Integrity of refolded and reoxidized gelatin-binding fragments of fibronectin.
The gelatin-binding region of fibronectin is easily isolated as a stable and functional 42-kDa fragment (42-kDa GBF) containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9, where the numbers designate the order of these modules in each of the two polypeptide chains. Each module forms an independently folded domain stabilized by two disulfide bonds. Reduction of disulfides caused large changes in the intrinsic fluorescence and abolished the gelatin-binding activity of 42-kDa GBF and two nonoverlapping gelatin-binding subfragments, 30-kDa GBF (I6-II1-II2-I7) and 21-kDa GBF (I8-I9). However, high yields of active material could be regenerated, without diluting the protein, by dialysis into GdmCl followed by slow overnight removal of GdmCl while maintaining the redox potential with a mixture of oxidized and reduced glutathione. Fluorescence spectroscopic analysis indicated that the tertiary structure and thermodynamic stability of the refolded fragments were similar to those of the originals. The refolded fragments were quantitatively indistinguishable from the originals with respect to their dissociation constants for binding to a fluorescent-labeled collagen fragment. The results suggest that all or most of the cystines, a total of 24 in 42-kDa GBF, are correctly paired in the refolded products and that the tertiary structure was completely recovered. The fact that the 30- and 21-kDa fragments bind with a similar affinity proves the existence of at least two nonoverlapping sites in 42-kDa GBF that recognize gelatin.
Ingham
K C
KC
Biochemistry Laboratory, American Red Cross, Biomedical Research and Development, Rockville, Maryland 20855.
Brew
S A
SA
D002352:Carrier Proteins; D003094:Collagen; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; D005353:Fibronectins; D005780:Gelatin; D003094:Collagen