A Copper-binding Immunoglobulin from a Myeloma Patient

A copper l immunoglobulin complex isolated from a myeloma patient with hypercupremia has been investigated in an attempt to localize and characterize the copper-binding site. Cleavage of the purified IgGl type immunoglobulin with papain yielded peptides which were identified as Fab and F, fragments by immunodiffusion. The fragments were separated by DEAE-cellulose chromatography and the Fab fragment then was purified to homogeneity by chromatography on Sephadex G-100 and protein A-Sepharose. Copper is bound only to the F,b fragment. In contrast, the F, fragment, but not the F,b fragment, binds to concanavalin A, suggesting that the carbohydrate moiety on the F, fragment is not involved in the binding of copper. Light and heavy chains were isolated by gel filtration of the products formed upon reaction of the immunoglobulin with mercaptoethanol and iodoacetamide. Acid hydrolysates of light chain, heavy chain, and Fab fragment have been analyzed for amino acid content. The copper l Fab complex, like the copper*IgG complex, is colorless with no detectable visible absorption peak. Dialysis of the copper 0 Fab against cyanide yields apo-F,b; reconstitution with cupric ions yields a complex with 1 copper atom/F,b fragment. The isolated and reconstituted copper l F,b complexes are EPR-nondetectable before and after treatment with ferricyanide. Comparison of the copper l Fab and apo-F,b in terms of fluorescence spectra, circular dichroism spectra, and immunological properties revealed no structural differences due to copper binding. Reaction with dithiodipyridine demonstrates the presence of one exposed sulfhydryl group in apo-F,b but not in isolated or reconstituted copper l F,h complexes. The dithiodipyridinereacted apo-F,b no longer binds copper, indicating that this sulfhydryl group might be involved in copper binding. A preliminary experiment indicates that the involved sulfhydryl is labeled upon reaction with [14C]iodoacetamide.