J. Membrane Biol. 46, 255-282 (1979)
Kinetics of the Potential-Sensitive Extrinsic Probe Oxonol VI in Beef Heart Submitochondrial Particles J.C. Smith and Britton Chance Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Received 6 November 1978
Summary. The interaction of the potential-sensitive extrinsic probe oxonol VI with beef heart submitochondrial particles has been investigated under time resolved and equilibrium conditions. The time course of the probe absorption spectrum red shift induced by ATP or NADH injection into a suspension of submitochondrial particles in a dye solution is biphasic, consisting of a faster process described by a second-order rate law with k2~3 x 105 M-1 sec -1. For the ATP pulse experiments, the slower process follows first-order kinetics with kl ~0.3 sec -*. In oxygen pulse experiments to an anaerobic dyeparticle system, the slower process is not significantly developed due to rapid depletion of the oxygen, but the faster process follows second-order kinetics with the same rate constant as for the ATP and NADH cases. Evidence for permeation of the submitochondrial particle membrane by oxonol VI has been obtained; the slower process is interpretable as describing the permeation of the membrane bilayer. The results of the time-resolved work are consistent with a mechanism involving a redistribution of the dye from the bulk phase to the particle membrane. The value of the second-order rate constant for passive binding of the dye to submitochondrial particles is not compatible with a mechanism proposed to explain the microsecond probe response times in bilayer and excitable membrane experiments nor are such rapid signals observed in the oxonol VI-submitochondrial particle system.
The use of extrinsic probes in the study of biological membrane phenomena has received considerable impetus from the discovery of several related classes of dyes that are potential sensitive. These probes usually fall into the cyanine, merocyanine, or oxonol class of dyes. Dyes of the preceding three types consist of two ring structures joined by a conjugated carbon chain. Since the wavelength of maximum absorption increases with the length of the carbon chain, it has been possible to synthesize probes the spectral absorption and emission spectra of which do not significantly overlap the principle bands of intrinsic pigments of biological membranes. The visible absorption band of these dyes is
0022-2631/79/0046-0255 $05.60 9 Springer-Verlag New York Inc. 1979
256
J.C. Smith and B. Chance
usually quite intense, enabling the use of micromolar concentrations of these probes in membrane studies, thereby minimally perturbing the system under investigation. Most but not all of these probes are fluorescent which, in addition to providing additional sensitivity to conventional membrane suspension work, makes possible work on nontransmitting samples, such as intact organs by the use of surface fluorimetry (Chance, Mayevsky & Smith, 1976). It is often possible to markedly alter the magnitude of the spectral responses of these probes to membrane energization by modifying substituent groups of the molecule which are not part of the optical chromophore but which control such factors as charge and membrane binding affinity. Using the giant axon from the squid, Loligo peali, Cohen et al. (1974) and Ross et al. (1977) have screened a large number of dyes and have identified a number of promising potential-sensitive probes which have been systematically improved based on the criterion of increased signal to noise ratio in experiments involving the application of potential gradients across the axon membrane by means of microelectrodes. In a number of cases it has been possible to calibrate the spectral response of certain cyanine dyes such as diS-C3-5 using valinomycininduced diffusion potentials in the system under investigation. Using this approach, Laris, Bahr and Chaffee (1975) have measured the membrane potential in mitochondria, Hoffman and Laris (1974) and Sims et al. (1974) have measured the resting potential in the red cell, and Renthal and Lanyi (1976) have obtained the potential in Halobacterium halobium. Reviews on the design and use of extrinsic probes of membrane potential in a number of systems have appeared by Waggoner (1976) and by Cohen and Salzberg (1978). Waggoner, Wang and Tolles (1977) have also investigated the mechanism of the rapid response of certain cyanines and oxonols to a train of voltage pulses applied across a black lipid membrane. The dye merocyanine 540 has been employed by Salama and Morad (1976) as a probe of the action potential in the heart. Studies (Chance et al., 1974; Chance & Baltscheffsky, 1975) in our laboratory have been focused on a homologous series of oxonol dyes derived from the oxonol V probe, the structural, spectral properties, and energy-linked spectral changes of which have been described by Smith et al. (1976). Transient increases in the probe response are induced by agents such as NH3 (Smith & Chance, 1976) and nigericin (Bashford & Thayer, 1977) which are known to abolish the ApH component of the electrochemical gradient. These findings indicate that these oxonols
