Zemser M, Friedman M, Katzhendler J, Greene L L, Minsky A, Gorinstein S
Department of Pharmaceutical Chemistry, School of Pharmacy, Hebrew University of Jerusalem, Israel.
J Protein Chem. 1994 Feb;13(2):261-74. doi: 10.1007/BF01891984.
The effects of ovalbumin (OVA) denaturation using urea, guanidinium chloride (GdnHCl), sodium dodecyl sulphate (SDS), cetylpyridinium chloride (CPC), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 5 different cationic detergents with various side chains, HCl, and CH3COOH were observed. Progressive unfolding in ovalbumin was measured as a function of fluorescent light intensity, peak response and shift in the maximum of emission. Kinetic measurements demonstrated that the rate of denaturation usually followed a double exponential decay pattern, but at small concentrations of urea and acids first-order reaction was indicated. The reversibility of the unfolding-folding transitions was confirmed from tryptophan fluorescence and circular dichroism (CD) measurements. Differences in secondary structure were observed and changes of alpha-helical content were calculated. Polyacrylamide gel electrophoresis (PAGE) with and without sodium dodecyl sulphate (SDS-PAGE) showed differences in the structure of native and denatured ovalbumin. Native protein samples in PAGE demonstrated smaller number and larger mobilities of subunits than denatured ones with different reductants, such as SDS and 2-mercaptoethanol (2 ME). Scanning of SDS protein patterns showed the appearance of aggregated forms in region of 45 kD.
观察了使用尿素、氯化胍（GdnHCl）、十二烷基硫酸钠（SDS）、十六烷基氯化吡啶（CPC）、3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐（CHAPS）以及5种不同侧链的阳离子去污剂、盐酸和乙酸对卵清蛋白（OVA）变性的影响。通过荧光强度、峰值响应和发射最大值的位移来测量卵清蛋白的逐步展开。动力学测量表明，变性速率通常遵循双指数衰减模式，但在低浓度尿素和酸的情况下显示为一级反应。通过色氨酸荧光和圆二色性（CD）测量证实了去折叠-折叠转变的可逆性。观察到二级结构的差异并计算了α-螺旋含量的变化。有或没有十二烷基硫酸钠的聚丙烯酰胺凝胶电泳（SDS-PAGE）显示了天然和变性卵清蛋白结构的差异。在PAGE中，天然蛋白质样品显示出的亚基数量比使用不同还原剂（如SDS和2-巯基乙醇（2ME））的变性样品少，迁移率更大。SDS蛋白质图谱扫描显示在45 kD区域出现聚集形式。
