Calorimetric Studies of Heat of Respiration of Mitochondria

Abstract

The construction of an adiabatic microcalorimeter using a rapid-response microthermistor (t1/2 = 50 ms) as a detector is described. The apparatus uses an air-tight 5 ml Dewar flask as the reaction cell, and permits simultaneous recording of temperature change and O2 consumption in a reaction medium at a full-scale sensitivity of < 0.01.degree. and stability of < 0.0003.degree./60 s in the measurement of temperature change. Reactant solution could be added to the reaction medium in the cell at any time during the course of a reaction. The apparatus was calibrated by means of the reaction of HCl and NaOH, and those catalyzed by peroxidase and glucose oxidase. Time courses of heat production and O2 consumption by rat liver mitochondria in various states of respiration with succinate or glutamate as a substrate were recorded. In respiration uncoupled by 2,4-dinitrophenol (DNP), the apparent enthalpy change per gram atom of O2 consumed (.DELTA.Ha) was in agreement with the heat of combustion of these substrates. In state 4 respiration .DELTA.Ha was reduced to 54 or 51% of that in DNP-uncoupled respiration with succinate or glutamate as a substrate, respectively. In state 3 respiration .DELTA.Ha was further reduced. On adding DNP to mitochondria after exhaustion of O2, heat production was observed in anaerobiosis which was quantitatively in agreement with that expected from the reaction of hydrolysis of ATP, which had been formed in the preceding state 3 respiration, caused by DNP-stimulated ATPase activity of mitochondria.