STUDIES ON SUCCINIC DEHYDROGENASE

II. ISOLATION AND PROPERTIES OF THE DEHYDROGENASE FROM BEEF HEART*

BY THOMAS P. SINGER,t EDNA B. KEARNEY, AND PAUL BERNATH

(From the Edsel B. Ford Institute for Medical Research, Henry Ford Hospital, Detroit, Michigan)

(Received for publication, April 16, 1956)

During the past 2 years succinic dehydrogenase has been isolated from animal tissues as a soluble, essentially homogeneous protein. It has been shown to be a ferroflavoprotein with an unusually tightly bound flavin com- ponent, and evidence has been presented for the identity of the enzyme with “fumaric hydrogenase” (l-5). The first paper in this series (6) re- ported methods for the assay of the primary dehydrogenase and for its extraction in soluble form from a variety of animal tissues and from micro- organisms. The present paper deals with the purification of the dehydro- genase from beef heart mitochondria and surveys some of its salient cata- lytic properties and protein characteristics.

Materials and Methods

Phenazine methosulfate was synthesized by a modification’ of the method of Dickens and McIlwain (7). Antimycin A and BAL2 were the kind gifts of Dr. Frank M. Strong and Dr. Henry A. Lardy, respectively. The cal- cium phosphate gel was an aged preparation (1 to 3 months old) (8), and all the other reagents were commercial preparations of high purity. Glass- distilled water was used throughout this work. Double distilled water from commercial block tin-lined stills or reservoirs inactivated the enzyme rapidly and irreversibly, although it was found suitable for mitochondrial preparations after passage through Dowex 50 resin, Hf cycle.

Total and inorganic iron were estimated by an unpublished modification of the o-phenanthroline method (9), elaborated by Dr. H. Beinert of the

* This investigation was supported by grants from the National Heart Institute, the National Institutes of Health, United States Public Health Service, and the American Heart Association, and by a contract between the Office of Naval Re- search, United States Navy, and the Edsel B. Ford Institute for Medical Research, contract No. NR 123-337. A preliminary report has appeared (1).

t Established Investigator of the American Heart Association. 1 Mimeographed copies of the procedure are available upon request. 2 The following abbreviations are used: BAL, l,%-dithiopropanol; FMN, flavin

mononucleotide; FAD, flavin adenine dinucleotide; Tris, tris(hydroxymethyl)amino- methane.

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Institute for Enzyme Research.’ Spectrographic analyses were performed by the American Spectrographic Laboratories, San Francisco, and lipoic acid was kindly determined by Dr. W. Razzell and Dr. I. C. Gunsalus. For electrophoretic analyses and diffusion constants, the Perkin-Elmer model No. 38-A apparatus was employed. Sedimentation velocity was measured in a Specialized Instruments Corporation analytical ultracentrifuge. The dehydrogenase was assayed as previously described (6).

1 unit of succinic dehydrogenase activity is defined as 1 c.mm. of O2 up- take per minute under standard assay conditions, and specific activity is defined as units per mg. of protein. Protein was determined by the biuret method (10) with the following coefficients (optical density at 540 rnp, 1 cm. light path, given by a solution of 1 mg. of protein per 3 ml. of reaction mix- ture in the presence of 1.5 ml. of biuret reagent) : first (NH&S04 precipitate, 0.095; gel eluate and all stages thereafter, 0.110. These factors were de- termined on thoroughly dialyzed preparations of known dry weight. In particulate preparations and samples containing Tris buffer, protein was measured by dry weight.

Results

IsoZution of Dehydrogenase

The use of mitochondria instead of whole tissue for the isolation of the dehydrogenase offers the advantage of a highly concentrated source mate- rial, free from many interfering substances which would be difhcult to re- move from soluble enzyme preparations. Their use, however, also entails at least two disadvantages. First, the isolation of mitochondria in large quantity and of constant composition is not a simple problem. Second, the success of the fractionation of the soluble enzyme, after extraction from mitochondrial material, depends primarily on the quality of the mitochon- dria. Since the dehydrogenase occurs in a considerably higher stage of purity in mitochrondrial extracts than in those of whole tissues, minor var- iations in the isolation of the particles may result in contamination with particles whose proteins later interfere with the fractionation of the dehy- drogenase in soluble extracts. The procedure for the isolation of mito- chondria has been modified from a large scale adaptation (11) of the pro- cedure of Schneider (12).

