Thermal stability of human plasma fibronectin and its constituent domains.
Human plasma fibronectin undergoes thermal denaturation with a midpoint between 62 and 64 degrees C. The irreversible transition is characterized by an increase in the intensity and wavelength of intrinsic tryptophan fluorescence, by an increase in the ability to enhance the fluorescence of 1,8-anilinonaphthalene sulfonate, and by an increase in the fluorescence polarization of covalently attached fluorescein. Addition of molecules which bind to fibronectin with high affinity, e.g. gelatin or heparin, had no stabilizing effect. This was attributed to the presence of multiple domains, all of which must be stabilized to prevent denaturation and aggregation. Further support for this interpretation came from studies of six different proteolytic fragments of fibronectin which collectively span almost the entire molecule. Cell-binding fragments derived from the central regions of the chain were least stable, exhibiting behavior similar to that of the whole protein. Fragments derived from the C-terminal regions were more stable by 7-8 degrees C, and those derived from the N-terminal region showed no thermal transition by any of the fluorescence parameters up to 85 degrees C in some experiments. A fluorescein-labeled 60-kilodalton gelatin-binding fragment which had been heated to 70 degrees C produced an increase in polarization upon addition of gelatin with Kd = 1.3 X 10(-7) M, similar to that of an unheated control. The intrinsic fluorescence spectra of the fragments had maxima which decreased progressively from 335 nm at the N terminus to 313 nm at the C terminus. These observations further elaborate the multidomain structure of human plasma fibronectin and reveal significant differences between the tertiary structure and stabilities of the various domains.
D002918:Chymotrypsin / Q000378:metabolism; D005353:Fibronectins / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism; D005454:Fluorescence Polarization; D006358:Hot Temperature; D006801:Humans; D010196:Pancreatic Elastase / Q000378:metabolism; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013861:Thiocyanates / Q000378:metabolism
D005353:Fibronectins; D005452:Fluoresceins; D013861:Thiocyanates; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D016650:Fluorescein-5-isothiocyanate