JP4731733B2 - Determination of homocysteine in cysteine coexisting samples - Google Patents

Description

ｖ：反応速度、Ｓ：基質濃度、Ｖmax：基質飽和における最大反応速度、Ｋｍ：ミカエリス定数（基質親和性の逆数）
【００１９】
に従う場合において、システインに対するＶmaxが低く、低濃度のシステインで容易に基質飽和するが、ホモシステインに対するＶmaxは高いためホモシステインでは基質飽和されず、更に、システインに対するＫｍよりホモシステインに対するＫｍの方が小さい（親和性が高い）ため実質的にシステインの影響なくホモシステインとの反応が進む酵素のことである。
【００２０】
本発明で用いる、システインよりもホモシステインに対する作用が強いホモシステイン変換酵素は、以下に述べる種々の方法によって得ることができる。
ホモシステイン分解酵素であってホモシステイン置換酵素でもあるＬ−メチオニンγ−リアーゼは、この酵素を産生する微生物、例えばシュードモナス属の細菌等から公知の方法により得ることもでき、また和光純薬株式会社等から市販されており、これらＬ−メチオニンγ−リアーゼから上記条件に合う酵素を選別して用いればよい。
【００２１】
ホモシステイン置換酵素であるＯ−アセチルホモセリン−リアーゼやγ−シアノ−α−アミノ酪酸合成酵素（ＧＣＳ）は、公知の酵素を用いることもでき（特開２０００−２２８９９８号公報）、また、該酵素を産生する様々な微生物等から公知の方法（例えば、Ｏｚａｋｉ等，Ｊ．Ｂｉｏｃｈｅｍ．，９１，１１６３−１１７１（１９８２）、Ｙａｍａｇａｔａ，Ｊ．Ｂｉｏｃｈｅｍ．，９６，１５１１−１５２３(１９８４)、Ｂｒｚｙｗｃｚｙ等，Ａｃｔａ Ｂｉｏｃｈｉｍｉｃａ Ｐｏｌｏｎｉｃａ，４０（３），４２１−４２８（１９９３）等）により得ることもできる。また、γ−シアノ−α−アミノ酪酸合成酵素（ＧＣＳ）としては、市販の酵素、例えば池田食研株式会社製のバチルス属由来のものも使用可能であり、これらの酵素から上記条件に合う酵素を選別して用いればよい。池田食研株式会社製の上記ＧＣＳは特開２０００−２２８９９８号公報に記載されている。
【００２２】
また、これら上記の酵素をコードする遺伝子を酵素を産生する微生物から取り出し、この遺伝子またはこの遺伝子情報を参考にして修飾した遺伝子、酵素のアミノ酸配列情報を参考にして作成した遺伝子などを、遺伝子工学的手法を用いて微生物等に発現させ、次いで上記条件に合う酵素を選別して得ることもできる。
【００２３】
例えば、この遺伝子工学的手法を用いて発現し選別された酵素としては、耐熱性菌であるサーマス・サーモフィラス菌株由来のＯ−アセチルホモセリン−リアーゼがあり、池田食研株式会社から市販されている。
【００２４】
該酵素について、その遺伝子組換え法の手法は、例えば下記の通りである。
Ｏ−アセチルホモセリン−リアーゼのアミノ酸配列及びそれをコードする遺伝子の塩基配列を解析した報告は多数存在する。例えば、公開データベースの集合体であるGenomeNet WWW server（https://www.genome.ad.jp/）にある「LIGAND」で、EC番号「4.2.99.10」をキーワードとして検索すると、「ec:4.2.99.10 O-Acetylhomoserine (thiol)-lyase; Methionine synthase」というエントリーが見出され、Pseudomonas aeruginosa、Candida jejuni、Bacillus halodurans、Deinococcus radiodurans、Thermotoga maritime Halobacterium、Saccharomyces cerevisiae、Schizosaccharomyces pombeなどの微生物等が有するＯ−アセチルホモセリン−リアーゼのアミノ酸配列及びその遺伝子の塩基配列データを、閲覧する事が可能である。
【００２５】
これらのデータから、幾つかのＯ−アセチルホモセリン−リアーゼで保存されている特有の塩基配列を知る事が出来る。この情報を元に、サザンハイブリダイゼーションに用いるプローブ或いはPolymerase Chain Reaction （PCR）に用いるプライマーをデザインし、サーマス・サーモフィラスの染色体DNAを対象にサザンハイブリダイゼーション或いはPCRを行う事により、サーマス・サーモフィラスのＯ−アセチルホモセリン−リアーゼ遺伝子を特定し、取得する事が可能である。
【００２６】
Ｏ−アセチルホモセリン−リアーゼの構造遺伝子と、この遺伝子の発現を調節し得るプロモーター領域を含む適当な宿主細胞内で複製可能なプラスミドDNAとからなる組換え体DNAは、常法（例えばジェイ・サムブルック、イー・エフ・フリッチ、ティー・マニアティス、モレキュラー・クローニング：ア・ラボラトリー・マニュアル第2版、コールド・スプリング・ハーバー・ラボラトリー・プレス社刊（1989)［J.Sambrook, E.F.Fritsch, T.Maniatis, Molecular Cloning: A Laboratory Manual 2nd ed, Cold Spring Harbor Laboratory Press（1989)］等参照）により作製する事が出来る。
【００２７】
大腸菌K12株等の適当な宿主細胞を、上記の組換え体DNAで形質転換し、Ｏ−アセチルホモセリン−リアーゼを産生する形質転換された宿主細胞を得る事が出来る。この形質転換体を適当な条件で培養し、産生するＯ−アセチルホモセリン−リアーゼを得る方法としては、一般の微生物等の培養方法及び蛋白質の精製方法が利用出来る。即ち、この形質転換体を培養し、培養液を遠心分離して得られる形質転換体を破砕し、遠心分離等によって破砕液から上清液を得る。この上清液中に含まれるＯ−アセチルホモセリン−リアーゼを、塩析、透析、イオン交換クロマトグラフィー、疎水吸着クロマトグラフィー、ゲル濾過、アフィニティークロマトグラフィー、電気泳動等の適当な精製操作を組み合わせる事によって精製して、本発明に使用し得るＯ−アセチルホモセリン−リアーゼを調製することができる。
【００２８】
さらに、本発明に使用されるシステインよりもホモシステインに対する作用が強いホモシステイン変換酵素には、酵素活性を保っていれば上記の酵素を修飾したものも含まれる。
【００２９】
本発明の定量法において試料に添加するシステインとしては、ホモシステイン変換酵素によるホモシステイン測定の際においてシステインであればよく、システインのほかに、ホモシステイン測定の条件下またはホモシステイン測定に影響を与えない条件でシステインを遊離するシステイン誘導体が挙げられる。システイン誘導体としては例えばシスチン等でもよい。
本発明の定量法において試料に添加するシステインの量は、試料中に存在するシステインの影響を実質的に無視できる程度、即ち本発明で使用するシステインよりもホモシステインに対する作用が強いホモシステイン変換酵素の基質飽和量となる量程度を加えればよい。このシステインの添加量は、ホモシステイン置換酵素の場合、ホモシステインに対する特異性が高く、システインよりホモシステインに強く作用する酵素ほど少なくてよいが、試料中のシステイン濃度が、通常１μＭ〜１０ｍＭ程度、好ましくは１０μＭ〜１ｍＭ程度となる量である。例えば、ホモシステイン置換酵素がサーマス・サーモフィラス菌株由来のＯ−アセチルホモセリン−リアーゼの場合では酵素反応の場において終濃度が２０μＭ以上、より好ましくは１００〜５００μＭとなるように添加すればよい。ホモシステイン分解酵素あるいはＬ−メチオニンγ−リアーゼを用いる場合も、システインの添加量は、ホモシステイン置換酵素の場合とほぼ同様である。
