Inhibition of succinic dehydrogenase by polysulfonated compounds

Inhibition of succinic dehydrogenase by polysulfonated compounds

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 68, 432451 (1957) Inhibition of Succinic Dehydrogenase by Polysulfonated Compounds A. 0. M. Stoppani a...

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Inhibition of Succinic Dehydrogenase by Polysulfonated Compounds A. 0. M. Stoppani and J. A. Brignone From the Department University

of Biochemistry, School of Medicine, of Buenos Aires, Argentina



10, 1956

Succinoxidase, the enzyme system that catalyzes succinate oxidation by molecular oxygen, is inhibit’ed by suramin [hexasodium sym.-bis(maminobenzoyl-m-amino-p-methylbenzoyl- 1-napththylamine-4,6,8-trisulfonate) carbamide], succinic dehydrogenase being apparently the component of succinoxidase more sensitive to suramin (1, 2). The experiments to be reported in this paper show that suramin and its component 1-naphthylamine-4,6, S-trisulfonic acid inhibit succinic dehydrogenase like competitive inhibitors. Since the studies of Hopkins et al. (3) it is known that succinate competitors protect succinic dehydrogenase from thiol reagents. On these grounds the possible interference of suramin in the action of several sulfhydryl detectors has been investigated taking into consideration that in the treatment of trypanosomiasis suramin has been used jointly with chemotherapeutic agents which are known to react with thiols (4-6). EXPERIMENTAL METHODS Enzyme Preparations Succinoxidaae was obtained from pig heart muscle as described by Slater (7). The precipitation of the enzyme was carried out eit,her (a) at pH 5.2, the precipitate being spun off at 2500 r.p.m. and 4” in the refrigerated International Centrifuge PR2; or (b) by centrifugation for 15 min. in the Servall SSl centrifuge at 15,000 X g. The precipitated succinoxidnse was suspended in phosphate buffer 0.04 M or in borate buffer 0.03 M, at pH 7.3 according to Bonner (8). The latter preparations are referred to as succinoxidase-borate. The concentration of succinoxidase in each preparation was estimated by the weight of the protein dried at 100” according to Slater (7). The specific activity of the enzyme preparations is represented by the Qo, &l. 02 taken up/hr. by 1.0 mg. protein). Cytochrome c (0.34% iron) was prepared according to Keilin and Hartree (9).







Em yme Activities Succinoxidase and succinic dehydrogenase activities were measured according to Slater (7). In some experiments the phosphate buffer was replaced by borate (Palitszch buffer) plus ethylenediamine tetraacetate or histidine [cf. (Sj]. Succinic dehydrogenase was also measured anaerobically nit.h ferricyanide as oxidant ilo). All measurement,s were carried out at 37’.

Rea.gents The following have been used: suramin (trade name: moranyl) from Rhodia Argentina (Poulenc Freres) ; acaprin [l, 3-bis(6-quinolyl)ureadimetho?cysulfate] from Winthrop Chemical Co., Inc.; trypan red and trypan blue, from G. Grtibler 8: Co.; Chlorazol Fast Pink and (Jhlorazol Sky Blue from Imperial Chemical Industries; melantherine BH, Chlorant.ine Red 8BN and .4cid Black KN from Ciba S. A.; 1-naphthylamine-J,6,8-trisulfonic and 1-naphthylamine-3,6,%trisulfonic acids from Bayer A. G. (the latter was conveniently recrystallized). a-Amino+ hydroxyphenylarsenoxide hydrochloride was obtained from Parke Davis & Co.; iodoacetic acid from British Drug Houses; osalacet,ic from H. bl. C!hemical Co. Ltd.; 2,3-dimercaptopropanol from Boots Pure Drug Co. and p-chloromercuriphenol, from British Drug Houses. p-Chloromercuribenzoic acid (ll), o-iodosobenzoic acid (12), chloroacetophenone (13)) and methyldichloroarsine (14) were prepared according to the references stated in parent,heses. Glutathione was oxidized by shaking with air a solution of the reduced form (from Schwarz Laboratories Inc.) until the oxygen consumption ceased. The reagents were neutralized to pH 7.3. At this pH, methyldichloroarsine hydrolyzes giving methylnrsenoxide to which the results will be referred. Chloroacetophenone was dissolved in freshly dist.illed peroxide-free dioxane and added in 5.0.~1. solution. The controls received the same amount, of dioxane. Suramin was hydrolyzed by treating 128 mg. of the drug with 6.0 ml. of 33.3% (w/w) hydrochloric acid in a sealed tube, at loO”, for 6 hr. The suspension was evaporated to dryness under reduced pressure, and the residue was dissolved with distilled n-ater at pH 7.4.

In.terjerence of SuCfona.tQdCompounds and Thiol Reagents The interference of suramin (or related compounds) with the action of thiol reagents has been assayed by “previous incubation” experiments where concentrated succinoxidase was treated at, 37” (a) with suramin (or other protectors), (b) the same plus thiol reagents, and (c) with thiol reagent alone. The inhibited preparations as well as the respective controls were diluted with phosphate buffer or distilled water, and an aliquot was placed in the Warburg flask to measure the activity of succinoxidase or succinic dehydrogenase. In this way the concentration of reversible inhibitors of succinic dehydrogenase (like suramin, etc.) was greatly diminished at the time of measuring the enzyme activity. The action of suramin on succinoxidase inactivation with BAL was tested shaking the concentrated enzyme suspension with B.4L, BAL plus suramin, or suramin alone. The enzyme suspensions and the respective controls were then diluted, and succinoxidase was measured as described.





