title: Revisiting Mitochondrial pH with an Improved Algorithm for Calibration of the Ratiometric 5(6)-carboxy-SNARF-1 Probe Reveals Anticooperative Reaction with H+ Ions and Warrants Further Studies of Organellar pH.
abstract: Fluorescence measurements of pH and other analytes in the cell rely on accurate calibrations, but these have routinely used algorithms that inadequately describe the properties of indicators. Here, we have established a more accurate method for calibrating and analyzing data obtained using the ratiometric probe 5(6)-carboxy-SNARF-1. We tested the implications of novel approach to measurements of pH in yeast mitochondria, a compartment containing a small number of free H+ ions. Our findings demonstrate that 5(6)-carboxy-SNARF-1 interacts with H+ ions inside the mitochondria in an anticooperative manner (Hill coefficient n of 0.5) and the apparent pH inside the mitochondria is ~0.5 unit lower than had been generally assumed. This result, at odds with the current consensus on the mechanism of energy generation in the mitochondria, is in better agreement with theoretical considerations and warrants further studies of organellar pH.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106, Warsaw, Poland.
authors.forename: Tomasz Michał
authors.initials: TM
authors.lastname: Żurawik
authors.affiliation: Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, F. Joliot-Curie 14A, 50-383, Wrocław, Poland.
authors.forename: Adam
authors.initials: A
authors.lastname: Pomorski
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106, Warsaw, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Belczyk-Ciesielska
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106, Warsaw, Poland.
authors.forename: Grażyna
authors.initials: G
authors.lastname: Goch
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106, Warsaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Niedźwiedzka
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106, Warsaw, Poland.
authors.forename: Róża
authors.initials: R
authors.lastname: Kucharczyk
authors.affiliation: Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, F. Joliot-Curie 14A, 50-383, Wrocław, Poland.
authors.forename: Artur
authors.initials: A
authors.lastname: Krężel
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106, Warsaw, Poland.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Bal
mesh_terms: D000465:Algorithms; D001578:Benzopyrans; D015374:Biosensing Techniques; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000378:metabolism*; D009284:Naphthols; D011522:Protons; D012235:Rhodamines; D015003:Yeasts / Q000378:metabolism
chemical_list: D001578:Benzopyrans; D005456:Fluorescent Dyes; D009284:Naphthols; D011522:Protons; D012235:Rhodamines; C068301:carboxy-seminaphthorhodaminefluoride