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Electrometric monitoring of ferricyanide reduction in respiration and photosynthesis
ANALYTICAL BIOCHEIVIISTRY 3 2 ,
396--401 (1969)
Electrornetric Monitoring of Ferricyanide Reduction in Respiration and Photosynthesis~ A. H. CASWELL 2 A N n B. C. PRESSMAN ~ Department o] Biophysics and Physical Biochemistry, Johnson Research Foundation, University o] Pennsylvania, Philadelphia, Pennsylvania 19104
Received June 9, 1969 A range of redox reagents is able to mediate electron flow at intermediate stages of the electron transport chain of mitochondria or chloroplasts. One of the most extensively used of such reagents is the ferricyanide/ferrocyanide couple, since it is able to operate at substrate concentrations with little deleterious effect on the organelle studied. The monitoring of redox changes of this couple as electrons are transferred to or from the reagent has been carried out by a number of techniques. The pH change associat.ed with ferricyanide reduction and oxygen evolution in chloroplasts has served as a measure of the rate of ferricyanide reduction in the Hill reaction (1). This method, however, lacks specificity, since there are several sources of pH change occurring in photosynthetic and respiratory reactions. Spikes, Lumry, Eyring, and Wayrynen (2) have employed potentiometric measurements to monitor changes in the redox potential of ferricyanide/ferroeyanide as ferricyanide is reduced in the Hill reaction. Whittaker and Redfearn (3) extended potentiometric studies to monitor ferricyanide reduction in submitoehondrial particles. McCarty and Jagendorf (4) have employed potentiometric measurements to observation of ferricyanide reduction in chloroplasts using a pH stag to hold the potential constant as ferricyanide is added to the medium to balance the ferricyanide converted to ferrocyanide in the course of reaction. Pressman (5) observed ferricyanide reduction in mitochondria spectrophotometrically through the disappearance of the ferricyanide absorption band at 420 mu and Estabrook (6) extended the technique using continuous measurements with a double-beam spectrophotometer. Polarography appears not to have been employed in measurements of ferricyanide reduction either in photosynthesis or in 1Supported by U. S. Public Health Service Grants GM 12202, 571-GM-277, and K3-GM-3626. Present address: Pa,panieolaou Cancer Research Institute, 1155 N. W. 14 Street, Miami, Florida 33136. 396
MONITORING FERRICYANIDE REDUCTION
397
respiration, although it has been used to follow the reduction of viologen dyes by chloroplasts illuminated with a modulated light source (7). Electrometric techniques have been employed in following changes of redox potential of intermediates in electron transfer processes through addition of catalytic concentrations of redox mediators (8-10). The aim of this paper is to examine the merits and disadvantages of eleetrometrie methods of following reduction of substrate levels of ferrieyanide in relation to other techniques. METHODS
Mitochondria were prepared by the method of Schneider (11). Chloroplasts were prepared by the method of Izawa and Hind (12) and suspended in Trieine buffer, pH 8.2. The polarographic or potentiometric measurements were carried out using a vibrating platinum electrode soldered to the reed of a Brown Instruments Co. (Philadelphia) converter (9). The potentiometrie method depends upon the direct measuremerit of the potential generated from the ferroeyanide/ferrieyanide couple compared with the potential of a calomel half-cell according to the following equation: RT E = E.~ - - - 7
In
[ferrocyanide] [ferrieyanide] -- Ec~i
