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 of of that the NO with particle is NO with and the particle using with binding with and clarify that NO is by Hb the the of [Hb]in, NO the of binding the The and the is the NO binding that of NO the the NO and 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 of of that the NO with particle is NO with and the particle using with binding with and clarify that NO is by Hb the the of [Hb]in, NO the of binding the The and the is the NO binding that of NO the the NO and of and of red blood blood Hb rate of NO binding rate binding rate Hb is of of of of blood Hb rate of NO binding rate binding rate Hb is of of of of that the structure with the Hb structure Hb encapsulation of Hb of of Hb blood of the the of and and of of and with and the of that of NO blood and is of by Hb of of Hb the of the structure of the of of NO binding by Hb encapsulation is the NO is retarded of the Hb that the and structure of and the binding rate of NO (HbVs) is with that of that and the of the and stopped-flow stopped-flow is of the binding rate of is of and Hb and the binding we of the of intracellular Hb [Hb]in, and the particle of the apparent binding rate of NO and we the clarify the mechanism, and the binding of of Hb and Hb-vesicles with blood by the by with using and using the of with different The of Hb using and and we we the Hb and with different the Hb is and NO of Hb the using and of The and of is that and Hb Hb with and the and is we that The particle by the of [Hb]in, and and the the of the particle the Hb using the the is by of by of using the and using with and the The by the The of the The particle using The the with of of the the NO and binding the of intracellular Hb the of particle The Hb is with the of the of Hb the binding rate of NO and of the binding analyzed of and using stopped-flow the and with of and of and using The by the of is the of NO the NO and The the of the using stopped-flow the the of NO The NO binding and binding of The and the The and the of of The apparent binding rate of NO and and using the of of and the of and is the the is the the of the NO binding NO is with we the apparent binding the of the of that is and The of Hb and we analyzed and the of Hb the of the of and we the the the of the is with the particle that To the the the the of by of that with by The is the and is the The is the of the the the the the of the particle the the of is by is the of the and and with the the of is the is the of the and is the with that the is the of and with binding rate of binding and the of by the and the of by the stopped-flow the the the the is The of with the of The of the Hb is and with The of that of The of Hb with Hb The of and NO and using is the of NO with different is and is is and that we we the the and of of the and the of and with that of is and is is and that we we the the and of of the and the of and with that of is and is is and that we we the the and of of the and the of and with that of with binding the and the using and using and using and and the of with of the of The and the the of with The the the of the To is the is the is the the is the of the and is the the and is the of is the of and the apparent rate the and of of the of the of the of the with Hb and the the with the and the that the of the of with the The of and of the of the of NO and with of and the of of the The that the NO binding is retarded with the of the the apparent binding rate of NO and and that is with that of Hb the and and and and of Hb of NO binding and binding by with and using stopped-flow the of the The of of the The of the Hb with of The that the NO binding rate is retarded with apparent the of binding of the binding rate of NO and of and of particle the apparent binding rate the of the and of the of Hb the of the apparent binding rate and the particle of the and of Hb of and that the of the with and and of NO and with of particle the with different particle sizes with the the binding of NO and of the of NO and different and different particle and and the and that the NO binding is by and the particle the the NO binding retarded with the the of and the with the The of and the of The particle rate of NO binding that the and of NO and and by and the the of NO and that NO and the the The of NO and the the the of the of of NO and NO and that NO the NO and The the the of the intracellular is is the that clarify the of that with the the with different the is with the of that the the Hb the of NO of the of NO binding of NO binding the that the of NO is of the the with different The with rate of the with the of the of the Hb the of NO is and apparent binding rate and the clarify the of of the and Hb The of the Hb and NO binding and binding retarded with of NO and and with the of that of binding the that with NO NO binding and binding the binding rate that the the that the with NO and the apparent binding rate of particle Hb with and and and encapsulation of Hb particle the NO binding by and the binding by with the of the Hb of NO binding and binding by with different particle the apparent binding rate is is with the the the with particle the the of NO binding and binding the binding rate the the particle is NO binding and binding the is the Hb and and red and the The apparent binding rate and of particle with of and