JP4925384B2 - Method for quantifying N-terminal glycated protein - Google Patents

Description

【００２９】
39.2： 4AA-TOOS のミリモル吸光係数
2：過酸化水素２分子から4-AA、TOOSが縮合した色素１分子を生じるこことによる係数
10：反応時間（分）
1000：総反応液量（μL)
67：酵素溶液量（μL)
【００３０】
本発明における、遊離されたＮ末端の糖化アミノ酸またはペプチドを測定する方法としては除蛋白後糖化アミノ酸、糖化ペプチドの還元能を利用して色素等を発色させて定量しても良く、また好ましくは除蛋白することなくクロマトグラフィー、抗原抗体反応、酵素反応等の手段を用いて定量しても良く、最も好ましくは糖化アミノ酸、または糖化ペプチドに作用する酵素を用いて定量すれば良い。
【００３１】
糖化アミノ酸または糖化ペプチドに作用する酵素としては、前記プロテアーゼの作用により、被検液に含まれるＮ末端が糖化されたペプチド又は蛋白質から生成される糖化アミノ酸または糖化ペプチドに有効に作用し、実質的にＮ末端が糖化されたペプチド及び蛋白質が測定できる酵素であれば如何なるものを用いても良いが、αアミノ基が糖化されたアミノ酸または糖化ペプチドに効率的に作用する酵素が好ましく、αアミノ基が糖化されたアミノ酸に特異的に作用しεアミノ基が糖化されたアミノ酸には実質的に作用しない酵素が最も好ましい。また一般にこのようなαアミノ基が糖化されたアミノ酸に特異的に作用しεアミノ基が糖化されたアミノ酸には実質的に作用しない酵素は安定性が悪いことから、安定性の高いεアミノ基及びαアミノ基が糖化された糖化アミノ酸両方に良く作用する酵素を用いて測定しても良く、さらに安定性が高いεアミノ基が糖化されたアミノ酸に特異的に作用する酵素を用いてεアミノ基が糖化されたアミノ酸のみを消去し、安定性の高いεアミノ基及びαアミノ基が糖化された糖化アミノ酸の両方に良く作用する酵素を用いてαアミノ基が糖化されたアミノ酸のみを測定しても良い。
【００３２】
特異性の点から、最も好ましい糖化アミノ酸に作用する酵素の例としては、εアミノ基が糖化されたアミノ酸には作用しないαアミノ基糖化アミノ酸特異的な酵素、例えばフルクトシルアミノ酸オキシダーゼ（FAOD)(コリネバクテリウム（Corynebacterium)属由来（FERM P-8245)) が挙げられるがこれらの酵素は45℃で10分の処理で残存活性10％以下と安定性が悪く、実使用には好ましくない。一方εアミノ基及びαアミノ基が糖化された糖化アミノ酸の両方に良く作用する酵素であり安定性が高い酵素としてはギベレラ（Gibberella）属またはアスペルギルス（Aspergillus)属（例えばIFO-6365、-4242 、-5710 等）由来フルクトサミンオキシダーゼ、カンジダ（Candida)属由来フルクトシルアミンデグリカーゼ、ペニシリウム（Penicillium)属（例えばIFO-4651、-6581 、-7905 、-5748 、-7994 、-4897 、-5337 等）由来フルクトシルアミノ酸分解酵素、フサリウム（Fusarium）属（例えばIFO-4468、-4471 、-6384 、-7706 、-9964 、-9971 、-31180、-9972 等）由来、アクレモニウム（Acremonium）属由来又はデブリオマイゼス（Debaryomyces）属由来のケトアミンオキシダーゼ等の少なくとも糖化アミノ酸に作用するオキシダーゼが挙げられ、さらに好ましい例としてはプロテアーゼと共存した状態でも十分な活性を有する、遺伝子組み替えフルクトサミンオキシダーゼ（旭化成工業社製）が挙げられる。
【００３３】
糖化アミノ酸に作用する酵素の活性測定法を下記に示す。
＜＜糖化アミノ酸に作用する酵素の活性測定法＞＞
＜反応液の組成＞
50mM トリス-HCl緩衝液 pH7.5
0.03% 4-AA（和光純薬社製）
0.02% フェノール（和光純薬社製）
4.5U/ml POD（シグマ社製）
1.0mM FVal 若しくはαカルボベンズオキシε- D-フルクトシル-L- リジン
（ハシバらの方法に基づき合成、精製した。Hashiba H,J.Agric.Food
Chem.24:70,1976, 以下FZLys と略す。）
【００３４】
上記の反応液1ml を小試験管に入れ、37℃で5 分間予備加温した後、適当に希釈した酵素液0.02mlを添加して攪拌し、反応を開始する。正確に10分間反応の後に0.5%のSDS を2ml 添加して反応を停止し、波長555nm の吸光度を測定する（As）。またブランクとして酵素液の代わりに蒸留水0.02mlを用いて同一の操作を行い吸光度を測定する（Ab）。酵素作用の吸光度（As）と盲検の吸光度（Ab）の吸光度差（As-Ab)より酵素活性を求める。別にあらかじめ過酸化水素の標準溶液を用いて吸光度と生成した過酸化水素との関係を調べておく。37℃、１分間に１μM の過酸化水素を生成する酵素量を1Uと定義し、下記の計算式(2) で酵素活性を求める。
【００３５】
【式２】

【００３６】
3.02：総反応液量(ml)
0.02：酵素溶液量(ml)
10：反応時間（分）
2：過酸化水素2 分子から4-AA、フェノールが縮合した色素１分子を
生じることによる係数
12.0：4AA-フェノールのミリモル吸光係数
【００３７】
本発明のＮ末端が糖化されたペプチド又は蛋白質を含有する被検液からＮ末端の糖化アミノ酸または糖化ペプチドを遊離する反応の液組成については、使用するＮ末端が糖化されたペプチド又は蛋白質を含有する被検液からＮ末端の糖化アミノ酸または糖化ペプチドを遊離するプロテアーゼの至適pHを考慮し、反応が効率よく進行するようにpH及びプロテアーゼ濃度を決定すればよい。
【００３８】
例えば前記パイロコッカス(Pyrococcus)属由来のデブロッキングアミノペプチダーゼ（Deblocking Aminopeptidase；タカラ社製）を用いる場合には、蛋白質分解活性がpH 6.5〜9.0 付近で強いことから、反応のpHは 6.5〜9.0 を選択することができる。またプロテアーゼ濃度は実際に使用される反応時間中に被検液中の蛋白質を十分に分解し得る濃度であれば良く0.01〜1000U/mlが好ましく、 0.1〜500U/ml がさらに好ましい。
【００３９】
糖化アミノ酸に作用する酵素を用いて断片化された糖化ペプチドまたは蛋白質を測定する反応の液組成については、使用する糖化アミノ酸に作用する酵素の至適pHを考慮し、反応が効率よく進行するようにpHを選択しその後糖化アミノ酸に作用する酵素量を決定すればよい。
【００４０】
例えば遺伝子組換フルクトサミンオキシダーゼ（旭化成工業社製）の場合、最大活性の50％以上の活性を示す領域がpH 6.5〜10と広く、反応のpHは 6.5〜10を選択できる。また酵素添加濃度は実際に使用される反応時間中に被検液から生成された糖化アミノ酸を十分に測定し得る濃度で有れば良く、 0.01U〜1000U/mlが好ましく、0.1U〜500U/ml がより好ましく、0.5U〜100U/mlが最も好ましい。
【００４１】
以上のことから、本発明に於ける糖化蛋白質定量用組成物としては、デブロッキングアミノペプチダーゼ（Deblocking Aminopeptidase）、ジペプチジルアミノペプチダーゼ（Dipeptidyl Aminopeptidase) 、ロイシンアミノペプチダーゼ(Leucine Aminopeptidase）、N-アシルアミノアシル−ペプチドハイドロラーゼ（N-Acylaminoacyl-peptide hydrolase）又はヘミセルラーゼ（Hemicelullase)を含有するもの、又はデブロッキングアミノペプチダーゼ（Deblocking Aminopeptidase)、ジペプチジルアミノペプチダーゼ(Dipeptidyl Aminopeptidase)、ロイシンアミノペプチダーゼ(Leucine Aminopeptidase）、N-アシルアミノアシル−ペプチドハイドロラーゼ（N-Acylaminoacyl-peptide hydrolase）又はヘミセルラーゼ（Hemicelullase)及び糖化アミノ酸に作用する酵素を含有するものとして調製すれば良く、例えば液状品、液状品の凍結物あるいは凍結乾燥品として提供できる。
【００４２】
