Refolding of trypsin-subtilisin inhibitor from marine turtle eggwhite.
Trypsin-subtilisin inhibitor from marine turtle eggwhite refolded quantitatively from its fully reduced state at pH 8.5 in the presence of reduced and oxidized glutathione. The refolding process was studied by following the accompanying changes in inhibitory activity, fluorescence, sulfhydryl group titer, and hydrodynamic volume. The refolding process followed second-order kinetics with rate constants of 4.80 x 10(2) M-1 sec-1 for trypsin-inhibiting domain and 0.77 x 10(2) M-1 sec-1 for subtilisin-inhibiting domain of the inhibitor at 30 degrees C and their respective activation energies of the refolding process were 15.9 and 21.6 kcal/mol. Fluorescence intensity of the reduced inhibitor decreased with time of refolding until it corresponded to the intensity of the native inhibitor. The inhibitor contained 1-2% alpha-helix, 40-42% beta-sheet, and 57-58% random coil structure. Refolded inhibitor gave a circular dichroic spectrum identical to that of the native inhibitor. A number of principal intermediates were detected as a function of the refolding time. Size-exclusion chromatography separated the intermediates differing in hydrodynamic volume (Stokes radius). The Stokes radius ranged from 23 A (fully reduced inhibitor) to 18.8 A (native inhibitor). Results indicated the independent refolding of two domains of the inhibitor and multiple pathways of folding were followed rather than an ordered sequential pathway.
Chaudhuri
T K
TK
Department of Chemistry, Bose Institute, Calcutta, India.
Sinha
N K
NK
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D004226:Dithioerythritol / Q000737:chemistry; D004527:Egg Proteins / Q000737:chemistry; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013381:Subtilisins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000378:metabolism; D014426:Turtles
D004220:Disulfides; D004527:Egg Proteins; D013439:Sulfhydryl Reagents; D014361:Trypsin Inhibitors; D004226:Dithioerythritol; D013381:Subtilisins