Preparation of Beef Heart Mitochondria-Fresh beef hearts from young prime grade cattle, quartered and chilled in ice at the slaughterhouse, were thoroughly freed from fat and connective tissue and passed through a meat grinder (all operations at 3-5”). Lots of 400 gm. were immediately blended with 1200 ml. of sucrose-phosphate (85 gm. of sucrose and 1.85 gm. of

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T. P. SINGER, E. B. KEARNEY, AND P. BERNATH 601

KzHPOl per liter) in a special, high capacity blendor for 45 seconds. The blendor was of the overhead type, designed to fit a 7800 ml. stainless steel beaker, equipped with a 0.2 horsepower, 18,000 r.p.m. motor, a shaft 25 cm. long, and a three-pronged blade which was kept extremely sharp. The pH was adjusted to 8.6 to 8.8 by the addition of 5 to 5.3 ml. of 6 N KOH. The homogenate was immediately centrifuged for 10 minutes at 1800 r.p.m. in the International Equipment Company refrigerated centrifuge No. SR-3 by using the No. 632 cups (1000 X g at the bottom of the tube, two and one-half blendings per centrifugation). The supernatant fluid was de- canted through a double layer of cheese-cloth, care being taken not to dis- turb the sedimented nuclei, and, after dilution with 7 liters of 0.9 per cent KCl, the mitochondria were collected in the Sharples centrifuge (50,000 r.p.m., flow rate of 400 to 450 ml. per minute). The Sharples bowl was changed after the collection of mitochondria from 2000 gm. of minced heart. The sedimented layer, after resuspension by homogenization in a Waring blendor in twice its volume of a solution containing 48.1 gm. of su- crose, 6.6 gm. of KCl, and 0.45 gm. of anhydrous disodium succinate per liter, was frozen overnight.

Preparation of Acetone Powder-Treatment of the mitochondrial suspen- sion with tert-amyl alcohol in the cold extracts a certain amount of pro- tein material without bringing succinic dehydrogenase into solution and thereby simplifies the subsequent purification of the dehydrogenase.4

The thawed mitochondrial suspension was briefly blended at low speeds to assure even resuspension, and 0.111 times its volume of tert-amyl al- cohol was added. After standing 1 hour at 0’ with occasional stirring, for each liter of the mitochondrial suspension 10 ml. of 0.5 M K2HP04 were added, and the suspension was centrifuged for 15 minutes at 21,000 r.p.m. (59,000 X g at the bottom of the tube) in the No. 21 rotor of the Spinco model L ultracentrifuge. The clear yellow supernatant solution was care- fully decanted and the residue from each five Spinco tubes was homogenized in the Waring blendor with 800 ml. of acetone at -10”. The contents of two Waring bowls were stirred with an additional 4 liters of acetone for 5 minutes and then rapidly filtered by suction through a 31 cm. Biichner funnel. The moist filter cake was washed with 500 ml. of acetone, resus- pended in 500 ml. of acetone, blended, and filtered again, and the filter cake copiously washed with ether. Residual solvent was removed by spreading the filter cake on heavy paper in the cold room before a fan, and was then dried in a high vacuum at room temperature for 30 minutes. The

* The blendor was designed by Mr. W. Handrow of the Institute for Enzyme Re- search. Blueprints are available from the authors upon request.

4 Treatment with tertiary amyl alcohol is an adaptation of a step in the procedure of Green et al. (13) but serves a different purpose here.

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602 SUCCINIC DEHYDROGENASE

resulting light tan powder weighed 40 to 60 gm. per 12 kilos of heart mince.

Extraction of Dehydrogenase-The yield of enzyme on extraction is a function of the efficiency of the blending and stirring and varies from 80 to 100 per cent.

A 2 per cent suspension of the acetone powder in O.OG ht Tris buffer, pH 8.9 (pH at room temperature), was first blended at 0” for 45 seconds and then vigorously stirred for 30 minutes. The clear or slightly opalescent yellowish solution obtained on centrifugation at or above 4000 X g for 25 minutes contained the dehydrogenase in soluble form.

First Protamine and (.NH4)$04 Precipitations-Treatment of the extract with protamine results in considerable purification and in removal of im- purities which appear to combine with the enzyme and to render it highly labile. When used in excess, protamine precipitates the dehydrogenase. For best yield and purity, it is advisable to determine for a given acetone powder the amount of protamine which gives 60 to 70 per cent precipita- tion of protein, measured by light absorption at 280 rnp, and not more than 20 to 35 per cent precipitation of the enzyme, the usual range being 4.5 to 5 ml. of 0.3 per cent protamine sulfate per 100 ml. of extract.

The extract obtained above was treated with 4.5 ml. of 0.3 per cent pro- tamine sulfate in 0.03 M phosphate, pH 7.6, and after being stirred for 10 minutes, it was centrifuged at moderate speed to give a clear yellowish supernatant fluid, which contains most of the enzyme, and a heavy brown pellet. The solution was brought to 0.50 saturation with solid (NH&S04, stirred for 30 minutes, and centrifuged for 25 minutes at 4000 X g. The precipitate containing 40 to 50 per cent of the activity of the first extract was resuspended in a minimal volume of 0.005 M phosphate, pH 7.6, and dialyzed for 1 hour against a large volume of the same buffer and then for 1 hour against 0.002 M phosphate, pH 7.6, in casings of t inch diameter. The precipitated protein was removed by brief centrifugation at 18,000 X g, yielding a deep brown solution of the enzyme.

