title: Encapsulation of concentrated hemoglobin solution in phospholipid vesicles retards the reaction with NO, but not CO, by intracellular diffusion barrier.
abstract: 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 mum) showing retardation even for CO binding. The obtained k(on)('(NO)) and k(on)('(NO)) 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.
authors.affiliation: Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Sakai
authors.affiliation: Graduate School of Advanced Science and Engineering, Waseda University, Tokyo 169-8555, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Sato
authors.affiliation: Kobelco Research Institute, Inc., Kobe 651-2271, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Masuda
authors.affiliation: Graduate School of Advanced Science and Engineering, Waseda University, Tokyo 169-8555, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Takeoka
authors.affiliation: Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan. Electronic address: eishun@waseda.jp.
authors.forename: Eishun
authors.initials: E
authors.lastname: Tsuchida
mesh_terms: D002248:Carbon Monoxide / Q000378:metabolism*; D003198:Computer Simulation; D004058:Diffusion; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism*; D010316:Particle Size; D010743:Phospholipids / Q000378:metabolism*; D012996:Solutions; D013053:Spectrophotometry; D013997:Time Factors; D053835:Unilamellar Liposomes / Q000378:metabolism*
chemical_list: D006454:Hemoglobins; D010743:Phospholipids; D012996:Solutions; D053835:Unilamellar Liposomes; D009569:Nitric Oxide; D002248:Carbon Monoxide