JPH0371045A - Method for detecting amino acid derivative with high sensitivity - Google Patents
Method for detecting amino acid derivative with high sensitivityInfo
- Publication number
- JPH0371045A JPH0371045A JP20722489A JP20722489A JPH0371045A JP H0371045 A JPH0371045 A JP H0371045A JP 20722489 A JP20722489 A JP 20722489A JP 20722489 A JP20722489 A JP 20722489A JP H0371045 A JPH0371045 A JP H0371045A
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- ptc
- aminofluorescein
- formula
- amino
- 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 abstract description 22
- 230000035945 sensitivity Effects 0.000 title claims abstract description 14
- 150000003862 amino acid derivatives Chemical class 0.000 title claims abstract description 9
- 150000001413 amino acids Chemical class 0.000 claims abstract description 10
- -1 phenylthiocarbamyl Chemical class 0.000 claims abstract description 10
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 239000002253 acid Substances 0.000 claims abstract description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 6
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims abstract description 6
- 230000003301 hydrolyzing effect Effects 0.000 claims abstract 2
- 238000001514 detection method Methods 0.000 claims description 10
- QKFJKGMPGYROCL-UHFFFAOYSA-N phenyl isothiocyanate Chemical compound S=C=NC1=CC=CC=C1 QKFJKGMPGYROCL-UHFFFAOYSA-N 0.000 claims description 7
- 229940117953 phenylisothiocyanate Drugs 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims 6
- 238000003776 cleavage reaction Methods 0.000 claims 1
- 238000005558 fluorometry Methods 0.000 claims 1
- 238000007363 ring formation reaction Methods 0.000 claims 1
- 230000007017 scission Effects 0.000 claims 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract description 6
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 3
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 abstract 2
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 1
- DGTNSSLYPYDJGL-UHFFFAOYSA-N phenyl isocyanate Chemical compound O=C=NC1=CC=CC=C1 DGTNSSLYPYDJGL-UHFFFAOYSA-N 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- GZAJOEGTZDUSKS-UHFFFAOYSA-N 5-aminofluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(N)=CC=C21 GZAJOEGTZDUSKS-UHFFFAOYSA-N 0.000 description 5
- 238000007796 conventional method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 238000001506 fluorescence spectroscopy Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N methyl alcohol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 3
- CXISPYVYMQWFLE-VKHMYHEASA-N Ala-Gly Chemical compound C[C@H]([NH3+])C(=O)NCC([O-])=O CXISPYVYMQWFLE-VKHMYHEASA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 108010047495 alanylglycine Proteins 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000004020 conductor Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- VFWCMGCRMGJXDK-UHFFFAOYSA-N 1-chlorobutane Chemical compound CCCCCl VFWCMGCRMGJXDK-UHFFFAOYSA-N 0.000 description 1
- PQMRRAQXKWFYQN-UHFFFAOYSA-N 1-phenyl-2-sulfanylideneimidazolidin-4-one Chemical compound S=C1NC(=O)CN1C1=CC=CC=C1 PQMRRAQXKWFYQN-UHFFFAOYSA-N 0.000 description 1
- 201000002929 Carnitine palmitoyltransferase II deficiency Diseases 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- IPIGTJPBWJEROO-UHFFFAOYSA-B thorium(4+);tetraphosphate Chemical compound [Th+4].[Th+4].[Th+4].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O IPIGTJPBWJEROO-UHFFFAOYSA-B 0.000 description 1
Landscapes
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、タンパク質のアミノ末端からのアミノ酸配列
決定法に応用するアミノ酸誘導体の同定方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for identifying amino acid derivatives that is applied to a method for determining amino acid sequences from the amino terminus of proteins.
本発明は、Ｉ’ＴＣ−アミノ酸とアミノフルオレセイン
あるいはローダミン系アミノ化合物をＤＣＣ存在下にお
いて反応させ、ＰＴＣ−アミノ酸７２ド誘導体とし、蛍
光光度法によって高感度に検出するものである。In the present invention, an I'TC-amino acid and an aminofluorescein or rhodamine-based amino compound are reacted in the presence of DCC to form a PTC-amino acid 72 derivative, which is detected with high sensitivity by fluorescence photometry.