さらに本発明に基づく糖化蛋白質を定量する酵素反応組成には、例えば界面活性剤、塩類、緩衝剤、pH調整剤や防腐剤などを適宜選択して添加しても良い。
【００４３】
界面活性剤としてはポリオキシエチレンアルキルエーテル類〔例えばポリオキシエチレン(10)オクチルフェニルエーテル（トリトンX-100)、ポリオキシエチレン(23)ラウリルエーテル等〕、ポリオキシエチレンソルビタン脂肪酸エステル類〔例えばポリオキシエチレンソルビタンモノラウレート（ツイーン20) 、ポリオキシエチレンソルビタンモノパルミテート（ツイーン40) 等〕、ポリオキシエチレングリセリン脂肪酸エステル類、ポリオキシエチレンソルビット脂肪酸エステル類、ポリオキシエチレンアルキルフェニルホルムアルデヒド縮合物、ポリオキシエチレンひまし油、ポリオキシエチレンステロール類、ポリオキシエチレンポリオキシプロピレンアルキルエーテル類、ポリオキシエチレンラノリン類、ポリオキシエチレンアルキルアミン・脂肪酸アミド類、ポリオキシエチレンアルキルエーテルリン酸・リン酸塩類、ポリオキシエチレンアルキルエーテル硫酸塩類、ポリグリセリン脂肪酸エステル類、グリセリン脂肪酸エステル類、プロピレングリコール脂肪酸エステル類、ソルビタン脂肪酸エステル類、Ｎアシルアミノ酸塩類、アルキルエーテルカルボン酸塩類、アルキルリン酸塩、Ｎアシルタウリン酸塩、スルホン酸塩、アルキル硫酸、酢酸ベタイン型両性界面活性剤、イミダゾリン型両性界面活性剤、レシチン誘導体（以上日光ケミカルズ社製）、アデカトール720N、アデカトールB-795 、アデカトールSO-120、アデカノールB-795(以上旭電化工業社製）、ポリエチレングリコール類、ポリエチレングリコールラウリルエーテル、ポリエチレングリコールイソオクチルフェニルエーテル、ポリプロピレングリコール、ポリビニルアルコール、トリトン X-305、トリトンX-114 、トリトン X-405、トリトンWR-1339(以上ナカライテスク社製）等が例示される。これらの界面活性剤は、0.01〜10％、好適には0.05〜５％の濃度で用いることが好ましい。
【００４４】
塩類としては、例えば塩化リチウム、塩化ナトリウム、塩化カリウム、塩化マンガン、塩化コバルト、塩化亜鉛、塩化カルシウム等が挙げられる。これらの塩類は、１mM〜5M、好適には10mM〜1Mの濃度で用いることが好ましい。緩衝液としては、例えばトリス−塩酸緩衝液、グリシン-NaOH 緩衝液、燐酸緩衝液、グッドの緩衝液等が挙げられ、これらは10mM〜2M、好適には20mM〜1Mの濃度で用いることが好ましい。防腐剤には、例えばアジ化ナトリウムがあり、0.01〜10％、好適には0.05〜１％を適宜添加すればよい。
【００４５】
また本発明に基づく糖化蛋白質の定量方法における糖化アミノ酸に作用する酵素作用の検出は、例えばデヒドロゲナーゼを用いた場合には補酵素の変化量を、例えば補酵素として NADを用いて生成される変化の量として還元型補酵素である還元型 NADをその極大吸収波長域である340nm 付近の波長にて比色計で測定する等公知の技術を用い直接定量するか、あるいは生じた還元型補酵素を各種ジアフォラーゼ、またはフェナジンメトサルフェート（以下、 PMSと略す。）、メトキシ PMS、ジメチルアミノベンゾフェノキサジニウムクロライド（メルドラブルー）等の電子キャリアー及びニトロテトラゾリウム、WST-1 、WST-8(以上同人化学研究所社製）に代表される各種テトラゾリウム塩等の還元系発色試薬を用い間接的に定量してもよく、またこれ以外の公知の方法により直接、間接的に測定してもよい。
【００４６】
また例えばオキシダーゼを用いた場合には、酸素の消費量または反応生成物の量を測定することが好ましい。反応生成物として、例えばフルクトシルアミノ酸オキシダーゼを用いた場合には反応により過酸化水素及びグルコソンが生成し、過酸化水素及びグルコソン共に公知の方法により直接、間接的に測定することができる。
【００４７】
上記過酸化水素の量は、例えばパーオキシダーゼ等を用いて色素等を生成させ、発色、発光、蛍光等により定量しても良く、また電気化学的手法によって定量しても良く、カタラーゼ等を用いてアルコールからアルデヒドを生成せしめて、生じたアルデヒドの量を定量しても良い。
【００４８】
過酸化水素の発色系は、パーオキシダーゼの存在下で4-AA若しくは3-メチル -2-ベンゾチアゾリノンヒドラゾン(MBTH)等のカップラーとフェノール等の色原体との酸化縮合により色素を生成するトリンダー試薬、パーオキシダーゼの存在下で直接酸化、呈色するロイコ型試薬等を用いることができる。
【００４９】
トリンダー型試薬の色原体としては、フェノール誘導体、アニリン誘導体、トルイジン誘導体等が使用可能であり、具体例としてN, Nジメチルアニリン、N, N- ジエチルアニリン、2,4-ジクロロフェノール、N-エチル-N-(2-ヒドロキシ-3- スルホプロピル）-3, 5-ジメトキシアニリン(DAOS)、N-エチル-N- スルホプロピル-3,5ジメチルアニリン(MAPS)、N-エチル-N-(2-ヒドロキシ-3- スルホプロピル）-3, 5-ジメチルアニリン(MAOS)、N-エチル-N-(2-ヒドロキシ-3- スルホプロピル）-m- トルイジン(TOOS)、N-エチル-N- スルホプロピル-m- アニシジン(ADPS)、N-エチル-N- スルホプロピルアニリン(ALPS)、N-エチル-N- スルホプロピル−3, 5- ジメトキシアニリン(DAPS)、N-スルホプロピル-3, 5-ジメトキシアニリン(HDAPS) 、N-エチル-N- スルホプロピル-m- トルイジン(TOPS)、N-エチル-N-(2-ヒドロキシ-3- スルホプロピル)-m-アニシジン(ADOS)、N-エチル-N-(2-ヒドロキシ-3- スルホプロピル）アニリン(ALOS)、N-(2- ヒドロキシ-3- スルホプロピル）-3, 5-ジメトキシアニリン(HDAOS) 、N-スルホプロピル−アニリン(HALPS) ( 以上同人化学研究所社製）等が挙げられる。
【００５０】
またロイコ型試薬の具体例としては、o-ジアニシジン、o-トリジン、3, 3ジアミノベンジジン、3, 3, 5, 5- テトラメチルベンジジン（以上同人化学研究所社製）、N- (カルボキシメチルアミノカルボニル）-4, 4-ビス（ジメチルアミノ）ビフェニルアミン(DA64)、10-(カルボキシメチルアミノカルボニル）-3,7- ビス（ジメチルアミノ）フェノチアジン(DA67)（以上和光純薬社製）等が挙げられる。
【００５１】
さらに蛍光法には、酸化によって蛍光を発する化合物、例えばホモバニリン酸、4-ヒドロキシフェニル酢酸、チラミン、パラクレゾール、ジアセチルフルオレスシン誘導体等を、化学発光法には、触媒としてルミノール、ルシゲニン、イソルミノール、ピロガロール等を用いることができる。
【００５２】
カタラーゼ等を用いてアルコールからアルデヒドを生成せしめて、生じたアルデヒドを定量する方法としては、ハンチ反応を用いる方法や、MBTHとの縮合反応により発色させる方法、若しくはアルデヒドデヒドロゲナーゼを用いる方法等が挙げられる。
グルコソンの定量はジフェニルアミン等の公知のアルドース試薬を用いて定量すればよい。
【００５３】
また過酸化水素を電極を用いて測定する場合、電極には、過酸化水素との間で電子を授受することのできる材料である限り特に制限されないが、例えば白金、金、銀等が挙げられ、電極測定方法としてはアンペロメトリー、ポテンショメトリー、クーロメトリー等の公知の方法を用いることができ、さらにオキシダーゼまたは基質と電極との間の反応に電子伝達体を介在させ、得られる酸化、還元電流或いはその電気量を測定しても良い。電子伝達体としては電子伝達機能を有する任意の物質が使用可能であり、例えばフェロセン誘導体、キノン誘導体等の物質が挙げられる。またオキシダーゼ反応により生成する過酸化水素と電極の間に電子伝達体を介在させ得られる酸化、還元電流またはその電気量を測定しても良い。
【００５４】
かくして調製された本発明の定量用組成物によって、被検液中の糖化ペプチド又は糖化蛋白質を定量するには、定量用組成物に被検液 0.001〜0.5ml を加え、37℃の温度にて反応させ、レートアッセイを行う場合には、反応開始後の一定時間後の２点間の数分ないし数十分間、例えば３分後と４分後の１分間、または３分後と８分後の５分間における変化した補酵素、溶存酸素、過酸化水素又はその他生成物の量を直接または間接的に前記の方法で測定すれば良く、エンドポイントアッセイの場合には反応開始後一定時間後の変化した補酵素、溶存酸素、過酸化水素又はその他生成物の量を同様に測定すれば良い。この場合既知濃度の糖化アミノ酸、ペプチド又は蛋白質を用いて測定した場合の吸光度等の変化と比較すれば被検液中の糖化ペプチドおよび糖化蛋白質の量を求めることができる。
