JPH0511266B2 - - Google Patents
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- Publication number
- JPH0511266B2 JPH0511266B2 JP59201827A JP20182784A JPH0511266B2 JP H0511266 B2 JPH0511266 B2 JP H0511266B2 JP 59201827 A JP59201827 A JP 59201827A JP 20182784 A JP20182784 A JP 20182784A JP H0511266 B2 JPH0511266 B2 JP H0511266B2
- Authority
- JP
- Japan
- Prior art keywords
- fluorescence
- amino acid
- nanoseconds
- dimethylamino
- fluorescein
- 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.)
- Expired - Lifetime
Links
- 238000000034 method Methods 0.000 claims description 11
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 9
- 238000003277 amino acid sequence analysis Methods 0.000 claims description 8
- UGWULZWUXSCWPX-UHFFFAOYSA-N 2-sulfanylideneimidazolidin-4-one Chemical compound O=C1CNC(=S)N1 UGWULZWUXSCWPX-UHFFFAOYSA-N 0.000 claims description 6
- 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 claims description 6
- 238000001917 fluorescence detection Methods 0.000 claims description 4
- -1 4-(N,N-dimethylamino)-1-naphthylthiohydantoin Chemical compound 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 2
- 238000004811 liquid chromatography Methods 0.000 claims description 2
- SMZHGTXTWIAKGS-UHFFFAOYSA-N 4-isothiocyanato-n,n-dimethylnaphthalen-1-amine Chemical compound C1=CC=C2C(N(C)C)=CC=C(N=C=S)C2=C1 SMZHGTXTWIAKGS-UHFFFAOYSA-N 0.000 claims 1
- 238000000354 decomposition reaction Methods 0.000 claims 1
- 235000001014 amino acid Nutrition 0.000 description 11
- 229940024606 amino acid Drugs 0.000 description 11
- 150000001413 amino acids Chemical class 0.000 description 11
- 239000003480 eluent Substances 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 238000006731 degradation reaction Methods 0.000 description 7
- 150000002540 isothiocyanates Chemical class 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 238000005259 measurement Methods 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- QKFJKGMPGYROCL-UHFFFAOYSA-N phenyl isothiocyanate Chemical compound S=C=NC1=CC=CC=C1 QKFJKGMPGYROCL-UHFFFAOYSA-N 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000010828 elution Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229940117953 phenylisothiocyanate Drugs 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- JJVXHDTWVIPRBZ-VKHMYHEASA-N (2r)-2-[carboxy(methyl)amino]-3-sulfanylpropanoic acid Chemical compound OC(=O)N(C)[C@@H](CS)C(O)=O JJVXHDTWVIPRBZ-VKHMYHEASA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 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
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001685 time-resolved fluorescence spectroscopy Methods 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
Landscapes
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
【発明の詳細な説明】
本発明はたん白質、ペプチドの４−（Ｎ，Ｎ−
ジメチルアミノ）−１−ナフチルイソチオシアネ
ート（DNTC）あるいはフルオレセインイソチ
オシアネート（FITC）を用いたエドマン分解反
応で生じるアミノ酸のイソチオシアネート誘導
体、すなわちチオヒダントインの高感度測定によ
るアミノ酸配列分析法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to protein, peptide 4-(N,N-
This invention relates to an amino acid sequence analysis method using highly sensitive measurement of thiohydantoin, an isothiocyanate derivative of an amino acid produced in the Edman degradation reaction using dimethylamino)-1-naphthyl isothiocyanate (DNTC) or fluorescein isothiocyanate (FITC).
アミノ酸配列分析法において現在、最も多く使
用されている方法はフエニルイソチオシアネート
（PITC）をエドマン分解試薬として用い、生成
したフエニルチオヒダントインアミノ酸（PTH
アミノ酸）を紫外吸収検出器を検出器とする高速
液体クロマトグラフイーで分離、同定するもので
ある。しかし、この方法ではPTHアミノ酸の吸
収極大における分子吸光係数が小さいため（ε＝
約16000：269nm）感度が充分でなく、ピコモル
以下の微量たん白質、ペプチドのアミノ酸配列分
析への適用は困難である。最近では感度の向上の
ために、発螢光エドマン試薬としてDNTCや
FITCが使用されるようになつた。DNTCや
FITCをもちいたエドマン分解反応で生じるアミ
ノ酸のイソチオシアネート誘導体は発螢光物質で
あり、PTHアミノ酸に比較すると高感度に測定
できる。 Currently, the most commonly used method for amino acid sequence analysis uses phenyl isothiocyanate (PITC) as an Edman degradation reagent to generate phenylthiohydantoin amino acids (PTH
Amino acids) are separated and identified using high-performance liquid chromatography using an ultraviolet absorption detector. However, in this method, the molecular extinction coefficient at the maximum absorption of PTH amino acids is small (ε=
(approximately 16,000:269 nm) is not sensitive enough, making it difficult to apply to amino acid sequence analysis of trace amounts of proteins and peptides of picomole or less. Recently, in order to improve sensitivity, DNTC and other fluorescent Edman reagents have been used.
