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A novel rapid‐reaction spectrophotometric method for monitoring monovalent anion exchange by human erythrocyte band 3
Author(s) -
Salhany James M.
Publication year - 1998
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/15216549800202552
Subject(s) - thiocyanate , chemistry , dids , iodide , band 3 , methemoglobin , chloride , absorbance , kinetics , hemoglobin , ion , inorganic chemistry , chromatography , biochemistry , membrane , erythrocyte membrane , organic chemistry , physics , quantum mechanics
Thiocyanate (SCN‐) uptake into human erythrocytes and resealed ghosts was measured by monitoring the intracellular reaction of SCN‐ with methemoglobin using a dual wavelength stopped‐flow apparatus. The cellular reaction was considerably slower than the reaction of SCN with methemoglobin in solution, indicating that SCN‐ diffusion and not chemical reaction was rate limiting. This view was confirmed by showing that the uptake rate followed saturation kinetics ( Km〈9 mM), thus indicating that SCN‐ transport involves a facilitated diffusion process. Addition of DIDS ( 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate) totally inhibited SCN‐ uptake, thus identifying band 3 as the sole facilitator. Substitution of iodide or sulfate for trans chloride, slowed SCN‐ uptake by 4‐fold and 35‐fold respectively. Reducing the trans chloride concentration from 150 to 2 mM decreased the extent of the reaction, and slowed the observed rate by about 2‐fold. These results define a new approach for the continuous monitoring of monovalent anion exchange by human erythrocyte band 3.