JPH03160370A - Hydrolysis of protein - Google Patents
Hydrolysis of proteinInfo
- Publication number
- JPH03160370A JPH03160370A JP30002289A JP30002289A JPH03160370A JP H03160370 A JPH03160370 A JP H03160370A JP 30002289 A JP30002289 A JP 30002289A JP 30002289 A JP30002289 A JP 30002289A JP H03160370 A JPH03160370 A JP H03160370A
- Authority
- JP
- Japan
- Prior art keywords
- acid
- tube
- test tube
- protein
- indole
- 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
- 230000007065 protein hydrolysis Effects 0.000 title claims description 3
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 claims abstract description 32
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 29
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000002253 acid Substances 0.000 claims abstract description 27
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 claims abstract description 16
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000011259 mixed solution Substances 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000007787 solid Substances 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 53
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 44
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 22
- 239000012071 phase Substances 0.000 claims description 14
- 230000003301 hydrolyzing effect Effects 0.000 claims description 7
- 238000010574 gas phase reaction Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000007790 solid phase Substances 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 238000012360 testing method Methods 0.000 abstract description 26
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 17
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 abstract description 14
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 abstract description 14
- 230000007062 hydrolysis Effects 0.000 abstract description 13
- 150000001413 amino acids Chemical class 0.000 abstract description 8
- 239000003708 ampul Substances 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 5
- 238000010438 heat treatment Methods 0.000 abstract description 5
- 238000001816 cooling Methods 0.000 abstract description 3
- 230000008016 vaporization Effects 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 238000001035 drying Methods 0.000 abstract description 2
- XTEGARKTQYYJKE-UHFFFAOYSA-N chloric acid Chemical compound OCl(=O)=O XTEGARKTQYYJKE-UHFFFAOYSA-N 0.000 abstract 4
- 229940005991 chloric acid Drugs 0.000 abstract 4
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 abstract 2
- 108010016626 Dipeptides Proteins 0.000 abstract 1
- 239000007792 gaseous phase Substances 0.000 abstract 1
- 229950003188 isovaleryl diethylamide Drugs 0.000 abstract 1
- 238000007789 sealing Methods 0.000 abstract 1
- -1 trifluoro accetate Chemical compound 0.000 abstract 1
- 125000002114 valyl group Chemical group 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 22
- 238000011084 recovery Methods 0.000 description 11
- 235000001014 amino acid Nutrition 0.000 description 7
- 239000007791 liquid phase Substances 0.000 description 7
- 239000000523 sample Substances 0.000 description 6
- 102000016943 Muramidase Human genes 0.000 description 5
- 108010014251 Muramidase Proteins 0.000 description 5
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 5
- 229960000274 lysozyme Drugs 0.000 description 5
- 239000004325 lysozyme Substances 0.000 description 5
- 235000010335 lysozyme Nutrition 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 238000011109 contamination Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000012916 structural analysis Methods 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-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
- 102000036675 Myoglobin Human genes 0.000 description 1