従来、タンパク質のアミノ末端からのアミノ酸配列決定
法であるエドマン法（ＰＴＣ法）における最終段階のア
ミノ酸誘導体の同定には、第１図に示すように、フェニ
ルチオヒダントイン（ＰＴＩ（）アミノ酸誘導体を紫外
吸光光度法によって検出する方法が用いられてきた。
（参考文献１）〔発明が解決しようとする課題〕
従来のＰＴＩＩ誘導体を紫外線吸光光度法ににより検出
する方法は、検出の手段としては簡便であるが、近年、
求められている極微量タンパク質またはペプチドの高感
度な分析には適さない。Conventionally, in the final step of identifying amino acid derivatives in the Edman method (PTC method), which is a method for determining amino acid sequences from the amino terminus of proteins, phenylthiohydantoin (PTI Spectrophotometric detection methods have been used.
(Reference Document 1) [Problems to be Solved by the Invention] The conventional method of detecting PTII derivatives by ultraviolet absorption spectrophotometry is a simple detection method, but in recent years,
It is not suitable for the highly sensitive analysis of ultra-trace amounts of proteins or peptides that is required.
一方、放射性同位体で標識した化合物を用いる高感度な
検出法が提案されているが、人体および環境へ及ぼす影
響を考慮すると汎用性は低い。On the other hand, highly sensitive detection methods using compounds labeled with radioactive isotopes have been proposed, but their versatility is low when considering the effects on the human body and the environment.
そこで、本発明はより高感度かつ簡便にアミノする。Therefore, the present invention provides amino acids with higher sensitivity and ease.
本実施例においては、ＰＴＣ−アミノ酸と４−アミノフ
ルオレセインを反応さセてＰＴＣ−アミノ酸アミドフル
オレセインとし、高感度にそれを検出する基本的方法を
示す。This example shows a basic method for reacting PTC-amino acid and 4-aminofluorescein to form PTC-amino acid amide fluorescein and detecting it with high sensitivity.
ここでは、簡単のためにタンパク質のかわりに、アラニ
ン（Ａｌａ）とグリシン（Ｇｌｙ）からなるジペプチド
（Ａｌａ−Ｇｌｙ）を用いた。Here, for simplicity, a dipeptide (Ala-Gly) consisting of alanine (Ala) and glycine (Gly) was used instead of protein.
（ｉｌ＾ＴＺ−Ａｌａの合成と精製（参考文献２）反応
は（■およびＭ式に従って進行する。(il^Synthesis and purification of TZ-Ala (Reference 2) The reaction proceeds according to (■ and M formula).
Ａｌａ−Ｇｌｙを５０％ピリジン水溶液に溶解し、２Ｍ
水酸化ナトリウム溶液でＰｌ＋ ８．６に調整する。続
いて、フェニルイソチオシアネート（ＰＩＴＣ）を加え
る。Dissolve Ala-Gly in 50% pyridine aqueous solution and make 2M
Adjust Pl+ to 8.6 with sodium hydroxide solution. Subsequently, phenylisothiocyanate (PITC) is added.
ＰｒＴＣの添加と共にＰｌ＋が降下するので、２Ｍ水酸
化ナトリウム溶液を加えてＰｌ＋を８．６に保つ。ＰＩ
（の変動が停止した後、溶液を４０℃で１時間加熱する
。Since Pl+ drops with the addition of PrTC, 2M sodium hydroxide solution is added to keep Pl+ at 8.6. P.I.
(After the fluctuations have stopped, heat the solution at 40 °C for 1 h.
その後、反応液をヘンゼンで洗浄する。水相に溶解した
ヘンゼンをＮ２ガスで除去し、ＩＭｌｌｃＪを加え酸誘
導体を検出する新しい方法を提供しようとするものであ
る。Thereafter, the reaction solution is washed with Hensen. The present invention aims to provide a new method for detecting acid derivatives by removing Hensen dissolved in the aqueous phase with N2 gas and adding IMllcJ.
