Positive cooperativity in the hapten binding by the VL dimer of protein 315

The hapten binding behavior of the dimer of the variable domains, (V_l)₂, derived from the light chains of protein 315 was investigated by difference absorption spectrophotometric titrations. The stoichiometry of binding of $\text{ε-N-(2,4-}\text{dinitrophenyl)-}\text{l}\text{-lysine}$ (DNPL) and 4-(α-N-alanine)-7-nitrobenz-2-oxa-1,3-diazole is two haptens per (Vl)₂. Positive cooperativity was observed in this binding. Such positive cooperativity has been reported previously for the light chain dimers of protein 315 with their native interchain disulfide bridge preserved or cleaved by reduction and alkylation. The hapten binding was analysed as in the previous cases according to the allosteric model of Monod et al. (1965). This model assumes that the protein exists in two conformations, and the equilibrium between them shifts upon hapten binding. The parameters of the hapten binding and of the allosteric transition of (Vl)₂ are similar to those of L_{2 ncov} showing that the C_l domains play a rather limited role in the behavior of the latter protein. In contrast, these parameters are drastically different from those reported for the L_{2 cov}. This constitutes a further illustration of the importance of the disulfide bond in affecting the protein's conformation and hapten binding.The detailed analysis of the titrations of (V_l)₂ and of L_2nocov with DNPL led to the calculation of the difference absorption spectra between the DNPL complexes formed by each of the two conformers of either protein and the free hapten. The distinct dissimilarity of these two difference spectra illustrates the different environments of the dinitrophenyl ring in the binding sites of each conformer.