システインまたはシステイン誘導体を添加する方法は、その溶液や粉末を直接試料に添加しても、ホモシステイン測定試薬の成分とともに試料に添加してもよい。
【００３０】
本発明の定量法において、前記酵素の添加量は、試料中のホモシステインを検出するのに充分な量であればよく、特に限定されない。ホモシステインが微量にしか含まれない試料の場合は、ホモシステインを完全に消費することができる最低必要量以上であることが好ましい。例えば、血漿中の総ホモシステインを測定する場合、例えば還元試薬で処理した血漿に、１〜１０００単位の酵素を加え、ｐＨ４．５〜１１．５の緩衝液中、５〜５０℃で、１〜１０分間反応させればよい。さらに好ましくは、１〜１００単位の酵素を加え、ｐＨ５〜１０の緩衝液中、２５〜４５℃で、２〜１０分間反応させる。
【００３１】
緩衝液は、上記したｐＨ範囲で緩衝作用を有するものから選ばれ、トリス緩衝液、グッド緩衝液、リン酸、炭酸、硼酸、などの無機酸塩由来の緩衝液、酢酸、クエン酸などの有機酸塩由来の緩衝液などがある。好ましくは、ホモシステインとシステインの反応性の差を大きくし、ホモシステインに対する特異性を向上できるような緩衝液が選ばれる。例えば、トリス緩衝液、リン酸緩衝液等が挙げられる。
【００３２】
本発明の定量法において、ホモシステインのγ位メルカプト基を求核試薬（アニオン）で置換する反応を触媒するホモシステイン置換酵素を用いる場合、求核試薬としては、該酵素の基質となるものであれば何であってもよく、特に限定されない。例えば、無機塩類のアニオン、有機化合物塩類のアニオン、チオール基を有する化合物および該化合物のチオレートアニオンや、水酸基を有する化合物および該化合物のアルコキシルイオンなどがあげられる。好ましくは、ホモシステインとシステインの反応性の差を大きくし、ホモシステインに対する特異性を向上できるような求核試薬、例えば２−メルカプトエタノール、チオグリセロール、ジチオスレイトール（ＤＴＴ）、シアンイオン等が選ばれ、特に好ましくは２−メルカプトエタノール、チオグリセロール等が挙げられる。なお、対カチオンは特に限定されない。
【００３３】
試料中のホモシステインは、ＳＨ基同士の酸化反応、または、ＳＳ結合との交換反応によって容易に縮合し、特に血漿や血清などの生体試料中ではＳＨ基を有する低分子化合物や蛋白質とＳＳ結合（ジスルフィド結合）して存在（コンジュゲート）する場合が多い。このため、これらの検体中に存在しているコンジュゲートも含めてホモシステインを総ホモシステインとして測定する必要がある場合には、コンジュゲートしているホモシステインを、化学反応や酵素反応によって強制的に遊離させ、この処理液中のホモシステインを測定する。本発明において定量されるホモシステインには、このようにして遊離されたホモシステインも含まれる。コンジュゲートしているホモシステインを遊離させる化学反応としては、チオール類、ホスフィン類や水素化硼素類などの還元剤を用いて行うことができる。例えば、Ｊｏｃｅｌｙｎによって報告されているもの（Ｍｅｔｈｏｄｓ ｏｆ Ｅｎｚｙｍｏｌｏｇｙ，１４３，２４３−２５６（１９８７））が利用できる。
【００３４】
中でも、ジチオスレイトール（ＤＴＴ）、ジチオエリスリトール（ＤＴＥ）、２−メルカプトエタノール、還元型グルタチオン、チオグリコール酸、メタンチオール、２−メルカプトエタン、システイン、チオグリセロール、システアミン、チオフェノール類などＳＨ基を有する低分子化合物を試料に過剰に加えて、これらとのＳＳ交換反応を利用し、穏やかにホモシステインを遊離させる方法が好ましい。例えば、血漿中の総ホモシステインを測定する場合、例えば、血漿に、１〜２０ｍＭのＤＴＴを加えて還元すればよい。
【００３５】
更にチオール類は、容易にコンジュゲート状態からホモシステイン及びシステインを特異的に遊離し、かつ、ホモシステイン置換酵素の求核試薬も兼ねることができるため、そのまま酵素反応も行うことができ好ましい。特に好ましくは、ホモシステインに対する特異性を高めるチオール類が選ばれ、例えば、チオグリセロール、２−メルカプトエタノールなどが挙げられる。
【００３６】
本発明のホモシステインの測定において、前記酵素を作用させることにより発生する硫化水素の検出法としては、公知の方法を含め何であってもよく特に限定されない。例えば、特開２０００−１６６５９７号公報、特開２０００−２２８９９８号公報、特開２０００−３３８０９６号公報、Clinical Chemistry, 46, 1686〜1688 (2000)等に記載の方法が挙げられる。
特に、臨床的に血清や血漿中のホモシステインを測定する場合、現在、広く普及している臨床検査用の汎用測定機に適用できる比色法による検出法が好ましい。
【００３７】
硫化水素を直接定量する方法のみならず、硫化水素に起因する硫化物イオンを定量する方法、あるいは更に発色させて定量する方法であってもよい。例えば、（１）酸性条件下で酢酸鉛紙の黒変を検出する方法、（２）亜鉛を用いて、生成する硫化亜鉛の沈殿を検出する方法、（３）アルカリ条件下、ニトロプルシッドナトリウムとの反応により、硫黄イオンとして紫色に発色させる方法、（４）強酸性下で、Ｎ，Ｎ−ジメチル−ｐ−フェニレンジアミンと塩化第二鉄を用いてメチレンブルーを生成させ青色発色を検出する方法、（５）Ｎ，Ｎ−ジブチル−ｐ−フェニレンジアミンとフェリシアン化カリを用いて蛍光色素を生成させ、その蛍光強度を検出する方法（Ｃｌｉｎｉｃａｌ Ｃｈｅｍｉｓｔｒｙ ４６， １６８６−１６８８（２０００））、（６）２−（５−ブロモ−２−ピリジルアゾ）−５−[Ｎ−ｎ−プロピル−Ｎ−（３−スルフォプロピル）アミノ]フェノール・ナトリウム塩（５−Ｂｒ−ＰＡＰＳ）と塩化第一鉄を用いて色素を生成させ、その発色強度を検出する方法（特開２０００−３３８０９６号公報）などが挙げられる。
【００３８】
本発明には、システインを含む、システイン共存試料中のホモシステイン定量用試薬も含まれる。システインは、試料中のシステインの影響が回避されるのに充分な量であればよく、前記記載の濃度で含まれる。また、システインよりもホモシステインに対する作用が強いホモシステイン変換酵素を含む、システイン共存試薬中のホモシステイン定量用試薬も本発明に含まれる。該酵素としては、前記のホモシステイン変換酵素が挙げられ、前記例示の酵素が好ましい。含まれる該酵素の量については、前記と同じ程度でよい。また、本発明の試薬に含まれるシステインは、ホモシステイン測定の際においてシステインであればよく、即ち前記のシステインを遊離するシステイン誘導体でもよい。
システイン、システインよりもホモシステインに対する作用が強いホモシステイン変換酵素、及び該変換酵素の作用により発生する硫化水素を検出するための検出試薬を含む、システイン共存試料中のホモシステインを測定するためのホモシステイン測定キットも本発明に含まれる。ホモシステイン測定用キットは、具体的には、システインの影響を回避するためのシステイン、システイン誘導体またはその溶液、ホモシステインから硫化水素を遊離させるためのホモシステイン変換酵素、硫化水素を検出するための検出試薬から構成される。システインよりもホモシステインに対する作用が強いホモシステイン変換酵素、硫化水素を検出するための検出試薬としては、前記した酵素及び試薬が挙げられ、好ましくは前記例示したものが挙げられる。特に検出試薬としてはＮ，Ｎ−ジアルキル−ｐ−フェニレンジアミンが好ましい。