FITC has come into use. DNTC and
The isothiocyanate derivatives of amino acids produced by the Edman degradation reaction using FITC are fluorescent substances and can be measured with high sensitivity compared to PTH amino acids.
しかし、近年遺伝子工学や生物化学ではさらに
高感度のアミノ酸配列分析法が要求されている。 However, in recent years, even more sensitive amino acid sequence analysis methods have been required in genetic engineering and biochemistry.
従つて、本発明者らは高感度のアミノ酸配列分
析法を鋭意検討した結果、高速液体クロマトグラ
フイーの溶離液の螢光測定においては、一般の螢
光検出法におけるバツクグラウンド要因であるレ
イリ散乱、ラマン散乱、セル壁での散乱、溶媒の
不純物によるバツクグラウンド螢光が光パルス照
射後ナノ秒以下で減衰してしまうのに対し、
DNTCあるいはFITCを用いたエドマン分解反応
で生じるアミノ酸のイソチオシアネート誘導体の
螢光は、光パルス照射後、２〜10ナノ秒まで延び
ていることを見出した。 Therefore, as a result of intensive research into highly sensitive amino acid sequence analysis methods, the present inventors found that in the fluorescence measurement of eluents in high-performance liquid chromatography, Rayleigh scattering, which is a background factor in general fluorescence detection methods, has been found. , background fluorescence due to Raman scattering, cell wall scattering, and solvent impurities attenuates within nanoseconds after irradiation with a light pulse.
We found that the fluorescence of isothiocyanate derivatives of amino acids produced in the Edman degradation reaction using DNTC or FITC extends for 2 to 10 nanoseconds after irradiation with a light pulse.
従つて、フローセルに光パルス照射後、２〜10
ナノ秒以上遅れて螢光を測定すればバツクグラウ
ンド螢光を除去できることになり、高感度のアミ
ノ酸配列の分析が可能となるものである。 Therefore, after irradiating the flow cell with a light pulse, 2 to 10
By measuring fluorescence with a delay of nanoseconds or more, background fluorescence can be removed, making it possible to analyze amino acid sequences with high sensitivity.
さらに、この場合FITCあるいはDNTCを用い
たエドマン分解反応で生じるアミノ酸のイソチオ
シアネート誘導体の螢光信号はある程度減少する
が、螢光検出器の励起光源（光パルス）として光
強度の大きいパルスレーザーを使用すると、一般
に、螢光検出法においては単に光源強度を大きく
し、発生する螢光強度を強くしても感度は余り向
上しないことが多いにもかかわらず充分に強い螢
光信号が得られ、かつバツクグラウンド要因も除
去することができることを見い出した。 Furthermore, in this case, although the fluorescence signal of the isothiocyanate derivative of the amino acid produced by the Edman degradation reaction using FITC or DNTC is reduced to some extent, a pulsed laser with high light intensity is used as the excitation light source (light pulse) of the fluorescence detector. In general, in the fluorescence detection method, a sufficiently strong fluorescence signal can be obtained even though the sensitivity often does not improve much even if the intensity of the generated fluorescence is increased simply by increasing the light source intensity. It has been found that background factors can also be removed.
すなわち、本発明はDNTCあるいはFITCを用
いてエドマン分解反応で生じるアミノ酸のイソチ
オシアネート誘導体をパルスレーザを光源とする
時間分解螢光法で測定する高感度のアミノ酸配列
分析法を提供することにある。 That is, the present invention provides a highly sensitive amino acid sequence analysis method that uses DNTC or FITC to measure isothiocyanate derivatives of amino acids produced in the Edman degradation reaction by time-resolved fluorescence spectroscopy using a pulsed laser as a light source.
以下、本発明を詳細に説明する。 The present invention will be explained in detail below.
