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J. Biol. Chem., Vol. 283, Issue 3, 1508-1517, January 18, 2008

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Encapsulation of Concentrated Hemoglobin Solution in Phospholipid Vesicles Retards the Reaction with NO, but Not CO, by Intracellular Diffusion Barrier^*

Hiromi Sakai, Atsushi Sato, Kaoru Masuda^¶, Shinji Takeoka¹, and Eishun Tsuchida²

From the Research Institute for Science and Engineering and Graduate School of Advanced Science and Engineering, Waseda University, Tokyo 169-8555, Japan and ^¶Kobelco Research Institute, Inc., Kobe 651-2271, Japan

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, , of HbV at [Hb]_in = 1 g/dl was 2.6 x 10⁷ 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, decreased to 0.9 x 10⁷ M^-1 s^-1, which was further decreased to 0.5 x 10⁷ M^-1 s^-1 with enlarging particle diameter from 265 to 452 nm. For CO binding, which is intrinsically much slower than NO binding, 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 µm) showing retardation even for CO binding. The obtained and 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.

Received for publication, September 12, 2007 , and in revised form, November 13, 2007.

^* This work was supported in part by Health and Labor Sciences Research Grants (Research on Regulatory Science of Pharmaceuticals and Medical Devices), Ministry of Health, Labor, and Welfare, Japan (to H. S. and E. T.) and by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science B16300162 (to H. S.) and 18500368 (to S. T.), and Global COE "Practical Chemical Wisdom" (to S. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku, Tokyo 169-8555, Japan.

² To whom correspondence should be addressed: Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku, Tokyo 169-8555, Japan. Tel.: 81-3-5286-3120; Fax: 81-3-3205-4740; E-mail: eishun{at}waseda.jp.

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