【００３９】
更に、対象とする検体によってキットの試薬の構成は変化し得る。ＳＨ基を有する低分子化合物や蛋白質とコンジュゲートしているホモシステインをも含めて測定する必要がある場合には、一般的には、コンジュゲート状態からホモシステインを遊離させるための還元試薬、例えば、前記したチオール類、ホスフィン類や水素化硼素類などを構成中に含んでもよい。ホモシステイン変換酵素がホモシステイン置換酵素である場合は、ホモシステインのγ位メルカプト基を置換しうる求核試薬も構成中に含まれる。しかし、ホモシステインのγ位メルカプト基を置換しうる求核試薬が、上記還元試薬も兼ねる場合、敢えて求核試薬を添加しなくてもよい。
更に、緩衝液を構成中に含むキットも本発明に含まれる。緩衝液としては、前記した緩衝液が挙げられ、好ましくは前記例示したものが挙げられる。上記試薬を緩衝液で溶解してキット中に含んでも良く、また緩衝液を別途付けてもよい。
【００４０】
これらの試薬を構成する上で、互いに干渉する成分が存在する場合には、さらに構成成分毎に分割し、細分化することもできる。逆に、これらの構成試薬を統合しても使用上問題がない場合には、これらを統合してもよい。例えば、システインを前記還元試薬として加えることで、システインの影響を回避するための別途添加を省略してもよい。
【００４１】
血漿を検体として総ホモシステインを測定するキットの場合、例えば、２−メルカプトエタノールとシステインを含む還元試薬、前記のＯ−アセチルホモセリン−リアーゼを含む酵素試薬、硫化水素をメチレンブルーとして検出するための発色剤から構成されるキットが好ましく挙げられる。このキットの場合の１キット当たりに使用される各成分の量は、キットのテスト数と、１テスト当たりの反応液量によって変化するが、１００テスト用（２０ｍＬ用）のキットの場合、例えば、２−メルカプトエタノールが０．２〜１０００μｍｏｌ、システインが１０〜１０００ｎｍｏｌ、Ｏ−アセチルホモセリンスルフヒドリラーゼが１〜１０００単位である。
【００４２】
【実施例】
以下に、比較例及び実施例により本発明をより具体的に説明するが、本発明はこれらに限定されるものではない。
【００４３】
比較例
システイン無添加によるホモシステインの測定
２５０ｍＭトリス塩酸緩衝液（ｐＨ８．５）５０μＬに、２５ｍＭ ２−メルカプトエタノール２０μＬ、２００Ｕ／ｍＬのサーマス・サーモフィラス菌株由来Ｏ−アセチルホモセリン−リアーゼ（池田食研製）１０μＬを添加後、検体として、倍々希釈した１００μＭのホモシスチン溶液（ホモシステインとしては２００μＭに相当し、終濃度では４０μＭに相当する）２５μＬと、蒸留水、または、６２５μＭのＬ−システイン２０μＬ（終濃度では１００μＭに相当する）を加え、３７℃で５分間反応した。さらに、蒸留水２５μＬ、１６ｍＭのＮ，Ｎ−ジメチル―ｐ―フェニレンジアミン塩酸塩の１Ｎ硫酸溶液８１．２５μＬと、１０ｍＭ塩化第二鉄の１Ｎ硫酸溶液１８．７５μＬを加え、３７℃で５分間反応した後、６７０ｎｍにおける吸光度を測定した。得られた吸光度とホモシステインの濃度の関係をプロットし、作成した検量線を図１に示す。
図１から、システインを添加した検体の検量線では、添加していないものよりも大きく嵩上げされ、強くシステインの影響を受けていることが分かる。
【００４４】
実施例１
システイン添加によるホモシステインの測定
上記比較例のトリス塩酸緩衝液に、さらに500μＭとなるようにＬ−システイン（終濃度では200μＭに相当する）を追加し、比較例と同様に反応させ、ホモシステインを測定した。得られた吸光度とホモシステインの濃度の関係をプロットし、作成した検量線を図２に示す。
図２に示したように、Ｌ−システインの追加によって、両方の検量線がほぼ同一直線状に重なった。このことは、緩衝液に追加されたＬ−システインによって、検体に含まれるシステインの影響を消去できることを意味し、この検量線を用いてホモシステインを定量することができる。
【００４５】
実施例２
モデルサンプルとして、ホモシスチン（ホモシステインの２量体）５０μＭ溶液（サンプルＡ）と、システイン１００μＭを添加したホモシスチン５０μＭ溶液（サンプルＢ）とを、比較例の方法と本発明の実施例１の方法で測定した。即ち、比較例の方法としては、サンプルＡとＢに更にシステインを添加することなく測定し、実施例１の方法としては、サンプルＡとＢに更に終濃度が２００μＭとなるようにシステインを添加して測定した。その結果を表１にまとめた。システインを含むサンプルＢのホモシステインの理論値は１００μＭであるが、比較例の方法では測定値が１５０．４μＭ、本発明の実施例１の方法では１０７．０μＭとなり、本発明の方法によるとほぼ理論値に近い値が得られ、システイン共存試料中のホモシステインの特異的測定が可能であることが示された。
【００４６】
【表１】

【００４７】
【発明の効果】
本発明の測定法を用いると、ホモシステインとシステインを含む試料中のホモシステイン量を、試料に基質飽和量となる量程度のシステインを添加し、システインよりもホモシステインに対する作用が強いホモシステイン変換酵素を用いて、システインの影響を受けることなく容易に測定することができる。この方法を用いて、臨床検体中のホモシステインを臨床検査用の汎用測定機で簡便に測定でき、動脈硬化症の診断にも使用できる。
【図面の簡単な説明】
【図１】検体中のシステインの影響を調べるホモシステインの検量線を示す。
【図２】システイン添加により、システインの影響を回避したホモシステインの検量線を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for quantifying homocysteine using an enzyme in a sample containing homocysteine and cysteine, and a reagent and a measurement kit used therefor.
[0002]
[Prior art]
As the sulfur atom-containing amino acids constituting proteins in living bodies, methionine, cysteine, cystine and the like are known, and each of them maintains homeostasis in a series of metabolic cycles in the living body. Normally, homocysteine is rapidly metabolized and is therefore hardly present in the living body at normal times.
[0003]
However, homocysteine accumulation may occur due to abnormalities of enzymes and cofactors involved in the metabolism (New England Journal of Medicine, 318, 1720-1728 (1988), Japanese Patent Laid-Open No. 7-49348).