【００５５】
【発明の実施の形態】
次に、本発明を実施例を挙げて具体的に述べるが、本発明は何らこれにより限定されるものではない。
【実施例１】
＜Ｎ末端が糖化されたペプチド又は蛋白質に作用しＮ末端の糖化アミノ酸又は糖化ペプチドを遊離するプロテアーゼのスクリーニング＞
スクリーニングは、プロテアーゼの絞込みを大量に行う１次スクリーニング、及び１次スクリーニングでピックアップされたものから合成基質よりフルクトシルアミンオキシダーゼを用いてフルクトシルバリンの精製を確認する２次スクリーニングの２段階に分けて行った。
【００５６】
(1) １次スクリーニング
＜反応液１＞
100mM トリス−HCl 緩衝液 pH8.5
2mM FValpNA
上記反応液１を96穴プレートに 200μL とり、プロテアーゼを含有する溶液50μL(高濃度の方が好ましい) を添加し室温で10分〜１晩反応させ黄色く着色したプロテアーゼを選択した。さらに着色が確認されたプロテアーゼは上記反応液900 μL をキュベットにとり、プロテアーゼを含有する溶液50μL を添加し500nm の吸光度変化ΔＡを測定した。また基質であるFValpNA の代わりに蒸留水を処方した反応液を作成し、同様の操作を行い吸光度変化ΔＡブランクを測定した。糖化アミノ酸誘導体へのプロテアーゼの作用はΔＡ−ΔＡブランクにより計算した。
【００５７】
(2) ２次スクリーニング
１次スクリーニングにて活性の認められたプロテアーゼ及び従来糖化ヘモグロビンへの作用が報告されていたプロテアーゼに関し下記の活性確認を行った。
＜反応液２＞
6mM FValpNA
15mM Tris-HCl buffer pH7.5 （和光純薬社製）
2U/ml FAOD(旭化成社製）
2U/ml POD（シグマ社製）
0.01% 4-AA(和光純薬社製）
0.01% TOOS(和光純薬社製）
【００５８】
上記反応液２を 933μL キュベットにとり37℃で５分間予備加温する。適当に希釈したプロテアーゼを含有する溶液67μL 添加し、撹拌して反応を開始し、555nm の10分間あたりの吸光度変化ΔＡを測定する。また基質であるFValpNA の代わりに蒸留水を処方した反応液を作成し、同様の操作を行い吸光度変化ΔＡブランクを測定した。糖化アミノ酸誘導体へのプロテアーゼの作用はΔＡ−ΔＡブランクにより計算した。
結果を表１に示す。
【００５９】
【表１】

【００６０】
表１に示したとおり意外なことにこれまで糖化ヘモグロビンの測定に使用できるといわれてきたプロテアーゼにはFValpNAからFValを遊離する活性は認められなかった。
【００６１】
一方パイロコッカス・フリオサス（Pyrococcus furiosus）属由来デブロッキングアミノペプチダーゼ（Deblocking aminopeptidase ；タカラ社製）、ブタ肝由来N-アシルアミノアシル−ペプチドハイドロラーゼ（N-Acylaminoacyl-peptide hydrolase ；タカラ社製）等のＮ末端がブロックされたペプチドや蛋白質に作用し、Ｎ端のブロックされたアミノ酸を遊離するプロテアーゼには強い作用が確認された。
【００６２】
同様にＮ末端から２アミノ酸を遊離するジペプチジルペプチダーゼ（Dipeptidyl peptidase)、たとえばウシ脾臓由来カテプシンＣやジペプチジルアミノペプチダーゼIV（Dipeptidyl aminopeptidaseIV）にも同様な作用が確認された。これは糖化アミノ酸をアミノ酸２分子としてプロテアーゼが認識し糖化アミノ酸を効率的に遊離するためと考えられた．
【００６３】
また前記の特殊なアミノペプチダーゼを除いたアミノペプチダーゼのなかではロイシンアミノペプチダーゼのみが合成基質からフルクトシルバリンを遊離する活性が認められその他のアミノペプチダーゼからは活性が確認できなかった。これはロイシンの側鎖がＮ末端に結合した糖と大きさが似ていることからロイシンアミノペプチダーゼのみに活性が確認できたものと推定された。
さらにヘミセルラーゼにも同様な活性が確認された。
【００６４】
【実施例２】
＜合成基質に対する反応曲線＞
＜反応液＞ 実施例１の反応液２に同じ
【００６５】
＜操作＞
実施例１に示された反応液２を 933μL キュベットにとり37℃で 500秒間予備加温する。適当に希釈したプロテアーゼを含有する溶液67μL 添加、撹拌し反応を開始し、555nm の吸光度を測定する。また基質であるFValpNA の代わりに蒸留水を処方した反応液を作成し、同様の操作を行い吸光度変化を測定した。FValpNAから得られる吸光度から水ブランクを差し引き反応曲線を算出した。
結果を図１に示す。
【００６６】
図１からわかるようにデブロッキングアミノペプチダーゼ（Deblocking aminopeptidase ；タカラ社製）、ブタ肝由来N-アシルアミノアシル−ペプチドハイドロラーゼ（N-Acylaminoacyl-peptide hydrolase ；タカラ社製）及びプチジルアミノペプチダーゼIV（Dipeptidyl aminopeptidaseIV) に関し、FValpNA からFValを生じFOD によって過酸化水素が経時的に生じる様子が観察された。
【００６７】
【実施例３】
＜糖化ヘモグロビンを基質とした場合の検量線＞
＜反応液１＞
10mM Tris緩衝液 pH8.5
10U/ml デブロッキングアミノペプチダーゼ（タカラ社製、日本）
【００６８】
＜反応液２＞
150 mM Tris緩衝液 pH8.5
15U/ml POD(シグマ社製）
12mM TOOS
8mM 4-AA（和光純薬社製）
2U/ml FOD(旭化成工業社製）
＜Hb基質溶液＞
ヒトヘモグロビンを４g/dlになるように蒸留水に溶解してHb基質溶液とした。
Hb基質溶液(4g/dl) の0.0 、0.5 、1.0 、1.5 、2.0 倍濃度の試料を用いた。
【００６９】
＜操作＞
上記反応液１ 240μl を試験管に取り37℃で５分間加温する。Hb基質溶液 8μl を添加し、攪拌して反応を開始し、正確に５分後にトリクロル酢酸にて除蛋白し、中和後この溶液に反応液２を80μL を添加した。反応液２添加前後の546nm の吸光度を測定し、その差を吸光度変化とした。また基質の代わりに蒸留水を用い同様の操作を行い、ブランク試料とした。
【００７０】
Hb基質溶液から得られた吸光度変化からブランク試料の吸光度変化を差し引き感度を計算した。またHb1.0 倍濃度は10回測定しCV値を計算した。その結果を図２に示す。
図２から分かるようにHb基質溶液は濃度に応じて良好な直線性が得られた。またHb1.0 倍濃度ではCV値＝2.5 ％と良好な再現性が確認されており、本発明の測定系を用いて糖化ヘモグロビンが選択的に、感度良く、また再現良く測定されていることが明らかとなった。
【図面の簡単な説明】
【図１】本発明の実施例２に基づく合成基質を用いた場合の反応曲線である。
【図２】本発明の実施例３に基づく糖化ヘモグロビンの検量線である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for quantifying the released glycated amino acid or peptide by liberating the N-terminal glycated amino acid or peptide using a protease from a test solution containing a peptide or protein glycated at the N terminus. The present invention relates to a composition used in the above. According to the present invention, glycated hemoglobin in a blood sample can be quantified accurately, simply and quickly.
