Chiou S H, Chen W
Laboratory of Crystallin Research, National Taiwan University, Taipei.
Biochem Int. 1992 Nov;28(3):401-12.
Crystallins from pigeon eye lenses were isolated and purified by gel-permeation and anion-exchange chromatographies and characterized by gel electrophoresis, amino acid analysis and Raman spectroscopy. alpha- and beta-Crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of gel filtration column. Structural analyses of total crude soluble proteins and purified alpha-, beta- and delta-crystallin fractions were made with respect to their amino acid compositions and characteristic near-IR Fourier-transform Raman spectra. The results indicate that the major secondary structures of alpha- and beta-crystallins are mainly anti-parallel beta-pleated sheet in nature as judged by the Raman signals at 1242 (amide III) and 1669-1670 cm-1 (amide I) whereas delta-crystallin consists of a significant content of alpha-helices as evidenced by the Raman signal at 1657-1660 cm-1 (amide I). The low intensity of S-S disulfide stretching vibration at 508-510 cm-1 coupled with the presence of S-H stretching at 2560-2580 cm-1 for alpha-, beta- and delta-crystallin pointed to the fact that sulfhydryl groups in most crystallins are resistant to air oxidation during the process of homogenization and protein extraction. It is also found that the relative Raman signal intensities of Tyr, Phe, and Trp residues in purified crystallins correlate very well with the data obtained from amino acid analysis. Especially noteworthy is the demonstrated usefulness of applying Raman techniques in the detection of the microenvironments of the aromatic amino acids such as Tyr and Trp in the native crystallins, which may prove useful in the study of contribution of these aromatic residues to crystallin packing and stability.
从鸽眼晶状体中分离并纯化了晶状体蛋白，采用凝胶渗透色谱法和阴离子交换色谱法进行分离，并通过凝胶电泳、氨基酸分析和拉曼光谱进行表征。通过单步尺寸排阻色谱法可获得相对纯的α-和β-晶状体蛋白，而从β-晶状体蛋白组分中分离δ-晶状体蛋白则需要额外的离子交换色谱步骤。与大多数已表征的脊椎动物物种不同，在凝胶过滤柱的第一个峰中，大量糖原以高分子形式被洗脱。对总粗可溶性蛋白以及纯化的α-、β-和δ-晶状体蛋白组分进行了结构分析，包括氨基酸组成和特征近红外傅里叶变换拉曼光谱。结果表明，根据1242 cm⁻¹（酰胺III）和1669 - 1670 cm⁻¹（酰胺I）处的拉曼信号判断，α-和β-晶状体蛋白的主要二级结构本质上主要是反平行β-折叠片层，而δ-晶状体蛋白由大量α-螺旋组成，这由1657 - 1660 cm⁻¹（酰胺I）处的拉曼信号证明。对于α-、β-和δ-晶状体蛋白，508 - 510 cm⁻¹处S - S二硫键伸缩振动强度较低，同时在2560 - 2580 cm⁻¹处存在S - H伸缩振动，这表明在匀浆和蛋白质提取过程中，大多数晶状体蛋白中的巯基对空气氧化具有抗性。还发现纯化的晶状体蛋白中酪氨酸、苯丙氨酸和色氨酸残基的相对拉曼信号强度与氨基酸分析数据非常吻合。特别值得注意的是，拉曼技术在检测天然晶状体蛋白中酪氨酸和色氨酸等芳香族氨基酸的微环境方面显示出实用性，这可能对研究这些芳香族残基对晶状体蛋白堆积和稳定性的贡献有用。
