Reaction Between Proteins and Diazotized Aromatic Amines in Neutral Solution. ∗

Abstract

In a series of 3 challenging papers entitled “Chemospecific Antigens” Klopstock and Selter 1 have raised a number of important questions dealing with the chemical basis of certain immune reactions. While several of their minor points will be dealt with in connection with other work now in progress, the present note concerns the underlying thought and principal conclusion of their work, namely, that no chemical interaction takes place in neutral solutions of diazotized aromatic amines and proteins or lipoids. It is held that such solutions are merely simple mixtures, and that “eine chemisch zu verstehende Substitution des Chemikals im Eiweissmolekül bei der chemospezifischen Komplexantigenbildung keine Rolle spielt.” Klopstock and Selter have failed entirely to report chemical control experiments in testing this conclusion, and the following are therefore submitted:

I. Conditions the same as in Paper II, p. 455, showing appearance of chemospecific antigen only after mixtures had stood in the cold 1 to 24 hours.

1% diazotized arsanilic acid, “neutralized to litmus.”

2 vols, rabbit serum (K. and S. used beef serum in this experiment, rabbit serum in others).

Since color formation could conceivably have been due to some other serum component than protein, a solution of 3-times recrystallized, dialyzed egg albumen (containing 0.018 g. per cc.) was next used.

II. No difference to the eye immediately after mixing.

Since the neutral diazo arsanilic acid solution darkened rapidly, as noted by Klopstock and Selter, making readings after several hours unsatisfactory, diazotized sulfanilic acid was substituted. This darkens comparatively little on standing in the cold, and, in addition, antisera to sulfanilic-azo-proteins were available. Moreover, since “neutrality to litmus” is a vague term an experiment was run at pH 6.8, but since it might be objected that this is toward the alkaline side of “neutral to litmus” and that actual coupling took place for this reason,† there are given below only the results at pH 6.3-6.4, at which blue litmus is faintly reddened.

III. Dialyzed egg albumen solution, 0.022 g. per cc, pH 6.3. 0.75% solution of diazotized sulfanilic acid, pH 6.4.