and The of of that of Hb with is that NO binding of Hb is retarded Hb is the binding of Hb by encapsulation with particle The the the apparent binding rate of NO and and of Hb using we that the of the the of by the binding rate of NO of Hb the of [Hb]in, the the intracellular Hb and the the particle of the of of binding by that the of cell and that is of the of the the apparent binding rate of NO and of Hb that the of the intracellular the of is by binding rate of NO Hb of [Hb]in, the intracellular Hb and the of NO binding by Hb encapsulation the of that NO binding of Hb is retarded by the membrane. stopped-flow of Hb the of the NO by Hb that the of the we clarify the of the that the and structure of and the NO binding of Hb and that the of the the and the that the Hb and the binding that of of the of and particle of the of is stopped-flow spectrophotometry. NO the of NO of stopped-flow of NO the of the rate of NO is that of the the the is NO and we the of the of NO with the and NO NO of is the of using the the of the Hb and we the of the and the that the of the of the binding of the we the is and the of particle that of the The the by the of is that the of the that of the of of the that the intracellular is the of NO binding the of stopped-flow and that the is the of that the is the of the and the Hb the the of the that the the NO binding using the the that the of binding we that the intracellular Hb the of the NO that that is by the intracellular Hb that NO the is the intracellular binding rate of of Hb is of NO and The binding is the with the the the of with the the rate is The that binding encapsulation that NO binding the of NO the of and NO the of of the the Hb is that with by and the of and Hb with of and and of that that the of Hb and of The the binding of intracellular and the with the Hb blood the of NO is by NO by and the rate of the and is is that of that the with intracellular that with the of Hb encapsulation the the of and that NO the is different the binding of with different Hb and different particle and the NO binding and binding of particle with that of the of The of and of particle Hb with of and the of using stopped-flow and the the of and the binding rate of the the of the the the of that the by the intracellular is by of with we the structure of of with that of the binding the of the of is The is that that the of binding of of intracellular with the NO The the the and the that the is the the is NO binding and of NO binding by Hb encapsulation the that with of of is that of is and is the the and of NO that the NO binding rate of of using the that of Hb with with of that NO Hb and and the of particle the cell the of the the the and the NO is the of by the of the NO NO and the particle and by we the of the intracellular and the of NO the of that the the and we that the binding of and the is the and rate the of and the binding of and and of the of of and of red blood blood Hb rate of NO binding rate binding rate Hb is of of of of blood Hb rate of NO binding rate binding rate Hb is of of of of that the structure with the Hb structure Hb encapsulation of Hb of of Hb blood of the the of and and of of and with and the of that of NO blood and is of by Hb of of Hb the of the structure of red blood cell intracellular Hb intracellular binding rate of apparent NO binding rate apparent binding rate Hb is of Hb of of NO of red blood cell intracellular Hb intracellular binding rate of apparent NO binding rate apparent binding rate Hb is of Hb of of NO of the of of NO binding by Hb encapsulation is the NO is retarded of the Hb that the and structure of and the binding rate of NO (HbVs) is with that of that and the of the and stopped-flow stopped-flow is of the binding rate of is of and Hb and the binding we of the of intracellular Hb [Hb]in, and the particle of the apparent binding rate of NO and we the clarify the mechanism, and the binding of of Hb and Hb-vesicles with blood by the by with using and using the of with different The of Hb using and and we we the Hb and with different the Hb is and NO of Hb the using and of The and of is that and Hb Hb with and the and is we that The particle by the of [Hb]in, and and the the of the particle the Hb using the the is by of by of using the and using with and the The by the The of the The particle using The the with of of the the NO and binding the of intracellular Hb the of particle The Hb is with the of the of Hb the binding rate of NO and of the binding analyzed of and using stopped-flow the and with of and of and using The by the of is the of NO the NO and The the of the using stopped-flow the the of NO The NO binding and binding of The and the The and the of of The apparent binding rate of NO and and using the of of and the of and is the the is the the of the NO binding NO is with we the apparent binding the of the of that is and The of Hb and we analyzed and the of Hb the of the of and we the the the of the is with the particle that To the the the the of by of that with by The is the and is the The is the of the the the the the of the particle the the of is by is the of the and and with the the of is the is the of the and is the with that the is the of and with binding rate of binding and the of by the and the of by the stopped-flow the the the the is The of with the of The of the Hb is and with The