[0004]
In recent years, homocysteine has been attracting attention as an indicator of new arteriosclerosis (especially heart and cerebrovascular) independent of traditional arteriosclerosis risk factors such as smoking, blood pressure, blood glucose, cholesterol, and neutral fat. Many clinical data showing the relationship between homocysteine blood levels and diseases have been reported (for example, New England Journal of Medicine, 324, 1149-1155 (1991)).
[0005]
Conventionally, as a method for measuring homocysteine in a sample, gas chromatography-mass spectrometry (JP-A-62-2212249, JP-A-7-49348) or high-performance liquid chromatography (HPLC) (Clinical Chemistry) is used. , 39, 1590-1597 (1993)) and the like, a fluorescent substance (Clinical Chemistry, 35, 1921-1927 (1989), Clinical Chemistry, 39, 263-) via the SH group of homocysteine. 271 (1993)) and radioactive substances (Clinical Chemistry, 31, 624-628 (1985)), and then these derivatives are separated and analyzed by high performance liquid chromatography. There was. However, these methods are not accepted as a clinical measurement method because the sample pretreatment is complicated, a dedicated analyzer is required, and the sample throughput is low.
[0006]
A method in which homocysteine is modified by a chemical method and immunologically measured using an antibody specific for this homocysteine derivative (Japanese Patent Publication No. 9-512634 (WO95 / 30151)), homocysteine and adenosine After treatment with S-adenosylhomocysteine hydrolase (EC 3.3.1.1), the amount of adenosine changed in this reaction is detected by a combination of enzyme reaction systems, or the product of this reaction is detected using an anti-adenosine antibody. In other words, a method for immunologically detecting and indirectly measuring the concentration of homocysteine (Japanese Patent Publication No. 8-506478 (WO93 / 15220)) is known. However, these measurement methods also have some problems such as complicated operations and the need for a special immunoassay device.
[0007]
Several methods for measuring homocysteine that detect hydrogen sulfide generated using an enzyme that acts on homocysteine to release hydrogen sulfide have been reported. As such enzymes, homocysteine-degrading enzymes and homocysteine-replacement enzymes are known, but both have problems as described later.
[0008]
Homocysteine-degrading enzymes liberate hydrogen sulfide and ammonia from homocysteine, and homocysteine desulfhydrylase (EC 4.4.1.2), homocysteinase, and the like are known. There is a known method for measuring homocysteine by degrading homocysteine using these enzymes, and colorimetrically detecting any of hydrogen sulfide, ammonia, or 2-oxobutyric acid in the product produced by this reaction. (WO98 / 077872). However, this enzyme reacts with cysteine and methionine in addition to homocysteine, and there is a problem with the specificity of the enzyme, so cysteine and methionine must be removed by pretreatment, which is not practical for use in clinical practice. It was complete (Japanese translations of PCT publication No. 2000-513589).
