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The radiochemical determination of succinate oxidation
I62
SHORT COMMUNICATTONS
4 P- JOLLY;S, C. ALA!S AND 7- JOLLY;S, ArC]~. Biochem. Bioptzys., 98 (~962) 56. 5 P- ,~OLL~S, C. ALAIS AND J. JOLLieS, Biochim. Biophys. Aeta, 69 (I963) 5z-t. 6 J. m. NORTHRUP, M. KUNITZ AND R. ~-VJ[.HERRIOTT, Crystalline Enzymes, C o l u m b i a U n i v e r s i t y Press, N e w York, I948, 2 n d ed. 7 W. HABERMANN, H. MATTE~I~E~MER, H. SKY-PEcK AND H. SINOHARA, Chimia, t 5 (t96~ } 339. 8 A. G. MACI
Received August 2zst, I964 Biochim. Biophys. Acta, 97 (I965) I 5 9 - I 6 2
sc 23 074
The radiochemical determination of succinate oxidation Present methods of estimating succinate oxidation depend on measurement of oxygen uptake via polarographic or manometric techniques. The method reported here combines the advantages of convenience and sensitivity afforded b y polarography with the potential for para!1el, simultaneous measurement provided b y manometry. When succinic acid is labeled with tritium in the 2 or 3 position, oxidation causes tritium to appear as tritiated water which can be sensitively assayed b y scintiliation counting. Since this method specifically measures the dehydrogenation of succinic acid, it m a y be used to quantitate succinate oxidation not only b y tile succinate oxidase of mitochondria but also b y D P N + during the energy-linked reversal of electron transport or b y the photosynthetic systems of certain bacteria. The determination is performed in the following manner: tritiated succinic acid (47 m e / m m o ! e ; New England Nuclear Corp.) was dissolved in distilled water and neutralized to p H 8-Io. This solution was placed on a x × Io cm DEAE-Sephadex A-25 coarse or medium anion-exchange column (CI- form) and 2o-25 ml of distilled water were run through the column. This treatment removed contaminants from the [aH]succinate which did not bind to the anion-exchange resin. After elution of the [3H]succinic acid with ! M KC1, solutions of convenient specific activity were prepared b y dilution of the radioactive soIution with carrier succinic acid. The determination of succinate oxidation was carried out b y incubation of [3H]succinic acid with mitochondria for io-2o rain. The reaction was stopped b y the addition of perchloric acid to a ievel of 2 %. The control or zero time vessel contained mitochondria treated with perchloric acid or sodium cyanide prior to the introduction of [3H]succinic acid. After the reaction was terminated, an aliquot of the supernatant solution (o.2-o.5 ml) was placed on a DEAE-Sephadex column which was then Muted with two successive 5-mi portions of water. An aIiquot of the combined eluate was assayed for 3H b y liquid scintillation counting. An internal standard was employed to permit a determination of absoIute tritium content. Mitoehondria were prepared from beef heart after GREEXa or rat liver according to SCt~m'EIDZR AND HOGEBOOM2, while rat-liver-submitochondrial particles were prepared according to the method of KIzzzY a~,~I) BRoxK a. Biochgm. Biophys. Actor, 97 (I965) x 6 2 - I 6 4
BIOCI-IIMICA ET BIOPHYSICA ACTA
163
Table I shows that the numbers of/~moles of succinate oxidized calculated from the specific activity of the succinate multiplied by 4 determines the number of/~atoms of oxygen utilized (see p. 5)TABLE I RADIOCHEMICAL AND MANOMETRIC DETERMINATION OF P / O RATIO E a c h s o d i u m vessel c o n t a i n e d 5.2 m g b e e f - h e a r t - m i t o c h o n d r i a l p r o t e i n , I O O / , m o l e s s o d i u m succ i n a t e (148 ooo d e s i n t e g r a t i o n s / m i n / # m o l e ) , IOO m M KC1, 16 miV[ KH~PO4, 0. 5 mlVi A D P , 45 K M u n i t s h e x o t d l l a s e (E C2.7.I.I) ( S i g m a t y p e III), i o o mlVf glucose, 5 ° m M Tris a n d 5 m M MgCI~. T h e r e a c t i o n w a s r u n a t p H 7.4 for 23 r a i n a t 3 °0 i n a v o l u m e of 2.3 m h
i 2
Net desintegrations/min as ~H~O*
#atoms oxygen (manometric)
#atoms oxygen (tritium assay)
PO~ esterified
395 ooo 402 ooo
9.8 11. 4
lO.7 lO.9
18. 4 17. 3
* N e t d e s i n t e g r a t i o n s / m i n is o b t a i n e d b y s u b t r a c t i n g d e s i n f e g r a t i o n s / m i l l zero t i m e f r o m t o t a l d e s i n t e g r a t i o n s / m i I 1 for i a n d 2.