Second Protamine and (NH&SO4 Precipitations-After determination of the protein content of the dialyzed enzyme by the biuret method, the solu- tion was diluted to a concentration of 5 mg. per ml. with 0.06 M Tris buffer, pH 8.9, and stirred with 5.5 ml. of 0.3 per cent protamine sulfate per 100 ml. of enzyme for 10 minutes. Any precipitate formed was removed by brief centrifugation at 10,000 X g and the supernatant solution was brought to 0.5 saturation with respect to (NH&S04. After 20 minutes stirring and 25 minutes centrifugation at 12,000 X g, the almost colorless supernatant solution was discarded and the precipitate, which redissolved readily in a small amount of 0.005 M phosphate buffer, pH 7.6, was dialyzed in 2 inch casings against a large volume of the same buffer for 2 hours and then for

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T. P. SINGER, E. B. KEARNEY, AND P. BERNATH 603

an additional 2 hours against 0.002 M phosphate, pH 76. The clear amber solution was kept frozen overnight. Continuation of the dialysis overnight resulted in slight loss of activity. The yield of enzyme in this step was 70 to 80 per cent.

Treatment with Calcium Phosphate Gel-After dilution of t,he solution to a protein concentration of 10 mg. per ml. (biuret method), it was stirred with 0.3 mg. of calcium phosphate gel per mg. of protein for 15 minutes. The gel was centrifuged and discarded and the supernatant solution, after redetermination of its protein content with use of a biuret coefficient of 0.095, was cautiously adjusted to pH 5.2 with 0.5 M acetic acid and stirred with 1.1 mg. of calcium phosphate gel per mg. of remaining protein. The gel, containing over 90 per cent of the remaining enzyme, was centrifuged and then eluted twice by homogenization and 15 minutes stirring with 0.3 M

phosphate, pH 7.6. The volume of eluent, per gm. of protein at the begin- ning of this step, was 50 ml. in the first and 33.3 ml. in the second elution. after brief centrifugation, the nearly colorless gel was discarded. Yield of enzyme, about 60 to 70 per cent.

Third (NH4)2S04 Precipitation-The eluate was treated dropwise with saturated, neutral (NH&SO4 solution to give 0.30 saturation. After 20 minutes stirring and centrifugation, the precipitate was discarded and the supernatant layer was brought to 0.46 saturation by further addition of saturated (NH&S04 solution. After 25 minutes stirring, the precipitated enzyme was centrifuged at or above 12,000 X g, redissolved in a minimal volume of 0.005 M phosphate-O.1 M NaCl buffer, pH 7.6, dialyzed at least 2 hours against the same buffer, and then frozen. Yield of enzyme, 50 to 55 per cent.

Ultracentrifugal Separation-In good preparations the enzyme was about 70 per cent pure at this stage and was devoid of colored impurities. The remaining impurity, which was ahnost entirely a light weight protein com- ponent, could be removed by fractional ultracentrifugation at the expense of some loss of enzyme. The solution from the last step was adjusted to a protein concentration of 10 to 15 mg. per ml. (biuret factor = 0.110) and centrifuged in 3 ml. tubes, fitted by means of special thin bottom Teflon microadapters into the No. 40 rotor of the Spinco model L ultracentrifuge, for 20 minutes at 144,000 X g (at the bottom of the tube). The clear solu- tion was separated from the slight film of denatured protein and the enzyme was recentrifuged in the same equipment for 4.5 hours. The colorless upper layer and the thin layer immediately over the pellet were removed and united, and the pellet was redissolved in any convenient buffer. In gen- eral, the ratio of enzyme in pellet and supernatant fluid was about 4: 1.

Table I summarizes the purification procedure, based on 60 gm. of ace- tone powder. This amount of starting material contains 250,000 to 300,000

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units of enzyme, but a deviation of 10 to 15 per cent in yield and specific activity from the values given is not unusual. Some inactivation fre- quently occurred during ultracentrifugation since, although the total pro- tein was satisfactorily recovered in the pellet and the supernatant fluid, some 20 to 30 per cent of the activity could not be accounted for. In such instances the activity observed in the final product was corrected for in- activation by the factor (total protein recovered)/(total activity recovered in ultracentrifugation).

TABLE I

Purijkation of Succinic Dehydrogenase

step -.

Acetone powder suspension. Tris extract...................................... After 1st (NH,)$SO, step.

(‘ 2nd “ ” _, . Geleluate........................................ After 3rd (NH,)&04 step,

“ differential ultracentrifugation.. -

Total units Specific activity* .-

280,000 3 256,000 10 134,000 147

92,600 174 64,800 200 35,100 230 27,200 302t

* Based on dry weight in the first two stages and on biuret coefficient of 0.110 thereafter.

t Corrected for inactivation during ultracentrifugation.