where E~ is the midpoint potential of the couple under the particular experimental conditions and Ec~l is the redox potential of the calomel halfcell. The bare platinum electrode equilibrates the electrical potential with the redox potential in solution. In order to record the potential, the outputs from the two half-cells are fed into the input of a pH meter connected to a potentiometric recorder. More recently a simple amplifier circuit was developed based on a FET a input operational amplifier (Analog Devices lZi2B, Cambridge, Mass.) which can drive a galvanometric recorder directly from the electrodes employed. The response of the electrode is most satisfactory if a reasonably large platinum surface is made available for reaction such as is provided by a spiral of platinum wire of diameter 0.36 mm X 15 mm length. Polarographic measurements depend on the flow of current when a potential is applied across a platinum electrode/calomel electrode pair. At a constant anodic potential at the pla~_.inum electrode, the current flowing is proportional to the concentration of the reduced species and the surface area of the exposed electrode. If the platinum electrode is made cathodic, then the electrode will respond to dissolved oxygen and FET, Field Effect Transistor,
398
C'ASWELL AND P R E S S h [ A N
so all measurements are made with the electrode positive with respect to the calomel and the concentration of the reduced species is followed. The electrode surface area should be chosen so as to give reasonable sensitivity but should not be so great t h a t the impedance at the platinum interface becomes comparable with t h a t through the solution. For the present experiments a spiral of platinum wire similar to t h a t used in potentiometrie measurements proved ideal. The current flow through the electrodes is amplified using a current-voltage converter containing a F E T input operational amplifier (Analog Devices 142B) and recorded directly. Mitochondria ~
Mdochondria
I~
0501:3
I00,5o-
E 200u "E 250::L5 0 0
(2D) ' ~ p
.
. ~
ADP
-
.350400-
FIG. 1. Potentiometric recording at 22° of ferricyanide reduction in mitoehondria when glutamate and malate (A) and suceinate (B) serve as substrates. Different mitoehondrial preparations were used for each trace. Incubation medium: 5 mM KCI; 5 mM Tris phosphate; 1 mM MgC12; 250 mM sucrose; 100 ~M NaCN; 500 #M K ÷ ferroeyanide; 500 /,M K + ferrieyanide; final pit 7.4. Additions to the medium: (A) Tris glutamate, 6 mM; Tris malate, 6 mM; mitoehondria, 1.5 mg protein/ml; ADP, 6:0 #M; antimycin A, 0.1 #g/ml. (B) Tris sueeinate, 6 mM; rotenone, 0.1 #g/ml; mitoehondria, 0.7 mg protein/ml; ADP, 60 #M. Figures in parentheses are the P :2:e ratios. RESULTS Figure 1 illustrates the potentiometric recordings obtained in mitochondria as ferricyanide is reduced through oxidation of metabolic substrates when oxygen reduction is inhibited by means of the terminal inhibitor, cyanide. The initial medium contained equal concentrations of ferrocyanide and ferrieyanide in order to provide substantial redox poising of the system. The ferroeyanide formed was calibrated from the equations of redox potential described above. The trace shows the increase in respiration associated with active electron transfer when added A D P
MONITORING FERRICYANIDE REDUCTION
399
is being phosphorylated by the mitochondria. The trace reverts to the base respiratory rate when the A D P is exhausted. Respiratory control, with ferricyanide as electron acceptor, is shown when either NAD-linked reagents or succinate are substrates, but the control with succinate as substrate is not as pronounced. For both substrates the respiratory control is less than that obtained when oxygen is electron acceptor. P : 2 e ratios for NAD-linked substrates are about 2 and for suceinate about 1 in accord with the findings of Pressman (5) and Estabrook (6). The potentiometric trace is unaffected by the presence of the terminal inhibitor, cyanide, which interferes with polarographic measurements. The traces are impressively free of noise and addition artifacts are not apparent; the ultimate sensitivity appears to exceed that of optical methods.
