Premium
In vitro renaturation of bovine β‐lactoglobulin A leads to a biologically active but incompletely refolded state
Author(s) -
Subramaniam Vinod,
Steel Duncan G.,
Gafni Ari
Publication year - 1996
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560051015
Subject(s) - guanidine , chemistry , denaturation (fissile materials) , lability , tryptophan , bovine serum albumin , phosphorescence , biochemistry , hydrochloride , escherichia coli , in vitro , protein tertiary structure , native state , fluorescence , tyrosine , amino acid , nuclear chemistry , physics , quantum mechanics , gene
Abstract When bovine β‐lactoglobulin (β‐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 enhancement of this ligand's fluorescence, was completely recovered. In contrast, the room‐temperature tryptophan phosphorescence lifetime of the refolded protein, a local measure of the residue environment, was ∽10 ms, significantly shorter than the phosphorescence lifetime of the untreated native protein (∽20 ms). The lability of the freshly refolded protein, as monitored by following the time course 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 contrast to the long term postactivation conformational changes detected 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 β‐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 conformation‐specific monoclonal antibodies to recognize native‐like structure, that long‐term changes occur in the protein conformation, compared with the native structure, on refolding.