Modification of the Amino Groups of Rabbit γG-Globulin: I. The Effect on Skin Sensitizing Activity in Guinea Pig Skin¹

We have demonstrated that a variety of amino reagents markedly influence the ability of rabbit γG-antibody to mediate passive cutaneous anaphylaxis in guinea pig skin. The amino reagents which were screened and found to cause inactivation included acetic anhydride, potassium cyanate, picryl sulfonate, phthalic anhydride, and N-acetylhomocysteine thiolactone. The possibility that the inactivation might be due to modification of the antibody-combining site was excluded by equilibrium dialysis or fluorescence enhancement or both.

The only amino reagent studied in detail which failed to produce marked inactivation was ethyl acetimidate. Ethyl acetimidate, unlike the amino reagents cited above, replaces the cationic ε-ammonium groups of lysine with the positively charged amidine function so that overall protein charge is not altered. While the failure of ethyl acetimidate to produce inactivation might suggest that an alteration of average protein charge is the basis for inactivation, experiments in which rabbit antibody was sequentially treated with ethyl acetimidate and picryl sulfonic acid indicate that this explanation may not be correct. Limited substitution of the amidinated protein by picryl groups, presumably at lysyl residues which had failed to react with ethyl acetimidate, resulted in complete loss of PCA reactivity even though protein charge was altered very little.