title: Tuning the hydrophobicity overcomes unfavorable deprotonation making octylamino-substituted 7-nitrobenz-2-oxa-1,3-diazole (n-octylamino-NBD) a protonophore and uncoupler of oxidative phosphorylation in mitochondria.
abstract: The environmentally sensitive fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole (NBD) is generally utilized to monitor dynamic properties of membrane lipids and proteins. Here we studied the behavior of a homologous series of 4-n-alkylamino-substituted NBD derivatives (NBD-Cn; n=4, 6, 8, 9, 10, 12) in planar lipid bilayers, liposomes and isolated mitochondria. NBD-C10 induced proton conductivity in planar lipid membranes, while NBD-C4 was ineffective. The NBD-Cn compounds readily provoked proton permeability of neutral liposomes being less effective in negatively charged liposomes. NBD-Cn increased the respiration rate and reduced the membrane potential of isolated rat liver mitochondria. Remarkably, the bell-shaped dependence of the uncoupling activity of NBD-Cn on the alkyl chain length was found in mitochondria in contrast to the monotonous dependence in liposomes. The effect of NBD-Cn on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by mitochondria swelling. Binding of NBD-Cn to mitochondria increased with n, as shown by fluorescence correlation spectroscopy. It was concluded that despite a pKa value of the amino group in NBD-Cn being about 10, i.e. far from the physiological pH range, the expected hindering of the uncoupling activity could be overcome by inserting the alkyl chain of a certain length.
authors.affiliation: A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1/40, Moscow 119991, Russia; Faculty of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory 1/3, Moscow 119991, Russia.
authors.forename: Stepan S
authors.initials: SS
authors.lastname: Denisov
authors.affiliation: A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1/40, Moscow 119991, Russia.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Kotova
authors.affiliation: A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1/40, Moscow 119991, Russia.
authors.forename: Ljudmila S
authors.initials: LS
authors.lastname: Khailova
authors.affiliation: A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1/40, Moscow 119991, Russia.
authors.forename: Galina A
authors.initials: GA
authors.lastname: Korshunova
authors.affiliation: A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1/40, Moscow 119991, Russia. Electronic address: antonen@genebee.msu.ru.
authors.forename: Yuri N
authors.initials: YN
authors.lastname: Antonenko
mesh_terms: D000818:Animals; D019069:Cell Respiration / Q000187:drug effects; D057927:Hydrophobic and Hydrophilic Interactions; D066298:In Vitro Techniques; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism; D010069:Oxadiazoles / Q000737:chemistry* / Q000494:pharmacology; D010085:Oxidative Phosphorylation; D010539:Permeability; D011522:Protons; D051381:Rats; D013329:Structure-Activity Relationship; D014475:Uncoupling Agents / Q000737:chemistry* / Q000494:pharmacology
chemical_list: C526864:7-nitrobenz-2-oxa-1,3-diazole; D008051:Lipid Bilayers; D008081:Liposomes; D010069:Oxadiazoles; D011522:Protons; D014475:Uncoupling Agents
keywords: Bilayer lipid membrane; Membrane potential; Mitochondria; NBD; Protonophore; Uncoupler