GLUTATHIONE-DEPENDENT PATHWAYS OF REFOLDING OF RNASE T-1 BY OXIDATIONAND DISULFIDE ISOMERIZATION - CATALYSIS BY PROTEIN DISULFIDE-ISOMERASE

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