本発明、上記の課題を克服しアミノ酸配列決定法の高感
度化を達成するために、アごノフルオレセインまたはロ
ーダミン系アミノ化合物などの蛍光性アミノ化合物を標
識化合物として用いた。In the present invention, in order to overcome the above-mentioned problems and achieve high sensitivity in amino acid sequencing, a fluorescent amino compound such as agonofluorescein or a rhodamine-based amino compound was used as a labeling compound.
以下に１．本発明の作用を従来法と比較して説明する。 Below are 1. The effect of the present invention will be explained in comparison with the conventional method.
本発明は、第１図に示す従来法のように、２〜アニリノ
チアゾリノン（ＡＴＺ）誘導体を酸で処理してＰＴＨ誘
導体として紫外吸光光度法で検出するのではなく、第２
図に示すように、ＰＴＣ−アミノ酸とアミノフルオレセ
インまたはローダミン系アミノ化合物などの蛍光性アミ
ノ化合物を反応させてＰＴＣ−アミノ酸アミドとし蛍光
法により高感度にアどノ酸誘導体を検出できる。The present invention does not treat a 2-anilinothiazolinone (ATZ) derivative with an acid and detect it as a PTH derivative by ultraviolet absorption spectrophotometry, as in the conventional method shown in FIG.
As shown in the figure, PTC-amino acid is reacted with a fluorescent amino compound such as aminofluorescein or a rhodamine-based amino compound to form PTC-amino acid amide, and an adonoic acid derivative can be detected with high sensitivity by fluorescence method.
以下、実施例に基づいて、本発明の詳細な説明てＰｌ＋
を２にするとＰＴＣ−Ａｌａ−、Ｇｌｙは白色沈澱とし
て得られる。The following is a detailed explanation of the present invention based on examples.
When PTC-Ala- and Gly are set to 2, PTC-Ala- and Gly are obtained as white precipitates.
ＴＦＡ
ＰＴＣ−へＩａ−Ｇｌｙ −ン へＴＺ−へｌａ
＋Ｇｌｙ −−−−−−ＮＤ上記ＰＴＣ−Ａｌａ−
Ｇｌｙをトリフルオロ酢酸（ＴＦＡ）に熔解し、５０℃
で５分間加熱する。反応後、溶液を完全番こ減圧乾固す
る。残渣にブチルクロライ１゛を加え、生成物を完全に
溶解し、セルロースカラムに通す。副生酸物及びｃｒｙ
はカラムに吸着される。TFA PTC-to Ia-Gly -n to TZ-to la
+Gly ------ND above PTC-Ala-
Dissolve Gly in trifluoroacetic acid (TFA) and heat at 50°C.
Heat for 5 minutes. After the reaction, the solution was completely dried under reduced pressure. Add 1 inch of butyl chloride to the residue to completely dissolve the product, and pass through a cellulose column. By-product acid and cry
is adsorbed on the column.
流出液を集めて減圧乾固するとＡＴＺ−Ａｌａが白色結
晶として得られる。The effluent is collected and dried under reduced pressure to obtain ATZ-Ala as white crystals.
（ｉｔｌ Ｐ Ｔ Ｃ−ア〔）酸の台底上記反応式（
■）に従って、ＡＴＺ−ア砧ノ酸を加水分解し、ＰＴＣ
−アミノ酸を合成する。(itl P T C-A [) The above reaction formula (
According to
-Synthesize amino acids.
ＡＴＺ−アミノ酸を５０％０％トリエチルアミン液に溶
解し、室温に３０分放置する。その後、減圧乾固すると
ＰＴＣ−Ａｈａが白色結晶として得られる。ATZ-amino acid is dissolved in 50% 0% triethylamine solution and left at room temperature for 30 minutes. Thereafter, the mixture is dried under reduced pressure to obtain PTC-Aha as white crystals.