of that of The of Hb with Hb The of and NO and using is the of NO with different is and is is and that we we the the and of of the and the of and with that of is and is is and that we we the the and of of the and the of and with that of is and is is and that we we the the and of of the and the of and with that of with binding the and the using and using and using and and the of with of the of The and the the of with The the the of the To is the is the is the the is the of the and is the the and is the of is the of and the apparent rate the of Hb and Hb-vesicles with blood by the by with using and using the of with different The of Hb using and and we we the Hb and with different the Hb is and NO of Hb the using and of The and of is that and Hb Hb with and the and is we that The particle by the of [Hb]in, and and the the of the particle the Hb using the the is by of by of using the and using with and the The by the The of the The particle using The the with of the binding analyzed of and using stopped-flow the and with of and of and using The by the of is the of NO the NO and The the of the using stopped-flow the the of NO The NO binding and binding of The and the The and the of of The apparent binding rate of NO and and using the of of and the of and is the the is the the of the NO binding NO is with we the apparent binding the of the of that is and The of Hb and we analyzed and the of Hb the of the of and we the the the of the is with the particle that To the the the the of by of that with by The is the and is the The is the of the the the the the of the particle the the of is by is the of the and and with the the of is the is the of the and is the with that the is the of and with binding rate of binding and the of by the and the of by the stopped-flow the the the the is The of with the of The of the Hb is and with The of that of The of Hb with Hb The of and NO and using is the of NO with binding the and the using and using and using and and the of with of the of The and the the of with The the the of the To is the is the is the the is the of the and is the the and is the The of is the of and the apparent rate the and of of the of the of the of the with Hb and the the with the and the that the of the of with the The of and of the of the of NO and with of and the of of the The that the NO binding is retarded with the of the the apparent binding rate of NO and and that is with that of Hb the and and and and of Hb binding rate of NO and of and of particle the apparent binding rate the of the and of the of Hb the of the apparent binding rate and the particle of the and of Hb of and that the of the with and and of NO and with of particle the with different particle sizes with the the binding of NO and of the of NO and different and different particle and and the and that the NO binding is by and the particle the the NO binding retarded with the the of and the with the The of and the of The particle rate of NO binding that the and of NO and and by and the the of NO and that NO and the the The of NO and the the the of the of of NO and NO and that NO the NO and The the the of the intracellular is is the that clarify the of that with the the with different the is with the of that the the Hb the of NO of the of NO binding of NO binding the that the of NO is of the the with different The with rate of the with the of the of the Hb the of NO is and apparent binding rate and the clarify the of of the and Hb The of the Hb and NO binding and binding retarded with of NO and and with the of that of binding the that with NO NO binding and binding the binding rate that the the that the with NO and the apparent binding rate of particle Hb with and and and encapsulation of Hb particle the NO binding by and the binding by with the of the Hb of NO binding and binding by with different particle the apparent binding rate is is with the the the with particle the the of NO binding and binding the binding rate the the particle is NO binding and binding the is the Hb and and red and the The apparent binding rate and of particle with of and and The of of that of Hb with NO and of of the of the of the of the with Hb and the the with the and the that the of the of with the The of and of the of the of NO and with of and the of of the The that the NO binding is retarded with the of the the apparent binding rate of NO and and that is with that of Hb the and and and and of Hb of NO and with of particle the with different particle sizes with the the binding of NO and of the of NO and different and different particle and and the and that the NO binding is by and the particle the the NO binding retarded with the the of and the with the The of and the of The particle rate of NO binding that the and The of NO and and by and the the of NO and that NO and the the The of NO and the the the To clarify the of that with the the with different the is with the of that the the Hb the of NO and apparent binding rate and the clarify the of of the and Hb The of the Hb and NO binding and binding retarded with of NO and and with the of that of binding the that with NO NO binding and binding the binding rate that the the that the with NO and the apparent binding rate of particle Hb with and and and encapsulation of Hb particle the NO binding by and the binding by with the of the Hb is that NO binding of Hb is retarded