Stopped Flow Apparatus for Time-Resolved Fourier Transform Infrared Difference Spectroscopy of Biological Macromolecules in 1H2O-猫眼课题宝

1H2O中生物大分子的时间分辨傅里叶变换红外差光谱停流装置

DOI：

发表时间：

2003

期刊：

Applied Spectroscopy

影响因子：

3.5

通讯作者：

中科院分区：

化学3区

文献类型：

作者：

R. Masuch;D. Moss

停流光谱是一种用于获取化学和生物化学反应中动态过程的动力学数据的成熟技术，傅里叶变换红外（FT-IR）技术可以提供生物大分子的特别丰富的结构信息。然而，这是一个相当大的挑战，设计一个FT-IR停止流动系统的光路长度足够低的工作与水（1H 2 O）的解决方案。本文介绍的系统专为最小样品体积（105 μL）设计，可同时进行FT-IR快速扫描和维斯测量。该系统采用微结构扩散混合器，在中等流量和压力条件下实现毫秒级时间尺度上的有效混合，允许在小于10 μm的池路径长度中进行测量。这使得可以在宽光谱范围内记录1H 2 O溶液的光谱。系统布局还设计用于动态和静态测量的组合，特别是获得关于系统死区时间期间发生的更快光谱变化的详细信息。FT-IR停流系统的详细表征，包括作为一个例子的细胞色素c的碱性构象转变的演示。

Stopped flow spectroscopy is an established technique for acquiring kinetic data on dynamic processes in chemical and biochemical reactions, and Fourier transform infrared (FT-IR) techniques can provide particularly rich structural information on biological macromolecules. However, it is a considerable challenge to design an FT-IR stopped flow system with an optical path length low enough for work with aqueous (1H2O) solutions. The system presented here is designed for minimal sample volumes (∼5 μL) and allows simultaneous FT-IR rapid-scan and VIS measurements. The system employs a micro-structured diffusional mixer to achieve effective mixing on the millisecond time scale under moderate flow and pressure conditions, allowing measurements in a cell path length of less than 10 μm. This makes it possible to record spectra in 1H2O solutions over a wide spectral range. The system layout is also designed for a combination of kinetic and static measurements, in particular to obtain detailed information on the faster spectral changes occurring during the system dead time. A detailed characterization of the FT-IR stopped flow system is presented, including a demonstration of the alkaline conformational transition of cytochrome c as an example.

Femtosecond spectral evolution of the excited state of bacterial reaction centers at 10 K.