Makino R, Tanaka T, Iizuka T, Ishimura Y, Kanegasaki S
J Biol Chem. 1986 Sep 5;261(25):11444-7.
Extracellular release of superoxide anion (O-2) and hydrogen peroxide (H2O2) during the respiratory burst of porcine and human neutrophils was studied by using diacetyldeuteroheme-substituted horseradish peroxidase as a trapping agent for these oxygen derivatives. The method permitted simultaneous measurement of oxygen consumption and formation of both O-2 and H2O2 in a single reaction mixture. When neutrophils were stimulated with phorbol myristate acetate in the presence of the heme-substituted peroxidase, a rapid accumulation of compound III, a complex of the enzyme with O-2, was observed accompanying an increase in oxygen consumption. During the process, amounts of compound III formed and oxygen consumed were stoichiometric, and no compound II, an indicator of H2O2 formation, was observed. These results establish that neutrophils stimulated with the phorbol ester produce exclusively O-2 as the primary oxygen metabolite and release it into the extracellular medium. When a limited amount of opsonized zymosan was used as the stimulus, compound III formation was also observed but it ceased at an early stage of oxygen consumption. When a sufficient amount of azide was included in the system, however, formation of compound II was noted in the later stage of oxygen consumption. The findings suggest that O-2, formed during phagocytosis, is converted to H2O2 within phagosomes and then diffuses out into the extracellular medium when its decomposition by catalase and/or peroxidases is blocked by azide.
利用二乙酰氘血红素取代的辣根过氧化物酶作为这些氧衍生物的捕获剂，研究了猪和人中性粒细胞呼吸爆发过程中超氧阴离子（O₂⁻）和过氧化氢（H₂O₂）的细胞外释放。该方法允许在单一反应混合物中同时测量氧气消耗以及O₂⁻和H₂O₂的生成。当中性粒细胞在血红素取代的过氧化物酶存在下用佛波酯肉豆蔻酸酯刺激时，观察到酶与O₂⁻的复合物化合物III迅速积累，同时氧气消耗增加。在此过程中，形成的化合物III的量和消耗的氧气量是化学计量的，并且未观察到作为H₂O₂生成指示剂的化合物II。这些结果表明，用佛波酯刺激的中性粒细胞仅产生O₂⁻作为主要的氧代谢产物并将其释放到细胞外介质中。当使用有限量的调理酵母聚糖作为刺激物时，也观察到化合物III形成，但在氧气消耗的早期阶段就停止了。然而，当系统中包含足够量的叠氮化物时，在氧气消耗的后期阶段注意到化合物II的形成。这些发现表明，吞噬作用过程中形成的O₂⁻在吞噬体内转化为H₂O₂，然后当其被过氧化氢酶和/或过氧化物酶分解被叠氮化物阻断时扩散到细胞外介质中。
