IN-VITRO RENATURATION OF BOVINE BETA-LACTOGLOBULIN-A LEADS TO A BIOLOGICALLY-ACTIVE BUT INCOMPLETELY REFOLDED STATE

When bovine beta-lactoglobulin (beta-LG) was refolded after extensive denaturation in 4.8 M guanidine hydrochloride (GuHCl), the functional activity of the protein, retinol binding, as measured by the enhanceme nt of this ligand's fluorescence, was completely recovered. In contras t, the room-temperature tryptophan phosphorescence lifetime of the ref olded protein, a local measure of the residue environment, was approxi mate to 10 ms, significantly shorter than the phosphorescence lifetime of the untreated native protein (approximate to 20 ms). The lability of the freshly refolded protein, as monitored by following the time co urse of its unfolding when incubated in 2.5 M GuHCl through the change in fluorescence intensity at 385 nm, was also determined and found to be increased significantly relative to untreated native protein. In c ontrast to the long term postactivation conformational changes detecte d previously in Escherichia coli alkaline phosphatase (Subramaniam V, Bergenhem NCH, Gafni A, Steel DG, 1995, Biochemistry 34:1133-1136), we found no changes in either the lability or phosphorescence decays of beta-LG during a period of 24 h. Our results are in agreement with the report by Hattori et al. (1993, J Biol Chem 268:22414-22419), using c onformation-specific monoclonal antibodies to recognize native-like st ructure, that long-term changes occur in the protein conformation, com pared with the native structure, on refolding.