Fep~hl+h~ O-5
~AD~P
FCCP
0.5-
"7(3 (3
c_ 1.0. LL
E
1.5
~q~IG.2. Polarographic recording at 20° of ferricyanide reduction in chloroplasts. Incubation medium: 40 mM trishydroxylmethylglycine (Trieine); 5 mM MgC12; 5 mM K + phosphate; 200 mM sucrose; final pII 8.2. Additions to the medium: K + ferricyanide (FeCy), 1.0 mM; chloroplasts (Chl), 67 ~g chlorophyll/ml; ADP, 80 /xM, p-trifluoromethoxy (carbonyl cyanide) phenylhydrazone (FCCP), 12 #M; 2-n-heptyl-4-hydroxylquinoline N-oxide (HOQNO), 25 ~M. Anodie potential at platinum electrode 0.4 V. -}-h, is illumination; --h, is darkness. The response time to changes in the rate of electron transport is of the order of 1-4 seconds. The disadvantage of potentiometric measurements is t h a t the trace is not a linear function of the reduction rate. The potentiostat technique employed by M e C a r t y and Jagendorf (4) is able to give a linear trace; this is obtained at the expense of a varying concentration of the redox couple in the medium, but a constant redox potential of the couple. In potentiometrie measurements, in distinction to optical measurements, there is no possible source of interference from light scattering changes of the mitochondria or absorbanee changes of mitoehondrial ehromophores.
400
CASWELL AND PRESSMAN
Figure 2 illustrates polarographic measurements of photosynthetic ferricyanide reduction accompanying oxygen evolution in the Hill reaction of chloroplasts. A slight artifact or slight reduction of ferricyanide is associated with chloroplast addition. Illumination initiates reduction of ferricyanide, which ceases immediately upon darkness. Addition of ADP causes the increase in reduction rate associated with the phenomenon of electron transport control exerted by the energy conservation reaction. Upon exhaustion of ADP, the reduction reverts to the base electron transport rate, which can be stimulated by the uncoupler FCCP. Cessation of reduction is then associated with complete utilization of ferricyanide. The inhibitory effect of HOQN0 is also illustrated. This polarographic trace was calibrated by observing the reduction of successive aliquots of ferricyanide and is linear with ferrocyanide concentration. The response is very rapid and the measurement is almost free of noise. Interference is produced only by other redox reagents that interact at the anodic platinum electrode and by electrode poisons such as cyanide. The linearity of the trace renders polarographic measurement preferable in this instance to potentiometric recordings. SUMMARY
Electrolnetric methods of monitoring ferricyanide reduction have the advantage over spectroscopic observation in that they are free from sources of interference such as variable absorbance or light scattering of particulate suspensions. Simple, inexpensive, and readily available apparatus is employed for the measurements. The choice of potentiometric or polarographic measurement depends on the requirement. Potentiometric measurements are free of interference from reagents such as cyanide and can be made extremely sensitive, but the trace is nonlinear and response is limited. Polarographic measurements give linear traces and immediate response, but are affected by electrode poisons. The use of the two methods is illustrated in electron transport in mitochondria and chloroplasts. REFERENCES 1. A. S. HOLT A~D C. S. F~ElVCH, Arch. Biochem., 9 (1946) 25. 2. J. D. SPIKES, ]~. LUMRY, H. EYRING, AND R. E. WAYRYiNEN, Arch. Biochem., 28 (1950) 48. 3. P. A. WHITTAKER AND E. R. REDFE~¢Rh', Biochim. Biophys. Acta, 131 (1967) 234. 4. R. E. MCCART¥ AND A. T. JAGENDORF, Plant Physiol., 40 (1965) 725. 5. B. C. PRESS~A-W, Biochim. Biophys. Acta, 17 (1955) 273. 6. R. W. EST~BROOK, d. B~ol. Chem., 236 (1961) 3051. 7. P. JOLIOT AND A. JOLIOT, Biochim. Biophys. Acta, 153 (1968) 625. 8. J. P. BAUMBERGER, Cold Spring Harbor Symp. Quant. Biol., 7 (1939) 195.
MONITORING
9. 1O. 11. 12.
FERRICYANIDE
REDUCTION
401
A. It. CASWELL AND B. C. PRESSHAN,Arch. Biochem. Biophys., 125 (1968) 318. A. H. CASWELL, J. Biol. Chem., 243 (1968) 5827. W. C. SCHNEIDER,J. Biol. Chem., 176 (1948) 259. S. IZAWA AND G. HIND, Biochim. Biophys. Acta, 143 (1967) 377.