257
R
R
OX-Vr, R= CHaCH2CH3 Fig. l. The structure of the neutral form of oxonol VI. At pH values used in this work, the dye is a symmetrical anion since the pK of the hydroxyl proton is approximately 4 are sensitive to AT' only, which is increased as ApH is abolished in order to maintain the electrochemical gradient constant. The derivative of oxonol V in which the phenyl groups bound to the isoxazolone rings have been replaced by propyl sidechains, oxonol VI 1, has proved to give much larger spectral shifts than any of the other members of our series in membrane preparations, such as photosynthetic bacteria chromatophores and submitochondrial particles in which the internal volume of the vesicle is positive with respect to the external phase. Oxonol VI has been used by Bashford and Thayer (1977) in a double probe experiment in which 9-aminoacridine was used as the probe of ApH, to obtain the value of the electrochemical gradient under equilibrium or steady-state conditions in submitochondrial particles. In this communication, the kinetics of oxonol VI spectral changes in submitochondrial particles have been studied, and the implications of the findings for the mechanism by which the energy-linked spectral changes occur in this system are considered. The structure of the neutral form of oxonol VI is shown in Fig. 1. It should be borne in mind, however, that the dye is a symmetrical anion at physiological pH since the pK for the dissociation of the hydroxyl proton is approximately four (Smith et al., 1976), and the resulting charge is delocalized over the conjugated system of the molecule. Materials and Methods
Submitochondrial particles were prepared by the procedure described by Hansen and Smith (1964) which is only briefly outlined here. Heavy beef heart mitochondria were 1 Abbreviations used. ANS: 1-analino-8-naphthalene sulfonate; ATP: adenosine 5'triphospbate, disodium salt, from equine muscle; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; HEPES : N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid; NADH:/Lnicotinamideadeninedinucleotide; Oxonol VI: bis [3-propyl-5-oxoisoxazol-4-yl]pentamethineoxonol.
258
J.C. Smith and B. Chance
suspended at approximately 20 mg/ml protein in an incubation mixture consisting of 0.25 M sucrose, 10mM K-HEPES at pH 7.5, l mM MgC12, 1 mM ATP, and 1 mM succinate. The suspension was stored frozen overnight and then thawed, and the mitochondria were isolated by centrifugation. The mitochondria were then resuspended at approximately 20 mg/ ml protein in the medium described above with the following exceptions: the MgC12 concentration was increased to 5 raM, and 10 mM MnC12 was included in the mixture. The resulting mitochondrial suspension was sonicated at approximately 50 W by using a model W 185 Heat Systems Ultrasonics, Inc., cell disrupter for 3540 sec. The submitochondrial particles were pelleted by ultracentrifugation of the supernatant obtained fl'om an initial centrifugation to remove mitochondrial debris. The resulting preparation exhibits respiratory control, The oxonol VI dye was synthesized by suitably modifying the procedure described by Smith et al. (1976) for the oxonol V probe. Relevant analytical data supporting the structure shown in Fig. 1 for the dye are given by Bashford et al. (1978b). Sucrose, MgC12, MnC12, and K2SO4 were obtained from J.T. Baker Chemical Co. and were of reagent grade purity. All other compounds were purchased from Sigma Chemical Co. Experimental conditions are described in the appropriate figure captions. Scanned absorption spectrum measurements were made using a Hitachi-Perkin Elmer model 356 spectrophotometer in the split beam mode. Rapid mixing experiments were carred out using a Johnson Foundation Model E (Chance, 1973) rapid mixing device with an 80:1 mixing ratio adapted to a Johnson Foundation 200 Hz time-sharing double beam spectrometer equipped with 500-mm Bausch and Lomb monochromators, The detector output was displayed on a type 564 Tektronix storage oscilloscope and a model 165 Perkin Elmer strip chart recorder as required. See Results for details. The titrations used in equilibrium binding investigations were monitored by a Beckman model UV 5270 spectrometer.
