Presentation on theme: "3-D reconstructions based on electron micrographs of isolated mitochondria taken with a large depth of field, at different tilt angles have indicated."— Presentation transcript:
3-D reconstructions based on electron micrographs of isolated mitochondria taken with a large depth of field, at different tilt angles have indicated that the infoldings of the inner mitochondrial membrane are actually variable in shape and are connected to the periphery and to each other by narrow tubular regions. A conventional view of mitochondrial structure is represented here.
Respiratory control ratio is the ratio of slopes after ADP addition and after ADP is all converted to ATP (b/a) P/O ratio is the moles of ADP divided by the moles of O consumed (based on c ) while phosphorylating the ADP. [O 2 ] time ADP added ADP all converted to ATP a b c State 3 State 4
An oxygen electrode (electrochemical sensor for oxygen) may be used to record the [O 2 ] in a closed vessel
Used for measuring the partial pressure of oxygen (sometimes referred to as ‘oxygen tension’) in the gas phase or, more commonly, dissolved solution. Applications: 1.Environmental studies (e.g. O 2 -levels in natural waters). 2.Sewage treatment (vital in monitoring the progress of bacterial attack). 3.Alcohol production (O 2 -levels in fermentation tasks need to be continuously monitored and controlled). 4.Medicine (invasive and non-invasive monitoring of a key physiological analyte).
Range of detection of O 2 : 10 -4 atm (i.e. 0.01%) – 1 atm (i.e. 100%). The key to continuing supremacy: a gas-permeable, ion-impermeable, membrane that separates the test system from the sensing electrode (the platinum cathode). No electrode passivation or poisoning that arise when the sensing electrode is placed in direct contact with the system (usually an aqueous solution) under test.
central platinum disc working electrode (cathode: O 2 diffusing through the membrane is reduced) The oxygen electrode cell silver ring counter and reference electrode (anode) Conduction: 3M potassium chloride solution Controller: apply a voltage to the platinum electrode that is sufficiently negative, with respect to the silver electrode, to reduce oxygen. Resultant current is proportional to P(O 2 ) voltage display in units of percentage saturation,to chart recorder or data logger.
Principles of Operation At platinum electrode:O 2 + 2 H 2 O + 4 e 4 OH - (1) At silver electrode:4 Ag + 4 Cl - -4 e AgCl (2) Overall electrochemical process: 4 Ag + O 2 + 2 H 2 O + 4 Cl - 4 AgCl + 4 OH - (3) The current, i d is related to the P(O 2 ): i d = 4·F·P m ·A·P(O 2 ) / b (4) F = Faraday’s constant (9.64 x 10 4 C.mol -1 ) P m =O 2 permeability of the Teflon membrane (typically 1.05 x 10 - 12 mol.atm -1.s -1 ) A = surface area of the Pt working electrode b = thickness of the Teflon membrane (typically 12.5 x 10 -4 cm) In air-saturated water, P(O 2 ) = 0.2 atm, the oxygen electrode would have a value for i d of 2 A approximately.
Calibrating the Oxygen Electrode The voltage (S) from the controller is proportional to the P(O 2 ) in the medium under test: S = K·P(O 2 ) K = proportionality constant. For 100% air saturated water: P(O 2 ) = 0.2095, atm 159 mmHg [O 2 ] = 2.4 x 10 -4 mol.dm -3 8 ppm 8 mg.dm -3
Oxygen Consumption of the Electrode The oxygen electrode consumes oxygen from the test medium reaction (see Equation 3). P(O 2 ) at the interface between the membrane and the test medium should be the same as that in the bulk of the medium continuous stirring. Problems with small volumes. Low volume solutions of high ionic strength (the actual concentration of O 2 in solution decreases with increasing salinity).
Temperature Sensitivity of the Oxygen Electrode The oxygen electrode is temperature sensitive and should be thermostatted whenever this is possible. No thermostatting: the signal is likely to increase and/or fluctuate with increasing or changing temperature
[O 2 ] time ADP added ADP all converted to ATP State 3 State 4 State 3 uncoupled Pre-state 4 Uncoupler added Succinated added State 4: coupled respiration (proton leak through inner membrane) State 3: respiration rate of phosphorylating mitochondria (substrate transporters, electron transport chain and phosphorylation system) State 3 uncoupled: uncoupled respiration (electron transport chain and substrate transporters)
475 nmoles O x 3 ml nmoles O/ml/mg protein = ---------------------------- ___ mm x 6 mg protein Fattore moltiplicativo (Fm) Fm x mm relativi al consumo di O 2 = v di consumo di O 2 (nmoles O/min/mg protein)