本発明に用いる装置は主要部が送液ポンプ、分
カウム、フローセルから構成されている液体クロ
マトグラフ部および主腰部が光源、フローセル、
分光部、電気回路部、演算回路から構成されてい
る。 The main part of the apparatus used in the present invention is a liquid chromatograph part consisting of a liquid pump, a liquid pump, a flow cell, and a main part consisting of a light source, a flow cell, and a liquid chromatograph part.
It consists of a spectroscopic section, an electric circuit section, and an arithmetic circuit.
第１図に基ずいて、試料（DNTCあるいは
FITCを用いたエドマン分解反応で生じるアミノ
酸の（イソチオシアネート誘導体）を分析する液
体クロマトグラフイの実施例を説明する。 Based on Figure 1, the sample (DNTC or
An example of liquid chromatography for analyzing amino acids (isothiocyanate derivatives) produced by Edman degradation reaction using FITC will be described.
第１図において、試料は２つの溶離液貯槽１，
２に貯えられた溶離液はグラジエント溶離装置３
により、次第にその組成比を、変えながら溶離液
送液ポンプ５により分離カラム６に導入される溶
離液中に試料導入装置４により注入され、分離カ
ラム６にて各成分に分離された後さらにフローセ
ル７に導かれ検出される。 In Figure 1, the sample is placed in two eluent reservoirs 1, 1,
The eluent stored in 2 is transferred to gradient elution device 3.
The sample introduction device 4 injects the eluent into the separation column 6 by the eluent feed pump 5 while gradually changing its composition ratio, and the sample is separated into each component by the separation column 6, and then further transferred to the flow cell. 7 and detected.
一般に、試料の成分の数が多い場合、単一の溶
離液で分離するよりも次第に溶離液組成を変化さ
せるグラジエント溶離のほうが分析時間が短縮で
きるため好ましい。 Generally, when a sample has a large number of components, gradient elution, in which the composition of the eluent is gradually changed, is preferable to separation using a single eluent because the analysis time can be reduced.
検出方法は、第２図に基ずいて説明する。第２
図において、４−（Ｎ，Ｎ−ジメチルアミノ）−１
−ナフチルヒダントインの分析の場合は窒素レー
ザー８、フルオレセインイソチオヒダントインの
場合は窒素レーザー８をボンビング源とする色素
レーザ９を光源として使用する。レーザ光の１部
はビームスプリツタ１０で取り出され、ピンフオ
トダイオド１１で検出し、遅延回路１５で遅延
後、サンプルホールド回路１７のトリガ信号とし
用い、また、ピンフオトダイオド１１で検出した
信号の１部はピークホールド回路１６によりホー
ルドし螢光強度補正用の参照信号とし用いる。レ
ーザー光の大部分はビームスプリツタ１０を通過
し、フローセル７に集光され、分離カラム１０に
て分離された各成分に照射される。フローセル７
から発生した螢光はレーザー光に対して直角方向
で集光され、さらに分光器１２で分光した後、マ
イクロチヤンネルプレート内蔵型高速光電子増倍
管１３で検出する。その信号はプリアンプ１４で
増幅され、遅延回路１５で設定された遅延時間で
サンプルホールドされ、演算回路１８で参照信号
によりレーザー光の光量変化を補償後、記録計１
９で記録される。 The detection method will be explained based on FIG. Second
In the figure, 4-(N,N-dimethylamino)-1
- In the case of analysis of naphthylhydantoin, a nitrogen laser 8 is used as a light source, and in the case of fluorescein isothiohydantoin, a dye laser 9 whose bombing source is a nitrogen laser 8 is used as a light source. A portion of the laser beam is taken out by the beam splitter 10, detected by the pin photo diode 11, delayed by the delay circuit 15, used as a trigger signal for the sample and hold circuit 17, and also detected by the pin photo diode 11. A portion of the signal is held by a peak hold circuit 16 and used as a reference signal for fluorescence intensity correction. Most of the laser light passes through the beam splitter 10, is focused on the flow cell 7, and is irradiated onto each component separated by the separation column 10. flow cell 7
The fluorescent light generated from the laser beam is focused in a direction perpendicular to the laser beam, further separated into spectra by a spectrometer 12, and then detected by a high-speed photomultiplier tube 13 with a built-in microchannel plate. The signal is amplified by the preamplifier 14, sampled and held at a delay time set by the delay circuit 15, and after compensating for changes in the amount of laser light using a reference signal in the arithmetic circuit 18, the signal is sent to the recorder 1.
Recorded at 9.
以下、実施例に基づいて、本発明を詳細に説明
する。 Hereinafter, the present invention will be explained in detail based on Examples.