Results and Data Analyses T h e o b s e r v a t i o n o f a red shift in the a b s o r p t i o n s p e c t r u m o f o x o n o l V I w h e n either p a s s i v e b i n d i n g ( B a s h f o r d e t a l . ,
1978b) or s u b s t r a t e
c o n s u m p t i o n by s u b m i t o c h o n d r i a l particles s u s p e n d e d in dye s o l u t i o n o c c u r s (Fig. 2) w a s u s e d as the basis for m o n i t o r i n g the t i m e c o u r s e o f the s p e c t r a l c h a n g e s to be d e s c r i b e d in this section. F l u o r e s c e n c e c h a n g e s c o u l d in p r i n c i p l e h a v e b e e n u s e d for this p u r p o s e ; the sense as well as the m a g n i t u d e o f the e m i s s i o n intensity c h a n g e , h o w e v e r , are f u n c t i o n s o f d y e - t o - p a r t i c l e ratio, w h i c h in the e x p e r i m e n t s to be d e s c r i b e d is a n e x p e r i m e n t a l variable. T h e c h a n g e s in t r a n s m i s s i o n are t h u s m o r e easily i n t e r p r e t e d . T h e a p p a r e n t isosbestic p o i n t at 603 n m (Fig. 2) p r o v i d e d a c o n v e n i e n t reference, while the large c h a n g e at 630 n m w a s used as the m e a s u r i n g p o i n t in the d o u b l e b e a m t r a n s m i s s i o n experiments. T w o basic t y p e s o f r a p i d m i x i n g e x p e r i m e n t s are r e p o r t e d in this c o m m u n i c a t i o n . T h e first a n d b y f a r the s i m p l e r to a n a l y z e is the passive b i n d i n g e x p e r i m e n t in w h i c h the m a j o r syringe o f the r a p i d m i x i n g device c o n t a i n s dye s o l u t i o n m i x e d w i t h s u b m i t o c h o n d r i a l particles via the min o r syringe. T h e t i m e c o u r s e o f the passive b i n d i n g p r o c e s s w a s suffi-
Potential-Sensitive Extrinsic Probes
259
free dye
r
\
SMP
~176
ATP,
006 [ I
005 i
0L
500
550
r 650
600 Mnm)
# ~
700
Fig. 2. The absorption spectrum of oxonol VI as the free dye, as slightly red shifted when the dye is bound to the submitochondrial particle membrane, the large red shift induced by ATP energization of the particles, and the complete reversal of the red shift by addition of the uncoupler CCCP. Medium: 0.25 M sucrose, 5 nan Na-HEPES at pH 7.5; concentrations: 1.2 gM oxonol VI, 0.25 mg/ml ATP-Mg § +-Mn ++ submitochondrial particle protein, 1.7 mM ATP-MgC12, 5 gM CCCP
-/c7 A~Q02
D~
II 2 0 msec Fig. 3. (A): The time course of the red shift measured as the difference in transmission at 603~530 nm caused by passive binding of oxonol VI to submitochondrial particles. Medium: 25 mM K2SO4, 10 mM K-HEPES at pH 7.5, 0.25 u sucrose. Concentrations: 15 gM oxonol VI, 3 gM CCCP, 0.09 mg/ml submitochondrial particle protein; time constant: 3 msec. (B): A diagram illustrating the determination of the apparent first-order rate constant from the oscilloscope trace using Eqs. (1) and (2). In this experiment kapp= 150 s e c - 1 The flow velocity trace is in the upper portion of the diagram, and the lower trace is the time course of the optical signal9 See text for details
c i e n t l y r a p i d t h a t t h e results c o u l d be a n a l y z e d in t e r m s o f the c o n t i n u o u s f l o w case ( C h a n c e , 1973) since the signal r e a c h e d a p l a t e a u b e f o r e t h e f l o w t h r o u g h t h e o b s e r v a t i o n t u b e ceased. A t y p i c a l p h o t o g r a p h o f a n oscilloscope record
of a passive
binding
experiment
is p r e s e n t e d in
260
J.C. Smith and B. Chance
Fig. 3a. T h e a p p a r e n t first-order rate c o n s t a n t kapp is given by (Chance, 1973) k~pp =(2.3/tin) log (Dz/D~). (1) T h e d i s p l a c e m e n t s Da a n d D 2 are defined in Fig. 3b; tm is related to t I which is o b t a i n e d f r o m the flow velocity trace (Fig. 3 b) by
t,. =
(v/v.)
