Studies on the Denaturation of Antibody Ii. the Effect of Protein Concentration on Ti4e Rate of Denaturation of Diphtheria Antitoxin by Urea

Although in a recent study (1) the susceptibility of diphtheria antitoxin to the denaturing action of urea was apparently established, the question of the fundamental nature of the reaction remains essentially unsolved. Since the action of urea probably involves a partial unfolding of the polypeptide chains of proteins, the lability of antibody in urea suggests that the antibody structure resides in a particular folding of these chains, as proposed by Pauling (2). I t is possible, however, to suggest other mechanisms for the observed inactivation; in particular, an aggregation of the protein molecules as a result of the denaturation might cause blocking off of unaltered antibody groups. Moreover complexing has frequently been observed by physical or immunological methods following denaturation of serum proteins by such agents as heat (3-6), ultraviolet light (7), and visible light in the presence of a photosensitizer (8, 9). With mixtures of serum albumin and globulin, urea denaturation leads to complexing demonstrable by electrophoretic analysis (10). I t is probable that if the inactivation of antibody were due to complexing, obviously a polymolecular process, this fact should be apparent from a study of the kinetics of the inactivation. I t is of course difficult to exclude by kinetic studies all possibility of a polymolecular step in the process, since it may be masked by a unimolecular rate-determining reaction. I t has been suggested, however, that complexing of the antibody would affect its behavior in the quantitative precipitation reaction (11). Thus complexing of solutions containing antibody and normal globulin might increase the amount of precipitable protein, since normal protein linked to the antibody would precipitate with it. The extent of this reaction should give some estimate of the degree of complexing accompanying the denaturation. In a previous paper (1) the rate of inactivation of antitoxin in urea was found to decrease more rapidly as the reaction progressed than would be required by the first order law; preliminary experiments, however, suggested that the rate of inactivation was independent of the initial antibody concentration. The present paper is concerned with further experiments on the effect of protein con-