Expressio?l, of Results The interference of suramin (and ot,her prot,ectors of succinic dehydrogenase) with the thiol reagents has been expressed by t.he percentage diminution of the action of the latter when allowed to react with succinoxidase in the presence of protectors. The protection (P) of succinic dehydrogenase is calculated with t.he equation P = lOO[(Z,, - Z,,,)/Z,,] (‘%) where II, is the percentage inhibition of succinic dehydrogenase by t,he thiol reagent and II, r the percentage inhibition in the presence of thiol reagent., surnmin, or other protectors. Under the conditions of “previous incubation” experiments suramin and the naphthylaminetrisulfonic acid alone did not affect the act.ivit.y of succinic dehydrogenase, and therefore the respective experimental values will be omitted in the tables. A similar equation has been applied to calculate the act.ion of suramin on succinoxidase inactivation with BAL.

Abbreviations When convenient, the following will be introduced: 3-amino-l-hydroxyphenylarsenoxide, mapharside; and 2,3-dimercaptopropanol, BAL. Unless stated otherwise, NATS refers to 1-naphthylamine4,6,%trisulfonate.


I 100

, 200

I 300


I 400



FIG. 1. Competitive inhibition of succinoxidase by suramin plotted according to Lineweaver and Burk (15) : 0.8 mg. heart. muscle succinoxidase (Qo2, 120). Succinoxidase activity measured manomet.rically in t.he presence of 0.13 M phosphate, pH 7.3 and 0.016 df succinate. Final volume 3.0 ml. 37”. Suramin: 0, 0.041 m&f; A, 0.011 mM and 0, none. u-1, reciprocal of the rate (~1. O&O min.) of SLWcinate oxidation. s-l, reciprocal of succinate molarity.







Succinoxidase and Succinic Dehydrogenasc Inhibitiota 1. Quantitative Treatme& of Succinoxidase Inh.ibition by Sura.min. The compet,itive action of suramin can be shown according to Lineweaver and Burk (15). If the reciprocal of the enzyme activity, free or inhibited by suramin at 0.041 or 0.011 mJI is plotted against F’s, t.he straight lines obtained fit Eq. (1) and intercept the ordinate at the same value (Fig. 1). In Eq. (1). 1’ is the activity of t,he enzyme uninhibited, v; same inhibited by suramin ; s, succinate concentration ; i, suramin concentration; K, and Ki the Michaelis-hlenten co&ants of succinate and suramin. In the experiments l,‘p = 1 1’ + l,‘T’(K,

+ i K,,!Ki)



represented in Fig. 1, K, was 1.32 X lop3 M and k’i were respecti\-ely 2.20 (line a-i) and 2.50 (line B) X 1OV AI. The addiCon of cytochrome c has been avoided in t.his experiments as it will be shown below that cytochrome c affects the action of suramin. According to and Bonner







FIG. 2. Inhibition of sucrinosidasc by suramin: 1.0 mg. heart muscle succinosidase (Qo, ,380). Similar conditions as in Fig. 1. V, rate (~1. 02/10 min.) of succinate oxidation by the free enzyme; and v, same in the presence of suramin. 0, suramin concentration versus enzyme inhibition (%); A, same, versus V/U.





(16) the heart muscle succinoxidase particles contain enough cytochrome c (endogenous) to give satisfactory activities for kinetic experiments. The relation between suramin concentration and percentage inhibition of succinoxidase is shown in Fig. 2, line A. If succinoxidase inhibition is represented by plotting ?‘/v against the inhibitor concentration (i), a straight line is obtained (line B). These dat,a fit Eq. (2) where the symis the slope and bols have the meaning already given and (K,/Ki)/s 1 + KJs the intercept of line B with the v axis (17). The value 535 obt,ained for K,/Ki from the slope of line B in Fig. 2 is fairly consistent with that, calculated w&h the values found for K, and Ki in Fig. 1, namely, 566. These values give a measure of the relative affinity of suramin and of succinate for succinic dehydrogenase. They were not varied when borate buffer was used (with ethylenediamine tetraacetate as enzyme activator) instead of phosphate.

V,h = 1 + K,/s + (K,IKi)



Equation (2) can be changed into Eq. (3) dividing both members by V and making (1 + KJs)/V = a and (K,,/K J/sV = b. 12is assumed t.o be uniby in Equ. (2). l/u = a + bi”


According to Eadie (18), ‘n gives the number of molecules of inhibitor which react with one enzyme active group. When the plot of l/v against i is a straight line, one molecule inhibitor is required for each “active group” of enzyme. From Fig. 2 can be t#hen concluded that one molecule of suramin combines with one active group of succinic dehydrogenase. Although suramin combines firmly with succinic dehydrogenase, the combination is reversible as shown by the following experiment. A concentration of 38.0 mg./ml. succinoxidase was added to suramin 1.5 mM, that is, a concentration which produced complete inhibition in a control experiment. After a few minutes incubation at 37”, the enzyme suspension was diluted tenfold, reducing suramin concentration to a level able to produce partial inhibition of succinoxidase at the concentration of 3.8 mg./ml. sucinoxidase. A 90% activity of succinoxidase in regard to the controls was then observed, that is, about t’he same as found in a control treated directly with the final concentration of suramin. In the presence of a high concentration of cytochrome c the action of suramin on succinoxidase diminishes significantly. Cytochrome c binds




TABLE InJluence

of Cytochrome




c on Succi~noxidase


by Suramin

0.70 mg succinoxidase; 0.13 M phosphate pH 7.3; 0.033 M succinate; 0.14 mlCl suramin. Final volume, 3.0 ml. 37”. Inhibition of succinoxidase by suramin 4L

Cytochrome c M X 10%

48.2 76.0 s3.0

1.3 0.7 0.0

sursmin to some extent making it, ineffective on succinic dehydrogenase (Table I). This may be due to the basic character of this protein and probably has little connections with t,he inhibition of the succinoxidase














./ A’ ./ .


