高等学校化学学报 ›› 2006, Vol. 27 ›› Issue (3): 432.

• 研究论文 • 上一篇    下一篇

胰蛋白酶与DNA相互作用的共振瑞利散射光谱及其分析应用研究

李珊,刘忠芳,刘绍璞   

  1. 西南大学化学化工学院,重庆 400715
  • 收稿日期:2005-05-17 出版日期:2006-03-10 发布日期:2006-03-10
  • 通讯作者: 刘绍璞(1940年出生),男,教授,博士生导师,主要从事分子吸收、 发射和散射光谱分析研究. E-mail: liusp@swu.edu.cn
  • 基金资助:

    国家自然科学基金(批准号: 20475045)资助.

Studies on the  Interaction of Trypsin with Some DNA and Their Analytical Application by Resonance Rayleigh Scattering Spectra

LI Shan,LIU Zhong-Fang,LIU Shao-Pu*   

  1. School of Chemistry and Chemical Engineering,Southwest University,Chongqing 400715,China
  • Received:2005-05-17 Online:2006-03-10 Published:2006-03-10

摘要:

当胰蛋白酶与鲱鱼精DNA(hsDNA)、 鲑鱼精DNA(sDNA)以及小牛胸腺DNA(ctDNA)之间发生相互作用时,共振瑞利散射(RRS)会显著增强,并产生新的RRS光谱. 三者有近似的光谱特征,其最大RRS峰分别位于307 nm(hsDNA和sDNA体系)和290 nm处(ctDNA体系),另一散射峰位于350 nm处,其散射强度(ΔI)与DNA或者胰蛋白酶的浓度成正比,因此可用于蛋白质和DNA的相互测定. 当用胰蛋白酶测定DNA时,hsDNA,sDNA和ctDNA的检测范围分别为1.4×10-3~2.3, 2.1×10-3~2.5和3.5×10-3~1.9 μg/mL(ctDNA),它们的检出限分别为0.4,0.7和1.1 ng/mL. 当用hsDNA测定胰蛋白酶时,其线性范围为0.1~30.0 μg/mL,检出限为39.0 ng/mL. 研究了最佳的反应条件、 影响因素和结合产物的化学性质,考察了胰蛋白酶与DNA的结合比,推测了它们的结合方式,并以胰蛋白酶作RRS探针,发展了一种高灵敏、 简便和快速测定痕量DNA的新方法.

关键词: 胰蛋白酶; hsDNA; sDNA; ctDNA; 共振瑞利散射

Abstract:

When the trypsin interacts with herring sperm DNA(hsDNA),salmen sperm DNA(sDNA) and calf thymus DNA(ctDNA) to form the binding products,the Resonance Rayleigh scattering(RRS) are obvious enhanced remarkably and new RRS spectra appear. They have similar characteristics of RRS spectra,the maximum RRS peaks are at 307 nm(hsDNA,sDNA) and 290 nm(ctDNA),and the other peaks are  at 350 nm. As the scattering intensity is proportion to the concentration of DNA or trypsin respectively,the reaction can also be used as a new method for the determination of trypsin with DNA and for the determination of DNA with trypsin. When it  is used  for the determination of  DNA with trypsin,the linear range are 1.4×10-3—2.3 μg/mL(hsDNA),2.1×10-3—2.5  μg/mL(sDNA) and 3.5×10-3—1.9 μg/mL(ctDNA),and the detection limits are 0.4 μg/mL(hsDNA),0.7  μg/mL(sDNA) and 1.1 μg/mL(ctDNA),respectively. When it is used for the  determination of trypsin with hsDNA,the linear range is between 0.1 ng/mL and 30.0 μg/mL,and its detection limit is 39.0 ng/mL. In this  paper,the optimal interaction conditions,the affecting factors,the properties of analytical chemistry of the binding complex and the binding ratio of trypsin with DNA were investigated. The binding mode,binding ratio of trypsin with DNA and the reasons for the enhancement of RRS intensity are also discussed. A highly sensitive,simple and quick method for the determination of trace amount of DNA with trypsin as a RRS probe was developed.

Key words: Trypsin;   hsDNA;  sDNA;  ctDNA;  Resonance Rayleigh scattering

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