[0002]
[Prior art]
The measurement of glycated protein is very important in the diagnosis and management of diabetes. In particular, if the glycated hemoglobin in the blood is controlled to a value of 7% or less according to recent research, the risk rate for the onset and progression of complications is superior. It has been proved that it can be reduced rapidly, and is frequently used as an indispensable indicator in the clinical field.
[0003]
As quantitative methods for glycated hemoglobin, electrophoresis, ion exchange chromatography, affinity chromatography, immunization, and enzyme method are generally known. However, the electrophoresis method and the chromatography method require an expensive dedicated apparatus, and the processing speed is slow, so that it is not suitable for a clinical test for processing a large number of specimens. In addition, the immunization method has rapidly spread in recent years because the analysis method is comparatively simple and requires a short time, but since the antigen-antibody reaction is used, the accuracy is not necessarily high in terms of reproducibility and influence of coexisting substances. There is a problem that can not be said.
[0004]
Thus, an expensive dedicated apparatus is unnecessary, the processing speed is fast, high accuracy, simple, and inexpensive method for quantifying glycated hemoglobin includes an enzymatic method. Since the definition of glycated hemoglobin is hemoglobin in which the amino acid at the N-terminus of hemoglobin β chain is glycated, when measuring glycated hemoglobin using an enzyme, the N-terminal glycated amino acid from hemoglobin in which the N-terminal amino acid is glycated Alternatively, the peptide containing the N-terminal glycated amino acid must be released and quantified. In general, the glycation site of hemoglobin is thought to be about 60% α-amino group and about 40% ε-amino group, and the protein whose N-terminus of protein is blocked by glycation is protease. Resistant. The following (a) to (h) are known as examples in which an N-terminal glycated amino acid or a peptide containing an N-terminal glycated amino acid is released from hemoglobin in which the N-terminal amino acid is glycated.
[0005]
(a) Glycohemoglobin added with 8M urea and boiled for 20 minutes, treated with trypsin, penicillium(Penicillium) Method of quantification with genus-derived fructosyl amino acid oxidase (FAOD) (JP-A-8-336386).
(b) Aspergillus by treating glycohemoglobin with protease(Aspergillus) Method of quantification with genus-derived FAOD (JP-A-10-33177, JP-A-10-33180)
(c) Protease treatment and detection by FAOD (Japanese Patent Laid-Open No. 5-192193).
(d) A method for measuring glycated hemoglobin using endo-type and exo-type proteases (WO97 / 13872).
[0006]
(e) A method in which a peptide or protein in which the N-terminal valine is fructosylated is treated with serine carboxypeptidase (Japanese Patent Laid-Open No. 2001-57897).
(f) A protease capable of treating hemoglobin A1c with a protease capable of cleaving the carboxyl group side of the third leucine from the N-terminus of the β chain, and then cleaving histidyl leucine from the resulting fructosylvalyl histidyl leucine. A method for measuring hemoglobin A1c to be treated (Japanese Patent Laid-Open No. 2000-300294).
(g) Corynebacterium that liberates amino acid with α-amino group glycated from glycated protein(Corynebacterium) Genus and Pseudomonas(Pseudomonas) A method for liberating glycated amino acids using a novel enzyme derived from a genus and quantifying it (WO00 / 50579).
(h) Sphingobacterium that liberates amino acid with α-amino group glycated from glycated protein(Sphingobacterium)Genus, Sphingomonas(Sphingomonas)Genus, Comamonas(Comamonas) Genus, Mucor(Mucor) Genus and Penicillium(Penicillium) A method for liberating glycated amino acids using a novel enzyme derived from a genus and quantifying the same (WO00 / 61732).
[0007]
In the above methods (a) to (d), the ε and α amino groups are measured using FAOD that works well on both glycated glycated amino acids. It is not clear whether only measured glycated amino acids are measured.
[0008]
Further, according to the experiments by the present inventors, the protease used in (a) to (d) is a synthetic substrate that can confirm that the α-amino group acts on a glycated protein, for example, glycated valylparanitroanilide. (Hereinafter abbreviated as FValpNA). Therefore, it is considered that a known method for measuring glycated hemoglobin measures not a N-terminal glycated α-amino group, which should be measured originally, but a glycated ε-amino group.
[0009]
On the other hand, if the methods (e) to (h) are used, the N-terminal glycation site of glycated hemoglobin can be measured. However, in the methods (e) and (f), a peptide containing an N-terminal glycated amino acid is once excised with an endoprotease, and then decomposed into glycated amino acids using carboxypeptidase. Protease is required.
[0010]
(G) and (h) above are corynebacterium(Corynebacterium) Genus, Pseudomonas(Pseudomonas) Genus, Sphingobacterium(Sphingobacterium)Genus, Sphingomonas(Sphingomonas)Genus, Comamonas(Comamonas) Genus, Mucor(Mucor) Genus and Penicillium(Penicillium) Limited to genus-derived enzymes.
Deblocking Aminopeptidase, Dipeptidyl Aminopeptidase, Leucine Aminopeptidase, N-Acylaminoacyl-peptide hydrolase or Hemicelullase There has been no report on a method for quantifying a glycated peptide or glycated protein, in which N-terminal glycated amino acid or glycated peptide is liberated and quantified.
[0011]
[Problems to be solved by the invention]
The present invention has been made for the purpose of solving such problems in the quantification of peptides or glycated proteins in which the N-terminus is glycated. That is, an object of the present invention is to release a N-terminal glycated amino acid or peptide from a test solution containing a peptide or protein glycated at the N-terminus, and to determine a method for quantifying this and a composition for this quantification. It is to provide. A further object of the present invention is to provide a quantitative method and quantitative composition useful for clinical biochemical tests, particularly measurement of glycated hemoglobin in blood, in large quantities and at low cost.
[0012]
[Means for Solving the Problems]
Therefore, the present inventors synthesized FValpNA and the like for the purpose of searching for a protease that efficiently degrades the N-terminus of glycated hemoglobin. However, surprisingly, proteases that have been confirmed to have an action on glycated hemoglobin so far have not been able to confirm the activity of degrading this synthetic substrate. Therefore, it was considered that the color development by FAOD from glycated hemoglobin, which has been clarified so far, does not originate from glycated valine at the N-terminal, but measures the sugar bound to the ε-amino group.
