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M.R. Burgio, J.F. Koretz; Comparative Studies of the Biophysical Properties of Native, Urea Renatured, and Recombinant Bovine Alpha-Crystallins . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2372.
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Purpose: To evaluate biophysical similarities and differences between native, urea renatured, and recombinant α-crystallins. Previous results in our and other laboratories have indicated that there may be differences in both the structural and functional properties of these three versions of alpha-crystallin. The reasons for the differences, however, remain undefined. Methods: Samples of native α-crystallin purified from bovine calf lenses, urea denatured/renatured calf lens α-crystallin, and recombinant bovine αA and αB-crystallin mixed in a 3:1 molar ratio were studied using non-denaturing (composite) gel electrophoresis, far-UV CD spectroscopy, gel exclusion chromatography, and other methods. The thermal stability of each sample was also evaluated, as was chaperone-like activity using the insulin denaturation protocol. Results: Far-UV CD spectroscopy revealed significant secondary structural differences between the native α-crystallin and either the renatured or recombinant samples, while the latter two more closely resembled each other. Most of the observed difference was located in the positive ellipticity region below 200 nm. Significant differences were also found in the structural and functional consequences of heat incubation among the three samples, with native α-crystallin again differing from both other samples. The different structural effects of heat incubation were related to the temperature at which the quaternary thermal changes occurred, and in the magnitude of the subsequent temperature-dependent transitions. Conclusion: Renatured and recombinant versions of α-crystallin exhibit significant structural and functional differences from the native material which must be considered when using either as a model for native α-crystallin properties.
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