Single disulfide bond reduced papain exists in a compact intermediate state.
Partially reduced proteins and other chemically modified derivatives are very useful model systems to understand the protein folding in vivo. Upon reduction, proteins attain different conformations with varying degrees of compactness. The reduction of papain in the presence of 8 M urea leads to the partial reduction of one disulfide bond. This derivative (single disulfide reduced carboxymethylated 1RCM papain (3RCM papain)) was characterized by spectroscopic methods and the effect of this reduction on the unfolding of the protein was investigated. Under this partial reduction, papain exhibits more than half of the tertiary and most of the secondary structures relative to the non-reduced molecule (free cysteine reduced and carboxymethylated papain (1RCM papain)). Hydrophobic regions are exposed to the solvent as observed through 8-anilino-1-naphthalene sulfonic acid binding which was absent in the fully intact and unfolded protein, at neutral pH. Hydrodynamic studies indicated that 3RCM papain, under neutral conditions, possess expanded conformation as compared to the native protein. Tryptophan fluorescence quenching studies suggested the exposure of aromatic residues to solvent. Guanidine hydrochloride induced unfolding of this derivative, at neutral pH, showed a non-cooperative transition contrary to the cooperativity seen with intact protein. Thermal unfolding indicates that 3RCM papain is less stable compared to the intact protein. These findings suggest that partial reduction of papain has a significant effect on the unfolding behavior of papain.
Edwin
F
F
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, 221 005, Varanasi, India.
Jagannadham
M V
MV
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D010206:Papain / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000817:Anilino Naphthalenesulfonates; D004220:Disulfides; C027132:1-anilino-8-naphthalenesulfonate; D010206:Papain