2x- .J’ /

FIG. 3. Succinic dehydrogenase inhibition by suramin: 0.93 mg. succinoxidase, Qo? (KO, are equivalent to 102), 323. Succinic dehydrogenase activity measured in the presence of 0.029 111ferricyanide, 0.02 111bicarbonate, and 0.016 M succinate; 3.0 ml final volume; 5% CO* in Nz in the gas phase. V, rate bl. CO&O min.) of succinate oxidation by free succinic dehydrogense; and v, same in the presence of inhibitor.





TABLE Comparatille


OJ Succinoxidase

1.8 mg. succinoxidase-borate ((Jo,, ml. 37”. pH 7.3. EDTA: ethylenediamine

II by Srcrattrin

and Hydrolyzed



Phosphate 0.12 M Borate buffer 0.08 Phosphate 0.13 M Borate buffer 0.08 drolyzed)


420). Succinate 0.033 ilf. Final volume 3.0

+ suramin 0.25 mM M + EDTA 0.2 mM + suramin 0.25 mM + suramin 1.0 mill (hydrolyzed) 111+ EDT-4 0.2 mM + suramin 1.0 mM (hy-

Succinoaidase inhibition To

93.0 93.0 38.0 60.0

2. The Inhibition of Succinic Dehydrogenase by Suramin. When the action of suramin on succinic dehydrogenase is measured directly, that is with ferricyanide as oxidant, the results obtained are about the same as found when the dehydrogenase activity is measured through the cytochrome c-cytochrome oxidase system. The values obtained from the quotient V/v, plotted against suramin concent,ration [Eq. (2)], fit a straight line (Fig. 3) which confirms that one molecule of suramin is required to inhibit one act,ive center of succinic dehydrogenase. 3. Structwal Conditions =l#ecting Suramin. The integrity of suramin is essential for its effectiveness on succinic dehydrogenase. In confirmation of Town ef al. (2), the resuks of Table II show that acid-hydrolyzed suramin loses a great deal of it’s original inhibitory power, and that t’he inhibition produced becomes dependent on the composition of the medium. In fact, hydrolyzed suramin is less active in phosphate than in borate. The presence of I-naphthylamine4,B ,8-trisulfonate in the hydrolyzate, which is shown by its yellow color, led us to compare the action of the free sulfonic acid and suramin. 4. Inhibition of Succinic Dehydrogenase by I-Naphthylamine-4,6,8Trisulfonate. The action of naphthylamine trisulfonabe is proporhional to its concentration and is affect,ed by the presence of phosphate (Fig. 4). This suggests that naphthylamine trisulfonat,e is mainly responsible for the inhibitory action of hydrolyzed suramin. The protective action of phosphate appears to be specific as it cannot be replaced by sulfate of equivalent ionic strength. By plotting T’,/zr against inhibitor concentration straight lines are obtained, as with suramin (Fig. 4). Similar results were obtained in experiments where succinic dehydrogenase was measured with ferricyanide as oxidant. Therefore [Eqs. (2) and (3)] one active center of succinic dehydrogenase is inhibited by one molecule of naphthylamine trisulfonate. The relative position of the acid groups in naph-






- 1.0 f /‘. p-






I-NAPHTHYLAMINE 4:6:8-TRISULPHONATE CONCENTRATION(mM) Fro. 4. Inhibition of succinoxidase by I-naphthylamine-4,6,S-trisulfonate: 1 .O mg. succinoxidase-borate (Qo, , 300) in 0.08 hf borate plus 0.2 mM ethylenediamine tetraacetate (lines d and B), or in 0.13 kl phosphate (lines C and DI. V/u, same as in Fig. 1. n and A, inhibitor concentration versus enzyme inhibition (70). 0 and 0, same versus V/P.

t#hylamine trisulfonate is significant for succinic dehydrogenase inhibition as shown by the lesser effecbveness of the 3,6 ,%isomer (Table III). The action of napht,hylamine trisulfonate can he studied bett,er by using borate inst,ead of phosphate buffer. Succinosidase is less active in

Cmnprcr.atirvz Inhibition

TABLE of Succinoridase

III by Naphfhylaminetrisulfonic


1.2 mg. succinoxidase; borate buffer 0.08 M, pH 7.3; ethylenediaminetetraacetate 0.34 mM; cytochromec, 6.0 X 1O-KM and succinate0.033 M. Final volume, 3.0 ml. 37”. Succinoxidase Qoz

Additions None

1.Naptlth~laminr:.4,6,8-trisrllfonnte l-Naphth~la1uinr-3,6.8-tri~~~lfonate

3.4 mM 3.4 mM

4.55 132 240

Succinoxidase inhi$tion o

69.0 47.2



I 100






300 S4





FIG. 5. Competitive inhibition of succinoxidase by 1-naphthylamine-4,6,8trisulfonate plotted according to Lineweaver and Burk (15): 1.5 mg. succinoxidase-borate (Qo? ,320); 0.08 M borate plus 0.2 mM ethylenediamine tetraacetate; I-naphthylamine-4,6,Mrisulfonate: a, 0.165 mM; A 0.066 mM; 0 0.033 mlli; and 0, none. V-I and s-1, same as in Fig. 1.

borate (8, 19), and ethylenediamine tetraacetate or histidine is required t,o give full activity t,o succinoxidase-borate. None of these activators affect the action of naphthylamine trisulfonate. On the other hand, the inhibition is strongly dependent on the succinate concentration (Fig. 5) and can be described as competitive. In Fig. 5 the straight lines -4, B, and C fit Eq. (1) and extrapolated intercept the x-axis giving K: = 83.5, 33.1 and 18.3 X 1O-3 M, respectively. From these figures, 5.30, 5.70, and 5.40 X 10e6 AT have been obtained for K i , which consistency proves the compet,itive nature of the inhibition.