(2)
where V is the v o l u m e f r o m the p o i n t o f o b s e r v a t i o n to that o f mixing a n d V~ is the v o l u m e d i s c h a r g e d in time tf. T h e s e characteristic v o l u m e s m u s t be d e t e r m i n e d for the p a r t i c u l a r mixing device in use. Since the rate c o n s t a n t for the s e c o n d - o r d e r binding process was to be d e t e r m i n e d , the s u b r n i t o c h o n d r i a l particle c o n c e n t r a t i o n was a d j u s t e d such that the dye w o u l d be the r e a c t a n t in excess. U n d e r these c o n d i t i o n s , the a p p a r e n t f i r s t - o r d e r rate c o n s t a n t is r e l a t e d to the s e c o n d - o r d e r rate c o n s t a n t by
(3)
kapp • k2nd ~D
w h e r e Yo is the c o n c e n t r a t i o n o f the r e a c t a n t in excess, which in this case is the o x o n o l VI dye. A plot o f kap p vs. J(n will thus be a straight
25O
2OO _---. ),j
J5o
ioo
50
0
5
I0
15
20
25
OX-VT Concentration(/.zM)
Fig. 4. A plot of the apparent first-order rate constant kapp vs. dye concentration for the passive binding of oxonol VI to submitochondrial particles with the dye in excess. The second-order rate constant is obtained from the slope of the line as described by Eq. (3) in the text. Experimental conditions are given in the caption to Fig. 3a. The value of the second-order rate constant is given in Table 1. Data taken in the absence (e) and presence of 3 gM CCCP (A) fall on the same line, indicating the lack of endogenous substrate in the submitochondrial preparation
Potential-Sensitive Extrinsic Probes
261
line the slope of which gives k2n d. Such a plot for the passive binding experiment in which the submitochondrial particle concentration was held constant and that of the dye varied is presented in Fig. 4. The value of the second-order rate constant is presented in Table 1. In the second type of rapid mixing experiment, the major syringe of the rapid mixing device contained submitochondrial particles suspended in oxonol VI dye solution in which the passive binding process had reached equilibrium. That the latter process was complete could be easily ascertained by simply discharging the contents of the major syringe through the observation tube with the minor syringe empty and the valve connecting the minor syringe to the mixing chamber closed. A fiat line was obtained, indicating that no passive binding was in progress. After equilibrium was reached, various substrates or oxygen were mixed with the submitochondrial particle-oxonol VI suspension via the minor syringe under a number of conditions, some of them of a control nature, to be described subsequently. The time course of the spectral shift caused by substrate or oxygen addition was such that only a negligible optical change occurred during the time of flow, i.e., the stopped flow case was applicable for data analysis. Initial substrate pulse experiments were carried out with N A D H . The time course of the resulting signal for a typical experiment is shown in Fig. 5a. The analysis of these data are illustrated in Fig, 5b. Since on a log scale the data at long time was reasonably linear with time,
Table 1. Summaryof rate constants characterizingthe kinetics of oxonol VI in submitochondrial particles Remarks
Rate constants
Passive binding, dye in excess Oxygen pulse Decay of oxygen pulse-induced dye response NADH, faster process ATP pulse, faster process ATP pulse, slower process ATP pulse, faster process, K2SO4 absent ATP pulse, slower process, K2SO4 absent
9.53(•215 ~sec 1 2.40(_+0.40)x105M ~ sec-1 0.218 (+0.001) sec-1 2.70 (•215 105 M-1 sec 3.10 ( _+0.20) xlOSM lsec-1 0.33 (+0.04) sec 1 2.46 (_+0.20) x 105 M- 1 sec- 1 0.55 (_+0.15) sec
Medium except as noted: 0.25 M sucrose, 25 mM K2SO4, 10 mM K-HEPES, pH 7.6. For the second order rate constants, the concentration unit refers to oxonol VI concentration; the numbers in parantheses are the standard errors in the values of the rate constants derived from fits by linear regression. The mean values and standard deviation are reported for the slower process observed in the ATP pulse experiments.
262
J.C. Smith and B. Chance
lOOj_l o o o o
IO
T I
AA=O.I
I I
2OOmsec
!