[0013]
Accordingly, the present inventors have extensively screened proteases from the natural world using the synthetic substrate FValpNA and the like. As a result, the protease, dipeptidylaminopeptidase (Dipeptidyl) used for deblocking proteins and peptides whose N-terminals are blocked. It was found that Aminopeptidase), leucine aminopeptidase (Leucine Aminopeptidase), or hemicellulase has an activity of degrading FValpNA.
[0014]
However, proteases obtained from bacteria and fungi are complex to cultivate and purify, and are difficult to supply in large quantities at low cost as clinical reagents. On the other hand, deblocking aminopeptidase, dipeptidylaminopeptidase (deblocking aminopeptidase) Dipeptidyl Aminopeptidase), leucine aminopeptidase, N-acylaminoacyl-peptide hydrolase or hemicellulase could be supplied in large quantities at low cost.
[0015]
Furthermore, the present inventors have found that glycated hemoglobin can be measured with good reproducibility, accuracy, and simplicity by allowing an enzyme that acts on a glycated amino acid such as FAOD to act on these protease reaction solutions.
[0016]
That is, the present invention provides a test solution containing a peptide or protein having a glycated N-terminus, a deblocking aminopeptidase, a dipeptidyl aminopeptidase, a leucine aminopeptidase, -A method for quantifying the N-terminal glycated amino acid or glycated peptide by allowing acylaminoacyl-peptide hydrolase or hemicelullase to act, and for use in quantification Relates to the composition. The present invention is particularly a measurement method and a composition for quantification useful for quantifying glycated hemoglobin in a blood sample accurately, simply, rapidly and inexpensively using such an enzyme.
[0017]
Hereinafter, the configuration and preferred embodiments of the present invention will be described in more detail.
The test solution that can be used in the present invention may be any test solution containing a peptide or protein glycated at the N-terminus. Preferred is a test liquid containing a peptide containing the N-terminus of glycated hemoglobin or glycated hemoglobin, and more preferably whole blood, whole blood lysed blood, centrifuged red blood cells, washed red blood cells, and the like. In addition, a protease-treated solution in which separated erythrocytes or glycated hemoglobin is fragmented with a protease in advance can also be used as a preferred test solution.
[0018]
Proteases that can be used in the present invention include deblocking aminopeptidase, N-acylaminoacyl-peptide hydrolase (EC = 3.4.19.1), dipeptidyl aminopeptidase, Any enzyme may be used as long as it is leucine aminopeptidase or hemicelullase.
[0019]
Examples of preferred enzymes include Pyrococcus for Deblocking Aminopeptidase.(Pyrococcus)Deblocking aminopeptidase derived from the genus (Deblocking Aminopeptidase), N-acylaminoacyl-peptide hydrolase, N-acylaminoacyl-peptide hydrolase derived from pig liver EC = 3.4.19.1), dipeptidyl aminopeptidase (Cipepsin C; EC = 3.4.14.1; from Bovine Spleen; Sigma), for example, dipeptidyl aminopeptidase I (Dipeptidyl Aminopeptidase I) ), Dipeptidyl aminopeptidase II (Dipeptidyl Aminopeptidase II; EC = 3.4.14.2; located in various organ lysosomes), dipeptidyl aminopeptidase III (Angiotensinase; EC = 3.4.14.4) Red blood cells), dipeptidyl aminopeptidase IV (Dipeptidyl) Aminopeptidase IV; EC = 3.4.14.5; pig liver; manufactured by Sigma), leucine aminopeptidase (Leucine Aminopeptidase; EC = 3.4.11.1; pig kidney, cytosol; manufactured by Sigma), hemicellulase, for example, hemi Cellulase “Amano” (manufactured by Amano Pharmaceutical Co., Ltd.), Sumiteam X (manufactured by Shin Nippon Chemical Industry Co., Ltd.), hemicellulase N (manufactured by Tanabe Seiyaku Co., Ltd.), cellulosin HC (manufactured by Hankyu Bioindustry Co., Ltd.), hemicellulase (manufactured by Tokyo Chemical Industry Co., Ltd.) However, the present invention is not limited to these enzymes.
[0020]
Furthermore, in the present invention, when the N-terminal glycated amino acid or glycated peptide is released, the above-mentioned protease that acts on the peptide or protein glycated at the N terminus and releases the N-terminal glycated amino acid or glycated peptide is used alone. Of course, another endoprotease may be allowed to act before or simultaneously with the reaction of the protease. By doing so, the reaction rate for releasing the N-terminal glycated amino acid or glycated peptide can be increased.
[0021]
The synthetic substrate used for measuring the activity of a protease that acts on a glycated peptide or protein at the N-terminus of the present invention and releases the glycated amino acid or peptide at the N-terminus is an α-fructosyl amino acid derivative or an α-fructosyl peptide. Any substrate may be used as long as it is a synthetic substrate. α-Fructosyl refers to an Amadori compound that is produced non-enzymatically by binding an amino acid, an α-amino group at the N-terminal of a peptide, to glucose.
[0022]
When the synthetic substrate is a fructosyl amino acid derivative, a fructosyl valine derivative is most preferred, but other amino acids may be used, and the C-terminus of the amino acid is in the form of anilide, ester, etc. for the purpose of distinguishing the substrate from the product. It needs to be protected. For the purpose of facilitating the screening of proteases, the present inventors used paranitroanilide which exhibits a yellow color upon protease cleavage, but any other protecting group may be used. When other protective groups are used, for example, when the protease reaction alone leads to color development, the color development may be used for protease detection. When the protease reaction alone cannot lead to color development, the produced glycated amino acid is converted to FAOD. The protease may be detected by a known method after conversion to hydrogen peroxide.
[0023]
When the synthetic substrate is a fructosyl peptide, the sequence is preferably the same as that of the glycated hemoglobin N-terminus. For example, fructosyl valyl histidine (Val-His) to fructosyl valyl histidyl leucyl threonyl proline (Val- Most preferred is a fructosyl peptide of 2 to 5 residues up to (His-Leu-Thr-Pro) or a derivative of its peptide C-terminal, but peptides of other sequences may also be used.
[0024]
The synthetic substrate may be synthesized by fructosylation and purification of the amino acid derivative or peptide by a known method, for example, the method of Hashiba et al. (Hashiba H, J. Agric. Food Chem. 24:70, 1976). It can also be synthesized using other methods. C-terminal derivatization of amino acids or peptides may be derivatized by a known method or a commercially available product may be used.
[0025]
In the present invention, the activity of a protease that acts on a glycated peptide or protein at the N-terminus and releases a glycated amino acid or peptide at the N-terminus was measured by the following method. This activity measurement utilizes the fact that FAOD (Asahi Kasei Co., Ltd.) does not act on FValpNA but acts on fructosyl valine (hereinafter abbreviated as FVal) produced by the action of protease.
[0026]
<< Method for measuring protease activity >>
<Composition of reaction solution>
6mM FValpNA
15mM Tris-HCl buffer pH7.5 (Wako Pure Chemical Industries, Ltd.)
2U / ml FAOD (Asahi Kasei)
2U / ml peroxidase (hereinafter abbreviated as POD, manufactured by Sigma)
0.01% 4-aminoantipyrine (hereinafter abbreviated as 4AA; Wako Pure Chemical Industries, Ltd.)
0.01% N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (hereinafter abbreviated as TOOS; manufactured by Wako Pure Chemical Industries, Ltd.)
[0027]
Pre-warm 933 μL of the above reaction solution in a cuvette at 37 ° C. for 5 minutes. Add 67 μL of a solution containing an appropriately diluted protease, stir to start the reaction, and measure the change in absorbance ΔA per 10 minutes at 555 nm. A reaction solution formulated with distilled water instead of the substrate FValpNA was prepared, and the same procedure was followed to measure the absorbance change ΔA blank (hereinafter abbreviated as ΔAb). The amount of enzyme that produces 1 μM of glycated amino acid from a glycated amino acid derivative at 37 ° C. for 1 minute was defined as 1 U.
The calculation formula is as shown in Formula 1 below.
