466
FLAVOPROTEINS
[47] M a m m a l i a n
Succinate
[47]
Dehydrogenase
By BRIAN A. C. ACKRELL, EDNA B. KEARNEV, and THOMAS P. SINGER
This article deals with four aspects of mammalian succinate dehydrogenase: (1) a critical comparison of assay methods, (2) activation-deactivation of the enzyme, (3) the active site of the enzyme, and (4) comparison of the properties of various purified preparations, including recent improvements of procedures for isolating the reconstitutively active form in high yield and with a high turnover number. Assay of Succinate Dehydrogenase
Activity
Since the catalytic turnover of succinate dehydrogenase is faster than the rate-limiting step in the respiratory chain, artificial electron acceptors are usually used for assays of the enzyme in order to ensure that full activity is being measured (a necessity in determining kinetic constants and turnover numbers, for instance). Of these, phenazine methosulfate (PMS),la with either DCIP or cytochrome c as terminal oxidant, may be used with either particulate or soluble preparations. With particle preparations such as complex II and ETP, the ubiquinone homologs Q1 and Q2, as well as the ubiquinone analog DPB, may be used in place of PMS, with the same activity, and nearly the same activity may be measured by using the free-radical form of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD', also called Wurster's blue). With soluble preparations of the enzyme, assays with PMS + DCIP, with low concentrations of ferricyanide ("low Km" ferricyanide assay) or with TMPD', all at Vmax, show the same rate of succinate oxidation. The precautions recommended for each of these procedures, their pitfalls and limitations are briefly noted at the end of each method and are more fully discussed elsewhere. 2'3 i The original studies reported here were supported by Program Project HL-16251 from the National Institutes of Health and by Grant No. PCM 76-03367 from the National Science Foundation. ia Abbreviations: DCIP, 2,6-dichlorophenolindophenol; DPB, 2,3-dimethoxy-5-methyl-6pentyl-l,4-benzoquinone; MES, 2-(N-morpholino)ethanesulfonic acid; PMS, phenazine methosulfate; TTF, thenoyltrifluoroacetone. 2 T. P. Singer, Methods Biochem. Anal. 22, 123 (1974). 3 B. A. C. Ackrell, C. J. Coles, and T. P. Singer, FEBS Lett. 75, 249 (1977).
[47]
MAMMALIAN SUCCINATE DEHYDROGENASE
467
Phenazine Methosulfate Assay The enzyme, reduced by its substrate, succinate, is reoxidized by PMS, which is varied in concentration to obtain V. . . . Reoxidation of the reduced PMS is accomplished by either DCIP or cytochrome c, whose reduction is monitored spectrophotometrically. The characteristics of the recording spectrophotometer required (scale expansion, recorder speed, optical density offset) have been described." Polarographic or manometric measurements of the succinate-PMS reaction are not recommended, as they are less sensitive and the rate is limited by the oxygen concentration.
Reagents 1. Potassium phosphate buffer, 200 mM, pH 7.5 (at room temperature) 2. Sodium succinate, 200 raM, adjusted to pH 7.5 3. KCN, 100 mM 4. DCIP (General Biochemicals, Inc.), 0.05% (w/v) in 100 mM potassium phosphate, pH 7.5 5. Phenazine methosulfate (Sigma), 0.33% (w/v), in glass-distilled water. Store in amber or 'qow actinic" red glassware, frozen when not in use; protect from light during use.