Kinetics of the Potential-Sensitive Extrinsic Probe Oxonol VI in Beef Heart Submitochondrial Particles J.C. Smith and Britton Chance Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Received 6 November 1978
Summary. The interaction of the potential-sensitive extrinsic probe oxonol VI with beef heart submitochondrial particles has been investigated under time resolved and equilibrium conditions. The time course of the probe absorption spectrum red shift induced by ATP or NADH injection into a suspension of submitochondrial particles in a dye solution is biphasic, consisting of a faster process described by a second-order rate law with k2~3 x 105 M-1 sec -1. For the ATP pulse experiments, the slower process follows first-order kinetics with kl ~0.3 sec -*. In oxygen pulse experiments to an anaerobic dyeparticle system, the slower process is not significantly developed due to rapid depletion of the oxygen, but the faster process follows second-order kinetics with the same rate constant as for the ATP and NADH cases. Evidence for permeation of the submitochondrial particle membrane by oxonol VI has been obtained; the slower process is interpretable as describing the permeation of the membrane bilayer. The results of the time-resolved work are consistent with a mechanism involving a redistribution of the dye from the bulk phase to the particle membrane. The value of the second-order rate constant for passive binding of the dye to submitochondrial particles is not compatible with a mechanism proposed to explain the microsecond probe response times in bilayer and excitable membrane experiments nor are such rapid signals observed in the oxonol VI-submitochondrial particle system.
The use of extrinsic probes in the study of biological membrane phenomena has received considerable impetus from the discovery of several related classes of dyes that are potential sensitive. These probes usually fall into the cyanine, merocyanine, or oxonol class of dyes. Dyes of the preceding three types consist of two ring structures joined by a conjugated carbon chain. Since the wavelength of maximum absorption increases with the length of the carbon chain, it has been possible to synthesize probes the spectral absorption and emission spectra of which do not significantly overlap the principle bands of intrinsic pigments of biological membranes. The visible absorption band of these dyes is
0022-2631/79/0046-0255 $05.60 9 Springer-Verlag New York Inc. 1979
256
J.C. Smith and B. Chance
usually quite intense, enabling the use of micromolar concentrations of these probes in membrane studies, thereby minimally perturbing the system under investigation. Most but not all of these probes are fluorescent which, in addition to providing additional sensitivity to conventional membrane suspension work, makes possible work on nontransmitting samples, such as intact organs by the use of surface fluorimetry (Chance, Mayevsky & Smith, 1976). It is often possible to markedly alter the magnitude of the spectral responses of these probes to membrane energization by modifying substituent groups of the molecule which are not part of the optical chromophore but which control such factors as charge and membrane binding affinity. Using the giant axon from the squid, Loligo peali, Cohen et al. (1974) and Ross et al. (1977) have screened a large number of dyes and have identified a number of promising potential-sensitive probes which have been systematically improved based on the criterion of increased signal to noise ratio in experiments involving the application of potential gradients across the axon membrane by means of microelectrodes. In a number of cases it has been possible to calibrate the spectral response of certain cyanine dyes such as diS-C3-5 using valinomycininduced diffusion potentials in the system under investigation. Using this approach, Laris, Bahr and Chaffee (1975) have measured the membrane potential in mitochondria, Hoffman and Laris (1974) and Sims et al. (1974) have measured the resting potential in the red cell, and Renthal and Lanyi (1976) have obtained the potential in Halobacterium halobium. Reviews on the design and use of extrinsic probes of membrane potential in a number of systems have appeared by Waggoner (1976) and by Cohen and Salzberg (1978). Waggoner, Wang and Tolles (1977) have also investigated the mechanism of the rapid response of certain cyanines and oxonols to a train of voltage pulses applied across a black lipid membrane. The dye merocyanine 540 has been employed by Salama and Morad (1976) as a probe of the action potential in the heart. Studies (Chance et al., 1974; Chance & Baltscheffsky, 1975) in our laboratory have been focused on a homologous series of oxonol dyes derived from the oxonol V probe, the structural, spectral properties, and energy-linked spectral changes of which have been described by Smith et al. (1976). Transient increases in the probe response are induced by agents such as NH3 (Smith & Chance, 1976) and nigericin (Bashford & Thayer, 1977) which are known to abolish the ApH component of the electrochemical gradient. These findings indicate that these oxonols