The relat,ive affinities of suramin and naphthylamine trisulfonate for succinic dehydrogenase can be easily compared by the ratio of their Ki values. Taking for suramin Ki = 2.35 X 10e6 M (from Fig. 1) and for naphthylamine trisulfonate Ki = 5.47 X 10m6M (from Fig. 5), the succinic dehydrogenase combines 23 times more firmly with suramin or 11.5 times if the comparison is made with respect to an equal number of naphthylamine trisulfonate groups. 5. Selectivity qf Szira~min Action ot1 Succinoridase. Ia a series of comparative trials, the action of suramin was compared with that of compounds having in their structure ureide and/or polysulfonated groups. The inhibitions found (%) were as shown in parentheses: Espt. 1: 0.42 mM trypan red (39.2); 0.12 m&f Chlorazol Fast Pink (17.6); 0.42 mM Chlorazol Sky Blue (0.0); and 0.42 mM suramin (97.0). Expt. d: 0.15 mM trypan blue (6.6), and 0.15 m&f suramin (75.6). Expt. S: 0.33 mM acaprin (13.1) ; and 0.33 mM suramin (92.0). Expt. 4: 1.0 mM chlorantine red 8BN (47.5) ; 1.0 mM Acid Black NN (22.2) ; 1.0 mM melantherine BH (10.4) ; and 1.O mM suramin (100.0). Protection of Succinic Dehyzlrogenase Thiols Swank and Related Compounds


6. Experiments ,with Trivalent Arsenical Compowuls. Trivalent arsenical compounds inhibit succ.inic dehydrogenase, as measured aerobically with methylene blue in t,he presence of cyanide (20). This has been confirmed by measuring succinic dehydrogenase anaerobically with ferricyanide as oxidant.. Thus, in experiments with previous incubation, mapharside 0.40, 0.20, and 0.10 mM inhibited succinoxidase (16.0 mg./ ml.), 95.0, 50.0, and 23.8 %, whereas succinic dehydrogenase was inhibited 91.2, 52.8, and 22.4 %, respectively. Therefore, in succinoxidase treated wit,h arsenicals, succinic dehydrogenase becomes the limiting factor of the catalytic activit,y of the system, whichever the hydrogen acceptor is. When succino?iidase is treated with suramin and arsenicals, the action of the latter diminish as compared t,o the controls treated with arsenical alone (Table IV). The interference of suramin with the arsenical does not depend on the integrity of suramin as acid-hydrolyzed suramin prevent,s as well t.he action of arsenical compounds (Table IV). 1-Naphthylamine-4,6,8-trisulfonate also int,erferes with arsenicals (Table IV). The free sulfonic acid is even more effective than suramin which explains





TABLE Effect

of Suramin, Hydrolyzed Suramin on Succinic Dehydrogenase

IV and I-Saphlhylanrine-4,6,8-trisulfona.te Inhibition by drsenicals

Succinoxidase treated as shown for 30 min. at, 3i”, diluted with 6.0 ml. 0.2 M ph0sphat.e pH 7.3. Succinoxidaie activity of 0.3 ml. extract measured in the presence of 0.13 M phosphate pH 7.3, 5.3 X 10-b Al cyt.ochrome c and 0.014 M succinate. There was no inhibition of succinoxidase treat,ed only with suramin, hydrolyzed suramin. or NATS. Succinoxidase

mg./l.?j ml.

45.0 45.0 45.0 45.0 45.0 45.0 10.0 10.0 10.0



IMapharside 0.37 Mapharside 0.37 Mapharside 0.37 Methylarsenoxide Methylarsenoxide Methylarsenoxide hlapharside 0.35 Mapharside 0.35 Mapharside 0.35

= Protection IIof.nhibition succini~ I of succinic dehydro-

mM mM + suramin 0.57 mM mM f NATS 0.57 mM 0.94 mA4 0.91 mdi + suramin 0.38 mM 0.94 mM + NATS 0.38 mA4 mM mAl + suramin 0.57 mM mM + suramin (hydrolyzed) 0.57


g&se genase ---jR. %

72.5 2”.4 u 2.6 55.1 38.2 30.1 98.0 66.0 60.0


96.1 30.6 45.3 32.7 38.8

the strong protect,ion of surcinic dehydrogenase provided by hydrolyzed suramin. 7. Experiments wit.h 0.ridizin.g Agents. Surcinic dehydrogenase is inhibited by oxidized glutathione (21) and o-iodosobenzoate (20). Suramin and I-naphthylamine-4, A ,%trisulfonate prevent the inhibition by hoth, t,he latter being the more active protector (Tahle IT). 8. Experiments with, Alkylating Agents. Surrinic dehydrogenase is sensitive t,o a.lkylating agents like iodoacetate, chloroacetophenone, etc. (3, 22-21). Suramin and I-napht,hylamine-4 ,6,8-trisulfonate prot,ect, succinic dehydrogenase from inhibition by iodoacetate or chloroacetophenone (Table VI), and very small concentrations of sulfonic compounds prevent the action of far stronger concentrations of the alkylator. The relative position of the acid groups in naphthylamine t,risulfonate is significant for the prot,ection, as shown by the far less effectiveness of the 3,G, Gsomere. Slkylating agents react with thiols (25), and one acid equivalent is liberat.ed fol each SH bound. The reaction can be followed manometrically in bicarbonate buffer