Procedure. If the enzyme to be assayed is not in the fully activated state, it should first be treated as described in a subsequent section. If a fully activated preparation is to be assayed, the reaction is usually started by adding the enzyme to the complete reaction mixture. Each cuvette receives 0.75 ml of phosphate buffer, 0.3 ml of succinate, water to give a final volume of 3 ml, 0.1 ml of DCIP (to give an absorbance of 1.0 to 1.25 at 600 nm in I-cm light path) and varying amounts of PMS. The latter is varied in the range of 0.3 to 0.03 ml of PMS per 3 ml of final volume. The cuvettes are brought to the temperature of assay while protected from light and placed in the spectrophotometer; 0.03 ml of KCN is added, and the enzyme immediately thereafter to start the reaction. If the reaction is started by addition of the dyes instead of the enzyme, these should be at the temperature of assay, as the volumes added are significantly large. The amount of enzyme used should cause an absorbance change corresponding to 30-50% of the chart width in 1530 sec. An absorbance range of 0-0.2 to 0-0.4 absorbance unit full scale is recommended, with a recorder chart speed of 10-12 inches/min at high PMS concentrations and 5-6 inches/min at the lower dye concentrations. The temperature of assay can be chosen for convenience but is usually 30 ° or 38 °. If it is desired to determine the extent of activation of a given
468
VLAVOPROTEINS
[47]
preparation, the assays should be carried out at or below 15°, since succinate does not activate the enzyme significantly during the assay in this temperature range. Activity is calculated from double reciprocal plots of absorbance change vs PMS concentration, using the millimolar extinction coefficient of 19.1 at 600 nm for DCIP.
Comments. In order to assure that no electron flux to cytochrome c and Oz via the respiratory chain occurs in the assay of membrane-bound preparations, antimycin A (1 tzg per milligram of protein) is included in the assay mixture as well as the KCN. In intact mitochondria penetration of PMS is rate-limiting. To overcome this, 1-2 /zg of crude Naja naja venom or of partially purified phospholipase A2 from this venom 4 and 750 ~ CaC12 are added to the assay mixture, and the reaction is started with dyes to allow time for phospholipase action. While the determination of activity at Vmax with respect to PMS is essential in kinetic studies, in determination of the turnover number, in studies with inhibitors, and in comparisons of soluble and particulate enzymes, because the Km for PMS is altered on extraction of the enzyme and on treatment with certain inhibitors, if only a rough estimate of the activity is desired, or if the experimental conditions do not bring about a change in Kin, the highest level of PMS recommended may be used in lieu of varying dye concentration. When membrane-bound succinate dehydrogenase is being assayed, deviations from linearity in double reciprocal plots are seen, since DCIP is reduced without the mediation of PMS. This "direct" reduction contributes significantly only at the lower concentrations of PMS. Substitution of heart muscle cytochrome c (50/xM final concentration) for the DCIP solves this problem, since cytochrome c is not reduced in the reaction without PMS, in the presence of antimycin. Although a polarographic variant of the PMS method has been used by some workers, for reasons detailed elsewhere 2 this procedure is not recommended. Ferricyanide Assay Ferricyanide has been widely used for the assay of succinate dehydrogenase. The conventional assay is spectrophotometric and uses either a fixed, high ferricyanide concentration ( - 5 mM) or a series of high ferricyanide concentrations (1.7-10 mM), with extrapolation to Vmax. Under either of these conditions the activity with ferricyanide is less 4 T. Cremona and E. B. Kearney, J. Biol. Chem. 239, 2328 (1964).
[47]
MAMMALIAN SUCCINATE DEHYDROGENASE
469
(-'
•
~ ,'. . . : .
.;.
5= dZ -;~"== >,.=-~
~
V;
~=h"'-
= . ~ .~ ~ . ~
~
.
. ~
"-
-
.
._
~ ~ ~ ~"
K.E
='~
.
