1998/04/01 by Isabel Velasco‐Guillén, Senena Corbalán‐García, Juan C. Gómez‐Fernández +1
Materials Science · Chemistry · Biochemistry, Genetics and Molecular Biology · #Lanthanide and Transition Metal Complexes #Molecular Sensors and Ion Detection #Electron Spin Resonance Studies
paper · pdf · doi:10.1046/j.1432-1327.1998.2530339.x
The Ca2+-transporting ATPase has been labeled with N-cyclohexyl-N'-(4-dimethyl-amino-alpha-naphthyl)carbodiimide (NCD-4), a fluorescent carbodiimide which reacts with carboxyl groups of acidic residues. It has been reported that NCD-4 labels a transmembrane portion of the protein at the high-affinity calcium-binding sites. We have determined the depth of the calcium-sensitive probe by quenching the fluorescence by nitroxide-substituted fatty acids with its spin probe located at different carbons of the fatty acid chain (5, 7, 10, 12 and 16-nitroxide derivatives). We have found that all the calcium-sensitive fluorescence is quenched and that the efficiency of quenching decreases as the n-(4,4-dimethyl-3-oxazolinyloxy) (Doxyl) group is deeper in the membrane. We conclude that the NCD-4 label which is involved in the high-affinity calcium-binding site is located near the water/lipid interface. The fluorescence of the NCD-4 bound to that site can be quenched by acrylamide and Cu2+ but not by iodide, probably due to its anionic nature which will be repulsed by the abundance of negative charges of Glu and Asp residues of NCD-4 located at this site. The hydrophobic location of NCD-4 was confirmed by the fact that its fluorescence could be quenched by the spin label 2,2,6,6-tetramethyl-1-piperidine-N-oxyl but not by 4-hydroxy-2,2,6,6-tetramethyl-1-piperidine-N-oxyl which is much less hydrophobic.