Chen S X, Hardin C C, Swaisgood H E
Department of Food Science, North Carolina State University, Raleigh 27695-7624.
J Protein Chem. 1993 Oct;12(5):613-25. doi: 10.1007/BF01025126.
Incubation of beta-lactoglobulin with immobilized trypsin at 5-10 degrees C results in a time-dependent release of several fragments of the core domain in yields approaching 15%. Digests were fractionated by ion-exchange chromatography with a Mono Q HR 5/5 column and analyzed after disulfide reduction by polyacrylamide gel electrophoresis in sodium dodecylsulfate. Three fragments with approximate molecular weights of 13.8, 9.6, and 6.7 kD were identified. The fraction from ion-exchange chromatography yielding the 6.7 kD fraction after disulfide reduction was further characterized because it was most homogeneous and gave the highest yield. The C-terminal cleavage site of the 6.7 kD core fragment appeared to be Lys100 or Lys101 as determined by C-terminal amino acid analysis. The exact masses, after reduction with dithiothreitol, are 6195 and 6926 as determined by laser desorption mass spectrometry, corresponding to residues 48-101 and 41-100. Prior to reduction, beta-lactoglobulin C-terminal residues 149-162 are connected to these core domain fragments as shown by C-terminal analysis and mass spectrometry. Structural studies indicate that these 7.9 and 8.6 kD core domain fragments released by immobilized trypsin retain much of their native structure. CD spectra indicate the presence of antiparallel beta-sheet structure similar to the native protein but the alpha-helix is lost. Spectra in the aromatic region indicate the existence of tertiary structure. Moreover, structural transitions in urea are completely reversible as measured by CD spectra, although the extrapolated delta GDH20 and the urea concentration at the transition midpoint are lower than for the native protein. The core domain fragments also display a pH-dependent binding to immobilized trans-retinal as does intact protein. A single endotherm is obtained for both core domain fragments and native protein upon differential scanning calorimetry, but again, the domain is less stable as indicated by a transition peak maxima of 56.9 degrees C as compared with 81.1 degrees C for native protein.
在5-10摄氏度下，将β-乳球蛋白与固定化胰蛋白酶一起温育，会导致核心结构域的几个片段随时间释放，产率接近15%。消化产物用Mono Q HR 5/5柱进行离子交换色谱分离，并在二硫键还原后通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳进行分析。鉴定出了三个分子量约为13.8、9.6和6.7 kD的片段。对离子交换色谱中产生二硫键还原后6.7 kD片段的馏分进行了进一步表征，因为它最均一且产率最高。通过C端氨基酸分析确定，6.7 kD核心片段的C端切割位点似乎是Lys100或Lys101。用二硫苏糖醇还原后，通过激光解吸质谱法测定的精确质量分别为6195和6926，对应于残基48-101和41-100。如C端分析和质谱所示，在还原之前，β-乳球蛋白的C端残基149-162与这些核心结构域片段相连。结构研究表明，固定化胰蛋白酶释放的这些7.9和8.6 kD核心结构域片段保留了其大部分天然结构。圆二色光谱表明存在与天然蛋白质相似的反平行β-折叠结构，但α-螺旋消失了。芳香族区域的光谱表明存在三级结构。此外，尽管外推的ΔGDH₂₀和转变中点处的尿素浓度低于天然蛋白质，但通过圆二色光谱测量，尿素中的结构转变是完全可逆的。核心结构域片段也像完整蛋白质一样表现出对固定化反式视黄醛的pH依赖性结合。差示扫描量热法对核心结构域片段和天然蛋白质均得到一个吸热峰，但同样，该结构域的稳定性较差，其转变峰最大值为56.9摄氏度，而天然蛋白质为81.1摄氏度。
