The calibration and use of a calcium ion-specific electrode for kinetic studies of mitochondrial calcium transport
Yamazaki, R.K.; Mickey, D.L.; Story, M.
Analytical Biochemistry 93(2): 430-441
1979
ISSN/ISBN: 0003-2697 PMID: 464270 Document Number: 154180
A Ca ion-specific electrode was used to study Ca transport by isolated hepatic mitochondria. The methodology requires only a sensitive pH meter operated in the millivolt mode with the electrode. Free Ca ion concentrations may be followed continuously. Using incubation conditions which cause release of intramitochondrial Ca, the Ca electrode system may also be used to determine total intramitochondrial Ca. Techniques for the calibration of the electrode response are discussed. Free Ca ion concentrations were calculated from total Ca concentrations and the association constants for the binding species present in the assay medium. The observation that the electrode response is linear to submicromolar concentrations allows calculation of a linear least-squares fit of mV reading to computed free Ca ion concentration. A computer program written in basic for these computations is included in Appendix material. The half-maximal rate constant for mitochondrial Ca uptake occurred at a free Ca ion concentration of 6.5 .mu.M. The interaction or Hill coefficient for the process is 2.3, indicating positive cooperativity.