Effect of Surumin and Naphth~~lam.ine-~,6,8-tris~~~ljo~~~~le on Succinic Inhibition by Oxidan.ts Succinoxidase 16 mg./1.2 ml., was t,reated as shown, at 37” for 30 min. (with o-iodosobenzoate) or 60 min. (with oxidized glutathione). After dilution with 5.0 ml. 0.2 Ill-phosphate, pH 7.3, succinoxidase was measured as in Table I. There was no inhibition of succinoxidase treated only with suramin or N$TS. Surcinoxidase


o-Iodosobenzoat,e 0.86 mM o-Iodosobenzoate 0.86 mBI + sura.min 0.38 mdl o-Iodosobenzoat,e 0.86 mM + NATS 0.38 mJl o-Iodosobenzoate 0.86 mJl + suramin 0.19 mM o-Iodosobenzoate 0.86 mM + NATS 0.19 mdl Oxidized glutat.hione 0.02 m:U Oxidized glutat,hione 0.02 mM + suramin 0.005 mM Oxidized glutathione 0.02 m!ll + NATS 0.005 mJi

Inhibition of succinic dehydrogenase

12.0 4.5 32.9 30.5 47.8 0.0 0.0

Protection of succinic dehydrogenase

81.0 93.0 Ai. 50.8 100.0 100.0

where each new acid equivalent is shown by the liberation of one molecule of carbon dioxide. The alkylation of cysteine with iod0acetat.e (Table VII) followed in that way, is not affected by the presence of suramin or 1-naphthylamine-i,6,8trisulfonate, nor does the latter react with iodoacetate, which eliminates the remote possibility that their binding could explain the inactivity of iodoacetate in the presence of the polysulfonated ion. Consequently, suramin and l-naphthylamine-+6,8-trisulfonate protect. the thiols of succinic dehydrogenase indirectly, that is, either by making them less reactive or by hindering their access to the specific reagents.

9. Inhibition of Succinoxidase by Metals in the Presence of Suramin. When succinoxidase is incubated wit,h p-chloromercuribenzoate or mercuric ions in t.he presence of suramin, the inhibition is scarcely affected, in spite of the submaximal concentrations of inhibitors, and the shortness of their contact with the enzyme (Table VIII). Similar results are obtained with p-chloromercuriphenol. These negative results could be explained by the fact t,hat mercurials react with components of succinoxidase not. accessible to suramin, since Cook et al. (26, 27) have found that cytochrome oxidase is inhibited by mercurials. This explanation was excluded by measuring the succinic dehydrogenase anaerobically wit.h ferricyanide as oxidant,. The inhibition brought about by 0.5’2 mM p-chloromercuriphenol (52.0 %) was scarrely diminished by bhe presence of 0.65 mAi suramin (4i.2 % inhibition). This small protection is not







Effect of Surantin, 1 -Naphthylamine-4,6,8-trisulfonate and 1 -Naphthylamine-8,6,8trisulfonate on Succinic Dehydrogenase Inhibition by Alkylating Agents

Succinoxidase treated as shown for 30 min. at 37”, diluted with 6.0 ml. 0.2 M phosphate, pH 7.3 (Expts. 8, B, and D) or 4.0 ml. distilled water (Expt. Cl. Succinoxidase measured as in Table IV (Expts. 8, B, snd C) or succinic dehydrogenase measured in presence of succinate 0.013 211,methylene blue 0.005 X, and cyanide 0.008 M (Expt. C). NATS (4,6,8-), 1-naphthylamine-4,6,%trisulfonate; NATS (3,6,8-), naphthylamine-3,6,%trisulfonate. Inhibition of succinosidase t.reated onlv with suramin, NATS (4,6,8-), or NATS (3,6,8-), 0.



= I Succinoxidase

Erperi ment


TT , -


mEtz.‘” 35.4 35.4 35.4 35.4 35.4 35.4 16.0 16.0

A d -4 B B B c c C



46.0 46.0





Lodoacetate 10.8 mill Iodoacetate 10.8 m2ll + suramin 0.20 mM IodoacetaLte 10.8 mM + NATS 0.20 mM Iodoacetate 10.8 mM Iodoacetate 10.8 mJ1 + suramin 0.02 mM Iodoacet,ate 10.8 mM + NATS 0.02 mM Iodoacetat,e 20.0 mM Iodoacetate 20.0mM + NATS (4,6,8-) 0.17 mM Iodoacetate 20.0 mM + NATS (3,6,8-) 0.17 mM Chloroacetophenone 0.96 mU Chloroacetophenone 0.96 mM + suramin 0.009 mY Chloroacetophenone 0.96 mM + NSTS 0.009 mM TABLE


Protection )f succinic dehydrogenase -___

[nhibitio If succin dehydro genase




67.2 6.8 -2.7 62.5 35.0 17.5 100.0 45.1

103.5 44.0 72.0 44.9



68.2 38.8







of Cysteine by Iodoacetate in the Presence of S,uramin and I-~Vaphthylamine-4,6,8-trisulfonate

Each Warburg flask contains 0.2 ml. iodoacetate 0.34 M in the side bulb; 358 pg. moles cysteine in 2.8 ml. bicarbonate 0.016 Y in the main compartment and 5% COe in Nz in the gas space. 37”. Cysteine alkylation is measured by the COz evolved after mixing iodoacetate and cysteine. CO1 evolved Additions

2.5 min. d.

