A comparison of the isoelectrofocusing porperties of antibodies to DNP, DNP-glycylglycylglycine and DNP-p-aminobenzoylglutamate.

The charge of heterogeneity of antibodies to DNP-glycylglycylglycine (DNP-Gly-3) and DNP-p-aminobenzoylglutamate (DNP-pABG) has been investigated using preparative liquid isoelectric focusing techniques. The focusing profiles of the two antibody preparations are qualitatively similar, each containing four or five major peaks. The results are also similar to the profiles obtained earlier for DNP antibodies. The binding properties of refocused fractions from the electro-focusing separation of the two DNP-specific antibodies were investigated. Both the unfocused and refocused DNP-pABG antibody fractions were found to be functionally less heterogeneous than the corresponding DNP-Gly-3 fractions, suggesting that the site-filling capacity of the haptens contributed to the observed functional heterogeneity. A marked increase in K0 with increasing pI of the focused antibody fraction was observed for the DNP-pABG antibody preparation. A much smaller increase of K0 with pI was observed for DNP-Gly-3 antibodies and no observed change was found for DNP antibodies. These differences probably reflect the difference in the net charge on the three DNP hapten groups. The isoelectric focusing properties of the separated light and heavy chains from a DNP-antibody preparation and a DNP-pABG antibody preparation were also determined. The light chains in both instances focused as one or two major bands, while the profile of the heavy chain in both cases exhibited five to six major bands. These results are discussed in terms of the model proposed by Haselkorn, Friedman, Givol and Pecht (1974).