Detection of intermediate species in the refolding of bovine trypsinogen.
The mixed disulfide of bovine trypsinogen and glutathione was refolded at pH 8.6 and 4 degrees C with a mixture of 3 mM cysteine and 1 mM cystine catalyzing disulfide interchange. The folding process was monitored by analysis of quenched samples with isoelectric focusing and size-exclusion chromatography. Isoelectric focusing showed a progressive change from a pI of 5.2 for the mixed disulfide derivative to a pI of 9.3 for native trypsinogen. A number of principal intermediates were detected as a function of the refolding time. These intermediates were also separated and further characterized by size-exclusion chromatography on columns of TSK G2000 SW operated in the high-performance liquid chromatographic mode. Rechromatography of a series of sequential fractions taken from the parental peak was necessary to resolve and characterize the principal intermediates. The loss of glutathione moieties produced a partly folded structure with an apparent hydrodynamic volume (Stokes radius, Rs) of 33.9 A. These structures became compact with time, and more intermediates were detected between 33.9 and 29.2 A. Finally, a change in conformation, resembling a two-state transition, changed the molecules of Rs 29.2 to the compact structure of native trypsinogen (22.4 A). The rate of formation of the native structure was determined from the progress curves derived from isoelectric focusing and size-exclusion chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
Higaki
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D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000494:pharmacology; D003553:Cystine / Q000494:pharmacology; D004220:Disulfides; D005978:Glutathione / Q000378:metabolism; D008970:Molecular Weight; D011487:Protein Conformation; D014362:Trypsinogen / Q000302:isolation & purification / Q000378:metabolism*
D004220:Disulfides; D003553:Cystine; D014362:Trypsinogen; D005978:Glutathione; D003545:Cysteine