None Suramin 3.2 mM NATS 0.9 mM

70.0 65.0 70.0

after 5.0 min. d.

135.0 124.0 146.0




TABLE Inhibition




of Succi,noxidase by p-Chloronlercuribenzoate Ions in the Presence of Szcmmin

and Mercuric

Succinoxidase, 40.0 mg./1.35 ml., was treated as shown for 1 min. at 37” and diluted with 6.0 ml. of 0.2 llf phosphate, pH 7.3. Succinoxidase was measured as in Table IV. There was no inhibition of succinoxidase treated with suramin only. Succiooridase

p-Chloromercuribenzoate 0.76 p-Chloromercurihenzoate 0.76 p-Chloromercuribenzoate 0.25 p-Chloromercuribenzoate 0.25 Mercuric chloride 0.25 mM Mercuric chloride 0.25 mM +

Inhibition of succinogdase IC


mdl mM + surnmin mJf mM + suramin suramin

Protection succinyidase ,c

59.0 55.0 28.2 23.9 21.2 20.3

1.3 mM 1.3 m.ll

1.3 mM


6.8 15.2 4.2

significant when compared with that provided by suramin against arsenicals and alkylating or oxidizing agents. So far, with the exception of t,he mercurial compounds, suramin protects the thiols of succinic dehydrogenase like succinate and its compet’itors, namely, malonate and oxalacetate. The lack of protect.ion in regard to mercurials led us to examine t.he action of t,hese inhibitors in the presTABLE Effect

of Succinate

on Succinoxidase

IS Znhibition

by Thiol


Phosphate, 0.12 M, pH 7.3; 3.9 X 10-h hf cytochrome c and 0.013 M succinate. I, inhibitor added 10 min. before succinate and P, succinate and inhibitor added simultaneously. Succinosidase activity measured 10 min. after the addition of succinate zxpt. I) or 10 min. bhereafter (Expt. P). Qo2 succinoxidase, 445. __~ ___Succiooxidas


0.46 0.46 0.46 0.46 1.84 1.84 0.56 0.56 3.20 3.20

rotection of ruccinic delydrogeoase ,y succinate




p-Chloromercuribenzoate 0.01 mM p-Chloromercuribenzoate 0.01 mill 1 p-Chloromercuribenzoate 0.005 mM p-Chloromercuribenzoate 0.005 m&Z Mercuric chloride 0.028 mM Mercuric chloride 0.028 mM Mapharside 3.8 mM Mapharside 3.8 mM Iodoacetate 8.3 mM Iodoacetate 8.3 m!lZ



50.0 36.8 13.1 79.5 67.1 84.8 3.4

43.9 64.5 15.6 96.0


, -








ence of succinate, malonate, and oxalacetate and, on the other hand, the interference of succinate, oxalacetate, and malonat,e with the arsenicals and iodoacetate so as to have standards by which to compare the effectiveness of the different enzyme protect,ors. In the preincubation experin1ent.s of the t,ype already described, in which the incubation mixture was diluted before measurement of the enzyme activity, succinat,e, like suramin, did not influence the inhibition of succinic dehydrogenase b.y p-chloromercuribenzoate. However, in experiments in which there was no dilution of the react,ion mixture aft,er TABLE h’ffect

of Oxalaceta.te on Succinic




by Thiol


Succinoxidase (&02, 435) treated as shown at 37”. After dilution with 20 ml. (expt. with p-chloromercuribenzoate) 10 ml. (expt. with mercuric ions, p-chloromercuriphenol, and mapharside), or 6 ml. (expt. with methylarsenoxide) of 0.1 fif phosphat,e, pH 7.3, succinoxidase (SO) measured as in Table IV and succinic dehydrogenase (SD) according to Quast,el and Wheatley in 0.2-ml. extract,. The effect of p-chloromercuribenzoate, mercuric ions, and p-chloromercuriphenol in the presence of oxalacetate has been calculated in comparison to a control treated only with oxalacetate. Succinoxidase

Succinoridase treatment

nag./1 2 ml

46.0 46.0 46.0 28.4 28.4 28.4 42.0 42.0 42.0 46.0 46.0 46.0 28.4 28.4 28.4

p-Chloromercuribenzoate 0.5 mdf p-Chloromercuribenzoate 0.5 mnf + oxalacetate 1.3 rnJ!f 0xalaaetat.e 1.3 mnl Mercuric chloride 0.38 rnnl Mercuric chloride 0.38 mM + oxalacetate 3.4 mM Oxalacetate 3.4 mM p-Chloromercuriphenol 0.76 mdl p-Chloromercuriphenol 0.76 mM + oxalacetate 3.4 mnf Oxalacetate 3.4 rnfif Methlyarsenoxide 2.0 rndf Methlarsenoxide 2.0 mM + oxalacetate 1.3 rnlll Oxalacetate 1.3 mnr Mapharside 0.36 rnn1 Mapharside 0.36 milf + oxalacetate 1.3 mJl Oxalacetate 1.3 mAi

