Effects of ammonium sulfate on the unfolding and refolding of the variable and constant fragments of an immunoglobulin light chain

Abstract

The equilibria and kinetics of unfolding and refolding by guanidine hydrochloride of the VL and CL fragments of a type kappa immunoglobulin light chain were studied in the presence of ammonium sulfate using circular dichroism and tryptophyl fluorescence at pH 7.5 and 25 degrees C. The unfolding equilibria of the VL and CL fragments were described in terms of the two-state transition. The midpoints of unfolding in the absence of ammonium sulfate were at 0.9 and 1.2 M guanidine hydrochloride for the CL and VL fragments respectively. The transition curves were shifted to higher concentrations of guanidine hydrochloride by 1.4 and 1.6 M for the CL and VL fragments, respectively, per mole of ammonium sulfate. Unfolding reactions of the VL and CL fragments in 3 M guanidine hydrochloride followed first-order kinetics, and the rate constants for the two proteins were both greatly decreased by the presence of ammonium sulfate. The refolding reaction of the CL fragment in 0.3 M guanidine hydrochloride consisted of two phases, and the rate constants were increased a little by the presence of ammonium sulfate. The refolding reaction of the VL fragment in 0.3 M guanidine hydrochloride followed first-order kinetics, and the rate was not affected by the presence of ammonium sulfate. These results showed that ammonium sulfate stabilizes the CL and VL fragments mainly by decreasing the unfolding rate.

Conformation of Amyloid Fibrils of β2-Microglobulin Probed by Tryptophan Mutagenesis

“…2A). These observations are consistent with an immunoglobulin domain having a Trp residue at the corresponding position (19,20), in which the fluorescence of the conserved Trp is quenched by the disulfide bond. A cooperative change in fluorescence intensity was observed with an increase in the concentration of Gdn-HCl, suggesting that W39-␤2-m assumes the native fold in the absence of Gdn-HCl (see below).…”

“…To address the structure of amyloid fibrils, we have taken advantage of the unique properties of Trp fluorescence. One important feature of immunoglobulin domains is the presence of a buried Trp residue located close to the conserved disulfide bonds (18), leading to a strong quenching of Trp fluorescence (19,20). We noticed that, although ␤2-m has two Trp residues (i.e.…”

Multistep Denaturation and Hierarchy of Disulfide Bond Cleavage of a Monoclonal Antibody

“…The multistep denaturation of our antibody may quently boiled in SDS) clearly and distinctly revealed the existence of a free light chain of slightly lower aparise from different qualities of interaction between the various b-barrels in adjacent regions of the light chain parent molecular weight (24 kDa) than free light chains that had been fully reduced and boiled in SDS (28 kDa) and heavy chain, as supported by the studies of Goto et al (24) and Getzoff et al (25). Goto et al (24) studied (Fig.…”

“…In support of the first point is the finding that proteins bridges between H and L, tends to remain intact during electrophoresis in low levels (0.5 M) of urea. Our study that function to bind lipids (apolipoproteins) may bind eight SDS without denaturation (24). Kyte-Doolittle extends the above qualitative work by revealing the existence of HHL***L and that this species survives hydropathy plots of our antibody revealed only two regions of marked lipophilicity, and these were surmised brief exposure to 1% SDS (prior to loading) and electrophoresis in 0.1% SDS.…”

Cold denaturation of monoclonal antibodies

“…26,36,47,48 While studies to characterize the thermodynamic stability of full-length mAbs have not been reported, studies have been employed previously to determine the ∆G u of single domains of antibodies and various regions in the Fab and Fc portions of IgG1 antibodies. 34,35,49,50 For example, Rowe and Tanford estimated a ∆G u of 5.5 kcal mol -1 for the C L and V L domains of a human κ light chain derived from the human IgG1 myeloma protein Wes at pH 7.0 and 25°C in the presence of GuHCl. 35 Goto et al used the LEM to calculate ∆G u values of 3.8 kcal mol -1 and 4.7 kcal mol -1 for the C L and V L fragments, respectively.…”