Vaughn M W, Huang K T, Kuo L, Liao J C
Department of Chemical Engineering, University of California, Los Angeles, California 90095, USA.
J Biol Chem. 2000 Jan 28;275(4):2342-8. doi: 10.1074/jbc.275.4.2342.
It has been reported that free hemoglobin (Hb) reacts with NO at an extremely high rate (K(Hb) approximately 10(7) M(-1) s(-1)) and that the red blood cell (RBC) membrane is highly permeable to NO. RBCs, however, react with NO 500-1000 times slower. This reduction of NO reaction rate by RBCs has been attributed to the extracellular diffusion limitation. To test whether additional limitations are also important, we designed a competition test, which allows the extracellular diffusion limitation to be distinguished from transmembrane or intracellular resistance. This test exploited the competition between free Hb and RBCs for NO generated in a homogenous phase by an NO donor. If the extracellular diffusion resistance is negligible, then the results would follow a kinetic model that assumes homogenous reaction without extracellular diffusion limitation. In this case, the measured effective reaction rate constant, K(RBC), would remain invariant of the hematocrit, extracellular-free Hb concentration, and NO donor concentration. Results show that the K(RBC) approaches a constant only when the hematocrit is greater than 10%, suggesting that at higher hematocrit, the extracellular diffusion resistance is negligible. Under such a condition, the NO consumption by RBCs is still 500-1000 times slower than that by free Hb. This result suggests that intrinsic RBC factors, such as transmembrane diffusion limitation or intracellular mechanisms, exist to reduce the NO consumption by RBCs.
据报道，游离血红蛋白（Hb）与一氧化氮（NO）的反应速率极高（K(Hb)约为10(7) M(-1) s(-1)），且红细胞（RBC）膜对NO具有高度通透性。然而，红细胞与NO的反应速度要慢500 - 1000倍。红细胞对NO反应速率的这种降低归因于细胞外扩散限制。为了测试是否还有其他限制因素也很重要，我们设计了一个竞争试验，该试验可以区分细胞外扩散限制与跨膜或细胞内阻力。这个试验利用了游离Hb和红细胞对由NO供体在均相相中产生的NO的竞争。如果细胞外扩散阻力可以忽略不计，那么结果将遵循一个动力学模型，该模型假设没有细胞外扩散限制的均相反应。在这种情况下，测得的有效反应速率常数K(RBC)将与血细胞比容、细胞外游离Hb浓度和NO供体浓度无关。结果表明，只有当血细胞比容大于10%时，K(RBC)才接近一个常数，这表明在较高的血细胞比容下，细胞外扩散阻力可以忽略不计。在这种情况下，红细胞消耗NO 的速度仍然比游离Hb慢500 - 1000倍。这一结果表明，存在内在的红细胞因素，如跨膜扩散限制或细胞内机制，以减少红细胞对NO的消耗。