I luration 0f Enzyme previous inhibition incubation I’ min. %

rotection f succinic jehydroge”E.e


5% -

2 2

SD 79.0 SD 61.8

2 2 2

SD 10.9 SD 6i.5 SD 31.2


2 2 2

SD 30.2 SD 79.0 SD 60.0


2 30 30

SD 26.6 SO 75.2

30 30 30 30

so 0.0 s0100.0 SO 12.8 so 0.0

SO 24.6

21.8 -

67.2 81.2






t.he incuhakioll (Table IX), a high conwnt rst,ion of swcinate lo\rercd the effwt of p-(,hloromcrc.Ivit,el~z~~t~~ and mercuric* iolls, I)ut# less t ban the iiAil)ition l)y iodoawtutr and arscnic~als. ()I1 the othtbr halld, cvcli ill preincbuhation c~sprtimcnt,s, oxal:wct atc gave protwtioll against. p-vhloromerc,~ui~)etizoate, p-~hloromerc~uri~~~eIlol, ad mercwic ions (Table S). In these observations, allowance has to he made c,f osalacetate’s 0~11 inhibitory action which, aft,er dilut.ion of t,he reartion mixture, is still significant on account. of the high affinity of osalacetate for succinic dehydrogenase. The inhibitory action of oxalaretate reported in Table S is calculated in relation to t.he rontrol which received ILO additiolla, whereas the inhibition hy mercurials in the presence of oxalacet.atc is ralculat,ed in relation to the succinosidase preparation incubated with osalaretate. The interference of oxalacetate wit,h merrurials is however much smaller than with arsenicals. ITinally, malonate prevents the inhihition of succinic dehydrogenase by mercuric ions and arsenicals (in agreement with Barron and Singer’s observat,ions) hut not by p-chloromercuribenzoate. Thus, in esperiments carried out, as in Table ,X, 1.3 mM malonate diminished by 82.2 and 81.7 %, respectively succinic dehydrogenase inhibit,ion by 0.30 mX mapharside (100 % illhihition) and 0.11 mJl mercuric chloride (27.8 % inhibition). On the other hand, 3.1 mJ1 malonate scarrely afferted (3.5 % increase) succinic dehydrogenase inhibition (40.2%) hy 0.51 mM p-chloromercuribenzoate. It may be concluded from these experiments that the probection of succinic dehydrogenase against mercurial compounds is. in all cases, far less evident than with the other SH detectors. TABLE Succinoridase



with BAL

in the Z’resence of Suramin

Succinosidase, 2i.S mg.j’L.0 ml., was shaken with BAL for 15 min. at 37”; then dilut.ed with 5.0 ml. distilled lvater. Succinosidase was measured in 0.2-ml. estract as in Table IV. ____

None BAL 4.0 mdf Suramin 1.8 m:\l BAL 4.0 mM + ~uramin


17.0 1.8 mill




252 110 212 122

/ /

-56.2 49.4

_ /






10. I,l&activation of Succinorida,se with BAL in the Presence of Suramin. Oxidation of succinoxidase with BAL inactivat,es a component required for the coupling of succinic dehydrogenase to cytochrome c (28). Suramin could be expected to affect the action of BAL as competitors of succinate should prevent succinoxidase inactivation with BAL (20). There is some diminution by suramin of the inactivation of succinoxidase with BAL (Table XI) though t#he protection of the BliL-sensitive factor by suramin becomes insignificant when compared with the protection of succinic dehydrogenase. Thus 0.006 pg. moles of suramin/mg. succinoxidase prevent 90.0% of the inhibition by iodoacetate (Table VI), and 0.015 pg.moles/mg. prevent 69.3 % of t,he action of mapharside (Table III), whereas 0.129 pg. moles of suramin/mg. sucainoxidase diminished only by 12.1% the action of BAL (Table XI). DISCUSSIONS Considering the values of Ki of malonate (17) and oxalacetate (29) given in the literature (5.4-9.8 X lo-” 171and 1.5 X 1O-6 M, respectively) suramin (K; = 2.3 X lo-” M) appears to be an effective competit,ive inhibitor of succinic dehydrogenase. This action of suramin depends on the presence of 1-naphthylamine-4,6, B-trisulfonate groups at the ends of the molecule. The st#ructure of the polysulfonated ion is of the utmost, importance as among the compounds studied, seven (trypan red, trypan blue, Chlorazol Fast Pink, Chlorazol Sky Blue, melantherine BH, chlorantine red 8BN and Acid Black NN) have at their ends naphthdene nuclei substituted at different places with amino, hydroxyl, or sulfonic groups, but none has an action on succinic dehydrogenase like suramin has. The select,ivity of 1-naphthylamine-4,6 ,&trisulfonate for succinic dehydrogenase is further proved by the smaller effectiveness of the 8,6, &isomer as an inhibit,or of t#he enzyme and protector against thiol reagents. The length of the molecule does not seem to be critical as Wills and Wormall (1) found equal inhibition of succinoxidase with suramin and the symmetric ureide of m-amino-p-methylbenzoyl-l-naphthylamine-4,6,8-trisulfonate, that is suramin less two m-aminobenzoyl groups. Contrary to what could be expected from succinoxidase inhibition by ureas and urethans (24), the role of the ureide group of suramin is insignificant as similar ureides, like Chlorazol Fast Pink and acaprin, have far less acbion if any on succinoxidase. As suramin and 1-naphthylamine-4,6,8-trisulfonate do not react wit#h thiols, it appears likely that. a t,hiol group is on the active area of