Electrophoresis, Sedimentation, and Molecular Weight

In the pH range of 7.0 to 7.9 in phosphate and Tris buffers of ionic strength = 0.1, the enzyme migrated as a single boundary in the Tiselius apparatus (5). In preparations with a specific activity of 300, the total impurity detected was of the order of 4 to 7 per cent. At 0” in 0.1 M NaCl- 0.005 M phosphate, pH 7.1, the observed mobility was 2.8 X 1O-6 cm.2 volt-l sec.-‘.

Examinations of several preparations in the analytical ultracentrifuge at protein concentrations from 1 to 1.5 per cent revealed the presence of a single sedimenting boundary (Fig. 1) with an sm value of 6.5 S. From the latter figure and from a preliminary measurement of the diffusion con- stant (Dzo = 4.2 X lo-’ cm.2 sec.-‘), a molecular weight of 150,000 has been calculated. This value is probably too low since the sedimentation veloc- ity is not corrected to zero protein concentration and an estimated 8 per cent of impurity of low molecular weight was known to be present in the

6 We are grateful to Dr. D. Basinsky of the Department of Laboratories, Henry Ford Hospital, for permission to use the Spinco analytical ultracentrifuge.

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T. P. SINGER, E. B. KEARNEY, AND P. BERNATH 605

sample used in the diffusion measurement, both of which factors would tend to lower the calculated molecular weight.

The molecular weight from light scattering data is about 220,000, as kindly determined by Dr. Robert Steiner. The most accurate estimate of the molecular weight came from analyses of the iron content of the dehy- drogenase. Preparations isolated from fresh mitochondrial acetone powders were found to contain 1 gm. atom of Fe per 49,000 gm. of protein, whereas the dehydrogenase isolated from aged acetone powders (cf. below) contained 1 gm. atom of Fe per 100,000 gm. of protein (Table II). The

Fro. 1. Sedimentation pattern of succinic dehydrogenase in the ultracentrifuge. Protein concentration, 9.1 mg. per ml.; buffer, 0.1 M NaCl-0.005 M phosphate, pH 7.6; temperature, 4.65’; speed, 59,771 r.p.m.; bar angle, 45”. Centrifugation was contin- ued until the sedimenting peak reached the bottom of the cell. Each of the four expo- sures was spaced 16 minutes apart.

TABLE II Iron Content of Succinic Dehydrogenase

Iron content’

Freshenzyme ............................... 1 mole per 49,000 gm. 320330 Enzyme from aged acetone powder .......... 1 “ “ 105,000 gm. 110-115

* Total iron or iron liberated by cold 5 per cent trichloroacetic acid.

minimal molecular weight is, then, 100,000, and the probable provisional value of the molecular weight is 200,000. As will be reported in a later publications, this figure is also in fair agreement with the flavin content of the enzyme (1 mole per 200,000 gm. of protein). Thus, the dehydrogenase isolated from fresh starting material contains 4 atoms of Fe per mole of flavin, whereas from aged material an enzyme containing 2 atoms of Fe per mole of flavin may be isolated.

Stability

In the form of the mitochondrial acetone powder, the enzyme has been preserved for periods of 3 to 8 months at -20”. In the purified form t,he

B V. Massey, T. P. Singer, and E. B. Kearney, to be published.

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606 SUCCINIC DEHYDROGENASE

enzyme is decidedly unstable. The inactivation encountered on storage of highly purified preparations at - 20” (10 to 20 per cent in 24 hours, 20 to 30 per cent in 5 days) may be due, at least in part, to a gradual loss of iron. With reagents prepared in glass-distilled water, reversible -SH ox- idation appears to play no significant part in the inactivation encountered during purification or on storage, since Versene and glutathione failed to protect the enzyme.

Turnover Number

The specific activity of the best preparations (310 at pH 7.6, 38”, 0.02 M

succinate) corresponds to a Qe, of 18,000. When corrected to maximal substrate concentration (V,.,), the Qe, is 20,000. With a molecular weight of 200,000, this is equivalent to 3000 moles of succinate oxidized per mole of enzyme per minute, a value comparable to the turnover num- ber of other flavoproteins. The concentration of phenazine methosulfate used here (2 to 3 mg. per 3 ml. of reaction mixture) gives apparent sat- uration with respect to the dye, since higher concentrations are somewhat inhibitory. The true V,.,, calculated by the double reciprocal method, is some 12 to 15 per cent higher in fresh preparations of the enzyme. Consid- eration of this additional correction gives a Qo, of 23,500 and a turnover number of 3530. It may be further calculated that the acetone powder used as starting material for the isolation of the soluble enzyme contains about 1 gm. of succinic dehydrogenase per 60 gm.