- 108010062374 Myoglobin Proteins 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- XXDVDTMEVBYRPK-XPUUQOCRSA-N Val-Gln Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O XXDVDTMEVBYRPK-XPUUQOCRSA-N 0.000 description 1
- UPJONISHZRADBH-XPUUQOCRSA-N Val-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O UPJONISHZRADBH-XPUUQOCRSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ZNEWHQLOPFWXOF-UHFFFAOYSA-N coenzyme M Chemical compound OS(=O)(=O)CCS ZNEWHQLOPFWXOF-UHFFFAOYSA-N 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229960004635 mesna Drugs 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000003746 solid phase reaction Methods 0.000 description 1
- FWMUJAIKEJWSSY-UHFFFAOYSA-N sulfur dichloride Chemical compound ClSCl FWMUJAIKEJWSSY-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (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酸＆［ｌ戒分析の第一段
階であるタンパク賞の加水分解を行う方法に関する．
〔発明の概要〕
本発明は、タンパク質のア竃ノ酸組戒分析の第一段階で
ある加水分解操作において、固体表面上に吸着したタン
パク質を塩酸、トリフルオロ酢酸、チオグリコール酸お
よび水を含む混合溶液を気化させた酸混合蒸気との固相
・気相反応により加水分解し、この際、前記酸混合蒸気
の供給される空間内にインドールを共存させることによ
り、従来の塩酸とトリフルオロ酢酸を用いる気相法の有
していた高速−性、低汚染性および自動化の容易性に加
えて、現在までの方法では回収が不可能であったトリプ
トファンをも定量可能としたものである。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for hydrolyzing protein, which is the first step in amino acid analysis of protein. [Summary of the Invention] The present invention provides a method for treating proteins adsorbed on a solid surface with hydrochloric acid, trifluoroacetic acid, thioglycolic acid, and water in a hydrolysis operation that is the first step in the analysis of amino acid composition of proteins. The mixed solution is hydrolyzed by a solid phase/gas phase reaction with the vaporized acid mixed vapor, and at this time, by allowing indole to coexist in the space where the acid mixed vapor is supplied, conventional hydrochloric acid and trifluoroacetic acid can be dissolved. In addition to the high speed, low contamination, and ease of automation possessed by the gas-phase method using the method, this method also makes it possible to quantify tryptophan, which has been impossible to recover with conventional methods.
従来、タンパク質の加水分解は例えば、次のような方法
により行われていた．
第１の方法は共沸点塩酸を用いる液相法であり、最も古
くから用いられているものである（Ｓ．ムーア，Ｈ．ス
タイン メンツズ イン エンザイモロジ− Ｓ．Ｐ．
コロビソク，Ｎ．Ｏ．カブラ偏 １９６３年 １８１９
〜８３１ページ，アカデミックプレス ニューヨーク）
。まず試験管底の乾燥試料に蒸留した共沸点塩酸を加え
、この試験管を水冷しながら減圧下で封管する．次いで
、このアンプルを１０５℃〜１１０℃で２４時間〜１４
４時間加熱する．次にこのアンプルを開青し塩酸を蒸発
除去する．
第２の方法はメルカブトエタンスルホン酸を用いる液相
法であり、特にトリプトファンの定量を目的として用い
られたものである（Ｂ．ペンケＲ．ファレンツィ，Ｋ．
コバクス，アナリティカルバイオケ壽ストリー １９７
４年 ６０巻 ４５〜５０ページ）．操作法は第１の方
法に準じる。共沸点塩酸の代わりに３Ｍメルカプトエタ
ンスルホン酸を用い、１１０±２℃で２４時間〜７２時
間加熱する。アンプルを開管した後、試料溶液を中和す
る。Conventionally, protein hydrolysis has been carried out, for example, by the following methods. The first method is a liquid phase method using azeotropic hydrochloric acid and is the oldest method used (S. Moore, H. Steinments in Enzymology, S.P.
Kolobisok, N. O. Cabra bias 1963 1819
~831 pages, Academic Press New York)
. First, add distilled azeotropic hydrochloric acid to the dry sample at the bottom of the test tube, and seal the test tube under reduced pressure while cooling with water. This ampoule was then heated at 105°C to 110°C for 24 hours to 14 hours.
Heat for 4 hours. Next, open the ampoule and remove the hydrochloric acid by evaporation. The second method is a liquid phase method using mercabutoethanesulfonic acid, which was used specifically for the purpose of quantifying tryptophan (B. Penke, R. Farenzi, K.
Kovacs, Analytical Biochemistry Story 197
4th grade, volume 60, pages 45-50). The operating method is the same as the first method. 3M mercaptoethanesulfonic acid is used instead of azeotropic hydrochloric acid and heated at 110±2° C. for 24 to 72 hours. After opening the ampoule, neutralize the sample solution.