481
482
FLAVOPROTEINS
[4 7]
dalton Subunit, za'3~ as well as in flavin peptides obtained by proteolytic digestion. 39,40 A flavin pentapeptide of the structure Ser-His-Thr-Val-Ala
I Flavin
may be obtained by precipitating the enzyme with trichloroacetic acid, digestion with trypsin-chymotrypsin, and purification of the flavin peptide by chromatographic procedures. 4oA longer flavin peptide, containing 23 amino acids, may be obtained by tryptic digestion, followed by chromatography on Florisil, DEAE-cellulose, and phosphocellulose. 41 The amino acid sequences of both peptides have been determined. 41 The 8c~[(N)3-histidyl]-FAD moiety may be obtained by digestion of the pentapeptide with aminopeptidase? °'41 Acid digestion of the peptide results in the corresponding histidyl anhydroflavin. 4z Substrate Site
Identification of the amino acid environment at the substrate site has been difficult because no stable, covalent adducts of the enzyme with substrates or competitive inhibitors are available. Even oxaloacetate, which is extremely tightly bound to the deactivated form of the enzyme, is released on denaturation of the enzyme, 12.43,44presumably because the thiohemiacetal bond is stabilized by noncovalent interactions in the native conformation. The problem has been circumvented as follows. 44 It has been found that treatment of the enzyme with N-ethylmaleimide leads to rapid alkylation of one - - S H group on each of the 30,000- and 70,000-dalton subunits. Of these, reaction with the - - S H group on the large subunit is slower, and this is accompanied by loss of catalytic activity. Alkylation of this - - S H group and loss of activity are prevented by succinate, malonate, and oxaloacetate, whereas alkylation of the other cysteine residue is unaffected by these compounds. Thus, treatment of two succinate dehydrogenase samples with [14C]N-ethylmaleimide, one of which contains malonate as a protective agent, followed by removal of un39 E. B. Kearney, J. Biol. Chem. 235, 865 (1960). 4o j. Salach, W. H. Walker, T. P. Singer, A. Ehrenberg, P. Hemmerich, S. Ghisla, and U. Hartmann, Eur. J. Biochem. 26, 267 (1972). 41 W. C. Kenney, W. H. Walker, and T, P. Singer, J. Biol. Chem, 247, 4510 (1972). 4z D. E. Edmondson and T. P. Singer, FEBS Lett. 64, 255 (1976). 4.~D. B. Winter and T. E. King, Biochem. Biophys. Res. Commun. 56, 290 (1974). 44 W. C. Kenney and P. C. Mowery, in "Flavins and Flavoproteins" (T. P. Singer, ed.), p. 532. Elsevier, Amsterdam, 1976.
[48]
SUCCINATEDEHYDROGENASE
483
r e a c t e d i n h i b i t o r a n d p r o t e o l y t i c d i g e s t i o n , l e a d s to t h e a p p e a r a n c e o f a ~4C-labeled p e p t i d e in t h e u n p r o t e c t e d s a m p l e , w h i c h is n o t s e e n in the p r o t e c t e d o n e . I s o l a t i o n o f this p e p t i d e s h o u l d p r o v i d e t h e r e q u i s i t e m aterial f o r d e t e r m i n a t i o n o f th e a m i n o a c i d s e q u e n c e at t h e s u b s t r a t e b i n d i n g site. A s o f this w r i t i n g this s e q u e n c e has n o t b e e n a n a l y z e d b e c a u s e d i g e s t i o n w i t h v a r i o u s p r o t e o l y t i c e n z y m e s has y i e l d e d p e p t i d e s t h a t ar e f ar t o o large f o r s e q u e n c i n g by c o n v e n t i o n a l p r o c e d u r e s .