257
R
R
OX-Vr, R= CHaCH2CH3 Fig. l. The structure of the neutral form of oxonol VI. At pH values used in this work, the dye is a symmetrical anion since the pK of the hydroxyl proton is approximately 4 are sensitive to AT' only, which is increased as ApH is abolished in order to maintain the electrochemical gradient constant. The derivative of oxonol V in which the phenyl groups bound to the isoxazolone rings have been replaced by propyl sidechains, oxonol VI 1, has proved to give much larger spectral shifts than any of the other members of our series in membrane preparations, such as photosynthetic bacteria chromatophores and submitochondrial particles in which the internal volume of the vesicle is positive with respect to the external phase. Oxonol VI has been used by Bashford and Thayer (1977) in a double probe experiment in which 9-aminoacridine was used as the probe of ApH, to obtain the value of the electrochemical gradient under equilibrium or steady-state conditions in submitochondrial particles. In this communication, the kinetics of oxonol VI spectral changes in submitochondrial particles have been studied, and the implications of the findings for the mechanism by which the energy-linked spectral changes occur in this system are considered. The structure of the neutral form of oxonol VI is shown in Fig. 1. It should be borne in mind, however, that the dye is a symmetrical anion at physiological pH since the pK for the dissociation of the hydroxyl proton is approximately four (Smith et al., 1976), and the resulting charge is delocalized over the conjugated system of the molecule. Materials and Methods
Submitochondrial particles were prepared by the procedure described by Hansen and Smith (1964) which is only briefly outlined here. Heavy beef heart mitochondria were 1 Abbreviations used. ANS: 1-analino-8-naphthalene sulfonate; ATP: adenosine 5'triphospbate, disodium salt, from equine muscle; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; HEPES : N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid; NADH:/Lnicotinamideadeninedinucleotide; Oxonol VI: bis [3-propyl-5-oxoisoxazol-4-yl]pentamethineoxonol.
258
J.C. Smith and B. Chance
suspended at approximately 20 mg/ml protein in an incubation mixture consisting of 0.25 M sucrose, 10mM K-HEPES at pH 7.5, l mM MgC12, 1 mM ATP, and 1 mM succinate. The suspension was stored frozen overnight and then thawed, and the mitochondria were isolated by centrifugation. The mitochondria were then resuspended at approximately 20 mg/ ml protein in the medium described above with the following exceptions: the MgC12 concentration was increased to 5 raM, and 10 mM MnC12 was included in the mixture. The resulting mitochondrial suspension was sonicated at approximately 50 W by using a model W 185 Heat Systems Ultrasonics, Inc., cell disrupter for 3540 sec. The submitochondrial particles were pelleted by ultracentrifugation of the supernatant obtained fl'om an initial centrifugation to remove mitochondrial debris. The resulting preparation exhibits respiratory control, The oxonol VI dye was synthesized by suitably modifying the procedure described by Smith et al. (1976) for the oxonol V probe. Relevant analytical data supporting the structure shown in Fig. 1 for the dye are given by Bashford et al. (1978b). Sucrose, MgC12, MnC12, and K2SO4 were obtained from J.T. Baker Chemical Co. and were of reagent grade purity. All other compounds were purchased from Sigma Chemical Co. Experimental conditions are described in the appropriate figure captions. Scanned absorption spectrum measurements were made using a Hitachi-Perkin Elmer model 356 spectrophotometer in the split beam mode. Rapid mixing experiments were carred out using a Johnson Foundation Model E (Chance, 1973) rapid mixing device with an 80:1 mixing ratio adapted to a Johnson Foundation 200 Hz time-sharing double beam spectrometer equipped with 500-mm Bausch and Lomb monochromators, The detector output was displayed on a type 564 Tektronix storage oscilloscope and a model 165 Perkin Elmer strip chart recorder as required. See Results for details. The titrations used in equilibrium binding investigations were monitored by a Beckman model UV 5270 spectrometer.