第３の方法は、次田らにより報告された塩酸とトリフル
オロ酢酸とを含む＃混合蒸気を用いる高速気相法である
．この方法は特開昭６１ − １５１１５７号公報に開
示されている。塩酸とトリフルオロ酢酸を含む水溶液を
入れておいた試験管に、あらかじめ底に試料を乾固して
おいた小試験管を入れ、第１，第２の方法と同様に封督
する。１５８℃で２２．５分間〜４５分間加熱後、開督
し、小試験管内に残存する酸を減圧除去する．
〔発明が解決しようとする課題〕
共沸点塩酸を用いる液相法には、長時間の加熱が必要で
、疎水性タンパク質の分解が不十分であるという欠点が
あった．メルカブトエタンスルホン酸を用いる液相法も
また長時間の加熱を要し、更に加水分解後に中和するた
めのアルカリ添加操作を必要とする欠点があった．また
液相法共通の欠点として、酸溶液からの汚染が避けられ
ないことが挙げられる．特に試料が微量である際にこの
影響は顕著である．近年、次田らにより報告された高速
気相法は、以上のｔＪ４Ｂ・を克服しているが、共沸点
塩酸法と同様にトリプトファンの定量が不可能であると
いう欠点を有している．
（！ＩＢを解決するための手段〕
本発明は前記の欠点を除去するためになされたものであ
り、高速気相法を改良し、固定表面上に吸着したタンパ
ク賞を塩酸、トリフロオロ酢酸、チオグリコール酸およ
び水を含む混合溶液を気化させた酸混合蒸気との固相、
気相反応により加水分解し、この際前記酸混合蒸気の供
給される空間内にインドールを共存させることとした．
〔作用〕
上記のような加水分解法によれば、固体表面上に吸着さ
れたタンパク賞に、塩酸、トリフルオロ酢酸、チオグリ
コール酸および水を含む混合溶液を気化させた酸混合蒸
気が作用し、この酸混合蒸気の供給される空間内にイン
ドールの蒸気を共存させることによってトリプトファン
の分解反応の進行が抑制された条件下でタンパク質のペ
プチド結合の加水分解反応が進行する。また、この加水
分解反応は短時間で完了し、気相法であるため酸混合溶
液からのアξノ酸の汚染はない。The third method is a high-speed gas phase method using a mixed vapor containing hydrochloric acid and trifluoroacetic acid, which was reported by Tsugita et al. This method is disclosed in Japanese Patent Application Laid-Open No. 151157/1983. A small test tube with a sample previously dried at the bottom is placed in a test tube containing an aqueous solution containing hydrochloric acid and trifluoroacetic acid, and sealed in the same manner as in the first and second methods. After heating at 158°C for 22.5 to 45 minutes, the tube is opened and the acid remaining in the small test tube is removed under reduced pressure. [Problems to be Solved by the Invention] The liquid phase method using azeotropic hydrochloric acid has the disadvantage that long-term heating is required and hydrophobic proteins are insufficiently decomposed. The liquid phase method using mercabutoethanesulfonic acid also had the disadvantage of requiring long heating times and addition of an alkali for neutralization after hydrolysis. A common drawback of liquid phase methods is that contamination from acid solutions is unavoidable. This effect is particularly noticeable when the amount of sample is small. The high-speed gas phase method recently reported by Tsugita et al. overcomes the above tJ4B, but it has the same drawback as the azeotropic hydrochloric acid method in that it is impossible to quantify tryptophan. (!Means for solving IB) The present invention has been made to eliminate the above-mentioned drawbacks, and it improves the high-speed gas phase method and uses hydrochloric acid, trifluoroacetic acid, thiochloride, etc. Solid phase with acid mixed vapor obtained by vaporizing a mixed solution containing glycolic acid and water,
Hydrolysis was carried out by a gas phase reaction, and at this time, indole was allowed to coexist in the space to which the acid mixed vapor was supplied.
[Effect] According to the hydrolysis method described above, an acidic mixed vapor produced by vaporizing a mixed solution containing hydrochloric acid, trifluoroacetic acid, thioglycolic acid, and water acts on the protein adsorbed on the solid surface. By allowing indole vapor to coexist in the space where the acid mixed vapor is supplied, the hydrolysis reaction of the peptide bonds of proteins proceeds under conditions in which the progress of the tryptophan decomposition reaction is suppressed. Furthermore, this hydrolysis reaction is completed in a short time, and since it is a gas phase method, there is no contamination of the ξ-anoic acid from the acid mixed solution.