[0028]
[Formula 1]

[0029]
39.2: millimolar extinction coefficient of 4AA-TOOS
2: Coefficient based on which one molecule of 4-AA and TOOS is condensed from two hydrogen peroxide molecules
10: Reaction time (minutes)
1000: Total reaction volume (μL)
67: Enzyme solution volume (μL)
[0030]
In the present invention, as a method for measuring the released N-terminal glycated amino acid or peptide, it may be quantified by coloring a dye or the like using the reducing ability of the glycated amino acid or glycated peptide after deproteinization, and preferably It may be quantified using means such as chromatography, antigen-antibody reaction, enzyme reaction, etc. without deproteinization, and most preferably quantified using an enzyme that acts on a glycated amino acid or glycated peptide.
[0031]
As an enzyme that acts on a glycated amino acid or glycated peptide, it effectively acts on a glycated amino acid or glycated peptide generated from a peptide or protein in which the N-terminus contained in the test solution is glycated by the action of the protease. Any enzyme can be used as long as it can measure peptides and proteins whose N-terminal is glycated, but an enzyme that efficiently acts on a glycated amino acid or glycated peptide is preferred, Most preferred is an enzyme that specifically acts on glycated amino acids and does not substantially act on amino acids whose ε-amino group is glycated. In general, an enzyme having an α-amino group specifically acting on a glycated amino acid and an ε-amino group substantially not acting on a glycated amino acid has poor stability. And α-amino group may be measured using an enzyme that works well on glycated amino acids, and ε-amino acid may be measured using an enzyme that acts more specifically on glycated amino acids that have higher stability. Only amino acids whose α-amino group is saccharified are measured using an enzyme that erases only the amino acid whose group is glycated and works well on both highly stable ε-amino group and glycated amino acid whose α-amino group is glycated. May be.
[0032]
Examples of the enzyme that acts on the most preferred glycated amino acid in terms of specificity include α-amino group glycated amino acid-specific enzymes that do not act on glycated amino acids such as fructosyl amino acid oxidase (FAOD) ( Corynebacterium (Corynebacterium)These enzymes are not suitable for actual use because these enzymes have poor stability with a residual activity of 10% or less after treatment at 45 ° C. for 10 minutes. On the other hand, an enzyme that works well on both ε-amino group and glycated amino acid in which α-amino group is glycated and has high stability as Gibberella (Gibberella) Genus or Aspergillus (Aspergillus)Fructosamine oxidase, Candida (eg, IFO-6365, -4242, -5710 etc.)Candida)Genus fructosylamine deglycase, penicillium (Penicillium)Fructosyl amino acid degrading enzyme derived from a genus (eg IFO-4651, -6581, -7905, -5748, -7994, -4897, -5337, etc.)Fusarium) Genus (eg IFO-4468, -4471, -6384, -7706, -9964, -9971, -31180, -9972, etc.), Acremonium (Acremonium) Genus or Debriomides (Debaryomyces) An oxidase that acts on at least a glycated amino acid such as ketoamine oxidase derived from the genus, and a more preferable example is a genetically modified fructosamine oxidase (manufactured by Asahi Kasei Kogyo Co., Ltd.) that has sufficient activity even in the presence of a protease. .
[0033]
A method for measuring the activity of an enzyme acting on a glycated amino acid is shown below.
<< Method for measuring activity of enzyme acting on glycated amino acid >>
<Composition of reaction solution>
50 mM Tris-HCl buffer pH 7.5
0.03% 4-AA (Wako Pure Chemical Industries, Ltd.)
0.02% Phenol (Wako Pure Chemical Industries)
4.5U / ml POD (Sigma)
1.0 mM FVal or α-carbobenzoxy ε-D-fructosyl-L-lysine
(Synthesis and purification based on the method of Hashiba et al. Hashiba H, J. Agric. Food
Chem. 24: 70,1976, hereinafter abbreviated as FZLys. )
[0034]
Place 1 ml of the above reaction solution in a small test tube, preheat at 37 ° C for 5 minutes, add 0.02 ml of an appropriately diluted enzyme solution, and stir to start the reaction. After exactly 10 minutes of reaction, stop the reaction by adding 2 ml of 0.5% SDS and measure the absorbance at 555 nm (As). Also, the absorbance is measured by performing the same operation using 0.02 ml of distilled water instead of the enzyme solution as a blank (Ab). The enzyme activity is determined from the difference in absorbance (As-Ab) between the absorbance of the enzyme action (As) and the absorbance of the blind (Ab). Separately, the relationship between the absorbance and the generated hydrogen peroxide is examined in advance using a standard solution of hydrogen peroxide. The amount of enzyme that produces 1 μM hydrogen peroxide per minute at 37 ° C. is defined as 1 U, and the enzyme activity is calculated by the following formula (2).
[0035]
[Formula 2]

[0036]
3.02: Total reaction volume (ml)
0.02: Enzyme solution volume (ml)
10: Reaction time (minutes)
2: 1 molecule of dye condensed with 4-AA and phenol from 2 molecules of hydrogen peroxide
Factor due to what happens
12.0: mmol extinction coefficient of 4AA-phenol
[0037]
Regarding the liquid composition of the reaction for liberating the N-terminal glycated amino acid or glycated peptide from the test solution containing the peptide or protein glycated at the N-terminus of the present invention, the N-terminal glycated peptide or protein to be used is contained. In consideration of the optimum pH of the protease that liberates the N-terminal glycated amino acid or glycated peptide from the test solution, the pH and the protease concentration may be determined so that the reaction proceeds efficiently.
[0038]
For example, the Pyrococcus(Pyrococcus)When using a deblocking aminopeptidase derived from a genus (Deblocking Aminopeptidase; manufactured by Takara), the proteolytic activity is strong around pH 6.5 to 9.0, and thus the pH of the reaction can be selected from 6.5 to 9.0. The protease concentration may be any concentration that can sufficiently decompose the protein in the test solution during the reaction time actually used, and is preferably 0.01 to 1000 U / ml, more preferably 0.1 to 500 U / ml.
[0039]
Regarding the liquid composition of the reaction for measuring a glycated peptide or protein fragmented using an enzyme that acts on a glycated amino acid, the optimum pH of the enzyme that acts on the glycated amino acid used is taken into account so that the reaction proceeds efficiently. The pH may be selected and the amount of enzyme acting on the glycated amino acid may be determined.
[0040]
For example, in the case of genetically modified fructosamine oxidase (manufactured by Asahi Kasei Kogyo Co., Ltd.), the region showing 50% or more of the maximum activity is as wide as pH 6.5 to 10, and the reaction pH can be selected from 6.5 to 10. The enzyme addition concentration may be a concentration that can sufficiently measure the glycated amino acid produced from the test solution during the reaction time actually used, and is preferably 0.01 U to 1000 U / ml, preferably 0.1 U to 500 U / ml. ml is more preferred, and 0.5 U to 100 U / ml is most preferred.
[0041]
From the above, the composition for quantifying glycated protein in the present invention includes deblocking aminopeptidase, dipeptidyl aminopeptidase, leucine aminopeptidase, N-acylaminoacyl- Contains peptide hydrolase (N-Acylaminoacyl-peptide hydrolase) or hemicellullase, or deblocking aminopeptidase, dipeptidyl aminopeptidase, leucine aminopeptidase (Leucine Aminopeptidase), N -An acylaminoacyl-peptide hydrolase (N-Acylaminoacyl-peptide hydrolase) or hemicellulase (Hemicelullase) and an enzyme that acts on glycated amino acids may be prepared. It can be provided as a frozen product or lyophilized product.
[0042]
Further, for example, surfactants, salts, buffers, pH adjusters and preservatives may be appropriately selected and added to the enzyme reaction composition for quantifying the glycated protein according to the present invention.
