Domain separation precedes global unfolding of rhodanese

The enzyme rhodanese was investigated for the conformational transition ass ociated with its urea unfolding. When rhodanese was treated with 0 or 3 M u rea, the activity was not significantly affected. 4.25 M urea treatment led to a time-dependent loss of activity in 60 min. Rhodanese was completely i nactivated within 2 min in 6 M urea. The 1,1'-bi(4-anilino)naphthalene-5,5' -disulfonic acid fluorescence intensity was not significantly increased dur ing 0, 3, and 6 M urea equilibrations, and the fluorescence was dramaticall y increased with 4.25 M urea, indicating that hydrophobic surfaces are expo sed. After 0 and 3 RI urea equilibration, rhodanese was not significantly p roteolyzed with trypsin, Treatment with 4.25 M urea led to simultaneous for mation of major 12-, 15.9-, 17-, and 21.2-kDa fragments, followed by progre ssive emergence of smaller peptides, The N termini of the 17- and 21.2-kDa bands were those of intact rhodanese, The N terminus of the 15.9-kDa band s tarts at the end of the interdomain tether, The 12-kDa band beans with eith er residue 183 or residue 187, The size and sequence information suggest th at the 17- and 15.9-kDa bands correspond to the two domains. The 21.2- and 12-kDa bands appear to be generated through one-site tryptic cleavage, It i s concluded that urea disrupts interaction between the two domains, increas ing the accessibility of the interdomain tether that can be digested by try psin, The released domains have increased proteolytic susceptibility and pr oduce smaller peptides, which may represent subdomains of rhodanese.