Results and Data Analyses T h e o b s e r v a t i o n o f a red shift in the a b s o r p t i o n s p e c t r u m o f o x o n o l V I w h e n either p a s s i v e b i n d i n g ( B a s h f o r d e t a l . ,
1978b) or s u b s t r a t e
c o n s u m p t i o n by s u b m i t o c h o n d r i a l particles s u s p e n d e d in dye s o l u t i o n o c c u r s (Fig. 2) w a s u s e d as the basis for m o n i t o r i n g the t i m e c o u r s e o f the s p e c t r a l c h a n g e s to be d e s c r i b e d in this section. F l u o r e s c e n c e c h a n g e s c o u l d in p r i n c i p l e h a v e b e e n u s e d for this p u r p o s e ; the sense as well as the m a g n i t u d e o f the e m i s s i o n intensity c h a n g e , h o w e v e r , are f u n c t i o n s o f d y e - t o - p a r t i c l e ratio, w h i c h in the e x p e r i m e n t s to be d e s c r i b e d is a n e x p e r i m e n t a l variable. T h e c h a n g e s in t r a n s m i s s i o n are t h u s m o r e easily i n t e r p r e t e d . T h e a p p a r e n t isosbestic p o i n t at 603 n m (Fig. 2) p r o v i d e d a c o n v e n i e n t reference, while the large c h a n g e at 630 n m w a s used as the m e a s u r i n g p o i n t in the d o u b l e b e a m t r a n s m i s s i o n experiments. T w o basic t y p e s o f r a p i d m i x i n g e x p e r i m e n t s are r e p o r t e d in this c o m m u n i c a t i o n . T h e first a n d b y f a r the s i m p l e r to a n a l y z e is the passive b i n d i n g e x p e r i m e n t in w h i c h the m a j o r syringe o f the r a p i d m i x i n g device c o n t a i n s dye s o l u t i o n m i x e d w i t h s u b m i t o c h o n d r i a l particles via the min o r syringe. T h e t i m e c o u r s e o f the passive b i n d i n g p r o c e s s w a s suffi-
Potential-Sensitive Extrinsic Probes
259
free dye
r
\
SMP
~176
ATP,
006 [ I
005 i
0L
500
550
r 650
600 Mnm)
# ~
700
Fig. 2. The absorption spectrum of oxonol VI as the free dye, as slightly red shifted when the dye is bound to the submitochondrial particle membrane, the large red shift induced by ATP energization of the particles, and the complete reversal of the red shift by addition of the uncoupler CCCP. Medium: 0.25 M sucrose, 5 nan Na-HEPES at pH 7.5; concentrations: 1.2 gM oxonol VI, 0.25 mg/ml ATP-Mg § +-Mn ++ submitochondrial particle protein, 1.7 mM ATP-MgC12, 5 gM CCCP
-/c7 A~Q02
D~
II 2 0 msec Fig. 3. (A): The time course of the red shift measured as the difference in transmission at 603~530 nm caused by passive binding of oxonol VI to submitochondrial particles. Medium: 25 mM K2SO4, 10 mM K-HEPES at pH 7.5, 0.25 u sucrose. Concentrations: 15 gM oxonol VI, 3 gM CCCP, 0.09 mg/ml submitochondrial particle protein; time constant: 3 msec. (B): A diagram illustrating the determination of the apparent first-order rate constant from the oscilloscope trace using Eqs. (1) and (2). In this experiment kapp= 150 s e c - 1 The flow velocity trace is in the upper portion of the diagram, and the lower trace is the time course of the optical signal9 See text for details
c i e n t l y r a p i d t h a t t h e results c o u l d be a n a l y z e d in t e r m s o f the c o n t i n u o u s f l o w case ( C h a n c e , 1973) since the signal r e a c h e d a p l a t e a u b e f o r e t h e f l o w t h r o u g h t h e o b s e r v a t i o n t u b e ceased. A t y p i c a l p h o t o g r a p h o f a n oscilloscope record
of a passive
binding
experiment
is p r e s e n t e d in
260
J.C. Smith and B. Chance
Fig. 3a. T h e a p p a r e n t first-order rate c o n s t a n t kapp is given by (Chance, 1973) k~pp =(2.3/tin) log (Dz/D~). (1) T h e d i s p l a c e m e n t s Da a n d D 2 are defined in Fig. 3b; tm is related to t I which is o b t a i n e d f r o m the flow velocity trace (Fig. 3 b) by
t,. =
(v/v.)
