Tsai A M, van Zanten J H, Betenbaugh M J
Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Biotechnol Bioeng. 1998 Aug 5;59(3):273-80.
The objective of this study was to investigate the relationship between oxidized RNase A protein structure and the occurrence of protein aggregation using several spectroscopic techniques. Circular dichroism spectroscopy (CD) measurements taken at small temperature intervals were used to determine the protein's melting temperature, Tm, of approximately 65 degrees C in deionized water. A more detailed examination of the protein structure was undertaken at several temperatures around Tm using near- and far-UV CD and one-dimensional nuclear magnetic resonance (NMR) measurements. These measurements revealed the presence of folded structures at 55 degrees C and below, while denatured structures appeared at 65 degrees C and above. Concurrent static light scattering (SLS) measurements, employed to detect the presence of RNase A aggregates, showed that RNase A aggregation was observed at 65 degrees C and above, when much of the protein was denatured. Subsequent NMR time-course data demonstrated that aggregates forming at 75 degrees C and pH 7.8 were indeed derived from heat-denatured protein. However, aggregation was also detected at 55 degrees C when the spectroscopic data suggested the protein was present predominantly in the folded configuration. In contrast, heat denaturation did not lead to RNase A aggregation in a very acidic environment. We attribute this phenomenon to the effect of charge-charge repulsion between the highly protonated RNase A molecules in very acidic pH.
本研究的目的是使用多种光谱技术研究氧化核糖核酸酶A蛋白质结构与蛋白质聚集发生之间的关系。在小温度间隔下进行的圆二色光谱（CD）测量用于确定该蛋白质在去离子水中的熔解温度Tm，约为65℃。在Tm附近的几个温度下，使用近紫外和远紫外CD以及一维核磁共振（NMR）测量对蛋白质结构进行了更详细的研究。这些测量结果表明，在55℃及以下存在折叠结构，而在65℃及以上出现变性结构。同时进行的静态光散射（SLS）测量用于检测核糖核酸酶A聚集体的存在，结果表明，当大部分蛋白质变性时，在65℃及以上观察到核糖核酸酶A聚集。随后的NMR时间进程数据表明，在75℃和pH 7.8下形成的聚集体确实源自热变性蛋白质。然而，当光谱数据表明蛋白质主要以折叠构象存在时，在55℃也检测到了聚集。相比之下，在非常酸性的环境中，热变性不会导致核糖核酸酶A聚集。我们将这种现象归因于在非常酸性的pH值下，高度质子化的核糖核酸酶A分子之间的电荷-电荷排斥作用。
