We have performed 13C nuclear magnetic resonance (NMR) studies of the binding of tetra-L-alanine haptens, each selectively enriched with 90% 13C in a single methyl group, to Fab' fragments of purified sheep anti-poly(L-alanine) antibodies. Sheep were immunized with a poly(L-alanine)-human serum albumin conjugate, and purified immunoglobulin G antibodies were isolated by affinity chromatography. Two separate pools of purified, specific F(ab')2 fragments were prepared, and each pool was reduced and alkylated to give approximately 200 mg of purified Fab' fragments. Pool 1 Fab' fragments bound tetra-L-[3H]alanine with an average association constant (Ka) of 1.1 × 104 M−1 at 5 °C; pool 2 had a Ka value of 2.6 × 104 M−1 at 5 °C. The enthalpy of binding (ΔH) of pool 1 and pool 2 Fab' fragments was -5.6 and -7.9 kcal M-1, respectively; the entropy of binding (AS) was -1.8 and -8.2 cal mol−1 deg−1, respectively. Sips constants for the first and second pools were 0.90 and 0.80, respectively. Both preparations of antibody fragments were found to be very heterogeneous when analyzed by isoelectric focusing. 13C NMR difference spectra were obtained over a range of hapten-Fab' fragment ratios in order to observe alterations induced in the [13C]methyl resonances of the tetrapeptide by interaction with high- and low-affinity fragments. The higher affinity proteins induce essentially uniform downfield shifts of ~2.8 parts per million in all four methyl resonances of the bound peptide, and the lower affinity antibodies either affected fewer methyl groups or, in most cases, did not induce any shifts in the methyl resonances of the bound peptide. These downfield shifts appear to be due to van der Waals interactions between the methyl groups of the bound peptide and the binding sites of the higher affinity antibodies. The uniformity of the downfield shifts of all four methyl resonances of the bound peptide is consistent with relatively homogeneous hydrocarbon- like environments in the hapten's side-chain binding region of the higher affinity fragments. The direct correlation between the number of methyl groups of the bound hapten that exhibit this shift and the association constants of the antibody fragments suggests that the major difference between the higher and lower affinity Fab' fragments is the presence or absence of side-chain van der Waals interactions with the hapten. The NMR line-width data indicate that the methyl groups of the peptide are free to rotate in the antibody-hapten complexes whereas the entire backbone of the peptide appears to be firmly bound to both high-and low-affinity fragments. The chemical shift data of the N-terminal methyl group of the peptide are consistent with the amino group being protonated when bound to the Fab' fragments, which suggests that there may be electrostatic binding between the hapten at this position and the antibody binding sites. The chemical shift of the methyl resonance of the C-terminal residue suggests that the carboxyl group of tetra-L-alanine is protonated when bound to higher affinity antibodies and ionized when bound to lower affinity fragments.The tetrapeptide was found to be in the NMR “slow-exchange” limit with the Fab' fragments, and calculations indicated that the forward rate constants) of peptide binding is (are) relatively slow. The kinetic results suggest that there may be substantial orientational requirements for binding of the hapten to the antibodies.
|Original language||English (US)|
|Number of pages||10|
|State||Published - Jul 1 1980|
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