[0009]
A homocysteine-replacement enzyme substitutes a mercapto group of homocysteine in the presence of a nucleophilic reagent such as a thiol compound. O-acetylhomoserine lyase (O-acetylhomoserine sulfhydrylase or methionine synthase, EC4 2.9.10), γ-cyano-α-aminobutyric acid synthase (GCS) and the like are known. One enzyme may belong to both the O-acetylhomoserine lyase (EC 4.2.9.10) and γ-cyano-α-aminobutyric acid synthase (GCS) classes. Measurement methods for detecting hydrogen sulfide desorbed by these enzymes have also been proposed (Japanese Patent Laid-Open Nos. 2000-166597 and 2000-228998). However, since O-acetylhomoserine lyase is reactive not only with homocysteine but also with cysteine, first the total of homocysteine and cysteine is measured using this enzyme, and the measured value of cysteine is subtracted from this value. Although a method for calculating a cysteine value has been proposed (Japanese Patent Laid-Open No. 2000-270895), a complicated process using two kinds of enzymes is required, and it is expected to be difficult to use in an actual clinical site.
[0010]
[Problems to be solved by the invention]
An enzyme-based measurement method that quantifies a small amount of homocysteine in a sample in which cysteine coexists is useful in terms of operability, but is highly specific for homocysteine using a single enzyme reaction. The measurement method was not known.
[0011]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have determined that when cysteine is quantified in a sample in which cysteine coexists, a sufficient amount of cysteine is present in the sample. The present inventors have found that when a homocysteine converting enzyme having a stronger effect on homocysteine is allowed to act, the influence of cysteine present in the sample can be avoided, and the homocysteine in the sample can be specifically quantified.
[0012]
That is, the present invention
(1) A method for quantifying homocysteine in a sample in which homocysteine and cysteine coexist, wherein cysteine is added to the sample, and then a homocysteine converting enzyme having a stronger effect on homocysteine than cysteine is allowed to act. A method for the determination of homocysteine;
(2) The method for quantifying homocysteine according to the above (1), wherein the homocysteine converting enzyme having a stronger effect on homocysteine than cysteine is a homocysteine-replacement enzyme derived from the genus Thermus;
(3) The method for quantifying homocysteine according to (1) or (2) above, wherein the cysteine concentration in the sample after addition of cysteine is 1 μM to 10 mM;
(4) A reagent for quantifying homocysteine in a sample coexisting with cysteine, characterized by containing cysteine;
[0013]
(5) A reagent for quantifying homocysteine in a cysteine coexisting sample, comprising a homocysteine converting enzyme having a stronger effect on homocysteine than cysteine;
(6) Measure homocysteine in a sample coexisting with cysteine, including cysteine, a homocysteine converting enzyme having a stronger effect on homocysteine than cysteine, and a detection reagent for detecting hydrogen sulfide generated by the action of the converting enzyme Homocysteine measurement kit for
(7) The homocysteine measurement kit according to (6), wherein the detection reagent is N, N-dialkyl-p-phenylenediamine; and
(8) The present invention relates to the homocysteine measurement kit according to (6) or (7), comprising a buffer solution.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The present invention is characterized in that cysteine is added to a sample in which homocysteine and cysteine coexist, and then a homocysteine converting enzyme having a stronger effect on homocysteine than cysteine is allowed to act on the homocysteine in the cysteine coexisting sample. It is a quantitative method.
The sample used in the present invention is not particularly limited as long as homocysteine and cysteine coexist. For example, clinical samples such as serum, plasma, spinal fluid, saliva, urine; red blood cells, stool, tissue, cultured cells, etc. In addition, industrial products such as foods, quasi-drugs, pharmaceuticals, etc., and if these products are solid, extracts of these products, or industrial products that produce products using fermentation In some cases, fermented liquor necessary for the process inspection may be mentioned.
[0015]
The homocysteine converting enzyme having a stronger action on homocysteine than cysteine used for quantification of homocysteine of the present invention is an enzyme having a stronger action on homocysteine than cysteine which acts on homocysteine to produce hydrogen sulfide. It is not specifically limited, The enzyme chosen from the said well-known enzyme may be sufficient. Examples of such homocysteine converting enzyme include those having both properties, such as homocysteine degrading enzyme, homocysteine-replacement enzyme, and L-methionine γ-lyase (EC 4.4.1.11.).
[0016]
Preferably, a homocysteine substitution enzyme that catalyzes a substitution reaction with a thiol compound in the presence of a thiol compound and has a stronger action on homocysteine than cysteine, such as O-acetylhomoserine lyase (O-acetylhomoserine sulfhydrylase) Alternatively, methionine synthase, EC 4.2.9.10), γ-cyano-α-aminobutyric acid synthase (GCS), an enzyme having a stronger action on homocysteine than cysteine selected from L-methionine γ-lyase and the like can be mentioned. It is done. In particular, homocysteine-converting enzyme derived from the genus Thermus having high specificity to homocysteine, particularly O-acetylhomoserine lyase derived from the thermostable bacterium Thermus thermophilus described later, is preferable.
[0017]
The enzyme having a stronger action on homocysteine than cysteine in the present invention is the following Michaelis-Menten formula:
[0018]
[Formula 1]

v: reaction rate, S: substrate concentration, Vmax: maximum reaction rate at substrate saturation, Km: Michaelis constant (reciprocal of substrate affinity)
[0019]
The Vmax for cysteine is low and saturates the substrate easily at low concentrations of cysteine, but the Vmax for homocysteine is high, so the substrate is not saturated with homocysteine, and the Km for homocysteine is higher than the Km for cysteine. Because it is small (high affinity), it is an enzyme that reacts with homocysteine substantially without the influence of cysteine.
[0020]
The homocysteine converting enzyme used in the present invention and having a stronger effect on homocysteine than cysteine can be obtained by various methods described below.
L-methionine γ-lyase, which is a homocysteine-degrading enzyme and a homocysteine-replacement enzyme, can also be obtained by a known method from a microorganism that produces this enzyme, such as bacteria belonging to the genus Pseudomonas, and Wako Pure Chemical Industries, Ltd. The enzymes that meet the above conditions may be selected from these L-methionine γ-lyases and used.