396--401 (1969)
Electrornetric Monitoring of Ferricyanide Reduction in Respiration and Photosynthesis~ A. H. CASWELL 2 A N n B. C. PRESSMAN ~ Department o] Biophysics and Physical Biochemistry, Johnson Research Foundation, University o] Pennsylvania, Philadelphia, Pennsylvania 19104
Received June 9, 1969 A range of redox reagents is able to mediate electron flow at intermediate stages of the electron transport chain of mitochondria or chloroplasts. One of the most extensively used of such reagents is the ferricyanide/ferrocyanide couple, since it is able to operate at substrate concentrations with little deleterious effect on the organelle studied. The monitoring of redox changes of this couple as electrons are transferred to or from the reagent has been carried out by a number of techniques. The pH change associat.ed with ferricyanide reduction and oxygen evolution in chloroplasts has served as a measure of the rate of ferricyanide reduction in the Hill reaction (1). This method, however, lacks specificity, since there are several sources of pH change occurring in photosynthetic and respiratory reactions. Spikes, Lumry, Eyring, and Wayrynen (2) have employed potentiometric measurements to monitor changes in the redox potential of ferricyanide/ferroeyanide as ferricyanide is reduced in the Hill reaction. Whittaker and Redfearn (3) extended potentiometric studies to monitor ferricyanide reduction in submitoehondrial particles. McCarty and Jagendorf (4) have employed potentiometric measurements to observation of ferricyanide reduction in chloroplasts using a pH stag to hold the potential constant as ferricyanide is added to the medium to balance the ferricyanide converted to ferrocyanide in the course of reaction. Pressman (5) observed ferricyanide reduction in mitochondria spectrophotometrically through the disappearance of the ferricyanide absorption band at 420 mu and Estabrook (6) extended the technique using continuous measurements with a double-beam spectrophotometer. Polarography appears not to have been employed in measurements of ferricyanide reduction either in photosynthesis or in 1Supported by U. S. Public Health Service Grants GM 12202, 571-GM-277, and K3-GM-3626. Present address: Pa,panieolaou Cancer Research Institute, 1155 N. W. 14 Street, Miami, Florida 33136. 396
MONITORING FERRICYANIDE REDUCTION
397
respiration, although it has been used to follow the reduction of viologen dyes by chloroplasts illuminated with a modulated light source (7). Electrometric techniques have been employed in following changes of redox potential of intermediates in electron transfer processes through addition of catalytic concentrations of redox mediators (8-10). The aim of this paper is to examine the merits and disadvantages of eleetrometrie methods of following reduction of substrate levels of ferrieyanide in relation to other techniques. METHODS
Mitochondria were prepared by the method of Schneider (11). Chloroplasts were prepared by the method of Izawa and Hind (12) and suspended in Trieine buffer, pH 8.2. The polarographic or potentiometric measurements were carried out using a vibrating platinum electrode soldered to the reed of a Brown Instruments Co. (Philadelphia) converter (9). The potentiometrie method depends upon the direct measuremerit of the potential generated from the ferroeyanide/ferrieyanide couple compared with the potential of a calomel half-cell according to the following equation: RT E = E.~ - - - 7
In
[ferrocyanide] [ferrieyanide] -- Ec~i
where E~ is the midpoint potential of the couple under the particular experimental conditions and Ec~l is the redox potential of the calomel halfcell. The bare platinum electrode equilibrates the electrical potential with the redox potential in solution. In order to record the potential, the outputs from the two half-cells are fed into the input of a pH meter connected to a potentiometric recorder. More recently a simple amplifier circuit was developed based on a FET a input operational amplifier (Analog Devices lZi2B, Cambridge, Mass.) which can drive a galvanometric recorder directly from the electrodes employed. The response of the electrode is most satisfactory if a reasonably large platinum surface is made available for reaction such as is provided by a spiral of platinum wire of diameter 0.36 mm X 15 mm length. Polarographic measurements depend on the flow of current when a potential is applied across a platinum electrode/calomel electrode pair. At a constant anodic potential at the pla~_.inum electrode, the current flowing is proportional to the concentration of the reduced species and the surface area of the exposed electrode. If the platinum electrode is made cathodic, then the electrode will respond to dissolved oxygen and FET, Field Effect Transistor,
398
C'ASWELL AND P R E S S h [ A N
so all measurements are made with the electrode positive with respect to the calomel and the concentration of the reduced species is followed. The electrode surface area should be chosen so as to give reasonable sensitivity but should not be so great t h a t the impedance at the platinum interface becomes comparable with t h a t through the solution. For the present experiments a spiral of platinum wire similar to t h a t used in potentiometrie measurements proved ideal. The current flow through the electrodes is amplified using a current-voltage converter containing a F E T input operational amplifier (Analog Devices 142B) and recorded directly. Mitochondria ~
Mdochondria
I~
0501:3
I00,5o-
E 200u "E 250::L5 0 0
(2D) ' ~ p
.
