Abstract
IgA of L-type was purified from serum of a patient with multiple myeloma by zone electrophoresis on agar gel, followed by precipitation with 34% saturated ammonium sulfate. The purified IgA preparation was shown by ultracentrifugal analysis to be heterogenous with respect to the molecular size, but free from other classes of immunoglobulin as examined by immunoelectrophoresis with a horse anti-human serum.
The IgA preparation was reduced with various concentrations of 2-mercaptoethanol (2-ME) and then alkylated with 20% excess of iodoacetamide relative to the sulfhydryl concentration of 2-ME. Each reduced-alkylated IgA preparation was subjected to gel-filtration on a column of Sephadex G-200, equilibrated with borate-buffered saline of pH 8.0. The IgA preparations obtained after treatment with 0.01 M to 0.05 M 2-ME were separated into two peaks. The first peak seems to be aggregated IgA (IgAp) and the second major peak, monomeric IgA (2-MEIgAm). On the other hand, the IgA preparations obtained after treatment with 0.1 M to 0.5 M 2-ME were separated into three peaks: an additional peak appeared after the 2nd peak and designated as IgAs.
Heavy (H) and light (L) chains were prepared from 0.2 M 2-ME-IgAM by gel-filtration on Sephadex G-100 equilibrated with 1 M propionic acid. The L-chain was further purified by gelfiltration on Sephadex G-75 at pH 8.0. The IgAs was identified as the L-chain of IgA, since the IgAs was indistinguishable from the L-chain with respect to the antigenic structure, electrophoretic mobility, sedimentation characteristics and amino acid compositions. The C-terminal amino acid was serine for both, IgAs and L-chain. These results suggest that some of the L-chain are easily dissociable from the original IgA molecule at neutral pH after treatment with 2-ME and iodoacetamide. The amounts of IgAs released from the 2-ME-treated IgA increased 4% to 18%, as the concentration of 2-ME increased 0.05 M to 0.5 M. The IgAM preparations, obtained by gel-filtration after treatment with various 2-ME concentrations, were separated into H and L chains by gel-filtration on Sephadex G-100 in 1 M propionic acid. The IgAM preparations, obtained after treatment with 0.01 to 0.1 M 2-ME, were reduced with 0.2 M 2-ME prior to separation into H and L chains. Recoveries of the L-chain from the various 2-ME-IgAM preparations were determined from the elution patterns on the basis of optical density at 280 mμ. Recovery of the L-chain decreased from 41% to 30%, as the concentration of 2-ME increased from 0.01 M to 0.5 M.
Theoretical recoveries of the L-chain in IgA molecule were calculated to be 44% and 34%, if it is assumed that IgA molecule consists of 3 L-chains and 2 H-chains, and 2 L-chains and 2 Hchains, respectively. The calculation was based on the assumption that the molecular weights of the L and H chains are 22,000 and 63,000, respectively, and carbohydrate content of IgA is 11.9% which is located exclusively on the H-chain. Thus the present results suggested the possibility that the original IgA molecule consists of 3 L-chains and 2 H-chains and one of the L-chains is released by treatment with 2-ME at pH 8.0 at a concentration higher than 0.05 M.