[0021]
As O-acetylhomoserine lyase and γ-cyano-α-aminobutyric acid synthase (GCS), which are homocysteine-replacement enzymes, known enzymes can be used (Japanese Patent Laid-Open No. 2000-228998), and the enzymes Known from various microorganisms and the like (eg, Ozaki et al., J. Biochem., 91, 1163-1171 (1982), Yamagata, J. Biochem., 96, 1511-1523 (1984), Brzywczy et al., Acta Biochimica Polonica, 40 (3), 421-428 (1993), etc.). Moreover, as γ-cyano-α-aminobutyric acid synthase (GCS), commercially available enzymes, for example, those derived from the genus Bacillus manufactured by Ikeda Shokuken Co., Ltd. can be used, and these enzymes meet the above conditions. May be selected and used. The GCS manufactured by Ikeda Shokuken Co., Ltd. is described in Japanese Patent Application Laid-Open No. 2000-228998.
[0022]
In addition, a gene encoding the above-mentioned enzyme is taken out from the microorganism that produces the enzyme, and this gene, a gene modified with reference to this gene information, a gene created with reference to the amino acid sequence information of the enzyme, etc. are genetic engineering. It can also be obtained by expressing in a microorganism or the like using a general technique and then selecting an enzyme that meets the above conditions.
[0023]
For example, as an enzyme expressed and selected using this genetic engineering technique, there is O-acetylhomoserine lyase derived from Thermos thermophilus strain, which is a thermostable bacterium, and is commercially available from Ikeda Shokuken Co., Ltd.
[0024]
For the enzyme, the method of gene recombination is as follows, for example.
There are many reports analyzing the amino acid sequence of O-acetylhomoserine-lyase and the base sequence of the gene encoding it. For example, searching for “LIGAND” on the GenomeNet WWW server (https://www.genome.ad.jp/), which is a collection of public databases, using the EC number “4.2.99.10” as a keyword, “ec: 4.2 .99.10 O-Acetylhomoserine (thiol) -lyase; Methionine synthase '' is found, and Pseudomonas aeruginosa, Candida jejuni, Bacillus halodurans, Deinococcus radiodurans, Thermotoga maritime Halobacterium, Saccharomyces cerevisiae, Sies The amino acid sequence of acetylhomoserine lyase and the base sequence data of the gene can be browsed.
[0025]
From these data, unique base sequences conserved in some O-acetylhomoserine lyases can be known. Based on this information, the probes used for Southern hybridization or primers used for Polymerase Chain Reaction (PCR) are designed and Southern hybridization or PCR is performed on the chromosomal DNA of Thermos thermophilus. -It is possible to identify and acquire the acetylhomoserine-lyase gene.
[0026]
A recombinant DNA comprising a structural gene of O-acetylhomoserine-lyase and a plasmid DNA capable of replicating in a suitable host cell containing a promoter region capable of regulating the expression of this gene can be obtained by a conventional method (for example, Jay Sam). Brook, EF Flitch, Tea Maniatis, Molecular Cloning: A Laboratory Manual Second Edition, Cold Spring Harbor Laboratory Press (1989) [J. Sambrook, EFFritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual 2nd ed, Cold Spring Harbor Laboratory Press (1989)]).
[0027]
An appropriate host cell such as Escherichia coli K12 can be transformed with the above recombinant DNA to obtain a transformed host cell producing O-acetylhomoserine lyase. As a method for culturing this transformant under appropriate conditions to obtain the produced O-acetylhomoserine lyase, a general microorganism culture method and protein purification method can be used. That is, this transformant is cultured, the transformant obtained by centrifuging the culture solution is disrupted, and a supernatant is obtained from the disrupted solution by centrifugation or the like. By combining O-acetylhomoserine-lyase contained in the supernatant with appropriate purification operations such as salting out, dialysis, ion exchange chromatography, hydrophobic adsorption chromatography, gel filtration, affinity chromatography, electrophoresis, etc. It can be purified to prepare O-acetylhomoserine lyase that can be used in the present invention.
[0028]
Furthermore, homocysteine converting enzymes having a stronger effect on homocysteine than cysteine used in the present invention include those modified from the above enzymes as long as the enzyme activity is maintained.
[0029]
Cysteine to be added to the sample in the quantification method of the present invention may be cysteine at the time of homocysteine measurement by homocysteine converting enzyme. In addition to cysteine, it affects the conditions of homocysteine measurement or affects homocysteine measurement. Examples include cysteine derivatives that liberate cysteine under no conditions. The cysteine derivative may be, for example, cystine.
The amount of cysteine added to the sample in the quantification method of the present invention is such that the influence of cysteine present in the sample can be substantially ignored, that is, a homocysteine converting enzyme having a stronger effect on homocysteine than cysteine used in the present invention. What is necessary is just to add the amount which becomes the substrate saturation amount. In the case of homocysteine-replacement enzyme, the amount of cysteine added is high in specificity to homocysteine and may be less as an enzyme that acts on homocysteine more strongly than cysteine, but the cysteine concentration in the sample is usually about 1 μM to 10 mM, The amount is preferably about 10 μM to 1 mM. For example, when the homocysteine-replacement enzyme is O-acetylhomoserine lyase derived from the Thermus thermophilus strain, it may be added so that the final concentration is 20 μM or more, more preferably 100 to 500 μM in the enzyme reaction. In the case of using homocysteine-degrading enzyme or L-methionine γ-lyase, the amount of cysteine added is almost the same as in the case of homocysteine-substituted enzyme.
As a method of adding cysteine or a cysteine derivative, the solution or powder may be added directly to the sample or may be added to the sample together with the components of the homocysteine measuring reagent.
[0030]
In the quantification method of the present invention, the amount of the enzyme added is not particularly limited as long as it is an amount sufficient to detect homocysteine in the sample. In the case of a sample containing only a very small amount of homocysteine, the amount is preferably more than the minimum necessary amount that can completely consume homocysteine. For example, when measuring total homocysteine in plasma, for example, 1 to 1000 units of enzyme is added to plasma treated with a reducing reagent, and the pH is 1 to 5 at 50 ° C. in a buffer of 4.5 to 11.5. What is necessary is just to make it react for 10 minutes. More preferably, 1 to 100 units of enzyme is added, and the reaction is carried out at 25 to 45 ° C. for 2 to 10 minutes in a pH 5 to 10 buffer.
