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化学学报
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化学学报 ›› 2010, Vol. 68 ›› Issue (12): 1210-1216. 上一篇    下一篇

研究论文

用硫酸软骨素A作探针共振瑞利散射及共振非线性散射法测定蛋白质

刘健，刘忠芳，胡小莉，孔玲，刘绍璞   

  1. 西南大学化学化工学院 发光与实时分析教育部重点实验室 重庆 400715
  • 投稿日期:2010-01-26 修回日期:2010-03-28 发布日期:2010-04-27
  • 通讯作者: 刘绍璞 E-mail:liusp@swu.edu.cn
  • 基金资助:

    生物大分子之间相互作用的共振瑞利散射研究

Determination of Proteins by Resonance Rayleigh Scattering and Resonance Non-linear Scattering Methods with Chondroitin Sulfate A as Probe

LIU Jian, LIU Zhong-Fang, HU Xiao-Li, KONG Ling, LIU Shao-Pu   

  1. Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715
  • Received:2010-01-26 Revised:2010-03-28 Published:2010-04-27
  • Contact: Shaopu Liu E-mail:liusp@swu.edu.cn

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被引次数

52 | 7

在pH 1.8～2.5的Britton-Robinson (BR)缓冲介质中, 硫酸软骨素A (CSA)的硫酸酯基离解而以带多个负电荷的大阴离子存在, 而人血清白蛋白(HSA)、牛血清白蛋白(BSA)、糜蛋白酶(Chy)、溶菌酶(Lyso)和α-淀粉酶(α-Amy)等蛋白质处于其等电点(pI)之下, 则是带多个正电荷的大阳离子, 两者可借静电引力、氢键作用、疏水作用而结合形成复合物. 此时将引起共振瑞利散射(RRS)和二级散射(SOS)、倍频散射(FDS)等共振非线性散射(RNLS)的显著增强并出现新的散射光谱. 3种散射的散射增强(ΔIRRS, ΔISOS和ΔIFDS)均在一定范围内与蛋白质的浓度成正比, 方法具有高灵敏度. 三种方法对蛋白质的检出限分别为4.5～12.0 (g/L (RRS法)、8.9～15.8 (g/L (SOS法)和13.4～31.5 (g/L (FDS法), 其中以CSA-BSA体系灵敏度最高(检出限可达4.5 (g/L). 研究了反应体系的RRS, SOS和FDS的光谱特征、适宜的反应条件和影响因素, 讨论了反应机理、结合模式以及散射增强的原因. 并以CSA-BSA体系为例考察了共存物质的影响, 表明方法有良好的选择性. 方法可用于正常人血清及尿样中蛋白质的测定.

关键词: 硫酸软骨素A, 蛋白质, 共振瑞利散射, 二级散射, 倍频散射

In pH 1.8～2.5 Britton-Robinson (BR) buffer medium, negatively charged chondroitin sulfate A (CSA) can react with positively charged proteins, such as human serum albumin (HSA), bovine serum albumin (BSA), chymotrypsin (Chy), lysozyme (Lyso) and α-amylase (α-Amy), via electrostatic attraction, hydrogen bonding and hydrophobic interaction. This lead to the enhancement of resonance Rayleigh scattering (RRS) and resonance non-linear scattering (RNLS), including second order scattering (SOS) and frequency doubling scattering (FDS), and appearance of new spectra. The scattering intensities (ΔIRRS, ΔISOS and ΔIFDS) are directly proportional to the concentrations of protein in certain ranges. Each method has high sensitivity and the detection limits (3() are 4.5～12.0 (RRS), 8.9～15.8 (SOS) and 13.4～31.5 (FDS) (g/L for proteins. The CSA-BSA system is the most sensitive which the detection limit for BSA is 4.5 (g/L. In this article, the spectral characteristics, optimum conditions of the reaction and influencing factors have been investigated. The reaction mechanism and binding mode are discussed. Take RRS method of CSA-BSA system for example, the effects of coexisting substances on the reaction are investigated. The method can be applied to the determination of protein in serum and urine samples with satisfactory results.

Key words: chondroitin sulfate A, protein, resonance Rayleigh scattering, second order scattering, frequency doubling scattering