Optical measurement of aqueous potassium concentration by a hydrophobic indicator in lipid vesicles.
An assay was developed for K+ in aqueous solution at neutral pH. The method was based on the change in optical absorbance of the hydrophobic indicator 7-(n-decyl)-2-methyl-4-(3',5'-dichlorophen-4'-one)indonaphthl++ +-1-ol (MEDPIN) in phospholipid vesicles. Formation of a ternary complex between a valinomycin-K+ pair and the anionic form of MEDPIN in the bilayer resulted in an absorption band at 584 nm. K+ concentration was determined by monitoring the MEDPIN absorbance at 584 nm and MEDPIN quenching of lissamine rhodamine B sulfonylphosphatidylethanolamine (L-RhB-PE) fluorescence by an energy-transfer mechanism. Both the fluorescence intensity and lifetime of L-RhB-PE decreased by more than 25% upon addition of 50 mM K+. Kinetic studies using stopped-flow photometry showed a single-exponential reaction of MEDPIN and valinomycin in vesicles with aqueous K+ (maximum rate 1.7 s-1) that was dependent upon [valinomycin] and [K+]. The lipid surface charge was shown to influence the ratio of anionic to neutral MEDPIN at constant pH, and to alter the sensitivity of MEDPIN absorbance to aqueous [K+]. A 1:20 neutral/negative lipid mole ratio was optimal for K+ detection at pH 7.4. Spectroscopic and kinetic data suggest that the optical response of MEDPIN to K+ involves the formation of a ternary complex between K+, valinomycin and MEDPIN.
Roe
J N
JN
Department of Bioengineering, University of California, San Francisco 94143.
Szoka
F C
FC
Verkman
A S
AS
D003124:Colorimetry / Q000379:methods; D007202:Indicators and Reagents; D007700:Kinetics; D008081:Liposomes; D008962:Models, Theoretical; D009284:Naphthols; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D011188:Potassium / Q000032:analysis*; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods
D007202:Indicators and Reagents; D008081:Liposomes; D009284:Naphthols; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D012996:Solutions; C061520:7-(n-decyl)-2-methyl-4-(3',5'-dichlorophen-4'-one)indonaphthal-1-ol; D011188:Potassium