title: Different susceptibility of inter- and intra-chain disulfide bonds to reductive cleavage in native fibronectin and effect of their cleavage on conformation.
abstract: The two thread-like subunits (Mr approximately equal to 250 000) of the multidomain protein fibronectin are connected by a pair of inter-chain disulfide bridges in their C-terminal regions. In addition each chain contains 29 intra-chain disulfide bonds which are located in 12 type I and 2 type II structural domains in the N-terminal and C-terminal regions of the strands. The 15 to 17 type III domains in the central portion of the strands do not contain disulfide bonds. The susceptibility of inter-chain disulfide bonds to 10mM 1,4-dithiothreitol at pH 7.8 as quantitated by the rate of reductive cleavage of fibronectin into its subunits was found to be only 8-fold larger than that of the intra-chain bonds. Consequently at 90% completion of chain separation 30% of the intra-chain disulfides are also cleaved. The rate of inter-chain disulfide cleavage was found to be identical for fibronectin and a 140-kDa fragment comprising the C-terminal portions of the two subunits. This shows that the relatively high protection of the inter-chain disulfide bonds must originate from interactions between C-terminal domains which are probably also responsible for the V shaped arrangement of the two subunit strands. Changes of circular dichroism and thermal transition profiles for fibronectin and its C-terminal 140-kDa fragment indicated that already partial reduction of the intra-chain disulfide bonds alters the conformations of type I and II domains without affecting the type III domains.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D002942:Circular Dichroism; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000032:analysis*; D006801:Humans; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013997:Time Factors
chemical_list: D004220:Disulfides; D005353:Fibronectins