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[104] Means of terminating reactions
680
SPECIAL ASSAYS AND TECHNIQUES
[104]
protein bands showed greater mobility but the pattern of the protein bands remained the same. There are certain limitations in the method. In all cases, some protein remains at the origin which is either nonmobile or unable to penetrate the gel. There is also the question of the degree of dissociation achieved by the solubilizing system. The phenol-acetic acid-water solution of the complexes studied are completely transparent, and the protein remains suspended even after centrifugation at 100,000 g for 30 minutes. It is possible that the phenol-acetic acid system functions by minimizing the hydrophobic interactions. This solvent system is admittedly drastic. All enzymatic properties are probably destroyed and most of the prosthetic groups are either dissociated, altered, or destroyed. Thus only the primary structure of the native protein could be assured of remaining intact.
[ 1 0 4 ] Means of Terminating Reactions
By MARTIN KLINGENBERGand ERICH PFAFF Investigations on mitochondrial metabolism include measurements of the instantaneous level of metabolites or the functional state of cofactors. For this purpose the reactions have to be instantaneously terminated ("quenched") by rapid mixing with a "quenching" agent. The quenched extract is then available for further analysis of the metabolites. This extraction can be combined with simultaneous continuous recording of spectrophotometric absorption of metabolic components and continuous recording of other functions such as respiration, pH changes, etc. The requiremenfs for the appropriate quenching method are instantaneous termination of the reaction, which can be followed by an extraction that preserves the metabolites or the cofactors in their functional state. Acid Extraction 1 The most widely used quenching reaction is the addition of acid, mostly perchloric acid, which gives an effective inactivation of the enzymes and destruction of the mitochondria. Thus the cofactors and substances bound in the mitochondria are effectively released. The lability of some a-keto acids and phosphate compounds containing phos1M. Klingenberg, in "Methoden der enzymatischen Analyse" (H.oU. Bergmeyer, ed.), p. 528. Verlag Chemie, Weinheim/Bergstrasse, 1962.
[104]
MEANS OF TERMINATING REACTIONS
681
phate in anhydride or amide bond (creatine-arginine-phosphate) requires that the acid extracts are rapidly neutralized to avoid degradation. The reduced pyridine nucleotides are at the low pH rapidly degraded to the nicotinamide-deficient moiety. The acid extracts are, after neutralization, well suited for enzymatic tests 1 or for chromatographic analysis.2 The procedure is to add 3 M HC1Q to the mitochondrial suspension to give a final concentration of 0.5M HC104. The acid is added under rapid mixing. Immediate quenching is effective under a broad temperature range from about 0 ° to 40 °. Alkaline Extraction 1 For the extraction of D P N t I and T P N H the quenching reagent has to be alkaline; 2 M KOH in ethanol is added to the mitochondrial suspension to give a final concentration of 0.5 M KOH. The enzymatic reactions are immediately terminated in the alkaline extract. In some determination procedures a complete destruction of DPN and TPN in the alkaline extract is required for the further determination of the DPNH and TPNH. To achieve this and the complete irreversible inactivation of the enzymes, the alkaline extract must be kept at 25 ° for 30 minutes before it is neutralized. It has to be taken into account that after neutralization the reduced coenzymes can be slowly reoxidized by nonenzymatic reaction catalyzed by the mitochondrial extract. Lipophilic Extraction ~ For the extraction of a lipophilic component, such as ubiquinone, the following procedure is recommended; it preserves the ubiquinone at its steady state reduction oxidation level. The mitochondrial suspension is rapidly mixed with double its volume of 70% methanol and 30% petroleum ether. The mixture is well shaken for 30 seconds. The upper petroleum ether phase contains the oxidized and reduced ubiquinone. The extraction is instantaneous since the protein is precipitated by the petroleum ether and the ubiquinone is rapidly removed from its environment in mitochondria by the mediation of methanol. The extraction can be modified to allow simultaneous measurement of hydrophilic mctabolites, such as pyridine nucleotides. For this purpose it must be combined with an acid quenching in order to inactivate completely the D P N H oxidase, ATPase, etc. The extraction mixture contains 0.2 M HCIO, in 70% methanol and 30% petroleum ether (composed of 70 ml of methanol, 30 ml of petroleum ether, 1.5 ml of 70% HCI04). 2H. W. Heldt, M. Klingenberg, and K. Papenberg, Biochem. Z. 342, 508 (1965). 3A. KrSger and M. Klingenberg, Biochem. Z. 344, 317 (1966).
