Encapsulation of concentrated hemoglobin solution in phospholipid vesicles retards the reaction with NO, but not CO, by intracellular diffusion barrier
Encapsulation of concentrated hemoglobin solution in phospholipid vesicles retards the reaction with NO, but not CO, by intracellular diffusion barrier
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DOI：
10.1074/jbc.m707660200
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发表时间：
2008-01-18
影响因子：
4.8
通讯作者：
Tsuchida, Eishun
中科院分区：
文献类型：
--
作者：
Sakai, Hiromi;Sato, Atsushi;Tsuchida, Eishun
红细胞（RBC）结构的一个生理学意义是通过用细胞膜包封来延迟Hb的NO结合。为了阐明机制，我们分析了血红蛋白囊泡（HbVs）与不同的细胞内血红蛋白浓度，[Hb]，和不同的颗粒大小，使用停流分光光度法。在[Hb]（in）= 1 g/dl时，HbV的表观NO结合速率常数k（on）（（NO））为2.6 × 10（7）M-1 s（-1），几乎等于分子Hb的k（on）（（NO）），表明脂膜对NO结合没有障碍。随着[Hb]增加到35 g/dl，k（对NO）降低到0.9 × 10（7）M-1 s（-1），随着粒径从265 nm增大到452 nm，k（对NO））进一步降低到0.5 × 10（7）M-1 s（-1）。对于CO结合，其本质上比NO结合慢得多，k（on α ′（（CO）不随[Hb] in和颗粒直径而显著变化。使用扩散模拟加上元素结合反应得到的结果与这些趋势一致，并澄清，NO被迅速捕获的Hb从内表面区域的核心HbV在高[Hb]中，阻碍NO扩散到核心的HbV。相比之下，缓慢的CO结合允许进一步的CO-扩散到核心的时间。模拟外推到更大的颗粒（8 μ m）显示延迟，即使CO结合。所获得的k（对NH 2 '（（NO））和k（对NH 2'（（CO）产生的值类似于针对RBC报道的那些值。总之，细胞内，而不是细胞外，扩散屏障是主要的，由于快速NO结合，诱导NO从内表面到核心的快速下沉，阻碍进一步NO扩散和结合。
One physiological significance of the red blood cell (RBC) structure is that NO binding of Hb is retarded by encapsulation with the cell membrane. To clarify the mechanism, we analyzed Hb-vesicles (HbVs) with different intracellular Hb concentrations, [Hb] in, and different particle sizes using stopped-flow spectrophotometry. The apparent NO binding rate constant, k(on)'((NO)), of HbV at [Hb](in) = 1 g/dl was 2.6 x 10(7) M-1 s(-1), which was almost equal to k(on)((NO)) of molecular Hb, indicating that the lipid membrane presents no obstacle for NO binding. With increasing [Hb] in to 35 g/dl, k(on)'((NO)) decreased to 0.9 x 10(7) M-1 s(-1), which was further decreased to 0.5 x 10(7) M-1 s(-1) with enlarging particle diameter from 265 to 452 nm. For CO binding, which is intrinsically much slower than NO binding, k(on)'((CO)) did not change greatly with [Hb] in and the particle diameter. Results obtained using diffusion simulations coupled with elementary binding reactions concur with these tendencies and clarify that NO is trapped rapidly by Hb from the interior surface region to the core of HbV at a high [Hb] in, retarding NO diffusion toward the core of HbV. In contrast, slow CO binding allows time for further CO- diffusion to the core. Simulations extrapolated to larger particles (8 mu m) showing retardation even for CO binding. The obtained k(on)'((NO)) and k(on)'((CO)) yield values similar to those reported for RBCs. In summary, the intracellular, not extracellular, diffusion barrier is predominant due to the rapid NO binding that induces a rapid sink of NO from the interior surface to the core, retarding further NO diffusion and binding.