実施例−１
本実施例においては、本発明による加水分解を行う基本
的方法を示す．
第１図（ａｌに示すように、ジベブチドの１つであるバ
リルグルタミンＭｌを小試験管２に２．２ｎｍｏｌ取り
十分乾燥させる．次にこの小試験管２を中試験管３に入
れる．この中試験管３の底にはあらかしめ２００μｌの
７Ｍ塩酸、１０％トリフルオロ酢酸、２０％チオグリコ
ール酸および水を含む酸混合溶液４が入れられている．
この中試験管３と小試験管２とをそのまま試験管５に入
れる．この試験管５の底にぱあらかしめ、ｌｍｇのイン
ドール６が入れられている．そして、この試験管５を氷
で冷却しながら、アンプル形状に真空封管７を行う．封
管した試験管５をオイルバスに入れ２５分間加熱する．
加熱後、小試験管２を取り出し、真空デシケータで十分
塩酸、トリフルオロ酢酸あるいはチオグリコール酸を除
去する．乾燥後、小試験管２に１２０Ｍｅの０．０ＩＭ
塩酸を入れて加水分解生戊物を溶解し、ア旦ノ酸分析装
置で分析する。Example-1 In this example, a basic method for carrying out hydrolysis according to the present invention is shown. As shown in Figure 1 (al), take 2.2 nmol of valylglutamine Ml, one of dibebutide, into a small test tube 2 and dry it thoroughly.Next, put this small test tube 2 into a medium test tube 3. At the bottom of test tube 3, approximately 200 μl of acid mixed solution 4 containing 7M hydrochloric acid, 10% trifluoroacetic acid, 20% thioglycolic acid, and water was placed.
Place medium test tube 3 and small test tube 2 directly into test tube 5. 1mg of indole 6 is placed in the bottom of test tube 5. Then, while cooling the test tube 5 with ice, vacuum seal the tube 7 into an ampoule shape. Place the sealed test tube 5 in an oil bath and heat for 25 minutes.
After heating, take out small test tube 2 and thoroughly remove hydrochloric acid, trifluoroacetic acid, or thioglycolic acid in a vacuum desiccator. After drying, add 0.0 IM of 120Me to small test tube 2.
Add hydrochloric acid to dissolve the hydrolyzed raw material, and analyze with an amino acid analyzer.
第２図の曲線ａは一定時間（１０分）での種々の温度に
おける加水分解率をプロットしたものである。比較のた
め、共沸点塩酸を用いる方法により得られた結果を曲＆
Ｉｂ，次田らにより報告された高速気相法により得られ
た結果を曲線Ｃに示した．本実施例より明らかなように
、本発明による方法はタンパク質を加水分解する目的に
かなっており、更にその加水分解反応速度は反応温度を
上げると著しく加速され、共沸点塩酸を用いる方法のそ
れにまさり、高速気相法のそれにほぼ匹敵することがわ
かる．
本発明による加水分解法は、第１図（ｂｌに示すように
、第ｌ図ｔａ＋に示した方法における酸混合溶液４とイ
ンドール６との位置を交換して実施することもできる。Curve a in FIG. 2 is a plot of the hydrolysis rate at various temperatures over a fixed period of time (10 minutes). For comparison, the results obtained by the method using azeotropic hydrochloric acid are
Curve C shows the results obtained by the high-speed gas phase method reported by Ib, Tsugita et al. As is clear from this example, the method according to the present invention is suitable for the purpose of hydrolyzing proteins, and furthermore, the rate of hydrolysis reaction is significantly accelerated by increasing the reaction temperature, and is superior to that of the method using azeotropic hydrochloric acid. , which is almost comparable to that of the high-speed gas phase method. The hydrolysis method according to the present invention can also be carried out by exchanging the positions of the acid mixed solution 4 and indole 6 in the method shown in FIG. 1 (ta+), as shown in FIG. 1 (bl).