682
SPECIAL ASSAYS AND TECHNIQUES
[104]
After extraction the lower phase contains the acid-extractable, hydrophilie components such as D P N and adenine nucleotides; the upper phase contains the ubiquinone. The various components can be studied in these two extracts2 Separate Measurements of Intra- and Extramitochondrial Metabolic States
For the separate analysis of sediment and medium the method of sedimentation and separation by a sucrose gradient has been frequently used. 4 By this method the mitochondria do not stay in equilibrium with the medium in the separation layer. The metabolic state will change further in the sediment since the mitochondria are not immediately inactivated. The sucrose density layer should be appropriately replaced by a dextrane density layer in order to avoid too strong an increase of the osmolarity, which would also influence the mitochondria. This layer filtration method can be used only in cases where the internal substances of the mitochondria do not leak out during the washing process or change in the sedimented state. Termination of the reaction combined with instantaneous fixation can be accomplished with another type of centrifugal filtration. This method--in principle first described by Werkheiser and BartleyS--sepa rates the suspension and incubation zone of the mitochondria from the acid fixation layer by a silicone layer. The layers must be arranged in a suitable density gradient. During centrifugation, the mitochondria pass the silicone layer and become stripped of their surrounding medium. The difficulties encountered at first ~ have been overcome after a detailed investigation of the filtration and sedimentation process on the basis of which the method was newly developed/,s During centrifugal sedimentation on the phase limit medium/silicone, portions of the mitochondria temporarily accumulate. From here minute droplets (0 ~ 0.2 mm) sediment very fast (in far less than 3 seconds) through the silicone2 ' B. C. Pressman, J. Biol. Chem. ~T2, 967 (1958). s W. C. Werkheiser and W. Bartley, Biochem. J. 66, 79 (1957). 6 j. E. Amoore, Biochem. J. 70, 718 (1958). ~E. Pfaff, Diplomarbeit, University of Marburg, 1962. 8 E. Pfaff, Doctoral thesis, University of Marburg, 1965. ~At the entrance to the acid layer, mixtures of deproteinized mitochondria can form that are at first less dense than the silicone. If the amount of mitochondria exceeds a certain limit, less dense droplets may migrate back in the centrifugal field and cause contamination by acid of the upper suspension. The reverse migration of acid is avoided when the concentration of mitochondria is small enough. Furthermore, it is important also that the density of the acid sufficiently exceeds that of the silicone. On the other hand, retarded inactivation can be caused by a too high density of the acid.
[104]
MEANS OF TERMINATING REACTIONS
683
The following experimental procedure has been the one principally used by the authors. Siliconized centrifuge tubes made of Pyrex glass (~6-8 mm) are prepared for the swinging-bucket rotor SW-39, Spinco-Beckman Co. The centrifuge tubes are placed in a polyethylene adapter containing water. They are run up to 22,000 rpm (35,000 g) and then stopped. The following densities can be recommended for suspension of mitochondria in 0.25M sucrose (p----1.037) :acid layer containing 1.6 M HCI04, p = 1.08 g cm-3; silicone layer, p = 1.05 to 1.06 g cm-3. The experiment is started by the addition of the mitochondria to the incubation layer; in other cases the suspension may be layered on top of the silicone. Control experiments proved that the intramitochondrial metabolic state is preserved through the silicone until the inactivation. The method is therefore proved to yield the instantaneous metabolic pattern of mitochondria. The extramitochondrial fluid which accompanies the mitochondrial droplets can be corrected for by the use of labeled high molecular glucose (14C-etherated globular polyglucose or 14C-dextrane). The filtered amount is composed of approximately equal parts of mitochondrial and extramitochondrial volume. The shortest time attainable for the incubation and separation of the mitochondria is about 50 seconds after the start of incubation. This includes the time for preparing the rotor and acceleration of the centrifuge until it reaches 8000 rpm, when the mitochondria start to penetrate the silicone layer. For kinetic experiments requiring shorter times for the separation of mitochondria, the method can be modified to include an additional incubation layer (centrifugal-layer filtration) s,l° (cf. Fig. 1). Here the mitochondria are first present in an upper layer under which is a layer containing the reactive substances. The layers are isolated by a small amount of isolating medium. The density of the three layers is increased by the addition of dextran. During centrifugation the mitochondria migrate for only a short time through the reaction layer, the time of which can be calibrated. Incubation times as low as l0 seconds can be obtained. The lag time at the phase-limit medium-silicone can be excluded by placing an additional washing layer before the silicone. Termination of Reaction by Sieve Filtration Another approach for separating intra- and extramitochondrial metabolites is sieve filtration. Here the mitochondrial suspension is pressed ,o M. Klingenberg, E. Pfaff, and A. KrSger, in "Rapid Mixing and Sampling Techniques in Biochemistry" (B. Chance, R. tt. Eisenhardt, Q. H. Gibson, and K. K. Lonberg-Holm, eds.), p. 333. Academic Press, New York, 1965.
