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首页> 外文会议>International Conference on Food Science and Technology(ICFST) vol.1; 20031022-24; Wuxi(CN) >Molecular Weight of Proteins Determined by Superose 12 Column Chromatography May Not be Correct
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Molecular Weight of Proteins Determined by Superose 12 Column Chromatography May Not be Correct

机译：用Superose 12柱色谱法测定的蛋白质分子量可能不正确

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Our research on α-amylase inhibitors (αAIs) and other proteins indicated that true molecular weights cannot be determined by Superose 12 column chromatography. In this paper, we use data on the molecular weights of red kidney bean (RKB) and white kidney bean (WKB) α-amylase inhibitors (RKB αAI and WKB αAI) and five standard proteins (cyto-chrome c, Kunitz soybean trypsin inhibitor, chicken ovalbumin, bovine serum albumin, and human transferrin) to document this problem. Homogeneous RKB αAI from red kidney bean (Phaseolus vulgaris) seeds was used to determine molecular weight (MW) using three methods. The molecular weight of purified RKB αAI determined by Sephadex G-100 gel filtration, polyacrylamide gel electrophoresis and Superose 12 gel filtration methods were 49.0, 51.0 and 22.9 kD, respectively. Purified RKB αAI, along with five standard proteins (above), were used also to test the effect of ionic strength in the running buffer on molecular weight determination using the Superose 12 column. The elution volume and Vn/Vo for human transferrin, bovine serum albumin, chicken ovalbumin and Kunitz soybean trypsin inhibitor increased to a plateau when the ionic strength in the running buffer increased. The Vn/Vo for cytochrome c and molecular weight for RKB αAI initially increased from 0 to 0. 05 M NaCl but then decreased at higher NaCl concentrations (the apparent size of RKB αAI was affected more than was that of cytochrome c). The determined MW for RKB αAI and cytochrome c were different when different ionic strength buffers were used. In all cases for RKB αAI the MWs were 22-62% smaller than those determined by Sephadex G-100 gel filtration and by electrophoresis. The data indicate there is ionic interaction between the proteins and Sephadex 12 in low i-onic strength buffers and hydrophobic interaction at higher ionic strength buffers. The results gave bell-shaped plots; however, even at the top of the bell the molecular weights were also too low. Researchers should be cautious when using a Superose 12 column for molecular weight determinations.
机译：我们对α-淀粉酶抑制剂（αAIs）和其他蛋白质的研究表明，用Superose 12柱色谱无法确定真实的分子量。在本文中，我们使用有关红芸豆（RKB）和白芸豆（WKB）α-淀粉酶抑制剂（RKBαAI和WKBαAI）和五种标准蛋白质（细胞色素c，Kunitz大豆胰蛋白酶抑制剂）的分子量数据，鸡卵清蛋白，牛血清白蛋白和人转铁蛋白）来证明此问题。使用三种方法，使用来自红芸豆（菜豆）种子的同质RKBαAI测定分子量（MW）。通过Sephadex G-100凝胶过滤，聚丙烯酰胺凝胶电泳和Superose 12凝胶过滤方法测定的纯化RKBαAI的分子量分别为49.0、51.0和22.9 kD。纯化的RKBαAI和5种标准蛋白质（上文）也用于测试运行缓冲液中离子强度对使用Superose 12色谱柱测定分子量的影响。当运行缓冲液中的离子强度增加时，人转铁蛋白，牛血清白蛋白，鸡卵清蛋白和Kunitz大豆胰蛋白酶抑制剂的洗脱体积和Vn / Vo增加至平台。细胞色素c的Vn / Vo和RKBαAI的分子量最初从0增加到0. 05 M NaCl，但随后在更高的NaCl浓度下降低（RKBαAI的表观大小比细胞色素c受到更大的影响）。当使用不同的离子强度缓冲液时，RKBαAI和细胞色素c的测定分子量不同。在所有情况下，对于RKBαAI，分子量均比通过Sephadex G-100凝胶过滤和电泳法测定的分子量小22-62％。数据表明在低离子强度缓冲液中蛋白质与Sephadex 12之间存在离子相互作用，而在较高离子强度缓冲液中存在疏水相互作用。结果给出了钟形图。但是，即使在钟形的顶部，分子量也太低。使用Superose 12色谱柱进行分子量测定时，研究人员应谨慎行事。

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