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

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.