684
SPECIAL ASSAYS AND TECHNIQUES
[104]
Storage layer Isolating layer Incubation layer Silicone oil Acid FIO. 1. Scheme of centrifugal layer filtration. The layers have the following composition: the storage layer contains the mitochondria in 0.25 M sucrose medium (density p ----1.037 g/ml), the density of the 0.25 M sucrose medium in the isolating and the incubation layer is increased by the addition of 15.0 mg and 22.5 mg of dextran per milliliter, respectively. The silicone oil has a density of 1.065 and the acid (1.6 M HC1OD of 1.08. through a filter which m a y consist of a Celite layer, 11 Millipore filter, 8 or glass fiber filter. TM In this case the filtrates can be collected in acid and thus immediately deproteinized. This allows determination of the true metabolic state of the extramitochondrial space. However, the intramitochondrial metabolites m a y have greatly changed, since collecting the mitochondria on the filter surface separates them from their substrate and oxygen supply. Furthermore it is difficult to account reliably for the amount of medium trapped in the mitochondrial layer on the filter by high molecular compounds since these are also concentrated with the mitochondria on the filter surface. 8 The following method has been applied by the authors. Filters of the Millipore Corporation ( 0 ~ 0.65/z) have been used in combination with a stainless steel pressure filter holder (Millipore Corporation). An air pressure of up to 6 atmospheres is used. The filter holder was modified by a stirring device which allowed starting of the incubation by mixing within the filter chamber and immediate filtration in less than 2 seconds. A suspension of 4 ml of mitochondria with a total of 10 mg of protein can be filtered within less than a second.
1~G. F. Azzone and L. Ernster, J. Biol. Chem. 236, 1501 (1961). 12G. Brierley and R. L. O'Brien, J. Biol. Chem. 240, 4532 (1965).
SPECIAL ASSAYS AND TECHNIQUES
[104]
protein bands showed greater mobility but the pattern of the protein bands remained the same. There are certain limitations in the method. In all cases, some protein remains at the origin which is either nonmobile or unable to penetrate the gel. There is also the question of the degree of dissociation achieved by the solubilizing system. The phenol-acetic acid-water solution of the complexes studied are completely transparent, and the protein remains suspended even after centrifugation at 100,000 g for 30 minutes. It is possible that the phenol-acetic acid system functions by minimizing the hydrophobic interactions. This solvent system is admittedly drastic. All enzymatic properties are probably destroyed and most of the prosthetic groups are either dissociated, altered, or destroyed. Thus only the primary structure of the native protein could be assured of remaining intact.
[ 1 0 4 ] Means of Terminating Reactions
By MARTIN KLINGENBERGand ERICH PFAFF Investigations on mitochondrial metabolism include measurements of the instantaneous level of metabolites or the functional state of cofactors. For this purpose the reactions have to be instantaneously terminated ("quenched") by rapid mixing with a "quenching" agent. The quenched extract is then available for further analysis of the metabolites. This extraction can be combined with simultaneous continuous recording of spectrophotometric absorption of metabolic components and continuous recording of other functions such as respiration, pH changes, etc. The requiremenfs for the appropriate quenching method are instantaneous termination of the reaction, which can be followed by an extraction that preserves the metabolites or the cofactors in their functional state. Acid Extraction 1 The most widely used quenching reaction is the addition of acid, mostly perchloric acid, which gives an effective inactivation of the enzymes and destruction of the mitochondria. Thus the cofactors and substances bound in the mitochondria are effectively released. The lability of some a-keto acids and phosphate compounds containing phos1M. Klingenberg, in "Methoden der enzymatischen Analyse" (H.oU. Bergmeyer, ed.), p. 528. Verlag Chemie, Weinheim/Bergstrasse, 1962.