[48] E P R a n d Other Properties of Succinate Dehydrogenase
By TOMOKO OHNISHI and T s o o E. KING General Features of Succinate Dehydrogenase Various lipid-flee, soluble, succinate dehydrogenase preparations ( S D H ) 1 h a v e b e e n r e p o r t e d f r o m s e v e r a l l a b o r a t o r i e s ; all p r e p a r a t i o n s c a n be s u m m a r i z e d in T a b l e 12-14 w i t h c o d e s u s e d in this c h a p t e r . Abbreviations used in this article: Eh, redox potential: Era, midpoint redox potential; Fd, ferredoxin; HiPIP, high-potential iron protein; HMP, Keilin-Hartree heart muscle preparation; Q and QH2, ubiquinone and its reduced form; SDH, succinate dehydrogenase; SMP, submitochondfial particles: TTFA, trifluorotheonylacetone. 2 T. E. King, J. Biol. Chem. 238, 4037 (1963). 3 T. E. King, this series, Vol. 10, p. 322. Introduction of a water washing of the SDHabsorbed calcium phosphate gel can increase the purity of the product significantly. It is now routinely done in our laboratories. 4 T. Ohnishi, J. C. Salerno, D. B. Winter, C. A. Yu, L. Yu, and T. E. King, J. Biol. Chem. 251, 2094 (1976). '~ K. A. Davis, and Y. Hatefi, Biochemistry 10, 2509 (1971). M. L. Baginsky and Y. Hatefi, J. Biol. Chem. 244, 5313 (1969). r T. E. King, D. Winter, and W. Steel, in -Structure and Function of Oxidation-Reduction Enzymes" (A. Akeson and A. Ehrenberg. eds.), p. 519. Pergamon, Oxford, 1972. 8 D. F. Wilson and T. E. King, Biochim. Biophys. Acta 92, 173 (1964). This is a modification of the original method of Singer et al. The heart muscle preparation is used instead of mitochondria; logically HMP is a good choice as the starting material because HMP contains a powerful succinate oxidase system and, moreover, has been used successfully for other SDH preparations. Indeed, the SDH thus prepared contains flavin:iron ratio of 1:4: Singer and co-workers (see Bernath and Singer 1° and cross references cited therein) have sometimes obtained the ratio 1:2 instead of 1:4, using their starting material of acetone powder of mitochondria. H~p. Bernath and T. P. Singer, this series, Vol. 2, p. 597. H A. D. Vinogradov, E. V. Gavrikova, and V. G. Goloveshkina, Biochem. Biophys. Res. Commun. 65, 1264 (1975). v-,j. R. Kettman, Ph.D. thesis, Oregon State University, Corvallis, 1967. ~:~C. A. Yu, L. Yu, and T. E. King, Biochem. Biophys. Res. Commun. 78, 259 (1977). 14 C. A. Yu. L. Yu, and T. E. King, Biochem. Biophys. Res. Commun. 79, 939 (1977).
FLAVOPROTEINS
[47] M a m m a l i a n
Succinate
[47]
Dehydrogenase
By BRIAN A. C. ACKRELL, EDNA B. KEARNEV, and THOMAS P. SINGER
This article deals with four aspects of mammalian succinate dehydrogenase: (1) a critical comparison of assay methods, (2) activation-deactivation of the enzyme, (3) the active site of the enzyme, and (4) comparison of the properties of various purified preparations, including recent improvements of procedures for isolating the reconstitutively active form in high yield and with a high turnover number. Assay of Succinate Dehydrogenase
Activity
Since the catalytic turnover of succinate dehydrogenase is faster than the rate-limiting step in the respiratory chain, artificial electron acceptors are usually used for assays of the enzyme in order to ensure that full activity is being measured (a necessity in determining kinetic constants and turnover numbers, for instance). Of these, phenazine methosulfate (PMS),la with either DCIP or cytochrome c as terminal oxidant, may be used with either particulate or soluble preparations. With particle preparations such as complex II and ETP, the ubiquinone homologs Q1 and Q2, as well as the ubiquinone analog DPB, may be used in place of PMS, with the same activity, and nearly the same activity may be measured by using the free-radical form of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD', also called Wurster's blue). With soluble preparations of the enzyme, assays with PMS + DCIP, with low concentrations of ferricyanide ("low Km" ferricyanide assay) or with TMPD', all at Vmax, show the same rate of succinate oxidation. The precautions recommended for each of these procedures, their pitfalls and limitations are briefly noted at the end of each method and are more fully discussed elsewhere. 2'3 i The original studies reported here were supported by Program Project HL-16251 from the National Institutes of Health and by Grant No. PCM 76-03367 from the National Science Foundation. ia Abbreviations: DCIP, 2,6-dichlorophenolindophenol; DPB, 2,3-dimethoxy-5-methyl-6pentyl-l,4-benzoquinone; MES, 2-(N-morpholino)ethanesulfonic acid; PMS, phenazine methosulfate; TTF, thenoyltrifluoroacetone. 2 T. P. Singer, Methods Biochem. Anal. 22, 123 (1974). 3 B. A. C. Ackrell, C. J. Coles, and T. P. Singer, FEBS Lett. 75, 249 (1977).