. ~
ADP
-
.350400-
FIG. 1. Potentiometric recording at 22° of ferricyanide reduction in mitoehondria when glutamate and malate (A) and suceinate (B) serve as substrates. Different mitoehondrial preparations were used for each trace. Incubation medium: 5 mM KCI; 5 mM Tris phosphate; 1 mM MgC12; 250 mM sucrose; 100 ~M NaCN; 500 #M K ÷ ferroeyanide; 500 /,M K + ferrieyanide; final pit 7.4. Additions to the medium: (A) Tris glutamate, 6 mM; Tris malate, 6 mM; mitoehondria, 1.5 mg protein/ml; ADP, 6:0 #M; antimycin A, 0.1 #g/ml. (B) Tris sueeinate, 6 mM; rotenone, 0.1 #g/ml; mitoehondria, 0.7 mg protein/ml; ADP, 60 #M. Figures in parentheses are the P :2:e ratios. RESULTS Figure 1 illustrates the potentiometric recordings obtained in mitochondria as ferricyanide is reduced through oxidation of metabolic substrates when oxygen reduction is inhibited by means of the terminal inhibitor, cyanide. The initial medium contained equal concentrations of ferrocyanide and ferrieyanide in order to provide substantial redox poising of the system. The ferroeyanide formed was calibrated from the equations of redox potential described above. The trace shows the increase in respiration associated with active electron transfer when added A D P
MONITORING FERRICYANIDE REDUCTION
399
is being phosphorylated by the mitochondria. The trace reverts to the base respiratory rate when the A D P is exhausted. Respiratory control, with ferricyanide as electron acceptor, is shown when either NAD-linked reagents or succinate are substrates, but the control with succinate as substrate is not as pronounced. For both substrates the respiratory control is less than that obtained when oxygen is electron acceptor. P : 2 e ratios for NAD-linked substrates are about 2 and for suceinate about 1 in accord with the findings of Pressman (5) and Estabrook (6). The potentiometric trace is unaffected by the presence of the terminal inhibitor, cyanide, which interferes with polarographic measurements. The traces are impressively free of noise and addition artifacts are not apparent; the ultimate sensitivity appears to exceed that of optical methods.