（ｉｉｉ）カソプリング反応
（４−アミノフルオレセイン）０［１
上記反応式（■）が示すように、４−アミノフルオレセ
インのアミノ基とＰＴＣ−Ａｌａのカルボキシル基がＤ
ＣＣの作用により縮合し、ｌ’Ｔｃ−Ａｌａ ７ξノフ
ルオレセインが生成する。反応はクロホルムを溶媒とし
て、室温で１時間放置した。(iii) Casopring reaction (4-aminofluorescein) 0 [1 As shown in the above reaction formula (■), the amino group of 4-aminofluorescein and the carboxyl group of PTC-Ala are
Condensation occurs under the action of CC to produce l'Tc-Ala 7ξnofluorescein. The reaction was allowed to stand at room temperature for 1 hour using chloroform as a solvent.
（ｉｖｌ ＰＴＣ−Ａｌａ−アミノフルオレセインの
検出反応生成物を高速液体クロマ１−グラフ（ＩＩＰＬ
Ｃ）にかｉノ紫外分光光度法により検出した。その結果
を第３図に示す。図より明らかなように、ＰＴＣ−Ａｌ
ａと４−アミノフルオレセインのカノプリング反応鳳１
１
Ｕｌ＋
（■）
室温下１時間で完結し、ＰＴＣ−Ａｌａ−アミノフルオ
レセインとして検出される。(ivl PTC-Ala-aminofluorescein detection reaction product was analyzed using high performance liquid chroma 1-graph (IIPL)
C) Detected by ultraviolet spectrophotometry. The results are shown in FIG. As is clear from the figure, PTC-Al
Kanopring reaction of a and 4-aminofluorescein 1
1Ul+ (■) Completed in 1 hour at room temperature and detected as PTC-Ala-aminofluorescein.
ＨＰＬＣの溶離条件：カラムーーー３ＥＲＶＡ Ｃａｔ
、 ｍ４２３１８ＳＥＲＶＡＣＨＲＯ閂Ｐａｃｋｉｎｇ
（２５０ｍｍＸ４．６ｍｍ）、溶媒系１７％、 ０．０
１５Ｍ酢酸−酢酸すＩ・リウム緩衝液（Ｐ Ｈ５）８３
％アセトニトリル−メチルアルコール（４：１、Ｖ／Ｖ
）、流速−−−１，５ｍＮ／ｍｉｎ、 検出−−−−
−２６９ｎｍ。HPLC elution conditions: Column--3ERVA Cat
, m42318SERVACHRO Bar Packing
(250mmX4.6mm), solvent system 17%, 0.0
15M acetic acid-lium acetate buffer (PH5) 83
% acetonitrile-methyl alcohol (4:1, V/V
), flow rate---1,5 mN/min, detection------
-269nm.
ｖ）ＰＴＣ−＾１ａ−アミノフルオレセインの蛍光検出
上記のＨＰＩ、Ｃ法と蛍光分光法を使ってＰＴＣ−Ａｈ
ａ−アミノフルオレセインの検出を試みた。その結果を
第４図に示す。v) Fluorescence detection of PTC-^1a-aminofluorescein PTC-Ah using the HPI, C method and fluorescence spectroscopy described above.
An attempt was made to detect a-aminofluorescein. The results are shown in FIG.
本発明の検出感度と従来のフェニルチオヒダントインア
ミノ酸検出法ＣＰＴＩＩ法〉のそれを比較したところ、
次のようになった。本発明の検出感度が非常に高いとこ
ろがわかる。When the detection sensitivity of the present invention was compared with that of the conventional phenylthiohydantoin amino acid detection method CPTII method,
It turned out like this: It can be seen that the detection sensitivity of the present invention is extremely high.