また第１図ｆｃｌに示すように、酸混合溶液４を入れた
中試験管３とバリルグルタξン酸工を入れた小試験管と
を試験管５内に並べる形でおくことによっても実施でき
る．また第１図＋ｄｉに示すように、第１図（Ｃｌにお
ける酸混合溶液４とインドール６の位置を交換して実施
することもできる．
実施例−２
本実施例においては、本発明による方法におけるタンパ
ク質を構成するアミノ酸の回収率が、トリプトファンを
含有するタンパク質の組戒分析をする目的にかなうもの
であることを示す．実施例ｌと同様の方法で２０種のア
ミノ酸について加水分解の條件にさらし回収率を求めた
ところ第１表の結果を得た．比較のために次田らによっ
て検討された高速気相法（１５８℃）による結果も合わ
せて示した．第１表中の数値は１６６℃で加２８後の回
収率をアラニンの値を１００％として表している．この
表から明らかなように、本発明による方法は従来の気相
法によっては不可能であったトリプトファンを定量可能
としている．また同時に従来の方法では経時的に回収率
の減少するメチオニンを定置的に回収することができる
．
実施例−３
本実施例においては、チオグリコール酸の添加がトリプ
トファンの回収に効果的であることを示す．実験は実施
例１あるいは２と同様の方法を用いた．タンパク質試料
としてリゾチームを用い、チオグリコール酸の添加量だ
けを変化させた時のトリプトファンの回収率をまとめて
第３図に示した．この図から明らかなように、チオグリ
コール酸の添加によってトリブトファンの回収率は向上
し、２０％以上ではほぼ一定になることがわかる。It can also be carried out by placing a medium test tube 3 containing the acid mixture solution 4 and a small test tube containing barylglutanic acid in a test tube 5, as shown in FIG. In addition, as shown in Figure 1 + di, it is also possible to carry out the experiment by exchanging the positions of the acid mixed solution 4 and indole 6 in Figure 1 (Cl). This shows that the recovery rate of amino acids constituting proteins is suitable for the purpose of structural analysis of proteins containing tryptophan.Twenty amino acids were exposed to hydrolysis conditions in the same manner as in Example 1. When the recovery rate was determined, the results shown in Table 1 were obtained.For comparison, the results from the high-speed gas phase method (158°C) studied by Tsugita et al. are also shown.The values in Table 1 are 166 The recovery rate after addition at 28 °C is expressed with the value of alanine as 100%.As is clear from this table, the method according to the present invention makes it possible to quantify tryptophan, which was not possible using the conventional gas phase method. At the same time, methionine, whose recovery rate decreases over time in conventional methods, can be recovered in a stationary manner.Example 3 In this example, the addition of thioglycolic acid was effective in recovering tryptophan. The experiment was conducted using the same method as in Example 1 or 2. Figure 3 summarizes the tryptophan recovery rate when lysozyme was used as the protein sample and only the amount of thioglycolic acid added was changed. As is clear from this figure, the tributophane recovery rate improves with the addition of thioglycolic acid, and becomes almost constant above 20%.