[0043]
Surfactants include polyoxyethylene alkyl ethers [eg, polyoxyethylene (10) octylphenyl ether (Triton X-100), polyoxyethylene (23) lauryl ether, etc.], polyoxyethylene sorbitan fatty acid esters [eg, polyoxyethylene (10) octyl phenyl ether (Triton X-100), etc. Oxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Tween 40), etc.], polyoxyethylene glycerin fatty acid esters, polyoxyethylene sorbit fatty acid esters, polyoxyethylene alkylphenyl formaldehyde condensate, Polyoxyethylene castor oil, polyoxyethylene sterols, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene lanolins, polyoxyethylene alkylamine・ Fatty acid amides, polyoxyethylene alkyl ether phosphates / phosphates, polyoxyethylene alkyl ether sulfates, polyglycerin fatty acid esters, glycerin fatty acid esters, propylene glycol fatty acid esters, sorbitan fatty acid esters, N acylamino acids Salts, alkyl ether carboxylates, alkyl phosphates, N acyl taurates, sulfonates, alkyl sulfates, acetic acid betaine type amphoteric surfactants, imidazoline type amphoteric surfactants, lecithin derivatives (manufactured by Nikko Chemicals) , Adecatol 720N, Adecatol B-795, Adecatol SO-120, Adecanol B-795 (manufactured by Asahi Denka Kogyo Co., Ltd.), polyethylene glycols, polyethylene glycol lauryl ether, polyethylene glycol isooctylphenyl ester Examples include ether, polypropylene glycol, polyvinyl alcohol, Triton X-305, Triton X-114, Triton X-405, Triton WR-1339 (manufactured by Nacalai Tesque). These surfactants are preferably used at a concentration of 0.01 to 10%, preferably 0.05 to 5%.
[0044]
Examples of the salts include lithium chloride, sodium chloride, potassium chloride, manganese chloride, cobalt chloride, zinc chloride, calcium chloride and the like. These salts are preferably used at a concentration of 1 mM to 5 M, preferably 10 mM to 1 M. Examples of the buffer include Tris-HCl buffer, glycine-NaOH buffer, phosphate buffer, Good's buffer, etc., and these are preferably used at a concentration of 10 mM to 2 M, preferably 20 mM to 1 M. . Examples of the preservative include sodium azide, and 0.01 to 10%, preferably 0.05 to 1% may be added as appropriate.
[0045]
In addition, the detection of the enzyme action acting on the glycated amino acid in the method for quantifying glycated protein based on the present invention can be performed by measuring the amount of change in coenzyme when using, for example, dehydrogenase, The amount of reduced coenzyme, reduced NAD, can be directly quantified using a known technique such as a colorimeter at a wavelength near its maximum absorption wavelength of 340 nm, or the resulting reduced coenzyme can be Various diaphorases, or electron carriers such as phenazine methosulfate (hereinafter abbreviated as PMS), methoxy PMS, dimethylaminobenzophenoxazinium chloride (Meldra Blue), and nitrotetrazolium, WST-1, WST-8 Indirect quantification may be carried out using reducing color developing reagents such as various tetrazolium salts represented by Research Institute Co., Ltd. The measurement may be performed directly or indirectly by a known method.
[0046]
For example, when oxidase is used, it is preferable to measure the amount of oxygen consumed or the amount of reaction products. As the reaction product, for example, when fructosyl amino acid oxidase is used, hydrogen peroxide and glucosone are produced by the reaction, and both hydrogen peroxide and glucosone can be measured directly or indirectly by a known method.
[0047]
The amount of hydrogen peroxide may be determined by coloration, luminescence, fluorescence, or the like by generating a pigment using, for example, peroxidase, etc., or by an electrochemical method, using catalase or the like. Then, an aldehyde may be produced from alcohol, and the amount of aldehyde produced may be quantified.
[0048]
In the presence of peroxidase, the hydrogen peroxide coloring system produces dyes by oxidative condensation of couplers such as 4-AA or 3-methyl-2-benzothiazolinone hydrazone (MBTH) and chromogens such as phenol. Or a leuco-type reagent that directly oxidizes and colors in the presence of peroxidase.
[0049]
As the chromogen of the Trinder type reagent, phenol derivatives, aniline derivatives, toluidine derivatives, etc. can be used. Specific examples include N, N dimethylaniline, N, N-diethylaniline, 2,4-dichlorophenol, N- Ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N-sulfopropyl-3,5 dimethylaniline (MAPS), N-ethyl-N- ( 2-Hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N-ethyl-N- Sulfopropyl-m-anisidine (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline (DAPS), N-sulfopropyl-3, 5 -Dimethoxyaniline (HDAPS), N-ethyl-N-sulfopropyl-m-toluidine (TOPS), N-ethyl-N- (2-hydroxy- 3-sulfopropyl) -m-anisidine (ADOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline (ALOS), N- (2-hydroxy-3-sulfopropyl) -3, 5 -Dimethoxyaniline (HDAOS), N-sulfopropyl-aniline (HALPS) (manufactured by Dojin Chemical Laboratory Co., Ltd.) and the like.
[0050]
Specific examples of leuco-type reagents include o-dianisidine, o-tolidine, 3,3 diaminobenzidine, 3, 3, 5, 5-tetramethylbenzidine (manufactured by Doujin Chemical Laboratory Co., Ltd.), N- (carboxymethyl). Aminocarbonyl) -4,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67) (above Wako Pure Chemical Industries) Is mentioned.
[0051]
Furthermore, for fluorescence methods, compounds that emit fluorescence upon oxidation, such as homovanillic acid, 4-hydroxyphenylacetic acid, tyramine, paracresol, diacetylfluorescin derivatives, etc., are used as chemiluminescent methods, and luminol, lucigenin, isoluminol as catalysts. Pyrogallol or the like can be used.
[0052]
Examples of a method for producing aldehyde from alcohol using catalase or the like and quantifying the generated aldehyde include a method using a hunch reaction, a method of developing a color by a condensation reaction with MBTH, or a method using an aldehyde dehydrogenase. .
The glucosone may be quantified using a known aldose reagent such as diphenylamine.
[0053]
When hydrogen peroxide is measured using an electrode, the electrode is not particularly limited as long as it is a material that can transfer electrons to and from hydrogen peroxide. Examples thereof include platinum, gold, and silver. As an electrode measurement method, a known method such as amperometry, potentiometry, coulometry, etc. can be used. Further, an oxidation or reduction current is obtained by interposing an electron carrier in the reaction between oxidase or substrate and electrode. Alternatively, the amount of electricity may be measured. Any substance having an electron transfer function can be used as the electron carrier, and examples thereof include substances such as ferrocene derivatives and quinone derivatives. Alternatively, the oxidation or reduction current obtained by interposing an electron carrier between hydrogen peroxide generated by the oxidase reaction and the electrode, or the amount of electricity thereof may be measured.
[0054]
In order to quantify the glycated peptide or glycated protein in the test solution using the quantification composition of the present invention thus prepared, 0.001 to 0.5 ml of the test solution is added to the quantification composition, and the temperature is 37 ° C. When the reaction is performed and a rate assay is performed, several minutes to several tens of minutes between two points after a certain time after the start of the reaction, for example, 1 minute after 3 minutes and 4 minutes, or 3 minutes and 8 minutes The amount of changed coenzyme, dissolved oxygen, hydrogen peroxide or other products in the next 5 minutes may be measured directly or indirectly by the above method. In the case of an endpoint assay, a certain time after the start of the reaction. The amount of the changed coenzyme, dissolved oxygen, hydrogen peroxide or other product may be measured in the same manner. In this case, the amount of glycated peptide and glycated protein in the test solution can be determined by comparing with changes in absorbance or the like when measured using a known concentration of glycated amino acid, peptide or protein.
[0055]
DETAILED DESCRIPTION OF THE INVENTION
Next, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.
[Example 1]
<Screening for proteases that act on glycated peptides or proteins at the N-terminus and release glycated amino acids or glycated peptides at the N-terminus>
The screening consists of two stages: a primary screening in which a large amount of proteases are narrowed down, and a secondary screening in which fructosyl valine is purified from a synthetic substrate by using fructosylamine oxidase from those picked up in the primary screening. I went separately.