[47]
MAMMALIAN SUCCINATE DEHYDROGENASE
467
Phenazine Methosulfate Assay The enzyme, reduced by its substrate, succinate, is reoxidized by PMS, which is varied in concentration to obtain V. . . . Reoxidation of the reduced PMS is accomplished by either DCIP or cytochrome c, whose reduction is monitored spectrophotometrically. The characteristics of the recording spectrophotometer required (scale expansion, recorder speed, optical density offset) have been described." Polarographic or manometric measurements of the succinate-PMS reaction are not recommended, as they are less sensitive and the rate is limited by the oxygen concentration.
Reagents 1. Potassium phosphate buffer, 200 mM, pH 7.5 (at room temperature) 2. Sodium succinate, 200 raM, adjusted to pH 7.5 3. KCN, 100 mM 4. DCIP (General Biochemicals, Inc.), 0.05% (w/v) in 100 mM potassium phosphate, pH 7.5 5. Phenazine methosulfate (Sigma), 0.33% (w/v), in glass-distilled water. Store in amber or 'qow actinic" red glassware, frozen when not in use; protect from light during use.
Procedure. If the enzyme to be assayed is not in the fully activated state, it should first be treated as described in a subsequent section. If a fully activated preparation is to be assayed, the reaction is usually started by adding the enzyme to the complete reaction mixture. Each cuvette receives 0.75 ml of phosphate buffer, 0.3 ml of succinate, water to give a final volume of 3 ml, 0.1 ml of DCIP (to give an absorbance of 1.0 to 1.25 at 600 nm in I-cm light path) and varying amounts of PMS. The latter is varied in the range of 0.3 to 0.03 ml of PMS per 3 ml of final volume. The cuvettes are brought to the temperature of assay while protected from light and placed in the spectrophotometer; 0.03 ml of KCN is added, and the enzyme immediately thereafter to start the reaction. If the reaction is started by addition of the dyes instead of the enzyme, these should be at the temperature of assay, as the volumes added are significantly large. The amount of enzyme used should cause an absorbance change corresponding to 30-50% of the chart width in 1530 sec. An absorbance range of 0-0.2 to 0-0.4 absorbance unit full scale is recommended, with a recorder chart speed of 10-12 inches/min at high PMS concentrations and 5-6 inches/min at the lower dye concentrations. The temperature of assay can be chosen for convenience but is usually 30 ° or 38 °. If it is desired to determine the extent of activation of a given
468
VLAVOPROTEINS
[47]
preparation, the assays should be carried out at or below 15°, since succinate does not activate the enzyme significantly during the assay in this temperature range. Activity is calculated from double reciprocal plots of absorbance change vs PMS concentration, using the millimolar extinction coefficient of 19.1 at 600 nm for DCIP.