実施例−４
本実施例においては、本発明による加水分解法における
インドール蒸気の使用がトリプトファンの回収に効果的
であることを示す．実験は実施例ｌあるいは２と同様の
方法を用いた．タンパク質試料としてリゾチームを用い
、インドールの量を変化させた時のトリブトファンの回
収率をまとめて第４図に示した．この図から明らかなよ
うに、インドールの使用によってトリプトファンの回収
率は向上し０．１９以上の使用ではほぼ一定になること
がわかる．
実施例−５
本実施例においては、本発明による加水分解法を種々の
タンパク質に応用した例を示す．実験は実施例１と同様
の方法を用いた。用いたタンパク賞は以下の通りである
．
リオグロピン （第、２表ｆａｔに示す）リゾチーム
（第２表ｆｂ）に示す）パパ イ ン （第２表
ｆｃｌに示す）結果を第２表ｆａｔ〜ｆｃｌにまとめた
。ミオグロビンについては特に比較のために次田らが報
告している高速気相法と、共沸点塩酸を用いる液相法に
よる結果も合わせて第２表Ｔａｌ〜（Ｃ）にまとめた。Example 4 This example shows that the use of indole steam in the hydrolysis method of the present invention is effective in recovering tryptophan. The experiment used the same method as in Example 1 or 2. Figure 4 shows a summary of the recovery rates of tributophane when the amount of indole was varied using lysozyme as a protein sample. As is clear from this figure, the recovery rate of tryptophan improves with the use of indole and becomes almost constant when 0.19 or more is used. Example 5 This example shows an example in which the hydrolysis method of the present invention is applied to various proteins. The experiment used the same method as in Example 1. The protein awards used are as follows. Lioglopine (shown in Table 2 fat) Lysozyme (shown in Table 2 fb)) Papain (shown in Table 2 fcl) The results are summarized in Table 2 fat to fcl. Regarding myoglobin, the results of the high-speed gas phase method reported by Tsugita et al. and the liquid phase method using azeotropic hydrochloric acid are also summarized in Table 2 Tal~(C) for comparison.
表中のＴの項はタンパク質の配列分析から得られた理論
値であり、ＳｅｒとＴｈｒのカツコ内の数値はゼロ時間
外挿値である。この表から明らかなように、トリプトフ
ァンを含むアミノ酸Ｍｉ戒値は実験値と理論値がよく一
致しており、本発明によるタンパク質を加水分解する方
法が、タンパク質の正確な組戒分析を行うために非常に
有効であることがわかる。また、タンパク試料の回収率
は１００％であった。この結果から、本発明はタンパク
質を定量的に分解していることがわかる。従ってタンパ
ク質の定量にも用いることがわかる。The term T in the table is a theoretical value obtained from protein sequence analysis, and the values in brackets for Ser and Thr are zero time extrapolated values. As is clear from this table, the experimental value and the theoretical value of the amino acid Mi value containing tryptophan are in good agreement, and the method of hydrolyzing proteins according to the present invention is useful for performing accurate structural analysis of proteins. It turns out to be very effective. Moreover, the recovery rate of the protein sample was 100%. This result shows that the present invention quantitatively degrades proteins. Therefore, it can be seen that it can also be used for protein quantification.
第２表ｔａ＋
第
２表山》
第
２表（Ｃｌ
〔発明の効果〕
以上説明したように、本発明の方法によれば、従来の気
相加水分解法の有していた高速性、低汚染性および自動
化の容易性を損なうことなく、困難であったトリプトフ
ァンの定量を含むタンパク質のアミノ酸紐放分折を行う
ことができる．このように本発明による塩酸、トリフル
オロ酢酸、チオグリコール酸、水およびインドールを用
いるタンパク質の気相加水分解法には、従来にない優れ
た点があり、その工業的価値は大である．なお、ここで
使用した用語および説明は、本発明の特徴が維持される
限り、類似のものを除去するものでないことば言うまで
もない．Table 2 ta + Table 2 Mountain》 Table 2 (Cl [Effects of the Invention] As explained above, the method of the present invention provides high speed and low pollution compared to the conventional gas phase hydrolysis method. Amino acid string dissociation analysis of proteins, including the determination of tryptophan, which has been difficult, can be performed without compromising performance and ease of automation.In this way, hydrochloric acid, trifluoroacetic acid, thioglycolic acid, The gas phase hydrolysis method of proteins using indole and indole has unprecedented advantages and has great industrial value.The terms and explanations used here are used to maintain the characteristics of the present invention. Needless to say, this term does not exclude similar items as long as they are similar.