Iron Content

The total iron content of the dehydrogenase (Table II) is liberated as inorganic iron by boiling or by denaturation of the enzyme with trichloro- acetic acid. No hemin iron was detected in the preparation at any stage. Comparison of the color obtained in the o-phenanthroline reaction (9) in the presence and absence of reducing agents showed that the iron is present in the enzyme entirely in the ferrous form.

Since the acid-labile iron follows the activity closely through most of the purification and since the ratio of specific activity to iron is constant in the two types of preparation (4-Fe and 2-Fe type, Table II) with either phena- zine methosulfate or ferricyanide as terminal electron acceptors, it appears that both of these oxidants accept electrons at the level of the iron and not at the level of the flavin. The 4-Fe and 2-Fe enzymes behaved identically in most cases, i.e. behavior in the fractionation procedure, molecular weight, electrophoretic mobility, pH optimum, and phosphate requirement, etc., but differed from each other in the following respects. The 4-Fe enzyme is more sensitive to inhibition by substances which chelate with iron than its 2-Fe counterpart, and part of its iron content appears to be

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T. P. SINGER, E. B. KEARNEY, AND P. BERNATH 607

more or less readily lost; these observations will be reported in Paper III of this series. The two types of preparations also show different absorption spectra (Fig. 2). It is noteworthy that at wave lengths above 500 rnp,

1 I I I I I I I I 320 350 400 440 480 520 560 600 640

WAVELENGTH

Fro. 2. Comparison of the spectra of the 4-Fe (A) and 2-Fe (B) enzymes at a pro- tein concentration of 10.9 y per ml. and pH 7.6.

E

0.075 0.050 0.025

7 0.025 0.050 0.075 0.100

300 340 380 420 460 500 540 580 .

FIG. 3. Difference spectrum of the enzyme after reduction by succinate and the inhibition of the process by malonate. 2-Fe enzyme, 4 mg. per ml., at pH 7.6. Suc- cinate and malonate concentrations, 1.25 X 10-z M. Succinate alone bleached 18 per cent of the color at 460 mp. 0, after succinate; 0, after succinate plus malonate.

where the contribution of flavin to the color is negligible and where the iron components of other ferroflavoproteins absorb light (14)) the ratio of the colors of the two preparations agrees with the ratio of their iron content. Further, at 450 rnp, at the flavin maximum, the greater part of the color is still due to iron and not to flavin. As shown in Fig. 3, succinate causes a partial bleaching of the color, with a maximum centering around 460 ml.r; at this wave length, in the case of the 2-Fe enzyme, about 18 per cent of the

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608 SUCCINIC DEWDROGENASE

color is bleached by succinate and 63 per cent by hydrosulfite. Malonate competitively inhibits the bleaching by succinate.

E$ect of pH

The pH-activity relations of the enzyme are shown in Fig. 4. As pre- viously noted (15), the activity in phosphate buffers is considerably higher than in Tris, imidazole, histidine, or glycylglycine buffers; arsenate does not replace phosphate but inhibits the stimulatory effect, of phosphate. The enzyme used in the experiments summarized in Fig. 4 contained appre- ciable phosphate since it had been dialyzed against 0.005 M phosphate and because, under these conditions, the dehydrogenase binds a considerable

50

40

,30 9

so 0

l:b

65 7.0 z5 a.0 a.5 PH

FIG. 4. pH-activity curve of succinic dehydrogenase. Standard assay conditions, except for buffer, as noted. Each vessel contained 0.06 mg. of enzyme (third (NH,),SOd precipitate) and 150 pmoles of buffer as follows: 0, phosphate; l , imi- dazole; A, Tris. The pH values marked on the abscissa were measured at 38” in the complete reaction mixture.

amount, of inorganic phosphate. The stimulatory effect, of added phos- phate is nevertheless obvious, for concentrations of added phosphate as high as 0.05 M are required for maximal stimulation. In preparations iso- lated in the absence of added phosphate, the stimulation is about, 3-fold (15). It is seen in Fig. 4 that this effect cannot be explained as a shift of the pH optimum. No requirement for added cations has been observed.

