Kono M, Sen A C, Chakrabarti B
Eye Research Institute of the Retina Foundation, Boston, Massachusetts.
Biochemistry. 1990 Jan 16;29(2):464-70. doi: 10.1021/bi00454a022.
The conformational stabilities of bovine lens gamma-crystallin fractions II, IIIA, IIIB, and IVA and those modified with glutathione were compared by studying the thermal and guanidine hydrochloride (Gdn-HCl) denaturation behavior. The conformational state was monitored by both far-UV CD and fluorescence measurements. All the gamma-crystallins studied showed a sigmoidal order-disorder transition with varied melting temperatures. The thermal denaturation of these proteins is reversible up to a temperature 3 or 4 degrees C above T 1/2; above this temperature, irreversible aggregation occurs. The validity of a two-state approximation of both thermal and Gdn-HCl denaturation was tested for all four crystallins, and the presence of one or more intermediates was evident in the unfolding of IVA. delta GDH2O values of these crystallins range from 4 to 9 kcal/mol. Upon glutathione treatment IVA showed the maximum decrease in T 1/2 by approximately 9 degrees C and in delta GDH2O value by 29%; the smallest decrease in T 1/2 was for IIIA by 2 degrees C and in delta GDH2O by 15%. We have demonstrated that the glutathione reaction can dramatically reduce the conformational stability of gamma-crystallins and, thus, that the thermodynamic quantities of the unreacted crystallins can be used to evaluate the stability of these proteins when modified during cataract formation.
通过研究热变性和盐酸胍（Gdn-HCl）变性行为，比较了牛晶状体γ-晶状体蛋白组分II、IIIA、IIIB和IVA以及用谷胱甘肽修饰后的这些蛋白的构象稳定性。通过远紫外圆二色光谱（CD）和荧光测量监测构象状态。所有研究的γ-晶状体蛋白均呈现出具有不同解链温度的S型有序-无序转变。这些蛋白质的热变性在高于T1/2 3或4摄氏度的温度范围内是可逆的；高于此温度，会发生不可逆聚集。对所有四种晶状体蛋白测试了热变性和Gdn-HCl变性的二态近似的有效性，并且在IVA的解折叠过程中明显存在一种或多种中间体。这些晶状体蛋白的ΔGDH2O值范围为4至9千卡/摩尔。经谷胱甘肽处理后，IVA的T1/2最大降低约9摄氏度，ΔGDH2O值降低29%；T1/2降低最小的是IIIA，降低2摄氏度，ΔGDH2O降低15%。我们已经证明，谷胱甘肽反应可显著降低γ-晶状体蛋白的构象稳定性，因此，未反应的晶状体蛋白的热力学量可用于评估这些蛋白在白内障形成过程中发生修饰时的稳定性。
