Abstract

The noncovalent interaction of light (L) chain with heavy (H) chain or Fd isolated from a human myeloma protein Jo (IgG1, kappa) was studied by following circular dichroic (CD) change at 235 nm. The dimerization constants of Jo-L chain determined by measuring the CD change at 293 nm with protein concentration showed that the Jo-L chain exists as the monomeric form under the experimental conditions used for recombination with H chain. The second-order rate constants for the interaction between H and L chains were in good agreement with those for the interaction between Fd and L chain at various pH values. The binding behavior of L chain to Fd could be described by a single association constant. In the interpretation of the binding of L chain to H chain, however, it was necessary to assume that the binding of L chain to one of the two sites on H chain dimer (H2) decreases the affinity of the other site for L chain. The binding constant of the first L chain to H2 was the same as that of L chain to Fd. Renaturation processes of L chain, Fd, Fab(SS) fragment (with intact interchain disulfide bond), and Fab(RA) fragment (in which the interchain disulfide bond had been reduced and alkylated) from the denatured states in 0.5 or 1 M acetic acid on neutralization were studied. The renaturation of Fd occurred very rapidly, while that of L chain consisted of a very rapid process and a slow process which followed first-order kinetics. The renaturation process of Fab(SS) consisted of rapid and slow phases, of which the latter followed first-order kinetics. The renaturation process of Fab(RA) also consisted of rapid and slow phases, but the latter process followed second-order kinetics. The overall rate constant of renaturation of Fab(RA) was the same as that of the reformation of Fab(RA) from isolated Fd and L chain. On the basis of these facts, the kinetic mechanism by which Fd and L chain recombine to yield Fab(RA) can be described in terms of the scheme Fd + L in equilibrium Fd ... L leads to Fab(RA), where Fd ... L is an intermediate, and CD change is only observed in the second unimolecular process and not in the first bimolecular process.