Post-irradiation inactivation, protection, and repair of the sulfhydryl enzyme malate synthase

Summary

Malate synthase from baker's yeast, a trimeric sulfhydryl enzyme with one essential sulfhydryl group per subunit, was inactivated by 2 kGy X-irradiation in air-saturated aqueous solution (enzyme concentration: 0.5 mg/ml). The radiation induced changes of enzymic activity were registered at about 0, 30, 60 h after irradiation. To elucidate the role of OH^⋅,$O_2^{\bar \cdot }$, and H₂O₂ in the X-ray inactivation of the enzyme, experiments were performed in the absence or presence of different concentrations of specific additives (formate, superoxide dismutase, catalase). These additives were added to malate synthase solutions before or after X-irradiation. Moreover, repairs of inactivated malate synthase were initiated at about 0 or 30 h after irradiation by means of the sulfhydryl agent dithiothreitol. Experiments yielded the following results:

• 1. Irradiation of malate synthase in the absence of additives inactivated the enzyme immediately to a residual activity A_r = 3% (corresponding to a D₃₇ = 0.6 kGy), and led to further slow inactivation in the post-irradiation phase. Repairs, initiated at different times after irradiation, restored enzymic activity considerably. The repair initiated att = 0 led to A_r = 21%; repairs started later on resulted in somewhat lower activities. The decay of reparability, however, was found to progress more slowly than post-irradiation inactivation itself. After completion of repair the activities of repaired samples did not decrease significantly.

• 2. The presence of specific additives during irradiation caused significant protective effects against primary inactivation. The protection by formate was very pronounced (e.g., A_r = 72% and D₃₇ = 6 kGy for 100 mM formate). The presence of catalytic amounts of superoxide dismutase and/or catalase exhibited only minor effects, depending on the presence and concentration of formate.

• 3. Both the presence of specific additives during irradiation and the addition of additives after irradiation may alter the post-irradiation inactivation. Catalase turned out to be the most potent inhibitor of post-irradiation inactivation; superoxide dismutase showed an ambivalent behaviour, it accelerated or impeded post-irradiation inactivation; formate, when added after irradiation, exhibited a moderate protective effect.

• 4. The presence of specific additives, added before and/or after irradiation, influenced the repair behaviour to some extent. The highest activity achieved by repair amounted to about 90% of the activity of the corresponding unirradiated sample. The percentual gain of activity was found to be the greater the lower the residual activity of the enzyme was before initiation of repair.

• 5. The results clearly show that the inactivation of malate synthase during irradiation is mainly caused by OH^⋅, the post-irradiation inactivation mainly by H₂O₂. To a minor extent$O_2^{\bar \cdot }$ and H₂O₂ may be involved in the primary inactivation, and OH^⋅ and$O_2^{\bar \cdot }$ in the post-irradiation inactivation.