[104]
MEANS OF TERMINATING REACTIONS
681
phate in anhydride or amide bond (creatine-arginine-phosphate) requires that the acid extracts are rapidly neutralized to avoid degradation. The reduced pyridine nucleotides are at the low pH rapidly degraded to the nicotinamide-deficient moiety. The acid extracts are, after neutralization, well suited for enzymatic tests 1 or for chromatographic analysis.2 The procedure is to add 3 M HC1Q to the mitochondrial suspension to give a final concentration of 0.5M HC104. The acid is added under rapid mixing. Immediate quenching is effective under a broad temperature range from about 0 ° to 40 °. Alkaline Extraction 1 For the extraction of D P N t I and T P N H the quenching reagent has to be alkaline; 2 M KOH in ethanol is added to the mitochondrial suspension to give a final concentration of 0.5 M KOH. The enzymatic reactions are immediately terminated in the alkaline extract. In some determination procedures a complete destruction of DPN and TPN in the alkaline extract is required for the further determination of the DPNH and TPNH. To achieve this and the complete irreversible inactivation of the enzymes, the alkaline extract must be kept at 25 ° for 30 minutes before it is neutralized. It has to be taken into account that after neutralization the reduced coenzymes can be slowly reoxidized by nonenzymatic reaction catalyzed by the mitochondrial extract. Lipophilic Extraction ~ For the extraction of a lipophilic component, such as ubiquinone, the following procedure is recommended; it preserves the ubiquinone at its steady state reduction oxidation level. The mitochondrial suspension is rapidly mixed with double its volume of 70% methanol and 30% petroleum ether. The mixture is well shaken for 30 seconds. The upper petroleum ether phase contains the oxidized and reduced ubiquinone. The extraction is instantaneous since the protein is precipitated by the petroleum ether and the ubiquinone is rapidly removed from its environment in mitochondria by the mediation of methanol. The extraction can be modified to allow simultaneous measurement of hydrophilic mctabolites, such as pyridine nucleotides. For this purpose it must be combined with an acid quenching in order to inactivate completely the D P N H oxidase, ATPase, etc. The extraction mixture contains 0.2 M HCIO, in 70% methanol and 30% petroleum ether (composed of 70 ml of methanol, 30 ml of petroleum ether, 1.5 ml of 70% HCI04). 2H. W. Heldt, M. Klingenberg, and K. Papenberg, Biochem. Z. 342, 508 (1965). 3A. KrSger and M. Klingenberg, Biochem. Z. 344, 317 (1966).
682
SPECIAL ASSAYS AND TECHNIQUES
[104]
After extraction the lower phase contains the acid-extractable, hydrophilie components such as D P N and adenine nucleotides; the upper phase contains the ubiquinone. The various components can be studied in these two extracts2 Separate Measurements of Intra- and Extramitochondrial Metabolic States
For the separate analysis of sediment and medium the method of sedimentation and separation by a sucrose gradient has been frequently used. 4 By this method the mitochondria do not stay in equilibrium with the medium in the separation layer. The metabolic state will change further in the sediment since the mitochondria are not immediately inactivated. The sucrose density layer should be appropriately replaced by a dextrane density layer in order to avoid too strong an increase of the osmolarity, which would also influence the mitochondria. This layer filtration method can be used only in cases where the internal substances of the mitochondria do not leak out during the washing process or change in the sedimented state. Termination of the reaction combined with instantaneous fixation can be accomplished with another type of centrifugal filtration. This method--in principle first described by Werkheiser and BartleyS--sepa rates the suspension and incubation zone of the mitochondria from the acid fixation layer by a silicone layer. The layers must be arranged in a suitable density gradient. During centrifugation, the mitochondria pass the silicone layer and become stripped of their surrounding medium. The difficulties encountered at first ~ have been overcome after a detailed investigation of the filtration and sedimentation process on the basis of which the method was newly developed/,s During centrifugal sedimentation on the phase limit medium/silicone, portions of the mitochondria temporarily accumulate. From here minute droplets (0 ~ 0.2 mm) sediment very fast (in far less than 3 seconds) through the silicone2 ' B. C. Pressman, J. Biol. Chem. ~T2, 967 (1958). s W. C. Werkheiser and W. Bartley, Biochem. J. 66, 79 (1957). 6 j. E. Amoore, Biochem. J. 70, 718 (1958). ~E. Pfaff, Diplomarbeit, University of Marburg, 1962. 8 E. Pfaff, Doctoral thesis, University of Marburg, 1965. ~At the entrance to the acid layer, mixtures of deproteinized mitochondria can form that are at first less dense than the silicone. If the amount of mitochondria exceeds a certain limit, less dense droplets may migrate back in the centrifugal field and cause contamination by acid of the upper suspension. The reverse migration of acid is avoided when the concentration of mitochondria is small enough. Furthermore, it is important also that the density of the acid sufficiently exceeds that of the silicone. On the other hand, retarded inactivation can be caused by a too high density of the acid.