Efect of Substrate Concentration

At pH 7.6, 38”, with phenazine methosulfate as acceptor, the Km of suc- cinic dehydrogenase of beef heart for succinate is 1.3 X 10m3 M, in agree- ment with data in the literature (16) for rat, liver succinic oxidase at, this pH and temperature, and with values obtained by the authors for Keilin- Hartree preparations of succinic oxidase from heart (38”, pH 7.6, 02 as the terminal acceptor). At 21”, the K, of the purified soluble dehydrogenase

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T. P. SINGER, E. B, KEARNEY, AND P. BERNATH 609

in the presence of phenazine methosulfate is 5.2 X 10m4 M, in agreement with the data of Slater and Bonner (4.8 X 10m4 M (17)) and Thorn (5.1 X 10e4 M (18)) obtained at 20-25” with ferricyanide as acceptor. Lolver values in the literature (e.g. 1.8 X 1O-4 M (13); temperature and electron acceptor not stated) may be due to the use of electron acceptors which do not permit the functioning of the dehydrogenase at full rapacity. The K, for succinate is remarkably constant in succinic dehydrogenase from various sources in the presence of acceptors which react with the primary dehydrogenase. Thus, the K, values of the soluble dehydrogenase from Proteus vulgaris (6) and from bakers’ yeast’ are 1.3 X 1O-3 M and 1.05 X 10m3 M at pH 7.6, 38”, with phenazine met’hosulfate as acceptor.

At pH 7.6 and 38”, the competitive inhibition of the beef heart enzyme by malonate, expressed as the ratio K,: Ki, is about 30; at 21” the ratio is about 21. The values of this ratio in the literature range from 3 to about 60 (see Thorn (18)), depending upon the temperature, type of preparation, and electron acceptor used, as fully discussed elsewhere (18). The lowest ratios reported by Thorn are extrapolated values, based on the use of methylene blue as an acceptor, which introduces a factor of uncertainty in the ealeu- lations, since methylene blue cannot react directly with the dehydrogenase (6, 17). Unlike the K,, K,:K; for malonate apparently varies with the source of dehydrogenase, since in this laboratory a value of 93 was ob served7 for the yeast enzyme at 38”, in satisfactory agreement with the preliminary data of Krebs et al. (19). The K,: K; for fumarate at 38” is 0.68 in the case of the beef heart enzyme. Oxalacetate is a potent com- petitive inhibitor, as in mitochondrial preparations, but the inhibition has not yet been quantitatively characterized.

In agreement with observations on the particulate enzyme (20, 21), the dehydrogenase is unaffected by treatment with cyanide (5 X 1O-3 M),

antimycin A (5 y per ml.), or BAL (10-S M, followed by dialysis to remove excess thiol), all of which substances are thought to inactivate the succin- oxidase system above the level of the dehydrogenase. At the concentration used in the standard assay system (2.4 X 10e3 M), phenazine methosulfate inactivates the enzyme 90 per cent or more if added to the enzyme prior to the substrate, probably by oxidation of the -SH groups; previous addition of succinate prevents this effect (6). In further agreement with the behavior of particulate preparations, the enzyme is exceedingly sensi- tive to sulfhydryl reagents, particularly of the mercaptide-forming group (Table III). Even in a relatively crude preparation such as represented

7 T. P. Singer, N. Zastrow, V. Massey, and E. B. Kearney, to be published.

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610 SUCCINIC DEHYDROGENASE

in Table III, 1 X lo+ M p-chloromercuribenzoate gave 90 per cent inhibition in the presence of 150 y of enzyme; in more purified preparations the sensitivity to this reagent increased further. As in particulate prepa- rations (22), previous addit’ion of succinate protected the dehydrogenase considerably from the effect of -SH inhibitors (Table III). Cyanide, at a concentration of 1 X 1O-3 M, reversed the inhibition by p-chloromercuri- benzoate completely, as did treatment with 1O-3 M glutathione, followed by brief dialysis to remove the unchanged thiol. The ready reactivation of the enzymatically inactive p-chloromercuribenzoate compound of the de-

TABLE III

Effect of -SH Inhibitors on Succinic Dehydrogenase

Treatment

p-Chloromercuribenzoate, 1 X 10-e M Same as Experiment 1, but succinate added before inhibitor ;D-Chloromercuribenzoate, 1 X 1O-6 M, followed by 3.5 hrs.

dialysis Same as Experiment 3, but 1 X 1OP M HCN in assay

“ C‘ “ 3, (6 3 x IO-3 “ glutathione added be-

fore dialysis o-Iodosobenzoate, 2 X lo+ M Iodoacetamide, 1 X 10-s M

fm- cenf

91 33 90

0 0

100 71

Conditions: Enzyme, 150 y at first (NHJ)SO( stage. Standard assay conditions, except that, in Experiments 1 to 3 and 5 to 7,l X IOP M 8-hydroxyquinoline was used to overcome the effect of Hz02 produced in the reaction in the place of HCN (6). In Experiment 5, 10 minutes contact was allowed between enzyme and mercurial derivative prior to addition of the thiol.

hydrogenase by the HCN present in the standard assay mixture permitted the fractionation of the enzyme throughout the procedure outlined above as the mercurial derivative. It could be shown by this means that the mer- curial derivative and the active enzyme could be purified by the same procedure, with no apparent differences in solubility and stability between the two. The inhibition of the dehydrogenase by iron-chelating substances will be discussed in Paper III.

