Skip to main content
Article thumbnail
Location of Repository

Glutathione-dependent pathways of refolding of RNase T-1 by oxidation and disulfide isomerization: Catalysis by protein disulfide isomerase

By Margherita Ruoppolo, Robert B. Freedman, Piero Pucci and Gennaro Marino


Protein folding, associated with oxidation and isomerization of disulfide bonds, was studied using reduced and denatured RNase T-1 (rd-RNase T-1) and mixed disulfide between glutathione and reduced RNase T-1 (GS-RNase T-1) as starting materials. Folding was initiated by addition of free glutathione (GSH + GSSG) and was monitored by electrospray mass spectrometry (ES-MS) time-course analysis. This permitted both the identification and quantitation of the population of intermediates present during the refolding process. Refolding experiments were performed in the presence of different absolute concentrations of glutathione species while keeping the redox potential fixed, in order to evaluate the effect of the glutathione concentration on the distribution of the refolding intermediates. All the analyses indicate a pathway of sequential reactions in the formation of native RNase T-1 which occurs via the reiteration of two steps: (i) formation of a species containing both mixed disulfides with glutathione and free protein thiols, and (ii) formation of an intramolecular disulfide via thiol-disulfide interchange reaction between them. Refolding of rd-RNase T-1 and GS-RNase T-1 was also performed in the presence of protein disulfide isomerase (PDI). Addition of PDI led to a catalysis of each individual reaction of the entire process without altering the refolding pathway. Refolding reactions carried out at different absolute concentrations of glutathione proved that GSH and/or GSSG participate directly in the reaction catalyzed by PDI. On the basis of these experiments and previous results on the refolding of RNase A [Torella, C., Ruoppolo, M., Marine, G., & Pucci, P. (1994) FEES Lett. 352, 301-306], a hypothesis of a general pathway for folding of S-S containing proteins is proposed

Topics: QD, QP506
Year: 1996
DOI identifier: 10.1021/bi960755b
OAI identifier:
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • (external link)
  • Suggested articles

    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.