[0056]
(1) Primary screening
<Reaction liquid 1>
100 mM Tris-HCl buffer pH 8.5
2mM FValpNA
200 μL of the reaction solution 1 was placed in a 96-well plate, 50 μL of a protease-containing solution (higher concentration is preferred) was added, and the mixture was reacted at room temperature for 10 minutes to overnight to select a yellow colored protease. Further, 900 μL of the above reaction solution was placed in a cuvette, and 50 μL of a protease-containing solution was added to the protease confirmed to be colored, and the absorbance change ΔA at 500 nm was measured. A reaction solution formulated with distilled water instead of the substrate FValpNA was prepared, and the change in absorbance ΔA blank was measured in the same manner. The effect of the protease on the glycated amino acid derivative was calculated by ΔA-ΔA blank.
[0057]
(2) Secondary screening
The following activities were confirmed for proteases that were confirmed to be active in the primary screening and proteases that had been reported to have an action on glycated hemoglobin.
<Reaction liquid 2>
6mM FValpNA
15mM Tris-HCl buffer pH7.5 (Wako Pure Chemical Industries, Ltd.)
2U / ml FAOD (Asahi Kasei)
2U / ml POD (Sigma)
0.01% 4-AA (Wako Pure Chemical Industries, Ltd.)
0.01% TOOS (Wako Pure Chemical Industries)
[0058]
Place the above reaction mixture 2 in a 933 μL cuvette and preheat at 37 ° C. for 5 minutes. Add 67 μL of a solution containing an appropriately diluted protease, stir to start the reaction, and measure the absorbance change ΔA per 10 minutes at 555 nm. A reaction solution formulated with distilled water instead of the substrate FValpNA was prepared, and the change in absorbance ΔA blank was measured in the same manner. The effect of the protease on the glycated amino acid derivative was calculated by ΔA-ΔA blank.
The results are shown in Table 1.
[0059]
[Table 1]

[0060]
Surprisingly, as shown in Table 1, the activity that liberates FVal from FValpNA was not recognized in the protease that has been said to be usable for the measurement of glycated hemoglobin.
[0061]
Pyrococcus furiosus (Pyrococcus  furiosus) Genus-derived deblocking aminopeptidase (manufactured by Takara), porcine liver-derived N-acylaminoacyl-peptide hydrolase (N-Acylaminoacyl-peptide hydrolase; manufactured by Takara), etc. A strong action was confirmed for proteases that release N-terminal blocked amino acids.
[0062]
Similarly, dipeptidyl peptidase that releases 2 amino acids from the N-terminus, such as bovine spleen-derived cathepsin C and dipeptidyl aminopeptidase IV, was confirmed to have the same action. This is thought to be because the protease recognizes the glycated amino acid as two amino acid molecules and efficiently releases the glycated amino acid.
[0063]
Of the aminopeptidases excluding the special aminopeptidase, only leucine aminopeptidase was found to release fructosyl valine from the synthetic substrate, and no activity was confirmed from other aminopeptidases. This is presumed that the activity was confirmed only in leucine aminopeptidase because the side chain of leucine is similar in size to the sugar bonded to the N-terminus.
Furthermore, a similar activity was confirmed for hemicellulase.
[0064]
[Example 2]
<Reaction curve for synthetic substrate>
<Reaction solution> Same as reaction solution 2 in Example 1
[0065]
<Operation>
The reaction solution 2 shown in Example 1 is placed in a 933 μL cuvette and pre-warmed at 37 ° C. for 500 seconds. Add 67 μL of a solution containing an appropriately diluted protease, stir to start the reaction, and measure the absorbance at 555 nm. A reaction solution formulated with distilled water instead of the substrate FValpNA was prepared, and the change in absorbance was measured by the same operation. A reaction curve was calculated by subtracting a water blank from the absorbance obtained from FValpNA.
The results are shown in FIG.
[0066]
As can be seen from FIG. 1, deblocking aminopeptidase (Deblocking aminopeptidase; manufactured by Takara), porcine liver-derived N-acylaminoacyl-peptide hydrolase (produced by Takara) and petitidylaminopeptidase IV (Dipeptidyl) Regarding aminopeptidase IV), FVal was generated from FValpNA and hydrogen peroxide was observed over time by FOD.
[0067]
[Example 3]
<Calibration curve when glycated hemoglobin is used as substrate>
<Reaction liquid 1>
10 mM Tris buffer pH 8.5
10U / ml deblocking aminopeptidase (Takara, Japan)
[0068]
<Reaction liquid 2>
150 mM Tris buffer pH 8.5
15U / ml POD (Sigma)
12mM TOOS
8mM 4-AA (Wako Pure Chemical Industries)
2U / ml FOD (Asahi Kasei Kogyo)
<Hb substrate solution>
Human hemoglobin was dissolved in distilled water so as to be 4 g / dl to obtain a Hb substrate solution.
Samples of 0.0, 0.5, 1.0, 1.5, and 2.0 times the concentration of Hb substrate solution (4 g / dl) were used.
[0069]
<Operation>
Take 240 µl of the above reaction mixture in a test tube and warm at 37 ° C for 5 minutes. 8 μl of Hb substrate solution was added, and the reaction was started by stirring. After exactly 5 minutes, the protein was deproteinized with trichloroacetic acid, and after neutralization, 80 μl of reaction solution 2 was added to this solution. The absorbance at 546 nm before and after the addition of reaction solution 2 was measured, and the difference was defined as the change in absorbance. The same operation was performed using distilled water instead of the substrate to obtain a blank sample.
[0070]
The sensitivity was calculated by subtracting the absorbance change of the blank sample from the absorbance change obtained from the Hb substrate solution. The Hb 1.0-fold concentration was measured 10 times and the CV value was calculated. The result is shown in FIG.
As can be seen from FIG. 2, the Hb substrate solution showed good linearity depending on the concentration. In addition, a good reproducibility with a CV value of 2.5% was confirmed at a Hb 1.0-fold concentration, and glycated hemoglobin was selectively measured with high sensitivity and good reproducibility using the measurement system of the present invention. It became clear.
[Brief description of the drawings]
FIG. 1 is a reaction curve when a synthetic substrate based on Example 2 of the present invention is used.
FIG. 2 is a calibration curve of glycated hemoglobin based on Example 3 of the present invention.

Claims (6)

A test solution containing a peptide or protein in which the N-terminal valine is fructosylated is added to a deblocking aminopeptidase, dipeptidylaminopeptidase, leucine aminopeptidase (Leucine Apeptidase).
minopeptidase), N-Acylaminoacyl-pep hydrolase (N-Acylaminoacyl-pep)
tide hydrolase) or hemicellulase (Hemicelullase) was allowed to act to liberate the fructosyl valine in N-terminal, quantitative methods of peptide or glycated proteins valine N-terminal is fructosylated characterized by quantifying this . Determination method according to claim 1, wherein measured using an enzyme that acts quantitation of fructosyl valine free N-terminal glycated amino acids. Or peptides valine N-terminal is fructosylated is a fragment of the N-terminus of hemoglobin glycated, or claim 1 or 2 glycated proteins valine is fructosyl of the N-terminal is hemoglobin glycated Quantification method. Deblocking Aminopeptidase, Dipeptidyl Aminopeptidase, Leucine Aminopeptidase (Leucine)
Aminopeptidase), N-acylaminoacyl-peptide hydrolase (N-Acylaminoacyl-p
eptide hydrolase) or hemicellulase (Hemicelullase) quantitative composition of a peptide or protein valine N-terminal and comprising content is fructosyl the. Deblocking Aminopeptidase, Dipeptidyl Aminopeptidase, Leucine Aminopeptidase (Leucine)
Aminopeptidase), N-Acylaminoacyl-peptide hydrolase (N-Acylaminoacyl-p
eptide hydrolase) or hemicellulase (Hemicelullase) as well as peptides valine N-terminal comprising an enzyme that acts on glycated amino acid is fructosylated or protein quantitative composition. 6. The peptide according to claim 4 or 5, wherein the peptide in which the N-terminal valine is glycated is an N-terminal fragment of glycated hemoglobin , or the glycated protein in which the N-terminal valine is fructosylated is glycated hemoglobin. Composition for determination.

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