Reaction with Various Acceptors

The relative efficiency of various electron acceptors is the same for the 2-Fe and 4-Fe enzymes at the highest stage of purity as in crude, soluble preparations (6). With the turnover of succinate in the presence of excess phenazine methosulfate taken as 100, the rate with ferricyanide is 39 in the presence of serum albumin as a protective agent, 5 to 25 in the absence

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T. P. SINGER, E. B. KEARNEY, AND I’. BERNATH 611

of albumin; all other dyes tested and cytochrome c fail to act as electron acceptors at a significant rate. (A very slow reaction with methylene blue has been observed spectrophotometrically by Dr. Massey in this Labora- tory, which may be a direct react.ion with the enzyme, unlike the rapid reaction observed nit$h particulate preparations.) Molecular O2 reacts slowly (1/5000th the rate of phenazine methosulfate), accepting electrons directly from the flavin (5). The high specificity of the dehydrogenase for electron acceptors when acting in the forward direction is all the more curious, since in the reverse reaction the reduced forms of riboflavin, FMN, FAD, and diethylsafranin all act efficiently as electron donors to fumarate. In these instances the reaction with the dyes occurs at t,he level of the flavin and not of the iron, whereas in the oxidation of succinate both phenazine methosulfate and ferricyanide accept electrons from the iron moiety.

DISCUSSIOS

The demonstration that the oxidation of succinate to fumarate is cata- lyzed by a single discrete protein molecule, whose catalytic action shows all the important features usually associated with succinic dehydrogenase action, permits the identification of this protein as succinic dehydrogenase. The differences in the behavior of the highly purified, soluble dehydro- genase and particulate preparations are the relatively high specificity of the former for electron acceptors, which is a consequence of t,he complete removal of the various electron transport components, and the activation of the soluble enzyme by phosphate, possible reasons for which have been discussed elsewhere (15). It is evident from these considerations and from the data presented in the preceding paper that preparations of the enzyme capable of oxidizing succinate efficiently with methylene blue, brilliant cresyl blue, tetrazolium, or indophenol dyes as acceptors must contain additional factors, possibly members of the original cytochrome chain, which catalyze the reaction between the primary dehydrogenase and these dyes.

The classical studies of Keilin and Hartree (23) led to the view that the succinic oxidase system, even in cell-free preparations, exists as an or- ganized respiratory system wherein succinic dehydrogenase and the various members of the cytochrome system are bound in a functional, organized unit. This view received further support from the successful isolation of the succinic dehydrogenase-cytochrome system as a particulate entity by Green et al. (13, 24). Subsequent to the authors’ report of the isolation of the soluble dehydrogenase (25), Green and coworkers explored the pos- sibilities of alternative procedures for the isolation of the dehydrogenase. These included treatment of their particulate preparations with trypsin in the presence of bile salts and alkaline degradation thereof (24, 26, 27).

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612 SUCCINIC DEHYDROGENASE

The various preparations thus obtained were reported to contain succinic dehydrogenase activity in solution, but analysis revealed significant dif- ferences between them and succinic dehydrogenase. Thus, the products of proteolysis contained 1 to 4 moles of hemin per mole of flavin, whereas the alkaline degradation product yielded 1 atom of non-hemin iron (in- stead of 4)s and no hemin per mole of flavin. These preparations further differed among each other and from succinic dehydrogenase in their ac- ceptor specificity. It was concluded apparently that the products men- tioned represent different “types” or “species” of succinic dehydrogenase (24, 26, 27). The present authors are not inclined to this view, since neither in the isolation of succinic dehydrogenase from animal tissues nor from bakers’ yeast7 was any evidence encountered for the existence of more than one type of succinic dehydrogenase nor for a change in its acceptor specificity in the course of purification. Alternative explanations have not been precluded. Since none of the preparations of Green et al. have been isolated in a state approaching homogeneity, the possibility remains that they contain, besides succinic dehydrogenase itself, contaminating hemoproteins. Another possibility, favored by the present authors, is that the degradative procedure used by these workers may be incomplete, breaking the succinoxidase chain at various points above the level of the dehydrogenase, liberating fragments which indeed contain succinic dehy- drogenase, but along with other members of the succinoxidase chain, may or may not be structurally attached, possibly by a lipide matrix, to the dehydrogcnase itself (28). If this should be true, it would be desirable to avoid confusion by clearly indicating their non-identity with the ferro- flavoprotein succinic dehydrogenase.

SUMMARY

1. Succinic dehydrogenase has been isolated from beef heart mitochon- dria as a soluble protein in a state approaching homogeneity by physico- chemical criteria. The over-all purification is about loo-fold compared with a mitochondrial acetone powder.

2. The enzyme is a ferroflavoprotein cont,aining 4 atoms of ferrous (non- hemin) iron and a mole of flavin per mole of protein (200,000 gm.). The dehydrogenase may be isolated from aged starting material with 2 atoms of iron per mole and half the specific activity.

