J. Biol. Chem., Vol. 263, Issue 18, 8642-8645, Jun, 1988

Size-exclusion high performance liquid chromatography of native trypsinogen, the denatured protein, and partially refolded molecules. Further evidence that non-native disulfide bonds are dominant in refolding the completely reduced protein

AM al-Obeidi and A Light
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.

Size-exclusion high performance liquid chromatography was used to compare the Stokes radius of the mixed disulfide of trypsinogen refolded for 10 min with the Stokes radius of denatured trypsinogen in high concentrations of urea. After folding for 10 min, rechromatography of a collection of sequential fractions of an initial separation showed that the fractions display microheterogeneity as seen in the value of the Stokes radius of each fraction. These intermediate species differed in their Stokes radius, and each had a globular structure cross-linked by disulfide bonds. In contrast, when trypsinogen with the native disulfides intact was equilibrated at different concentrations of urea (0-8 M), a progressive increase in Stokes radius was observed with extent of unfolding. Rechromatography of a series of fractions collected at a specific urea concentration showed that each had the same Stokes radius as the fraction in the initial separation. Urea- denatured trypsinogen and partially refolded trypsinogen must therefore differ in the disulfide pairing that links regions of the polypeptide chain. These observations support the suggestion that non-native disulfide bonds are responsible for the many stable conformations that form early in the folding of the mixed disulfide of trypsinogen (Light, A., and Higaki, J.N. (1987) Biochemistry 26, 5556-5564). These intermediates initially are loose structures (large Stokes radius) that become more compact with time (decreasing Stokes radius). The intermediates must therefore undergo a continuing disulfide interchange until native disulfides form late in the process when the stable conformation of the native molecule is reached.
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