方法 検出感度
ＰＴＨ法（紫外光分光法） 〜１０１”ｍｏ１本発明
（蛍光分光法） 〜Ｉｆｍｏｌ（Ｉ）Ｐ Ｉ、Ｃの条
件）
カラム−−−ｓ旧５ｆｉｌＤＯＣＡＰＳＩｉ［、ＰＡＣ
Ｃ１８ＡＧ Ｔｙｐｅ（１５０ｍｍＸ４．４ｍｍ）、溶
媒系−１，，０ｍＭリン酸水素−すトリウム−リン酸−
水素すトリウム緩衝液（Ｐ Ｈ８，０）−メチルアルコ
ールのグラ−ジエン１〜検出 −励起波超ｄ８６ｎｍ、
蛍光波超５１３ｎｍ実施例２
本実施例においては、タンパク質構成アどノ酸それぞれ
のＰＴＣ−アミノ酸アミノフルオレセインの一済分離検
出した例を示す。Method Detection sensitivity PTH method (ultraviolet spectroscopy) ~101"mo1 Invention (fluorescence spectroscopy) ~Ifmol(I)PI Conditions for I, C) Column ---s old 5filDOCAPSIi [, PAC
C18AG Type (150mmX4.4mm), Solvent system-1,0mM hydrogen phosphate-storium phosphate-
Thorium hydrogen buffer (PH8,0) - Methyl alcohol gradientene 1 ~ Detection - Excitation wave ultra d86 nm,
Example 2 of fluorescence wavelength at 513 nm In this example, an example is shown in which PTC-amino acid aminofluorescein of each protein-constituting adonoacid was separated and detected.
実施例１に示した手順によってＰＴＣ−アミノ酸アミノ
フルオレセインを合威し、蛍光、分光検出のＨＰＬＣ条
件にて分離検出した。その結果を第５図に示す。図より
明らかなように、タンパク質構成成分である２０種類の
アミノ酸のフェニルチオ力ルハミルアごノフルオレセイ
ンｇＪ”７を体は良好に分離されている。PTC-amino acid aminofluorescein was synthesized according to the procedure shown in Example 1, and separated and detected under HPLC conditions of fluorescence and spectroscopic detection. The results are shown in FIG. As is clear from the figure, the body has successfully separated the 20 types of amino acids phenylthiolhamylamine fluorescein gJ''7, which are protein constituents.
本発明の主眼とするところは、タンパク質あるいはペプ
チドのアごノ末端からのアミノ酸配列決定におけるアミ
ノ酸誘導体の高感度な同定である。The main focus of the present invention is the highly sensitive identification of amino acid derivatives in amino acid sequencing from the chin-terminus of proteins or peptides.
本発明の最重要点は、蛍光性ア多ノ化合物と安定なＰＴ
Ｃ−アミノ酸を反応させＰＴＣ−アミノ酸の７５ノフル
オレセイン誘導体とし、散開性同位体を用いることなく
高感度にアミノ酸誘導体を検出できることにある。The most important point of the present invention is that fluorescent amino compounds and stable PT
The purpose is to react C-amino acid to form a 75-nofluorescein derivative of PTC-amino acid, and to detect the amino acid derivative with high sensitivity without using a diffuse isotope.
実施例においては、４−アミノフルオレセインを用いた
例のみを示したが、他の蛍光性アどノ化合物を使っても
可能であり、実施例に制約されることはない。In the Examples, only an example using 4-aminofluorescein was shown, but it is also possible to use other fluorescent agonists, and the invention is not limited to the Examples.
したがって、本発明によるアミノ携導体を高感度に検出
する方法は工業的価値が大である。Therefore, the method of detecting aminocarboxylic conductors with high sensitivity according to the present invention has great industrial value.
１、 Ｐ、Ｅｄｍａｎ、Ａｃｔａ ｃｈｅｍ、 ５ｃａ
ｎｄ、、削、 ７６Ｈ１９５６）２、 Ｓ、Ｂ、Ｎｅａ
ｄｌｅｍａｎ＆Ｓ、Ｖｅｒｌａｇ、 Ｍｏ１ｅｃｕｌａ
ｒ ＢｉｏｌｏｇｙＢｉｏｃｈｅｍｉｓｔｒｙ ａｎｄ
Ｂｉｏｐｈｙｓｉｃｓ ８． ２５３1, P. Edman, Acta chem, 5ca
nd,, cut, 76H1956) 2, S, B, Nea
dleman&S, Verlag, Mo1ecula
r BiologyBiochemistry and
Biophysics 8. 253
第１図は従来法の工程図、第２図は本発明の方法を示す
工程図、第３図Ｌｅｔ： ＰＴＣ−Ａｌａのアミノフル
オレセイン誘導体のＩＩ Ｐ Ｌ Ｃのチャー１〜（吸
光分光法）、第４図はＰＴＣ−Ａｌａのアミノフルオレ
セイン読導体のＨＰＬＣのチャート （蛍光分光法）、
第５図は２０種類のＰＴＣ−アミノ酸のアミノフルオレ
セイン誘導体混合物の一済ＨＰ１．Ｃのチャートである
。
以
上FIG. 1 is a process diagram of the conventional method, FIG. 2 is a process diagram showing the method of the present invention, and FIG. 3: Char 1 of II PLC of aminofluorescein derivative of PTC-Ala (absorption spectroscopy), Figure 4 is an HPLC chart (fluorescence spectroscopy) of PTC-Ala's aminofluorescein reading conductor.