3. Among the common electron acceptors, only the following function with the dehydrogenase, at the relative rates indicated in parentheses: phenazine methosulfate (loo), ferricyanide (39), 0, (0.02). The first two of these acceptors react via the iron moieties, whereas 02 seems to react directly with the flavin.

* The iron of succinic dehydrogenase is readily labilized by strong alkali.

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T. P. SINGER, E. B. KEARNEY, AND P. BERNtlTH 613

4. The Qo, has been measured as 20,000 and t,he turnover number as 3000 under the standard assay conditions.

5. The properties of the isolated dehydrogenase agree with those pre- viously described for mitochondrial and other particulate preparations of the enzyme, except for properties related to the absence of contaminating hemoproteins. At 38” the pH optimum is 7.7; the K, for succinate is 1.3 X 1O-3 M at 38” and 5.2 X 10V4 M at 21”. Oxalacetate, malonate, and fumarate are competitive inhibitors. Antimycin A and BAL do not in- hibit the dehydrogenase. The dehydrogenase is highly sensitive to sulf- hydryl reagents, p-chloromercuribenzoate inhibiting it in a reversible man- ner and the substrate protecting the enzyme from this type of inhibition.

BIBLIOGRAPHY

1. Singer, T. P., Kearney, E. B., and Zastrow, N., Biochim. et biophys. acta, 17, 154 (1955).

2. Kearney, E. B., and Singer, T. I’., Biochim. et biophys. acla, 17, 596 (1955). 3. Singer, T. P., Kearney, E. B., and Massey, V., Arch. Biochem. and Biophys., 60,

255 (1956). 4. Singer, T. P., Massey, V., and Kearney, E. B., Biochim. cl biophys. acta, 19, 200

(1956). 5. Singer, T. I’., Kearney, E. B., and Massey, V., in Gaebler, 0. H., International

symposium. Enzymes: units of biological structure and function, New York, 417 (1956).

6. Kearney, E. B., and Singer, T. P., J. BioZ. Chem., 219, 963 (1956). 7. Dickens, F., and McIlwain, H., Biochem. J., 39, 1615 (1938). 8. Singer, T. P., and Kearney, E. B., Arch. Biochem., 29, 190 (1950). 9. Sandell, E. B., Calorimetric determination of traces of metals, New York (1944).

10. Gornall, A. G., Bardawill, C. J., and David, M. M., J. Biol. Chem., 177,751 (1949). 11. Mahler, H. R., Wakil, S. J., and Bock, R. M., J. Biol. Chem., 204, 453 (1953). 12. Schneider, W. C., J. BioZ. Chem., 176, 259 (1948). 13. Green, D. E., Mii, S., and Kohout, 1’. M., J. Biol. Chem., 217, 551 (1955). 14. Mahler, H. R., and Elowe, D. G., J. Biol. Chem., 210, 165 (1954). 15. Kearney, E. B., Singer, T. I’., and Zastrow, N., Arch. Biochem. and Biophys., 66,

579 (1955). 16. Ackerman, W. M., and Potter, V. It., Proc. Sot. Exp. BioZ. and Med., 72, 1 (1949). 17. Slater, E. C., and Bonner, W. D., Jr., Biochem. J., 62, 185 (1952). 18. Thorn, M. B., Biochem. J., 64, 540 (1953). 19. Krebs, H. A., Gurin, S., and Eggleston, L. V., Biochem. J., 61, 614 (1952). 20. Slater, E. C., Biochem. J., 46, 130 (1949). 21. Potter, V. R., and Reif, A. E., J. BioZ. Chem., 194, 287 (1952). 22. Hopkins, F. G., Morgan, E. J., and Lutwak-Mann, C., Biochem. J., 32,1829 (1938). 23. Keilin, D., and Hartree, E. F., Proc. Roy. Sot. London, Series B, 129,277 (1940). 24. Green, D. E., and Beinert, H., Ann. Rev. Biochem., 24, 1 (1955). 25. Singer, T. P., and Kearney, E. B., Biochim. et biophys. acta, 16, 151 (1954). 26. Green, D. E., Basford, R. E., andillackler, B., in McElroy, W. I)., and Glass, B.,

Inorganic nitrogen metabolism, Baltimore, 628 (1956). 27. Green, D. E., Lecture presented at the Oxford Symposium on Mitochondria and

Other Protoplasmic Inclusions, Oxford, Sept. 19-23 (1955). 28. Edwards, S. W., and Ball, E. G., J. BioZ. Chem., 209, 619 (1954).

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BernathThomas P. Singer, Edna B. Kearney and Paul

HEARTDEHYDROGENASE FROM BEEF

AND PROPERTIES OF THEDEHYDROGENASE: II. ISOLATION

STUDIES ON SUCCINIC

1956, 223:599-613.J. Biol. Chem. 

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