ROOM-TEMPERATURE PHOSPHORESCENCE STUDY OF REFOLDING OF DISULFIDE-REDUCED RNASE T-1

Refolding kinetics of disulfide reduced ribonuclease T-1 (RNase T-1) w as studied by the recovery of room temperature phosphorescence emitted from a tryptophan residue (Trp 59) of the protein. When unfolded in a deoxygenated aqueous solution: the enzyme does not emit phosphorescen ce at room temperature, but as soon as the protein was transferred int o an anoxic refolding buffer solution, it began emitting phosphorescen ce within the mixing dead time (similar to 30 s) of our apparatus. The initial quick recovery of RNase T-1 phosphorescence was followed by a gradual increase in the phosphorescence lifetime lasting over several tens of minutes and then slight decrease to a final value comparable to that of native RNase T-1. The initial quick recovery of phosphoresc ence suggests that the tryptophan residue, being exposed to surroundin g water molecules when the protein is unfolded, is quickly isolated fr om the external water when the enzyme starts refolding, and the gradua l change in the phosphorescence lifetime implies that the peptide-chai n around Trp 59 slowly rearranges itself during the very slow trans-to -cis proline isomerization at Pro 39 and/or Pro 55 of the protein.