Comments. In order to assure that no electron flux to cytochrome c and Oz via the respiratory chain occurs in the assay of membrane-bound preparations, antimycin A (1 tzg per milligram of protein) is included in the assay mixture as well as the KCN. In intact mitochondria penetration of PMS is rate-limiting. To overcome this, 1-2 /zg of crude Naja naja venom or of partially purified phospholipase A2 from this venom 4 and 750 ~ CaC12 are added to the assay mixture, and the reaction is started with dyes to allow time for phospholipase action. While the determination of activity at Vmax with respect to PMS is essential in kinetic studies, in determination of the turnover number, in studies with inhibitors, and in comparisons of soluble and particulate enzymes, because the Km for PMS is altered on extraction of the enzyme and on treatment with certain inhibitors, if only a rough estimate of the activity is desired, or if the experimental conditions do not bring about a change in Kin, the highest level of PMS recommended may be used in lieu of varying dye concentration. When membrane-bound succinate dehydrogenase is being assayed, deviations from linearity in double reciprocal plots are seen, since DCIP is reduced without the mediation of PMS. This "direct" reduction contributes significantly only at the lower concentrations of PMS. Substitution of heart muscle cytochrome c (50/xM final concentration) for the DCIP solves this problem, since cytochrome c is not reduced in the reaction without PMS, in the presence of antimycin. Although a polarographic variant of the PMS method has been used by some workers, for reasons detailed elsewhere 2 this procedure is not recommended. Ferricyanide Assay Ferricyanide has been widely used for the assay of succinate dehydrogenase. The conventional assay is spectrophotometric and uses either a fixed, high ferricyanide concentration ( - 5 mM) or a series of high ferricyanide concentrations (1.7-10 mM), with extrapolation to Vmax. Under either of these conditions the activity with ferricyanide is less 4 T. Cremona and E. B. Kearney, J. Biol. Chem. 239, 2328 (1964).
[47]
MAMMALIAN SUCCINATE DEHYDROGENASE
469
(-'
•
~ ,'. . . : .
.;.
5= dZ -;~"== >,.=-~
~
V;
~=h"'-
= . ~ .~ ~ . ~
~
.
. ~
"-
-
.
._
~ ~ ~ ~"
K.E
='~
.
481
482
FLAVOPROTEINS
[4 7]
dalton Subunit, za'3~ as well as in flavin peptides obtained by proteolytic digestion. 39,40 A flavin pentapeptide of the structure Ser-His-Thr-Val-Ala
I Flavin
may be obtained by precipitating the enzyme with trichloroacetic acid, digestion with trypsin-chymotrypsin, and purification of the flavin peptide by chromatographic procedures. 4oA longer flavin peptide, containing 23 amino acids, may be obtained by tryptic digestion, followed by chromatography on Florisil, DEAE-cellulose, and phosphocellulose. 41 The amino acid sequences of both peptides have been determined. 41 The 8c~[(N)3-histidyl]-FAD moiety may be obtained by digestion of the pentapeptide with aminopeptidase? °'41 Acid digestion of the peptide results in the corresponding histidyl anhydroflavin. 4z Substrate Site
Identification of the amino acid environment at the substrate site has been difficult because no stable, covalent adducts of the enzyme with substrates or competitive inhibitors are available. Even oxaloacetate, which is extremely tightly bound to the deactivated form of the enzyme, is released on denaturation of the enzyme, 12.43,44presumably because the thiohemiacetal bond is stabilized by noncovalent interactions in the native conformation. The problem has been circumvented as follows. 44 It has been found that treatment of the enzyme with N-ethylmaleimide leads to rapid alkylation of one - - S H group on each of the 30,000- and 70,000-dalton subunits. Of these, reaction with the - - S H group on the large subunit is slower, and this is accompanied by loss of catalytic activity. Alkylation of this - - S H group and loss of activity are prevented by succinate, malonate, and oxaloacetate, whereas alkylation of the other cysteine residue is unaffected by these compounds. Thus, treatment of two succinate dehydrogenase samples with [14C]N-ethylmaleimide, one of which contains malonate as a protective agent, followed by removal of un39 E. B. Kearney, J. Biol. Chem. 235, 865 (1960). 4o j. Salach, W. H. Walker, T. P. Singer, A. Ehrenberg, P. Hemmerich, S. Ghisla, and U. Hartmann, Eur. J. Biochem. 26, 267 (1972). 41 W. C. Kenney, W. H. Walker, and T, P. Singer, J. Biol. Chem, 247, 4510 (1972). 4z D. E. Edmondson and T. P. Singer, FEBS Lett. 64, 255 (1976). 4.~D. B. Winter and T. E. King, Biochem. Biophys. Res. Commun. 56, 290 (1974). 44 W. C. Kenney and P. C. Mowery, in "Flavins and Flavoproteins" (T. P. Singer, ed.), p. 532. Elsevier, Amsterdam, 1976.
