Discrimination of different domains in fibronectin on the basis of their stability against urea denaturation.

Circular dichroism spectra, thermal transition profiles and proteolytic susceptibility showed that different regions in the multidomain protein fibronectin exhibit different sensitivity against urea denaturation. A 70-kDa fragment obtained by cathepsin D treatment which comprises the N-terminal part of the fibronectin chains, exhibited in 8M urea a spectrum, at 20 degrees C, identical to that of the native fragment and its thermal unfolding was shifted to lower temperatures by only 10 degrees C. The central portions of the fibronectin chains were remarkably unfolded under the same conditions as clearly demonstrated by the spectra and transition profiles of a cathepsin D-raised 125/140-kDa fragment which originates from this region. When fibronectin or its fragments were exposed to 4 or 8M urea at 4 degrees C and the urea subsequently dialysed off, the spectra and transition curves recorded were very similar to those of the native proteins. Nevertheless, this treatment introduced local conformational changes which resulted in the creation of three new cleavage sites for chymotrypsin. The most prominent one was found to be located in the central part of the middle region and no sites were created in the N-terminal 70-kDa region. In the conjunction with sequence information [Petersen et al. (1983) Proc. Natl. Acad. Sci. USA 80, 137-141] it may be concluded that the disulfide rich domains, made up by regions of internal homology of types I and II in the N-terminal portion of fibronectin, exhibit a remarkable conformational stability, whereas the disulfide free middle region which contains type III domains, is much less stable. Some domains in this region are particularly sensitive to urea denaturation and are irreversibly affected already by 4M urea at 4 degrees C. Therefore, the use of high urea concentrations for the elution of fibronectin from affinity columns may lead to an at least partially irreversible unfolding of some domains and a loss of functions associated with these structural elements.