Use of a membrane-bound fluorophore to characterize diffusion boundary layers around human erythrocytes.
A novel method is used to demonstrate the presence of diffusion boundary layers around erythrocytes following rapid mixing in a stopped-flow spectrophotometer and to estimate the apparent dimensions of the diffusion boundary layers. Pink erythrocyte ghosts labeled on their external surfaces with tetramethyl rhodamine isothiocyanate (TRITC) were mixed in a stopped-flow apparatus with 50 mM NaI in Ringer's solutions. I- is an effective collisional quencher of TRITC fluorescence. TRITC fluorescence after flow stopped decreased monoexponentially with time. The concentration of I- at the cell surface as a function of time was estimated from the dependence of TRITC fluorescence on I- concentration in steady-state experiments. The kinetics of the increase in I- concentration at the cell surface was fit to two diffusional models: a planar erythrocyte ghost bounded by planar diffusion boundary layer and a spherical erythrocyte surrounded by a spherical shell diffusion boundary layer. The planar model best fits the experimental data with a diffusion boundary layer 4.68 microns thick. Using the spherical model the experimental data is best fit by a 6.9 microns diffusion boundary layer.
D004058:Diffusion; D004910:Erythrocyte Membrane / Q000502:physiology*; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D012235:Rhodamines; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D012235:Rhodamines; C009434:tetramethylrhodamine isothiocyanate