実施例 １
第３図、第４図にそれぞれ０ナノ秒、５ナノ秒
の遅延時間を設定して測定した10フエムトモルの
フルオレセインチオヒダントインのクロマトグラ
ムを示す。溶離液は0.1Mリン酸緩衝液（PH7.0）
を用い５％から50％までのアセトニトリルによる
リニアグラジエント溶離法を用い、使用したカラ
ムはTSKgel ODS 120T（東洋曹達工業(株)製）、
流速は１ml／minで測定した。螢光測定は515nm
の螢光強度を測定した。遅延時間が０ナノ秒の場
合、ベースラインの雑音が大きいが、遅延時間を
５ナノ秒とすると散乱光および短寿命の螢光が除
去されるため、明瞭なクロマトグラムが得られ
る。Example 1 Figures 3 and 4 show chromatograms of 10 femtomoles of fluorescein thiohydantoin measured with delay times of 0 nanoseconds and 5 nanoseconds, respectively. Eluent is 0.1M phosphate buffer (PH7.0)
The columns used were TSKgel ODS 120T (manufactured by Toyo Soda Kogyo Co., Ltd.),
The flow rate was measured at 1 ml/min. Fluorescence measurement is 515nm
The fluorescence intensity was measured. When the delay time is 0 nanoseconds, the baseline noise is large, but when the delay time is 5 nanoseconds, scattered light and short-lived fluorescent light are removed, so a clear chromatogram can be obtained.
実施例 ２
第５図、第６図にそれぞれ０ナノ秒、10ナノ秒
の遅延時間を設定して測定した20フエムトモルの
４−（Ｎ，Ｎ−ジメチルアミノ）−１−ナフチルチ
オヒダントインのクロマトグラムを示す。分離条
件は第３図の条件と同様である。螢光測定は、窒
素レーザ光（337nm）で励起し450nmの螢光強度
を測定している。フルオレセインチオヒダントイ
ンの場合と同様、遅延時間10ナノ秒のクロマトグ
ラムでは短寿命の雑音螢光が除去され、大きな信
号／雑音比が得られる。Example 2 Chromatograms of 20 femtomoles of 4-(N,N-dimethylamino)-1-naphthylthiohydantoin measured with delay times of 0 nanoseconds and 10 nanoseconds shown in FIGS. 5 and 6, respectively. shows. The separation conditions are similar to those shown in FIG. Fluorescence measurement is performed by exciting with nitrogen laser light (337 nm) and measuring the fluorescence intensity at 450 nm. As with fluorescein thiohydantoin, a 10 ns delay time chromatogram eliminates short-lived noise fluorescence and provides a large signal-to-noise ratio.
以上述べたようにパルスレーザを光源とする時
間分解螢光器を用いた高速液体クロマトグラフイ
がフルオレセインチオヒダントインや４−（Ｎ，
Ｎ−ヂメチルアミノ）−１−ナフチルチオヒダン
トインを分析する高感度アミノ酸配列分析法に有
効であることが明らかである。 As mentioned above, high-performance liquid chromatography using a time-resolved fluorescence device using a pulsed laser as a light source is effective for fluorescein thiohydantoin, 4-(N,
It is clear that this method is effective for highly sensitive amino acid sequence analysis for analyzing N-dimethylamino)-1-naphthylthiohydantoin.
第１図および第２図は本発明に用いる装置の液
体クロマトグラフ部および螢光検出部の模式図。
第３図、第４図はフルオレセインチオヒダントイ
ン（10フエムトモルのアミノ酸）の螢光遅延時間
０ナノ秒、５ナノ秒で検出したクロマトグラム、
第５図、第６図は４−（Ｎ，Ｎ−ヂメチルアミノ）
−１−ナフチルチオヒダントイン（20フエムトモ
ノのアミノ酸）の螢光遅延時間０ナノ秒、10ナノ
秒で検出したクロマトグラムである。
１，２……溶離液貯槽、３……グラジエント溶
離装置、４……試料導入装置、５……送液ポン
プ、６……分離カラム、７……フローセル、８…
…窒素レーザ、９……色素レーザ、１０……ビー
ムスプリツタ、１１……ピンホトダイオド、１２
……分光器、１３……高速光電子増倍管、１４…
…プリアンプ、１５……遅延回路、１６……ピー
クホールド回路、１７……サンプルホールド回
路、１８……演算回路、１９……記録計、２１…
…アスパラギン酸、２２……グルタミン酸、２３
……アスパラギン、２４……セリン、２５……カ
ルボキシメチルシステイン、２６……グリシン、
２７……グルタミン、２８……ヒスチジン、２９
……アルギニン、３０……アラニン、３１……セ
リンの分解物、３２……チロシン、３３……スレ
オニン、３４……ブロリン、３５……メチオニ
ン、３６……バリン、３７……トリブトフアン、
３８……フエニルアラニン、３９……イソロイシ
ン、４０……ロイシン、４１……リジン。
FIGS. 1 and 2 are schematic diagrams of a liquid chromatograph section and a fluorescence detection section of the apparatus used in the present invention.