[104]
MEANS OF TERMINATING REACTIONS
683
The following experimental procedure has been the one principally used by the authors. Siliconized centrifuge tubes made of Pyrex glass (~6-8 mm) are prepared for the swinging-bucket rotor SW-39, Spinco-Beckman Co. The centrifuge tubes are placed in a polyethylene adapter containing water. They are run up to 22,000 rpm (35,000 g) and then stopped. The following densities can be recommended for suspension of mitochondria in 0.25M sucrose (p----1.037) :acid layer containing 1.6 M HCI04, p = 1.08 g cm-3; silicone layer, p = 1.05 to 1.06 g cm-3. The experiment is started by the addition of the mitochondria to the incubation layer; in other cases the suspension may be layered on top of the silicone. Control experiments proved that the intramitochondrial metabolic state is preserved through the silicone until the inactivation. The method is therefore proved to yield the instantaneous metabolic pattern of mitochondria. The extramitochondrial fluid which accompanies the mitochondrial droplets can be corrected for by the use of labeled high molecular glucose (14C-etherated globular polyglucose or 14C-dextrane). The filtered amount is composed of approximately equal parts of mitochondrial and extramitochondrial volume. The shortest time attainable for the incubation and separation of the mitochondria is about 50 seconds after the start of incubation. This includes the time for preparing the rotor and acceleration of the centrifuge until it reaches 8000 rpm, when the mitochondria start to penetrate the silicone layer. For kinetic experiments requiring shorter times for the separation of mitochondria, the method can be modified to include an additional incubation layer (centrifugal-layer filtration) s,l° (cf. Fig. 1). Here the mitochondria are first present in an upper layer under which is a layer containing the reactive substances. The layers are isolated by a small amount of isolating medium. The density of the three layers is increased by the addition of dextran. During centrifugation the mitochondria migrate for only a short time through the reaction layer, the time of which can be calibrated. Incubation times as low as l0 seconds can be obtained. The lag time at the phase-limit medium-silicone can be excluded by placing an additional washing layer before the silicone. Termination of Reaction by Sieve Filtration Another approach for separating intra- and extramitochondrial metabolites is sieve filtration. Here the mitochondrial suspension is pressed ,o M. Klingenberg, E. Pfaff, and A. KrSger, in "Rapid Mixing and Sampling Techniques in Biochemistry" (B. Chance, R. tt. Eisenhardt, Q. H. Gibson, and K. K. Lonberg-Holm, eds.), p. 333. Academic Press, New York, 1965.
684
SPECIAL ASSAYS AND TECHNIQUES
[104]
Storage layer Isolating layer Incubation layer Silicone oil Acid FIO. 1. Scheme of centrifugal layer filtration. The layers have the following composition: the storage layer contains the mitochondria in 0.25 M sucrose medium (density p ----1.037 g/ml), the density of the 0.25 M sucrose medium in the isolating and the incubation layer is increased by the addition of 15.0 mg and 22.5 mg of dextran per milliliter, respectively. The silicone oil has a density of 1.065 and the acid (1.6 M HC1OD of 1.08. through a filter which m a y consist of a Celite layer, 11 Millipore filter, 8 or glass fiber filter. TM In this case the filtrates can be collected in acid and thus immediately deproteinized. This allows determination of the true metabolic state of the extramitochondrial space. However, the intramitochondrial metabolites m a y have greatly changed, since collecting the mitochondria on the filter surface separates them from their substrate and oxygen supply. Furthermore it is difficult to account reliably for the amount of medium trapped in the mitochondrial layer on the filter by high molecular compounds since these are also concentrated with the mitochondria on the filter surface. 8 The following method has been applied by the authors. Filters of the Millipore Corporation ( 0 ~ 0.65/z) have been used in combination with a stainless steel pressure filter holder (Millipore Corporation). An air pressure of up to 6 atmospheres is used. The filter holder was modified by a stirring device which allowed starting of the incubation by mixing within the filter chamber and immediate filtration in less than 2 seconds. A suspension of 4 ml of mitochondria with a total of 10 mg of protein can be filtered within less than a second.
1~G. F. Azzone and L. Ernster, J. Biol. Chem. 236, 1501 (1961). 12G. Brierley and R. L. O'Brien, J. Biol. Chem. 240, 4532 (1965).