(2)
where V is the v o l u m e f r o m the p o i n t o f o b s e r v a t i o n to that o f mixing a n d V~ is the v o l u m e d i s c h a r g e d in time tf. T h e s e characteristic v o l u m e s m u s t be d e t e r m i n e d for the p a r t i c u l a r mixing device in use. Since the rate c o n s t a n t for the s e c o n d - o r d e r binding process was to be d e t e r m i n e d , the s u b r n i t o c h o n d r i a l particle c o n c e n t r a t i o n was a d j u s t e d such that the dye w o u l d be the r e a c t a n t in excess. U n d e r these c o n d i t i o n s , the a p p a r e n t f i r s t - o r d e r rate c o n s t a n t is r e l a t e d to the s e c o n d - o r d e r rate c o n s t a n t by
(3)
kapp • k2nd ~D
w h e r e Yo is the c o n c e n t r a t i o n o f the r e a c t a n t in excess, which in this case is the o x o n o l VI dye. A plot o f kap p vs. J(n will thus be a straight
25O
2OO _---. ),j
J5o
ioo
50
0
5
I0
15
20
25
OX-VT Concentration(/.zM)
Fig. 4. A plot of the apparent first-order rate constant kapp vs. dye concentration for the passive binding of oxonol VI to submitochondrial particles with the dye in excess. The second-order rate constant is obtained from the slope of the line as described by Eq. (3) in the text. Experimental conditions are given in the caption to Fig. 3a. The value of the second-order rate constant is given in Table 1. Data taken in the absence (e) and presence of 3 gM CCCP (A) fall on the same line, indicating the lack of endogenous substrate in the submitochondrial preparation
Potential-Sensitive Extrinsic Probes
261
line the slope of which gives k2n d. Such a plot for the passive binding experiment in which the submitochondrial particle concentration was held constant and that of the dye varied is presented in Fig. 4. The value of the second-order rate constant is presented in Table 1. In the second type of rapid mixing experiment, the major syringe of the rapid mixing device contained submitochondrial particles suspended in oxonol VI dye solution in which the passive binding process had reached equilibrium. That the latter process was complete could be easily ascertained by simply discharging the contents of the major syringe through the observation tube with the minor syringe empty and the valve connecting the minor syringe to the mixing chamber closed. A fiat line was obtained, indicating that no passive binding was in progress. After equilibrium was reached, various substrates or oxygen were mixed with the submitochondrial particle-oxonol VI suspension via the minor syringe under a number of conditions, some of them of a control nature, to be described subsequently. The time course of the spectral shift caused by substrate or oxygen addition was such that only a negligible optical change occurred during the time of flow, i.e., the stopped flow case was applicable for data analysis. Initial substrate pulse experiments were carried out with N A D H . The time course of the resulting signal for a typical experiment is shown in Fig. 5a. The analysis of these data are illustrated in Fig, 5b. Since on a log scale the data at long time was reasonably linear with time,
Table 1. Summaryof rate constants characterizingthe kinetics of oxonol VI in submitochondrial particles Remarks
Rate constants
Passive binding, dye in excess Oxygen pulse Decay of oxygen pulse-induced dye response NADH, faster process ATP pulse, faster process ATP pulse, slower process ATP pulse, faster process, K2SO4 absent ATP pulse, slower process, K2SO4 absent
9.53(•215 ~sec 1 2.40(_+0.40)x105M ~ sec-1 0.218 (+0.001) sec-1 2.70 (•215 105 M-1 sec 3.10 ( _+0.20) xlOSM lsec-1 0.33 (+0.04) sec 1 2.46 (_+0.20) x 105 M- 1 sec- 1 0.55 (_+0.15) sec
Medium except as noted: 0.25 M sucrose, 25 mM K2SO4, 10 mM K-HEPES, pH 7.6. For the second order rate constants, the concentration unit refers to oxonol VI concentration; the numbers in parantheses are the standard errors in the values of the rate constants derived from fits by linear regression. The mean values and standard deviation are reported for the slower process observed in the ATP pulse experiments.
262
J.C. Smith and B. Chance
lOOj_l o o o o
IO
T I
AA=O.I
I I
2OOmsec
!
