Bence-Jones proteins and light chains of immunoglobulins. 10. Luminescence studies on Bence-Jones proteins and light chains of immunoglobulins and their subunits

Abstract

To provide information on the tertiary structure of the antibody molecule we have investigated the luminescent properties of the light polypeptide chain of human immunoglobulins. The fluorescence and phosphorescence yields, spectra, lifetimes, and anisotropies of a large number of homogeneous light chains, i.e., Bence-Jones proteins and light chains derived from myeloma proteins, were measured. No two proteins gave identical tyrosyl or tryptophyl fluorescence spectra in comparative studies on over 75 proteins belonging to the four basic subgroups of kappa chains and of lambda chains. Spectral differences were apparent even among proteins exhibiting more than 85% amino acid sequence identity. The fluorescence yields of tyrosine and tryptophan vaired 10- and 100-fold, respectively; the Stokes' shift of tryptophan ranged from 328 to 365 nm, but that for tyrosine was apparently invariant (305-307nm). Emission as well as excitation spectra showed tyrosyl and tryptophyl redidues interact minimally or not at all. Fluorescence lifetimes of the tyrosyl and tryptophyl contributions were measured spearately, and the apparent natural lifetimes were calculated. Proteins could be grouped in accordance with similarities in fluorescence lifetimes and fluorescence yields; there was no evident relationship between these groupings and the light chain type (kappa or lambda), amino acid sequence, or tryptophan content. Also apparent were individual differences among kappa light chains and among lambda light chains in respect to their tyrosyl and trptophyl phosphorescence spectra and phosphorescence lifetimes. Certain proteins showed an atypical, short-lived tryptophan phosphorescence decay time. Such variance in the luminescent behavior of the tryptophyl residue(s) indicates a conformational interaction between the V and C domains of light chains. Selective proteolytic cleavage of the light chain into VL and CL fragments permitted the comparison to be made of the luminescent properties of the V and C domains with those of the whole protein. The V domain and intact protein have luminescent features in common, whereas the C domain possesses features distinctive from that of the native protein. Data derived from fluorescence anisotropy spectral studies of intact light chains and their VL-related fragments indicate that energy transfer between tryptophyl residues occurs in the C domain. The results of emission spectroscopic measurements performed at 220 and at 77 K indicate that the observed phophorescence of light chains is mainly from a tryptophyl residue contiguous to a disulfide link. The potential for interdomain interaction in light chains is evidenced by the finding that the orientation of the tryptophyl residue(s) in the V domain can influence the tryptophyl-disulfide ling interactions in the C domain; this interaction may account further for the extensive structural diversity of antibody molecules.

Local Dynamics and Their Alteration by Excipients Modulate the Global Conformational Stability of an lgG1 Monoclonal Antibody

“…Although the majority of conserved Trp residues can be categorized into these two defined environments (solvent‐exposed and solvent‐shielded), immunoglobulin fluorescence has diverse contributions arising from many distinct fluorophores with differences in their individual fluorescence spectra, lifetimes, and anisotropies43 indicative of differences in their microenvironments. Thus, selectively exciting a population of these Trp residues in immunoglobulins (where τ R ≥ τ F ) with radiation containing progressively lower energy quanta (i.e., increasing excitation wavelength toward the red edge of an absorption band) should enable selective excitation of different average populations of fluorophores that have an increasing ability to interact strongly with solvent molecules in the excited state.…”

Structural Studies of Human IgD Paraproteins

“…CD. analysis similarly failed to reveal conformational change on cleavage of immunoglobulin light chains to their variable and constant halves (Ghose & Jirgensons 1971b, Bjork et al 1971, whilst a similar study measuring changes in chemiluminescence did reveal conformational change (Longworth et al 1976). We have demonstrated a susceptibility to conformational change within the Fc^ region on exposure to mild denaturing conditions similar to those which induce cytophilic and complement binding activities in human IgG (Stanworth & Turner 1973).…”

Evidence for ligand-induced conformational changes in proteins from phosphorescence spectroscopy

“…In the course of our investigation of the emission properties of the Fab' fragment of the anti-galactan antibody J539 in the presence and absence of antigen, the short-lived component in the tryptophan phosphorescence decay previously observed by Longworth et al (1976) with Bence Jones proteins was apparent. In the present work we describe the changes in the decay time of the short-lived component not only in the immunoglobulin but in hen egg-white lysozyme as well, providing evidence for ligand-induced changes in tryptophan-disulfide interactions within these proteins.…”

“…the emission is dominated by the structured tryptophan component, and with excitation at 295 nm the spectrum consists entirely of tryptophan. Whereas no attempt was made to calculate the tryptophan phosphorescence quantum yield, comparison of the intensity with tryptophan methylester at the same indole concentration reveals that the efficiency, as with the fluorescence and phosphorescence of a large number of Bence Jones proteins is low (Longworth et al, 1976).…”

“…The only intrinsic perturbation known to result in a marked shortening of the phosphorescence lifetimes of both tyrosine and tryptophan at 77K is that with proximal disulfide bridges. A short-lived component in the phosphorescence decay of tryptophan at 77K was observed in lysozyme by Churchich (1966), in y-globulin fractions (King and Miller, 1975, and references therein), bovine a-lactalbumin (Miller and , and in a large number of Bence Jones proteins (Longworth et al, 1976). X-Ray crystallography has confirmed the proximity of disulfide groups to tryptophan side chains in both the lysozyme (Blake et al, 1967) and antibody (Poljak et al, 1973) structures.…”