Thermodynamics of thermal and athermal denaturation of .gamma.-crystallins: changes in conformational stability upon glutathione reaction

Abstract

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.degree. C above T1/2; 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 T1/2 by .apprx. 9.degree. C and in .DELTA.GDH2O value by 29%; the smallest decrease in T1/2 was for IIIA by 2.degree. 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.