[48]
SUCCINATEDEHYDROGENASE
483
r e a c t e d i n h i b i t o r a n d p r o t e o l y t i c d i g e s t i o n , l e a d s to t h e a p p e a r a n c e o f a ~4C-labeled p e p t i d e in t h e u n p r o t e c t e d s a m p l e , w h i c h is n o t s e e n in the p r o t e c t e d o n e . I s o l a t i o n o f this p e p t i d e s h o u l d p r o v i d e t h e r e q u i s i t e m aterial f o r d e t e r m i n a t i o n o f th e a m i n o a c i d s e q u e n c e at t h e s u b s t r a t e b i n d i n g site. A s o f this w r i t i n g this s e q u e n c e has n o t b e e n a n a l y z e d b e c a u s e d i g e s t i o n w i t h v a r i o u s p r o t e o l y t i c e n z y m e s has y i e l d e d p e p t i d e s t h a t ar e f ar t o o large f o r s e q u e n c i n g by c o n v e n t i o n a l p r o c e d u r e s .
[48] E P R a n d Other Properties of Succinate Dehydrogenase
By TOMOKO OHNISHI and T s o o E. KING General Features of Succinate Dehydrogenase Various lipid-flee, soluble, succinate dehydrogenase preparations ( S D H ) 1 h a v e b e e n r e p o r t e d f r o m s e v e r a l l a b o r a t o r i e s ; all p r e p a r a t i o n s c a n be s u m m a r i z e d in T a b l e 12-14 w i t h c o d e s u s e d in this c h a p t e r . Abbreviations used in this article: Eh, redox potential: Era, midpoint redox potential; Fd, ferredoxin; HiPIP, high-potential iron protein; HMP, Keilin-Hartree heart muscle preparation; Q and QH2, ubiquinone and its reduced form; SDH, succinate dehydrogenase; SMP, submitochondfial particles: TTFA, trifluorotheonylacetone. 2 T. E. King, J. Biol. Chem. 238, 4037 (1963). 3 T. E. King, this series, Vol. 10, p. 322. Introduction of a water washing of the SDHabsorbed calcium phosphate gel can increase the purity of the product significantly. It is now routinely done in our laboratories. 4 T. Ohnishi, J. C. Salerno, D. B. Winter, C. A. Yu, L. Yu, and T. E. King, J. Biol. Chem. 251, 2094 (1976). '~ K. A. Davis, and Y. Hatefi, Biochemistry 10, 2509 (1971). M. L. Baginsky and Y. Hatefi, J. Biol. Chem. 244, 5313 (1969). r T. E. King, D. Winter, and W. Steel, in -Structure and Function of Oxidation-Reduction Enzymes" (A. Akeson and A. Ehrenberg. eds.), p. 519. Pergamon, Oxford, 1972. 8 D. F. Wilson and T. E. King, Biochim. Biophys. Acta 92, 173 (1964). This is a modification of the original method of Singer et al. The heart muscle preparation is used instead of mitochondria; logically HMP is a good choice as the starting material because HMP contains a powerful succinate oxidase system and, moreover, has been used successfully for other SDH preparations. Indeed, the SDH thus prepared contains flavin:iron ratio of 1:4: Singer and co-workers (see Bernath and Singer 1° and cross references cited therein) have sometimes obtained the ratio 1:2 instead of 1:4, using their starting material of acetone powder of mitochondria. H~p. Bernath and T. P. Singer, this series, Vol. 2, p. 597. H A. D. Vinogradov, E. V. Gavrikova, and V. G. Goloveshkina, Biochem. Biophys. Res. Commun. 65, 1264 (1975). v-,j. R. Kettman, Ph.D. thesis, Oregon State University, Corvallis, 1967. ~:~C. A. Yu, L. Yu, and T. E. King, Biochem. Biophys. Res. Commun. 78, 259 (1977). 14 C. A. Yu. L. Yu, and T. E. King, Biochem. Biophys. Res. Commun. 79, 939 (1977).
