JP2010273610A - 金属プロトポルフィリン錯体の定量方法及びそれに用いる酵素センサー - Google Patents
金属プロトポルフィリン錯体の定量方法及びそれに用いる酵素センサー Download PDFInfo
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Abstract
【解決手段】蛍光色素で蛍光ラベル化されたヘムオキシゲナーゼを用いる金属プロトポルフィリン錯体の定量方法において、蛍光色素として、親水性で、かつ、その蛍光波長が金属プロトポルフィリン錯体の吸収波長と重なるような蛍光色素を用いることからなる金属プロトポルフィリン錯体の定量方法、及び、それに用いる酵素センサー。ヘムオキシゲナーゼとしては、ラットヘムオキシゲナーゼの18番目の残基をシステインに変異させ、該位置に、金属ポルフィリン錯体の吸収波長と重なるような蛍光波長をもつ親水性蛍光色素を導入したものであるのが好ましい。
【選択図】なし
Description
野生型ラットHO−1のC末端膜結合部位を除いて可溶性とした、リコンビナントタンパク質の発現プラスミドを母体とし、K18Cをコードする遺伝子を大腸菌へ形質転換した。その変異株を液体培地で培養し、菌体内にラットHO−1を大量発現させた。野生型ラットHO−1は分子構造にシステイン残基を一つも含まないが、K18Cへと変異させることで、唯一のチオール基を有することになる。
精製したK18Cのシステイン残基に、蛍光団を導入する操作を行った。K18C溶液に、数倍モル量のDTTを加えてシステイン部位を活性化した後、4℃で透析してDTTを取り除いた。次に、K18Cに対して5倍モル量の蛍光色素溶解液を加え、室温、アルゴンガス雰囲気下で4時間反応させた後、反応停止剤として2−メルカプトエタノールを添加した。
℃で72時間透析を行い、未反応の蛍光色素分子と2−メルカプトエタノールを完全に取り除いた。透析終了後、蛍光ラベル化されたK18Cを回収し、吸収スペクトルを測定した。分光光度計はV−650(ジャスコエンジニアリング株式会社製)を使用した。
実施例1で作製した3種類の蛍光ラベル化タンパク質が、野生型ラットHO−1と同等のヘム親和性を維持しているかについて試験を行った。蛍光測定用石英セルに2ml
の蛍光ラベル化タンパク質溶液を入れ、ヘム溶液を滴定して25℃における蛍光スペクトルを測定した。蛍光分光光度計はFP−6500(ジャスコエンジニアリング株式会社製)を使用した。使用したヘム溶液の濃度決定は、ピリジンヘモクロム法を適用した。
図5のヘム濃度0〜200nMの範囲を拡大し、線形近似式を追加したものを図7に示した。蛍光強度差とヘム濃度との間に良好な相関係数が得られたため、これを検量線とすれば、未知のヘム濃度の定量が可能である。よって、このヘムセンサータンパク質(酵素センサー)を用いて定量操作を行うには、あらかじめ200nM程度のHO−A350溶液に、0〜200nMの既知濃度のヘムを加えたときのF0−Fを数点測定し、それらの値から検量線を作成しておき、次に、未知濃度のヘムの蛍光強度差より濃度を算出すればよい。この実施例より、ヘム濃度が10nM以上であれば定量可能であることが示され、従来の方法であるピリジンヘモクロム法(およそ10μM以上で定量可能)の1000倍もの感度が実現された。
Claims (6)
- 蛍光色素で蛍光ラベル化されたヘムオキシゲナーゼを用いる金属プロトポルフィリン錯体の定量方法において、蛍光色素として、親水性で、かつ、その蛍光波長が金属プロトポルフィリン錯体の吸収波長と重なるような蛍光色素を用いることを特徴とする金属プロトポルフィリン錯体の定量方法。
- ヘムオキシゲナーゼが、ラットヘムオキシゲナーゼであることを特徴とする請求項1記載の金属プロトポルフィリン錯体の定量方法。
- ヘムオキシゲナーゼが、ラットヘムオキシゲナーゼの18番目の残基をシステインに変異させ、該位置に親水性蛍光色素を導入したものであることを特徴とする請求項1又は2記載の金属プロトポルフィリン錯体の定量方法。
- 蛍光色素の蛍光波長が金属プロトポルフィリン錯体の吸収波長と重なるような親水性蛍光色素で蛍光ラベル化されたヘムオキシゲナーゼからなる酵素センサー。
- ヘムオキシゲナーゼが、ラットヘムオキシゲナーゼである請求項4記載の酵素センサー。
- ヘムオキシゲナーゼが、ラットヘムオキシゲナーゼの18番目の残基をシステインに変異させ、該位置に親水性蛍光色素を導入したものである請求項5記載の酵素センサー。
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