Eel haptoglobin (Hp) was mixed with the freshly prepared and the aged component F of eel hemoglobin (Hb) to provide two kinds of Hp-Hb complex: one considered to be composed of one molecule each of Hp and Hb, and the other, of one molecule of Hp and three molecules of Hb as judged by the electrophoretic binding test.
After being isolated by Sephadex gel filtration, both complexes were analyzed for molecular weight and heme content. Results showed that they have the same molecular weight (about 160, 000) and the same heme content (4 moles/mole of complex), thus indicat-ing that both complexes are composed of one molecule each of Hp and Hb. On the other hand, the diffuse electrophoretic band which appeared in the bingding test of eel Hp with the aged component F, was identified as denatured Hb.
It was concluded from these results that the denaturing action of eel Hp upon the aged component F is responsible for the apparent increase in the binding ratio.