succinic dehydrogenase in such a position that the inhibitor molecules make it less accessible to the specific reagents. It is remarkable that being that suramin is a stronger competitor of succinate than naphthylamine trisulfonate, t,he latter is equally or more effective than suramin in the protection of the t,hiols of succinic dehydrogenase. This can be understood if it, is assumed t,hat the inhibitors may bind at different sites of the enzyme active area, and the binding of the naphthylamine trisulfonat,e thus could more effectively block t,he reaction of the sensitive thiol group. Suramin, like malonate and oxalacetate, is not very effective in the protection of succinic dehydrogenase against mercurials. Only succinate, in far greater concentra’tion, and oxalacet8ate, which has the strongest affinity for succinic dehydrogenase (29), reduce significantly the action of the organic mercurials. In this regard, mercuric ion must be singled out, as the bivalent anions oxalacetate and malonate could complex mercuric ions and protect succinic dehydrogenase by diminishing the inhibitor available t’o the enzyme. The particular behavior of the organic mercurials may be explained by their fast reaction wit.h succinic dehydrogenase (20) while arsenicals, alkylators, etc., require not less t,han 30 min. to produce complete inactivation of the Keilin and Hartree succinoxidase preparation (20,23). The -‘SH-combining compounds probably react only with that portion of the enzyme which is uncombined with succinate or the competitive inhibitors. Thus the reaction between the enzyme and arsenicals, oxidizing agents, or alkylating reagents, which is slow even when all the enzyme is in the free form, becomes very slow when most of the enzyme is combined. On the other hand, the rapid reaction between the mercurial and the free enzyme will mean that an appreciable fraction of the total enzyme will be rapidly inactivated, even when t,he concentraCon of free enzyme in equilibrium with enzyme combined with succinate or competitive inhibitor is low. ACKNOWLEDGMENTS We are grateful to E. R. Squibb and Sons, Argentina, for financial support, to Ciba S. A. for a fellowship to J. A. B, and to Bayer A. G. for the naphthylaminetrisulfonic acids. SUMMARY

1. Among several sulfonated aromatic ureides or diazo compounds, suramin was found to be the most powerful inhibitor of succinoxidase. 2. Suramin inhibits succinoxidase at, the level of succinic dehydro-




genase, fulfilling t,he requirements of t,he theory for competit,ive inhibiCon. One molecule of suramin inhibits one enzyme active center. 3. The suramin component I-naphthylamine-4,6 ,%trisulfonate inhibits succinic dehydrogenase competitively, but is less act,ive than suramin. One molecule of sulfonic acid inhibits one enzyme active center. The inhibition diminishes in the presence of phosphate ions. 4. Suc:inic dehydrogenase inhibition by trivalent arsenicals, and by alkylators and oxidants of thiol groups, is prevented by suramin, hydrolyzed suramin, and naphthylaminet,risulfonic acids. 5. Suramin scarcely affect,s succinic dehydrogenase inhibition by pchloromerruribenzoate, p-chloromercuriphenol, and mercuric ions. Succinat.e, malonate, and oxalacetate are also less effective in the protection of succinic dehydrogenase against mercurials. 6. Suramin slight,ly diminishes succinoxidase inactivation by 2,3dimercaptopropanol (BAL). The effect is not significant, when compared wit,h the protection of succinic dehydrogenase. REFERENCES 1. WILLS, E. D., AND WORMALL, A., Biochewc. J. 47, 158 (1950). B. W., WILLS, E. D., WILSON, E. J., AND WORMALL, A., Biochem. J. 47. 149 (1950). 3. HOPKINS, F. G., MORGAN, E. J., .~ND LuT~.~K-M.~NN, C., Bioch.em. J. 33, 1829 (1938). -t. MAcLE.~N, G., AND FAIRBAIRN, H., rinn. Trap. Med. Parasitol. 26, 157 (1932). 5. LOURIE, E. M., Ann. Trop. Med. Parasilol. 36, 113 (1942). 6. HAWKING, F., in “Growth Inhibition and Chemotherapy,” p. 85. Fondazione Emanuele Paternrj, Roma, 1953. 7. SLATER, E. C., Riochenz. J. 46, 1 (1949). 8. BONNER, W. D., JR., Biochem. J. 66, 274 (1951). 9. KEILIN, D., AND HARTREE, E. F., J. 36, 289 (1915). 10. QUASTEL, J. II., AND WHEATLET, A. H. M., Biochem. J. 33.936 (1938). 11. WHITMORE, F. C., AND WOOD~.~RP, G. E., Org. Syntheses i, 159 (1941j. 12. ASKENASY, P., AND MEYER, V.? Ber. 26, 1354 (1893). 13. KORTEN, H., .~ND SCHOLL, R., Ber. 34, 1901 (1901). l-2. BAEYER, A., Ann. 107, 269 (1898). 15. LINEWEAVER, H., .~ND BURK, D., J. Am. Chem. Sot. 66,658 (1934). 16. SLATER, E. C., AND BONNER, W. D., JR., Biochem. J. 62, 185 (1952). 17. THORN, M. B., Biochenz. J. 64, 540 (1953). 18. EADIE, G. S., J. Biol. Chem. 146,85 (1942). 19. BALL, E. G., AND COOPER, O., J. Biol. Chem. 160, 113 (1949). 20. SLATER, E. C., Biochern. J. 46, 130 (1949). 21. HOPKINS, F. G., AND MORGAN, E. J., Biochem. J. 32,611 (1938). 22. BARRON, E. S. G., AND SINGER, T. P., J. Biol. Chem. 167, 221 (1945). 2. TOWN,


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