Fep~hl+h~ O-5
~AD~P
FCCP
0.5-
"7(3 (3
c_ 1.0. LL
E
1.5
~q~IG.2. Polarographic recording at 20° of ferricyanide reduction in chloroplasts. Incubation medium: 40 mM trishydroxylmethylglycine (Trieine); 5 mM MgC12; 5 mM K + phosphate; 200 mM sucrose; final pII 8.2. Additions to the medium: K + ferricyanide (FeCy), 1.0 mM; chloroplasts (Chl), 67 ~g chlorophyll/ml; ADP, 80 /xM, p-trifluoromethoxy (carbonyl cyanide) phenylhydrazone (FCCP), 12 #M; 2-n-heptyl-4-hydroxylquinoline N-oxide (HOQNO), 25 ~M. Anodie potential at platinum electrode 0.4 V. -}-h, is illumination; --h, is darkness. The response time to changes in the rate of electron transport is of the order of 1-4 seconds. The disadvantage of potentiometric measurements is t h a t the trace is not a linear function of the reduction rate. The potentiostat technique employed by M e C a r t y and Jagendorf (4) is able to give a linear trace; this is obtained at the expense of a varying concentration of the redox couple in the medium, but a constant redox potential of the couple. In potentiometrie measurements, in distinction to optical measurements, there is no possible source of interference from light scattering changes of the mitochondria or absorbanee changes of mitoehondrial ehromophores.
400
CASWELL AND PRESSMAN
Figure 2 illustrates polarographic measurements of photosynthetic ferricyanide reduction accompanying oxygen evolution in the Hill reaction of chloroplasts. A slight artifact or slight reduction of ferricyanide is associated with chloroplast addition. Illumination initiates reduction of ferricyanide, which ceases immediately upon darkness. Addition of ADP causes the increase in reduction rate associated with the phenomenon of electron transport control exerted by the energy conservation reaction. Upon exhaustion of ADP, the reduction reverts to the base electron transport rate, which can be stimulated by the uncoupler FCCP. Cessation of reduction is then associated with complete utilization of ferricyanide. The inhibitory effect of HOQN0 is also illustrated. This polarographic trace was calibrated by observing the reduction of successive aliquots of ferricyanide and is linear with ferrocyanide concentration. The response is very rapid and the measurement is almost free of noise. Interference is produced only by other redox reagents that interact at the anodic platinum electrode and by electrode poisons such as cyanide. The linearity of the trace renders polarographic measurement preferable in this instance to potentiometric recordings. SUMMARY
Electrolnetric methods of monitoring ferricyanide reduction have the advantage over spectroscopic observation in that they are free from sources of interference such as variable absorbance or light scattering of particulate suspensions. Simple, inexpensive, and readily available apparatus is employed for the measurements. The choice of potentiometric or polarographic measurement depends on the requirement. Potentiometric measurements are free of interference from reagents such as cyanide and can be made extremely sensitive, but the trace is nonlinear and response is limited. Polarographic measurements give linear traces and immediate response, but are affected by electrode poisons. The use of the two methods is illustrated in electron transport in mitochondria and chloroplasts. REFERENCES 1. A. S. HOLT A~D C. S. F~ElVCH, Arch. Biochem., 9 (1946) 25. 2. J. D. SPIKES, ]~. LUMRY, H. EYRING, AND R. E. WAYRYiNEN, Arch. Biochem., 28 (1950) 48. 3. P. A. WHITTAKER AND E. R. REDFE~¢Rh', Biochim. Biophys. Acta, 131 (1967) 234. 4. R. E. MCCART¥ AND A. T. JAGENDORF, Plant Physiol., 40 (1965) 725. 5. B. C. PRESS~A-W, Biochim. Biophys. Acta, 17 (1955) 273. 6. R. W. EST~BROOK, d. B~ol. Chem., 236 (1961) 3051. 7. P. JOLIOT AND A. JOLIOT, Biochim. Biophys. Acta, 153 (1968) 625. 8. J. P. BAUMBERGER, Cold Spring Harbor Symp. Quant. Biol., 7 (1939) 195.
MONITORING
9. 1O. 11. 12.
FERRICYANIDE
REDUCTION
401
A. It. CASWELL AND B. C. PRESSHAN,Arch. Biochem. Biophys., 125 (1968) 318. A. H. CASWELL, J. Biol. Chem., 243 (1968) 5827. W. C. SCHNEIDER,J. Biol. Chem., 176 (1948) 259. S. IZAWA AND G. HIND, Biochim. Biophys. Acta, 143 (1967) 377.