[0031]
The buffer solution is selected from those having a buffering action in the pH range described above, Tris buffer solution, Good buffer solution, buffer solution derived from inorganic acid salts such as phosphoric acid, carbonic acid, boric acid, etc., and organic solvents such as acetic acid and citric acid. Examples include salts derived from acid salts. Preferably, a buffer solution that can increase the difference in reactivity between homocysteine and cysteine and improve the specificity for homocysteine is selected. For example, a Tris buffer, a phosphate buffer, etc. are mentioned.
[0032]
In the quantification method of the present invention, when a homocysteine substitution enzyme that catalyzes a reaction for substituting the γ-position mercapto group of homocysteine with a nucleophile (anion), the nucleophile is a substrate for the enzyme. It may be anything as long as it is not particularly limited. Examples thereof include anions of inorganic salts, anions of organic compound salts, compounds having a thiol group and thiolate anions of the compound, compounds having a hydroxyl group, and alkoxyl ions of the compound. Preferably, a nucleophile capable of increasing the difference in reactivity between homocysteine and cysteine and improving the specificity to homocysteine, such as 2-mercaptoethanol, thioglycerol, dithiothreitol (DTT), cyanide ion, etc. Particularly preferred are 2-mercaptoethanol, thioglycerol and the like. The counter cation is not particularly limited.
[0033]
Homocysteine in the sample easily condenses by oxidation reaction between SH groups or exchange reaction with SS bond, and particularly in biological samples such as plasma and serum, SS bonds with low molecular weight compounds and proteins having SH group. It is often present (conjugate) in the form of (disulfide bond). For this reason, when it is necessary to measure homocysteine as the total homocysteine, including conjugates present in these specimens, the conjugated homocysteine should be forced by chemical or enzymatic reactions. And homocysteine in the treatment solution is measured. The homocysteine quantified in the present invention includes homocysteine released in this manner. The chemical reaction for releasing the conjugated homocysteine can be carried out using a reducing agent such as thiols, phosphines and boron hydrides. For example, what is reported by Jocelyn (Methods of Enzymology, 143, 243-256 (1987)) can be used.
[0034]
Among them, SH group such as dithiothreitol (DTT), dithioerythritol (DTE), 2-mercaptoethanol, reduced glutathione, thioglycolic acid, methanethiol, 2-mercaptoethane, cysteine, thioglycerol, cysteamine, thiophenols, etc. A method of adding a low molecular weight compound in excess to a sample and gently releasing homocysteine using an SS exchange reaction with these is preferable. For example, when measuring total homocysteine in plasma, for example, 1-20 mM DTT may be added to plasma and reduced.
[0035]
Furthermore, thiols are preferable because they can easily liberate homocysteine and cysteine from a conjugate state easily and can also serve as a nucleophile of a homocysteine-substituting enzyme. Particularly preferably, thiols that increase the specificity for homocysteine are selected, and examples thereof include thioglycerol and 2-mercaptoethanol.
[0036]
In the measurement of homocysteine according to the present invention, the method for detecting hydrogen sulfide generated by the action of the enzyme is not particularly limited, including any known method. Examples thereof include the methods described in JP 2000-166597 A, JP 2000-228998 A, JP 2000-338096 A, Clinical Chemistry, 46, 1686-1688 (2000) and the like.
In particular, when clinically measuring homocysteine in serum or plasma, a detection method based on a colorimetric method that can be applied to general-purpose measuring instruments for clinical tests that are currently widely used is preferable.
[0037]
Not only a method of directly quantifying hydrogen sulfide, but also a method of quantifying sulfide ions caused by hydrogen sulfide, or a method of quantifying by further coloring. For example, (1) a method for detecting blackening of lead acetate paper under acidic conditions, (2) a method for detecting precipitation of zinc sulfide produced using zinc, (3) sodium nitroprusside under alkaline conditions (4) A method for detecting blue color development by generating methylene blue using N, N-dimethyl-p-phenylenediamine and ferric chloride under strong acidity (5) A method for producing a fluorescent dye using N, N-dibutyl-p-phenylenediamine and potassium ferricyanide and detecting the fluorescence intensity (Clinical Chemistry 46, 1686-1688 (2000)), (6) ) 2- (5-Bromo-2-pyridylazo) -5- [Nn-propyl-N- (3-sulfopropyl) amino] phenol sodium (5-Br-PAPS) and form dye using ferrous chloride, a method of detecting the color intensity (JP 2000-338096 JP) and the like.
[0038]
The present invention also includes a reagent for quantifying homocysteine in a sample coexisting with cysteine, including cysteine. Cysteine may be in an amount sufficient to avoid the influence of cysteine in the sample, and is contained at the concentration described above. Also included in the present invention is a reagent for quantifying homocysteine in a cysteine coexisting reagent, which contains a homocysteine converting enzyme having a stronger effect on homocysteine than cysteine. Examples of the enzyme include the homocysteine converting enzyme described above, and the enzymes exemplified above are preferred. The amount of the enzyme contained may be the same as described above. The cysteine contained in the reagent of the present invention may be cysteine in the homocysteine measurement, that is, a cysteine derivative that liberates the cysteine.
Homocysteine for measuring homocysteine in cysteine coexisting samples, including cysteine, a homocysteine converting enzyme having a stronger effect on homocysteine than cysteine, and a detection reagent for detecting hydrogen sulfide generated by the action of the converting enzyme A cysteine measurement kit is also included in the present invention. The kit for measuring homocysteine specifically includes cysteine for avoiding the effects of cysteine, cysteine derivatives or solutions thereof, homocysteine converting enzyme for releasing hydrogen sulfide from homocysteine, and for detecting hydrogen sulfide. Consists of detection reagents. Examples of the detection reagent for detecting homocysteine converting enzyme and hydrogen sulfide having a stronger effect on homocysteine than cysteine include the enzymes and reagents described above, and preferably those exemplified above. In particular, N, N-dialkyl-p-phenylenediamine is preferred as the detection reagent.