Figure 5 shows Ichiji HP1, a mixture of 20 types of PTC-amino acid aminofluorescein derivatives. This is a chart of C. that's all
Claims (2)
ＰＴＣ−アミノ酸）とアミノフルオレセインあるいはロ
ーダミン系アミノ化合物（ｂ）とを（ I ）式に従って
、シンクロヘキシルカルボジイミド（ＤＣＣ）存在下、
ＰＴＣ−アミノ酸アミド誘導体（ｃ）とし、該アミノ酸
誘導体（ｃ）を蛍光光度法により高感度に検出するアミ
ノ酸誘導体の高感度検出法 ▲数式、化学式、表等があります▼ Ｘ：アミノフルオレセイン あるいはローダミン誘導体 ▲数式、化学式、表等があります▼（ I ）式(1) Phenylthiocarbamyl derivatives of amino acids (a,
PTC-amino acid) and aminofluorescein or rhodamine-based amino compound (b) according to formula (I) in the presence of synchronohexylcarbodiimide (DCC),
High-sensitivity detection method for amino acid derivatives using PTC-amino acid amide derivative (c) and detecting the amino acid derivative (c) with high sensitivity using fluorometry▲Mathical formulas, chemical formulas, tables, etc. are available▼X: Aminofluorescein or rhodamine derivative ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) Formula
配列決定法（エドマン法）において、フェニルイソチオ
シアネート（ｄ）とタンパク質またはペプチド（ｅ）と
を（II）式に従って反応させ、フェニルチオカルバミル
タンパク質またはペプチド（ｆ）を生成させ、（III）
式に従って酸と反応させて環化切断し、（IV）式に従っ
て加水分解して得る請求項１記載のアミノ酸誘導体の高
感度検出法。 ▲数式、化学式、表等があります▼…………… ▲数式、化学式、表等があります▼………（II） ▲数式、化学式、表等があります▼……………‥‥（I
II） ▲数式、化学式、表等があります▼…‥‥（IV）(2) The PTC-amino acid (a) is obtained by reacting phenylisothiocyanate (d) with a protein or peptide (e) according to formula (II) in a normal amino acid sequencing method (Edman method). producing a mil protein or peptide (f), (III)
2. The highly sensitive method for detecting an amino acid derivative according to claim 1, which is obtained by reacting with an acid according to the formula, cyclization and cleavage, and hydrolyzing according to the formula (IV). ▲There are mathematical formulas, chemical formulas, tables, etc.▼…………… ▲There are mathematical formulas, chemical formulas, tables, etc.▼………(II) ▲There are mathematical formulas, chemical formulas, tables, etc.
II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼... (IV)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20722489A JPH0371045A (en) | 1989-08-09 | 1989-08-09 | Method for detecting amino acid derivative with high sensitivity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20722489A JPH0371045A (en) | 1989-08-09 | 1989-08-09 | Method for detecting amino acid derivative with high sensitivity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0371045A true JPH0371045A (en) | 1991-03-26 |
Family
ID=16536298
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP20722489A Pending JPH0371045A (en) | 1989-08-09 | 1989-08-09 | Method for detecting amino acid derivative with high sensitivity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0371045A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008063793A (en) * | 2006-09-06 | 2008-03-21 | Kurabo Ind Ltd | Mounting structure of building exterior decorative material |
-
1989
- 1989-08-09 JP JP20722489A patent/JPH0371045A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008063793A (en) * | 2006-09-06 | 2008-03-21 | Kurabo Ind Ltd | Mounting structure of building exterior decorative material |
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