Figures 3 and 4 are chromatograms of fluorescein thiohydantoin (10 femtomoles of amino acid) detected at fluorescence delay times of 0 and 5 nanoseconds.
Figures 5 and 6 show 4-(N,N-dimethylamino)
This is a chromatogram of -1-naphthylthiohydantoin (20 femtomonoamino acids) detected at fluorescence delay times of 0 nanoseconds and 10 nanoseconds. 1, 2... Eluent storage tank, 3... Gradient elution device, 4... Sample introduction device, 5... Liquid feed pump, 6... Separation column, 7... Flow cell, 8...
...Nitrogen laser, 9...Dye laser, 10...Beam splitter, 11...Pin photodiode, 12
...Spectrometer, 13...High-speed photomultiplier tube, 14...
... Preamplifier, 15 ... Delay circuit, 16 ... Peak hold circuit, 17 ... Sample hold circuit, 18 ... Arithmetic circuit, 19 ... Recorder, 21 ...
...Aspartic acid, 22...Glutamic acid, 23
... Asparagine, 24 ... Serine, 25 ... Carboxymethylcysteine, 26 ... Glycine,
27...Glutamine, 28...Histidine, 29
... Arginine, 30 ... Alanine, 31 ... Degradation product of serine, 32 ... Tyrosine, 33 ... Threonine, 34 ... Broline, 35 ... Methionine, 36 ... Valine, 37 ... Tributophan,
38...Phenylalanine, 39...Isoleucine, 40...Leucine, 41...Lysine.
Claims (1)
ルイソチオシアネートあるいはフルオレセインイ
ソチオシアネートを用いたエドマン分解反応で生
じる４−（Ｎ，Ｎ−ジメチルアミノ）−１−ナフチ
ルチオヒダントインあるいはフルオレセインチオ
ヒダントインを液体クロマトグラフイで分離し、
パルスレーザを光源とする時間分解螢光検出法で
検出することを特徴とするアミノ酸配列分析法。1 4-(N,N-dimethylamino)-1-naphthylthiohydantoin or fluorescein thiohydantoin produced in the Edman decomposition reaction using 4-(N,N-dimethylamino)-1-naphthylisothiocyanate or fluorescein isothiocyanate. Separated by liquid chromatography,
An amino acid sequence analysis method characterized by detection using a time-resolved fluorescence detection method using a pulsed laser as a light source.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20182784A JPS61233371A (en) | 1984-09-28 | 1984-09-28 | Analysis of amino acid sequence |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20182784A JPS61233371A (en) | 1984-09-28 | 1984-09-28 | Analysis of amino acid sequence |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS61233371A JPS61233371A (en) | 1986-10-17 |
| JPH0511266B2 true JPH0511266B2 (en) | 1993-02-15 |
Family
ID=16447550
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP20182784A Granted JPS61233371A (en) | 1984-09-28 | 1984-09-28 | Analysis of amino acid sequence |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS61233371A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4837726A (en) * | 1987-06-19 | 1989-06-06 | Applied Biosystems, Inc. | Quantitation of chromatographic information |
| JP2725731B2 (en) * | 1991-02-28 | 1998-03-11 | 株式会社島津製作所 | Quenching method for excess fluorescent reagent |
| WO1994002854A1 (en) * | 1992-07-27 | 1994-02-03 | Seiko Instruments Inc. | Method for high-sensitivity detection of amino acid derivative |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5847239A (en) * | 1981-09-14 | 1983-03-18 | Nisshin Denki Seisakusho:Kk | Liquid chromatograph apparatus |
-
1984
- 1984-09-28 JP JP20182784A patent/JPS61233371A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5847239A (en) * | 1981-09-14 | 1983-03-18 | Nisshin Denki Seisakusho:Kk | Liquid chromatograph apparatus |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS61233371A (en) | 1986-10-17 |
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