Revisiting Mitochondrial pH with anudImproved Algorithm for Calibration of theudRatiometric 5(6)-carboxy-SNARF-1 ProbeudReveals Anticooperative Reaction with H+udIons and Warrants Further Studies ofudOrganellar pH

摘要

Fluorescence measurements of pH and other analytes in the cell rely on accurate calibrations,udbut these have routinely used algorithms that inadequately describe the properties ofudindicators. Here, we have established a more accurate method for calibrating and analyzinguddata obtained using the ratiometric probe 5(6)-carboxy-SNARF-1. We tested the implicationsudof novel approach to measurements of pH in yeast mitochondria, a compartment containinguda small number of free H+ ions. Our findings demonstrate that 5(6)-carboxy-SNARF-ud1 interacts with H+ ions inside the mitochondria in an anticooperative manner (Hill coefficientudn of 0.5) and the apparent pH inside the mitochondria is ~0.5 unit lower than had been generallyudassumed. This result, at odds with the current consensus on the mechanism ofudenergy generation in the mitochondria, is in better agreement with theoretical considerationsudand warrants further studies of organellar pH.