Hb is the binding of Hb by encapsulation with particle The the the apparent binding rate of NO and and of Hb using we that the of the the of by the binding rate of NO of Hb the of [Hb]in, the the intracellular Hb and the the particle of the the of NO binding by Hb encapsulation the of that NO binding of Hb is retarded by the membrane. stopped-flow of Hb the of the NO by Hb that the of the we clarify the of the that the and structure of and the NO binding of Hb and that the of the the and the that the Hb and the binding that of of the of and particle of the of is stopped-flow spectrophotometry. NO the of NO of stopped-flow of NO the of the rate of NO is that of the the the is NO and we the of the of NO with the and NO NO of is the of using the the of the Hb and we the of the and the that the of the of the binding of the we the is and the of particle that of the The the by the of is that the of the that of the of of the that the intracellular is the of NO binding the of stopped-flow and that the is the of that the is the of the and the Hb the the of the that the the NO binding using the the that the of binding we that the intracellular Hb the of the NO that that is by the intracellular Hb that NO the is the intracellular binding rate of of Hb is of NO and The binding is the with the the the of with the the rate is The that binding encapsulation that NO binding the of NO the of and NO the of of the the Hb is that with by and the of and Hb with of and and of that that the of Hb and of The the binding of intracellular and the with the Hb blood the of NO is by NO by and the rate of the and is is that of that the with intracellular that with the of Hb encapsulation the the of and that NO the is different the binding of with different Hb and different particle and the NO binding and binding of particle with that of the of The of and of particle Hb with of and the of using stopped-flow and the the of and the binding rate of the the of the the the of that the by the intracellular is by of with we the structure of of with that of the binding the of the of is The is that that the of binding of of intracellular with the NO The the the and the that the is the the is NO binding and of NO binding by Hb encapsulation the that with of of is that of is and is the the and of NO that the NO binding rate of of using the that of Hb with with of that NO Hb and and the of particle the cell the of the the the and the NO is the of by the of the NO NO and the particle and by we the of the intracellular and the of NO the of that the the and we that the binding of and the is the and rate the of and the binding of and and of the of is that NO binding of Hb is retarded Hb is the binding of Hb by encapsulation with particle The the the apparent binding rate of NO and and of Hb using we that the of the the of by the binding rate of NO of Hb the of [Hb]in, the the intracellular Hb and the the particle of the the of NO binding by Hb encapsulation the of that NO binding of Hb is retarded by the membrane. stopped-flow of Hb the of the NO by Hb that the of the we clarify the of the that the and structure of and the NO binding of Hb and that the of the the and the that the Hb and the binding that of of the of and particle of the of is stopped-flow spectrophotometry. NO the of NO of stopped-flow of NO the of the rate of NO is that of the the the is NO and we the of the of NO with the and NO NO of is the of using the the of the Hb and we the of the and the that the of the of the binding of the we the is and the of particle that of the The the by the of is that the of the that of the of of the that the intracellular is the of NO binding the of stopped-flow and that the is the of that the is the of the and the Hb the the of the that the the NO binding using the the that the of binding we that the intracellular Hb the of the NO that that is by the intracellular Hb that NO the is the intracellular The binding rate of of Hb is of NO and The binding is the with the the the of with the the rate is The that binding encapsulation that NO binding the of NO the of and NO the of of the the Hb is that with by and the of and Hb with of and and of that that the of Hb and of The the binding of intracellular and the with the Hb blood the of NO is by NO by and the rate of the and is is that of that the with intracellular that with the of Hb encapsulation the the of and that NO the is different the binding of with different Hb and different particle and the NO binding and binding of particle with that of the of The of and of particle Hb with of and the of using stopped-flow and the the of and the binding rate of the the of the the the of that the by the intracellular is by of with we the structure of of with that of the binding the of the of is The is that that the of binding of of intracellular with the NO The the the and the that the is the the is NO binding and The of NO binding by Hb encapsulation the that with of of is that of is and is the the and of NO that the NO binding rate of of using the that of Hb with with of that NO Hb and and the of particle the cell the of the the the and the NO is the of by the of the NO NO and the particle and by we the of the intracellular and the of NO the of that the the and we that the binding of and the is the and rate the of and the binding of and and of the of of and the of