[0039]
Furthermore, the configuration of the reagent of the kit can vary depending on the target specimen. When it is necessary to measure including a low molecular weight compound having an SH group or homocysteine conjugated with a protein, generally, a reducing reagent for releasing homocysteine from the conjugated state, for example, The above-described thiols, phosphines, borohydrides, and the like may be included in the structure. When the homocysteine converting enzyme is a homocysteine substitution enzyme, a nucleophile capable of substituting the γ-position mercapto group of homocysteine is also included in the configuration. However, when the nucleophilic reagent capable of substituting the γ-position mercapto group of homocysteine also serves as the reducing reagent, it is not necessary to add the nucleophilic reagent.
Furthermore, a kit containing a buffer solution in the composition is also included in the present invention. Examples of the buffer solution include the above-described buffer solution, preferably those exemplified above. The reagent may be dissolved in a buffer solution and included in the kit, or a buffer solution may be added separately.
[0040]
In the case of constituting these reagents, when there are components that interfere with each other, they can be further divided and subdivided for each component. Conversely, if these constituent reagents are integrated without any problem in use, they may be integrated. For example, by adding cysteine as the reducing reagent, additional addition for avoiding the influence of cysteine may be omitted.
[0041]
In the case of a kit for measuring total homocysteine using plasma as a sample, for example, a reducing reagent containing 2-mercaptoethanol and cysteine, an enzyme reagent containing the above-mentioned O-acetylhomoserine-lyase, and color development for detecting hydrogen sulfide as methylene blue A kit composed of an agent is preferred. The amount of each component used per kit in the case of this kit varies depending on the number of tests of the kit and the amount of reaction solution per test, but in the case of a kit for 100 tests (for 20 mL), for example, 2-mercaptoethanol is 0.2 to 1000 μmol, cysteine is 10 to 1000 nmol, and O-acetylhomoserine sulfhydrylase is 1-1000 units.
[0042]
【Example】
Hereinafter, the present invention will be described more specifically with reference to comparative examples and examples, but the present invention is not limited to these examples.
[0043]
Comparative example
Measurement of homocysteine without addition of cysteine
After adding 10 μL of 25 mM 2-mercaptoethanol 20 μL, 200 U / mL Thermus thermophilus strain-derived O-acetylhomoserine-lyase (manufactured by Ikeda Shokuken) to 50 μL of 250 mM Tris-HCl buffer (pH 8.5), it was diluted as a specimen twice. 25 μL of 100 μM homocystin solution (corresponding to 200 μM as homocysteine and corresponding to 40 μM at the final concentration) and 20 μL of distilled water or 625 μM L-cysteine (corresponding to 100 μM at the final concentration) were added, and 37 The reaction was conducted at 5 ° C. for 5 minutes. Further, 25 μL of distilled water, 81.25 μL of 1N sulfuric acid solution of 16 mM N, N-dimethyl-p-phenylenediamine hydrochloride and 18.75 μL of 1N sulfuric acid solution of 10 mM ferric chloride were added, and the reaction was carried out at 37 ° C. for 5 minutes. After that, the absorbance at 670 nm was measured. The relationship between the obtained absorbance and the homocysteine concentration is plotted, and the prepared calibration curve is shown in FIG.
From FIG. 1, it can be seen that the calibration curve of the sample to which cysteine was added was larger than the sample without addition and was strongly influenced by cysteine.
[0044]
Example 1
Measurement of homocysteine by addition of cysteine
L-cysteine (corresponding to a final concentration of 200 μM) was further added to the Tris-HCl buffer of the comparative example so that the concentration was 500 μM, and the reaction was performed in the same manner as in the comparative example, and homocysteine was measured. The relationship between the obtained absorbance and the concentration of homocysteine is plotted, and the prepared calibration curve is shown in FIG.
As shown in FIG. 2, the addition of L-cysteine caused both calibration curves to overlap in substantially the same straight line. This means that the influence of cysteine contained in the specimen can be eliminated by L-cysteine added to the buffer, and homocysteine can be quantified using this calibration curve.
[0045]
Example 2
As a model sample, a homocystin (homocysteine dimer) 50 μM solution (sample A) and a homocystin 50 μM solution (sample B) added with cysteine 100 μM were used in the method of the comparative example and the method of Example 1 of the present invention. It was measured. That is, as a method of the comparative example, measurement was performed without adding cysteine to samples A and B, and as a method of Example 1, cysteine was further added to samples A and B so that the final concentration was 200 μM. Measured. The results are summarized in Table 1. The theoretical value of homocysteine in sample B containing cysteine is 100 μM, but the measured value is 150.4 μM in the method of the comparative example, and 107.0 μM in the method of Example 1 of the present invention. A value close to the theoretical value was obtained, and it was shown that the specific measurement of homocysteine in the cysteine coexisting sample is possible.
[0046]
[Table 1]

[0047]
【The invention's effect】
By using the measurement method of the present invention, homocysteine conversion in a sample containing homocysteine and cysteine is added to the sample so that the amount of cysteine is about the amount of substrate saturation, and has a stronger effect on homocysteine than cysteine. Using an enzyme, it can be easily measured without being affected by cysteine. Using this method, homocysteine in clinical specimens can be easily measured with a general-purpose measuring instrument for clinical tests, and can also be used for diagnosis of arteriosclerosis.
[Brief description of the drawings]
FIG. 1 shows a calibration curve of homocysteine for examining the effect of cysteine in a sample.
FIG. 2 shows a calibration curve of homocysteine in which the influence of cysteine is avoided by addition of cysteine.

Claims (4)

A method for quantifying homocysteine in a sample in which homocysteine and cysteine coexist , wherein cysteine is added to the sample so that the cysteine concentration in the sample after addition of cysteine is 1 μM to 10 mM, and then derived from a Thermus thermophilus strain A method for quantifying homocysteine, which comprises reacting O- acetylhomoserine- lyase . The cysteine, O-acetylhomoserine lyase derived from Thermus thermophilus strain , and a detection reagent for detecting hydrogen sulfide generated by the action of the lyase , for use in the method for quantifying homocysteine according to claim 1 Homocysteine measurement kit. The homocysteine measurement kit according to claim 2, wherein the detection reagent is N, N-dialkyl-p-phenylenediamine. The homocysteine measurement kit according to claim 2 or 3 , comprising a buffer.

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