Distribution of iodine among various parts of iodinated normal and monoclonal human IgG molecules

Abstract

The relative availability of groups subject to iodination in the H chain, the L chain and the Fd and Fc fragments of human normal IgG and several human monoclonal IgG proteins has been investigated by employing lactoperoxidase-catalyzed iodination. Normal IgG and four monoclonal IgG proteins, three IgGκ and one IgGλ, were iodinated to two or more levels of substitution. One of the IgGκ proteins was of the IgG2 subclass and the other three monoclonal proteins were of the IgGl subclass. The iodinated proteins were reduced and the resulting heavy and light chains were separated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The ratio of the extent of iodination of the heavy chain to that of the light chain (the H/L ratio) was different for each protein, even when total iodination was to the same extent. In general, the H/L ratio exceeded two even though the ratio of the number of tyrosines in the two chains is approx two in all cases. One monoclonal IgG with γ 2 chain, IgGκ Lin, showed a ratio of one at a low iodination level. Another IgG, IgGκ Col, was iodinated exclusively on the heavy chain at all the iodination levels examined (0.3–12 iodine atoms per IgG molecule). Nonenzymatic iodination of this protein was also carried out with hypoiodite to iodine levels of 20, 32.5 and 50 iodine atoms per IgG molecule. Again, iodination occurred almost exclusively on the heavy chain. A more detailed study of the differences between IgG Col and normal IgG was carried out by determining the distribution of iodine on the Fd and the Fc fragments, as well as on the light and heavy chains, after these proteins were iodinated enzymatically to 0.34 and 0.39 iodine atoms per molecule, respectively. The relative amounts of iodine in the Fc, and Fd and the light-chain portions were found to be 1.0, 0.48 and 0.0 for the IgG Col and 1.0, 1.3 and 0.70 for normal IgG.