Room Temperature Phosphorescence Study of Refolding of Disulfide Reduced RNase T1

Abstract

Refolding kinetics of disulfide reduced ribonuclease T 1 (RNase T 1 ) was 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 phosphorescence at room temperature, but as soon as the protein was transferred into an anoxic refolding buffer solution, it began emitting phosphorescence within the mixing dead time (∼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 phosphorescence suggests that the tryptophan residue, being exposed to surrounding water molecules when the protein is unfolded, is quickly isolated from the external water when the enzyme starts refolding, and the gradual change in the phosphorescence lifetime implies...