nmol of phenacyl bromide in acetone, adding 5% N-ethylmorpholine in acetone, and reacting at 50°C for 2 hours. O
DS column (Wakosil: manufactured by Wako Pure Chemical Industries, Ltd.) (0,4
6 x 25 cm) and separated by gradient elution with 0.1% cetonyl IJ in 0.1% phosphoric acid. Detection is 25
Performed at 4 nm. Phenacylated α- isolated in Figure 1
A chromatograph of acetylamino acid is shown. In Figure 1, the vertical axis is absorbance (254 nm) and the horizontal axis is time (minutes).
shows. In the figure, 1 is Asn, 2 is Gln, and 3 is S.
er, 4 is Gly, 5 is Thr, 6 is ^la,
7 is His, 8 is Pro, 9 is Tyr, 10 is V
al's, 11 is Met's, 12 is 11e's, 13 is Le
u's, 14 is ^sp, 15 is Phe, 16 is Glu
The elution peaks of each of the 7 enacylated α-acetyl amino acids are shown.
その結果、各α−アセチル化アミノ酸を、高分離状態で
分析することができた。As a result, each α-acetylated amino acid could be analyzed in a highly separated state.
実施例2
馬心臓シトクロームCのN末端アミノ酸の同定100
r+mol馬心臓シトクロームCのトリプシン消化物に
、アシルアミノ酸遊離酵素0.002ユニツトを添加し
、37℃、10時間反応させた。反応物を凍結乾燥後、
0.01 Mギ酸に溶解し、0.01 Mギ酸で平衡化
したSP−セファデックス(ファルマシア社)に添加し
、素通りした両分を集めた。素通り画分を凍結乾燥し、
実施例1と同様な方法でフェナシル化を行った。Example 2 Identification of N-terminal amino acids of horse heart cytochrome C 100
0.002 unit of acyl amino acid releasing enzyme was added to a tryptic digest of r+mol horse heart cytochrome C, and the mixture was reacted at 37°C for 10 hours. After freeze-drying the reaction product,
The solution was dissolved in 0.01 M formic acid and added to SP-Sephadex (Pharmacia) equilibrated with 0.01 M formic acid, and both portions that passed through were collected. Freeze-dry the pass-through fraction,
Phenacylation was carried out in the same manner as in Example 1.
実施例1と同様にODSカラムにより分離し、標準物質
との比較から、馬心臓シトクロームCのN末端をアセチ
ルグリシンと同定した。It was separated using an ODS column in the same manner as in Example 1, and from comparison with a standard substance, the N-terminus of horse heart cytochrome C was identified as acetylglycine.
実施例3
α−アシルアミノ酸分析用キットの作成α−アシルアミ
ノ酸分析用キットを作成した。Example 3 Creation of kit for α-acyl amino acid analysis A kit for α-acyl amino acid analysis was created.
化学修飾試薬としてフェナシルブロマイドのアセトン溶
液を用いた(表1)。An acetone solution of phenacyl bromide was used as a chemical modification reagent (Table 1).
表 1
1剤:フェナシルプロマイト1rnl
ア七トン溶液
(2μmol/ml)
■剤:α−アシルアミノ酸 100本遊離酵素
(0,002ユニツト)
■剤:IMリン酸ナトリウム 10m1緩衝液
(■銅溶解用)
100回分
100回分
100回分
〔発明の効果〕
以上、詳細に説明したとおり、本発明の方法及びキット
を用いることにより、簡便かつ、高感度に、α−アシル
アミノ酸の微量定量を行うことが可能となった。Table 1 1 agent: phenacylpromite 1rnl a7tone solution (2 μmol/ml) ■ agent: α-acyl amino acid 100 free enzymes (0,002 units) ■ agent: IM sodium phosphate 10 ml buffer (■ copper dissolution [Effects of the Invention] As explained above in detail, by using the method and kit of the present invention, it is possible to easily and sensitively quantify α-acylamino acids in trace amounts. It has become possible.
第1図は、化学修飾されたα−アシルアミノ酸の溶出パ
ターンを示すグラフである。FIG. 1 is a graph showing the elution pattern of chemically modified α-acyl amino acids.
Claims (1)
−アシルアミノ酸のカルボキシル基に疎水性基を含む化
学修飾基を導入することを特徴とするα−アシルアミノ
酸の分析方法。 2、請求項1記載の方法を用いて、α−アシルアミノ酸
の分析を行うための分析キットであって、α−アシルア
ミノ酸のカルボキシル基に疎水性基を含む化学修飾基を
導入することのできる化学修飾剤を含有することを特徴
とするα−アシルアミノ酸分析用キット。[Claims] 1. A method for analyzing α-acylamino acids, in which the α
-A method for analyzing α-acylamino acids, which comprises introducing a chemical modification group containing a hydrophobic group into the carboxyl group of the acylamino acids. 2. An analysis kit for analyzing α-acylamino acids using the method according to claim 1, which is capable of introducing a chemical modification group containing a hydrophobic group into the carboxyl group of α-acylamino acids. A kit for analyzing α-acyl amino acids, characterized by containing a chemical modifier.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP18416090A JPH0472559A (en) | 1990-07-13 | 1990-07-13 | Method and kit for analyzing alpha-acyl amino acid |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP18416090A JPH0472559A (en) | 1990-07-13 | 1990-07-13 | Method and kit for analyzing alpha-acyl amino acid |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH0472559A true JPH0472559A (en) | 1992-03-06 |
Family
ID=16148419
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP18416090A Pending JPH0472559A (en) | 1990-07-13 | 1990-07-13 | Method and kit for analyzing alpha-acyl amino acid |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH0472559A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2001186632A (en) * | 1999-12-24 | 2001-07-06 | Yazaki Corp | Electrical junction box |
JP2009016357A (en) * | 2008-09-16 | 2009-01-22 | Sumitomo Wiring Syst Ltd | Chain terminal |
-
1990
- 1990-07-13 JP JP18416090A patent/JPH0472559A/en active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2001186632A (en) * | 1999-12-24 | 2001-07-06 | Yazaki Corp | Electrical junction box |
JP2009016357A (en) * | 2008-09-16 | 2009-01-22 | Sumitomo Wiring Syst Ltd | Chain terminal |
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