On the mechanism of inactivation of antibody activity by cobalt-60 gamma radiation. I.

Experimentally determined relationships between 6°Co radiation dose and inactivation of combining activity of anti-PAB indicated that a relatively large number of reactions were associated with inactivation. A progressive increase in gross molecular disorganization with increasing radiation dose was indicated by a change in viscosity. Some degree of molecular rearrangement was also suggested by an increased availability of an average of two to three disulphide bonds for reduction after irradiation in presence of urea. The molecular rearrangement suggested by viscosity and disulphide analysis is probably related to the inactivation process, rather than to mere secondary radiation-induced damage of material already inactivated, since, they were first measurable at doses of 7 × 104 and 15 × 104 rads, respectively, as compared to Da7 of 35 × 104 rads. The irradiated material was found to contain, in addition to a grossly denatured fraction insoluble at low ionic strength, some subtly denatured fractions. These fractions were demonstrable by such sensitive techniques as elimination patterns in rabbits and rates of tryptic hydrolysis. Augmented inactivation above that resulting from irradiation alone, was demonstrated to follow mild post-irradiation heat treatment (40-41 ° for 3 hr). This increased heat-induced inactivation was not accompanied by detectable changes in viscosity or in the number of disulphide bonds available for reduction. It appeared to relate to the presence of molecular species, in irradiated antibody solution, that had undergone almost complete disorganization of combining sites, since attempted stabilization of combining site by p-iodobenzoic added before heat treatmentcould not protect against the additional loss of activity. The degree of inactivation relating to the alternative possibility of free radical reactions with a critical group(s) in the combining site was evaluated by blocking experiments with specific haptens and was found to be insignificant.