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

PLoS One. 2016 Aug 24;11(8):e0161353. doi: 10.1371/journal.pone.0161353. eCollection 2016.

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.

MeSH terms

  • Algorithms
  • Benzopyrans*
  • Biosensing Techniques
  • Fluorescent Dyes
  • Hydrogen-Ion Concentration*
  • Mitochondria / metabolism*
  • Naphthols*
  • Protons*
  • Rhodamines*
  • Yeasts / metabolism

Substances

  • Benzopyrans
  • Fluorescent Dyes
  • Naphthols
  • Protons
  • Rhodamines
  • carboxy-seminaphthorhodaminefluoride

Grants and funding

The work was supported by Narodowe Centrum Nauki (National Science Center of Poland) grants: UMO-2013/11/B/NZ1/02102 to RK, UMO-2012/07/E/NZ1/01894 to AK and UMO-2011/01/B/ST4/06144 to WB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.