Protein infrared spectroscopy.doc

5 Protein infrared spectroscopy 蛋白质 红外 光谱学Infrared spectroscopy is based on the infrared absorption of molecules and is, compared with crystallography, a relatively simple and inexpensive tool for the global characterization of molecular conformations and conformational changes of proteins and other biomolecules. 红外光谱法 是基于 分子的红外吸收，与晶体学相比，它是一个相对简单和便宜的工具，用于 总体描述 蛋白质 及 其它生物大分子 的 分子构象 和 构象变化Depending on the measurement technique, scanning infrared (IR) spectrometers, Fourier transform infrared (FTIR) spectrometers, and single wavelength infrared apparatuses are distinguished (see Sect. 5.1).根据测量方法，扫描红外光谱仪，傅里叶变换红外光谱仪，和 单波长的红外装置 被区分开来（见5.1节）。

Typically the most interesting spectral region for biomolecules is ν equals four hundred to four thousand reciprocal centimeter, where the wavenumber, ν, is defined as ν is identical to one over wavelength.对于生物分子，典型地 最令人关注的 光谱区间 是 v = 400 - 4000 cm-1,这里的波数，v 被定义为 ：v 恒等于 波长的倒数Infrared activity requires a change of dipole moment upon excitation (Fig. 5.1). 红外活性 振动 需要 一个 受激发的 偶极矩 的变化（如图 5.1）Fig. 5.1 Example of infra-red active and non-active vibrations. 图5.1 红外活性 和 非活性 振动 的例子Note that infra-red activity requires a change of dipole moment 注意，红外活性振动需要一个偶极矩的变化。

图中的箭头表示振动方向分子的振动形式可以分为两大类：伸缩振动和弯曲振动伸缩振动是指原子沿键轴方向的往复运动，振动过程中键长发生变化弯曲振动是指原子垂直于化学键方向的振动可以看到，下图表示红外非活性振动，由于是对称伸缩振动，分子中各个共价键偶极矩的矢量和不变，所以无法产生红外吸收光谱For proteins the amide chromophore absorption in the region of one thousand five hundred reciprocal centimeter to one thousand seven hundred reciprocal centimeter ( approximately equals six micrometers wavelength ) is particularly important for the assessment of secondary structure content and structural changes.对蛋白质而言，氨基生色团 的 吸收光谱 大约在1500 cm-1 - 1700 cm-1 附近（≈ 6 μm 波长） 对于评估 蛋白质的 二级结构 及其 结构变化 是特别重要的。

Regarding the resolution of protein secondary structure, the information content of IR and FTIR spectroscopy is comparable with that of circular dichroism , and regarding the resolution of features of the tertiary structure of proteins, IR and FTIR are often inferior, and yet IR is much easier to apply on a fast time scale and for remote sensing.关于 蛋白质二级结构 的 分辨率 ，红外和傅里叶红外光谱的 信息内容 与 圆二色谱 不相上下，而关于蛋白质 三级结构特征的分辨率，红外和傅里叶红外光谱 往往就 稍逊一筹了，不过，红外光谱 运用在 快速时间尺度 和 遥感方面 则容易得多5.1 Spectrometers and devices光谱仪 及其 装置5.1.1 Scanning infrared spectrometers 扫描式红外光谱仪Early IR spectrometers (Fig. 5.2) were constructed similarly to scanning UV virgule VIS absorption spectrometers. 早期的红外光谱仪（如图5.2）被构造成 类似于 扫描式 紫外可见吸收光谱仪（Ultra Violet Visible）。

Fig. 5.2 Example of a scanning infrared (IR) spectrometer.图5.2 扫描式红外光谱仪的例子The monochromator separates the radiation of the IR source into its different wavelengths and selects one wavelength at a time. 单色仪 把 红外线源 的连续光 分离成 不同波长的单色光，并且每次选择一种波长的光A beam splitter separates the monochromatic beam into sample beam and reference beam. 一个分束器把单色的光束分成样本光束和参考光束The absorption coefficient, according to the chemical and structural properties of the sample molecules, is calculated using the detected intensity quotient between both beams, the path length, and the sample concentration吸收系数，是按照 样本分子的化学和结构特性，利用检测到的 两种光束的 强度比值、 路径长度 以及 样本浓度计算出来的。

The emission of the source, for example, a thermal source operated at one thousand degrees, is passed through a monochromator selecting a single wavelength. 发射源，例如，一个被控制在1000℃的热源，是通过一个选择某种单一波长的单色仪而发出的The monochromatic beam is split into two beams – one having the sample in the path.这种单色光被分离成两束光，其中一束光的路径上有样本A shutter passes through only one of the two beams at a time.一块可移动的快门一次只让两束光中的其中一束通过Both beams are alternatingly detected by an IR detector, for example, a pyroelectric detector, and compared with each other. 这两束光依次被一个红外探测器如热释电探测器 检测，并与另外一束光作比较。

The optical density of the sample is calculated from the logarithm of the intensity quotient.样本的光密度是由两束光的强度之比再取对数计算得到的The use of light modulation is quite indispensable since the problem of background radiation is much more severe than in UV/VIS spectrometers.光束调制的运用是绝对必要的，因为背景辐射问题比在紫外可见光谱仪中要严重得多背景辐射的波长一般是7.35 cm，更靠近红外线波段，与紫外线波段要远一些Spectra are recorded by scanning the wavelength region of interest.通过扫描那些感兴趣的波长范围而将光谱记录下来This scanning principle of operation is still widely used in IR spectrometers with time resolutions in the femtosecond to nanosecond region, where infrared lasers serve as IR source这种扫描的操作原理仍然被广泛的运用于 红外光谱仪，其时间分辨率 在飞秒到纳秒范围内，由红外激光作为红外源。

5.1.2 Fourier transform infrared (FTIR) spectrometers傅里叶变换红外光谱仪FTIR spectrometers (Figs. 5.3–5.7) use the technique of Michelson interferometry and have the advantage of using a larger part of the emission of the IR source during the measurement of a spectrum, compared with scanning IR spectrometers that are based on monochromators which select only one wavelength at a time.傅里叶变换红外光谱仪（图5.3–5.7）运用迈克逊干涉度量学技术，在光谱测量时，与基于一次只选择一个波长的单色光的扫描式红外光谱仪相比，它的优势是The better usage of radiation improves the inherent signal-to-noise ratio, esp。