第１図＋ａ）〜ｌｄｌはそれぞれ本発明に用いる実験容
器の断面図、第２図は本発明の共沸点塩酸を用いる液相
法および高速気相法によるバリルグルタミン酸の温度一
加水分解率曲線図、第３図はりゾチーム中のトリブトフ
ァンの回収率に与えるチオグリコール酸の添加量の影響
図、第４図はリゾチーム中のトリプトファンの回収率に
与えるインド−ル共存量の影響図である。
・バリルグルタミン酸
・小試験管
・中試験管
・酸混合溶液
・試験管
・インドール
・真空封管Figure 1 +a) to ldl are cross-sectional views of experimental vessels used in the present invention, respectively, and Figure 2 is a temperature-hydrolysis rate curve of valylglutamic acid obtained by the liquid phase method and high-speed gas phase method using azeotropic hydrochloric acid of the present invention. , FIG. 3 is a graph showing the influence of the amount of thioglycolic acid added on the recovery rate of tributophane in lysozyme, and FIG. 4 is a graph showing the influence of the amount of coexisting indole on the recovery rate of tryptophan in lysozyme.・Barylglutamic acid・Small test tube・Medium test tube・Acid mixed solution・Test tube・Indole・Vacuum sealed tube
Claims (5)
酸、トリフルオロ酢酸（ＣＦ＿３ＣＯＯＨ）、チオグリ
コール酸（ジメチルサルファイド−α，α′−ジカルボ
ン酸）および水を含む酸混合溶液を気化させた酸混合蒸
気（気相）との固相−気相反応により前記タンパク質を
加水分解することを特徴とするタンパク質の加水分解方
法。(1) A mixed acid solution containing protein (solid phase) adsorbed on a solid surface, hydrochloric acid, trifluoroacetic acid (CF_3COOH), thioglycolic acid (dimethylsulfide-α,α′-dicarboxylic acid), and water is vaporized. A method for hydrolyzing proteins, characterized in that the protein is hydrolyzed by a solid phase-gas phase reaction with a mixed acid vapor (gas phase).
存させることを特徴とする特許請求の範囲第１項記載の
タンパク質の加水分解方法。(2) The method for hydrolyzing proteins according to claim 1, characterized in that indole is allowed to coexist in the space to which the acid mixed vapor is supplied.
０℃の間にあることを特徴とする特許請求の範囲第１項
記載のタンパク質の加水分解方法。(3) The temperature of the acid mixed vapor is at least 130°C to 20°C.
The method for hydrolyzing proteins according to claim 1, characterized in that the temperature is between 0°C.
２Ｍ、トリフルオロ酢酸１〜５０％、チオグリコール酸
１〜３０％であることを特徴とする特許請求の範囲第１
項記載のタンパク質の加水分解方法。(4) The composition of the acid mixed solution is at least 3 to 1 part of hydrochloric acid.
2M, trifluoroacetic acid 1-50%, thioglycolic acid 1-30%
Protein hydrolysis method described in section.
１０ｍｇであることを特徴とする特許請求の範囲第１項
または第２項記載のタンパク質の加水分解方法。(5) The amount of indole coexisting in the space is 0.1~
The method for hydrolyzing protein according to claim 1 or 2, wherein the amount is 10 mg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP30002289A JPH03160370A (en) | 1989-11-17 | 1989-11-17 | Hydrolysis of protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP30002289A JPH03160370A (en) | 1989-11-17 | 1989-11-17 | Hydrolysis of protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH03160370A true JPH03160370A (en) | 1991-07-10 |
Family
ID=17879767
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP30002289A Pending JPH03160370A (en) | 1989-11-17 | 1989-11-17 | Hydrolysis of protein |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03160370A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0552852A (en) * | 1991-08-28 | 1993-03-02 | Seiko Instr Inc | Method of determining aminoacid sequence from carboxylic terminal of protein or peptide |
| WO2000063232A1 (en) * | 1999-04-17 | 2000-10-26 | Genevac Limited | Methods and apparatus for preventing sample loss |
-
1989
- 1989-11-17 JP JP30002289A patent/JPH03160370A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0552852A (en) * | 1991-08-28 | 1993-03-02 | Seiko Instr Inc | Method of determining aminoacid sequence from carboxylic terminal of protein or peptide |
| WO2000063232A1 (en) * | 1999-04-17 | 2000-10-26 | Genevac Limited | Methods and apparatus for preventing sample loss |
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