The data of Table II show that when the succinate oxidase system is blocked by cyanide, the amount of tritium appearing as 3H~0 is inhibited by 96 %. Table n also shows significant amounts of 3H20 are not liberated in subsequent DPN+-linked oxidation of Krebs cycle acids derived from succinate oxidation since rotenone effectively blocks DPN+-linked oxidations. In our hands, this method is capable of measuring succinate oxidase activity in as little as 20/~g of mitochondrial protein. TABLE II EFFECT OF CYANIDE AND ROTENONE E a c h vessel c o n t a i n e d 1.2 m g of r a t d i v e r - m i t o c h o n d r i a l p r o t e i n , i o # m o l e s s o d i u m s u c c i n a t e (9.48" lO5 d e s i n t e g r a t i o n s / m i n / / * m o l e ) , xoo m2Vf KC1, 13 m M KH~PO4, o. 5 mM A D P , 45 K M u n i t s h e x o k i n a s e (Sigma t y p e I I I ) , IOO m M glucose, 5o mlVf Tris a n d 5 m M MgC12. T h e d e t e r m i n a t i o n s w e r e c o n d u c t e d a t p H 7-5 for 3o m i n a t a t e m p e r a t u r e of 3 °o i n a v o l u m e of 3.15 ml.
I 2 3 4 5
System
Net desintegrations/min × zo -5
#atoms oxygen used (manomelric)
#moles succinate oxidized (radiochemical)
Mitoehondria Mitochondria M i t o c h o n d r i a + 1.2-1o -5 M r o t e n o n e 1Viitoehondria + 1.2- l o -5 M r o t e n o n e M i t o c h o n d r i a + 2. 4. l o -3 M lqaCiN
6.39 7 .28 7.18 5.75 o.288
2.63 2.63 2.98 2.22 o
2.70 3.08 3.o 4 2.42 o.I2
Table III shows that it is possible to measure phosphate esterification using [a2p] orthophosphate and succinate oxidation utilizing [3H]succinate in the same vessel. Because of differences in the energy of/3 particles emitted from sH and 3~p, radiochemical determination of esterified phosphate may be conducted in the presence of tritium. Manometric calibration has shown that the number of /~moles of succinic acid oxidized by beef-heart or rat-liver mitochondria may be calculated by assuming the Biochim. Biophys. Acta, 97 (1965) I 6 2 - I 6 4
16 4
SHORT COMMUNICATIONS
specific activity to be 0.25 of its measured value. The observation that the e~ective specific activity of the succinic acid is o.25 of the measured activity may be attributed to two factors - statistical elimination of label and isotope effects. TABLE III MEASUREMENT OF P / O RATIOS IN SUBMITOCHONDRIAL PARTICLES USING 3 ~ AND 32p E a c h vessel c o n t a i n e d 0.76 rag r a t - l i v e r - s u b m i t o c h o n d r i a I p r o t e i n , 4 mM s o d i u m s u c c i n a t e (69oooo desintegrations/min/#mole), I6 mM K H 2 P O 4 (1.6. i o a counts/min/~tmoie); o15 m M A D P ; 45 KN[ u n i t s h e x o k i n a s e (Sigma t y p e ][II); Ioo m M glucose; 5 ° mM Tris a n d 5 mM MgCl 2. T h e r e a c t i o n w a s r u n a t p H 7.4 for 2o rain a t 28°.C.
No.
Total desinlegrations/min all20
/~moles /*moles P,i succinate oxidized esleri]bed
P/O
I 2
98 × IOs 117 X IOs
0.57 o.68
0.23 0.20
o.I3o o.I35
The [aH]succinate used in these experiments was randomly labeled in the 2 or 3 position. Thus, statistical elimination of two hydrogens from succinic acid would result in the loss of tritium only hail the time. The additional apparent reduction of the measured specific activity b y a factor of 2 might be due to an isotope effect, not unexpected when one replaces 1H by ~H. When uptake of oxygen by mitochondria is blocked with cyanide, no significant exchange between protons of water and protons at the 2 and 3 positions of succinic acid occurs. This demonstrates that in mitochondria, the protons on the flavoprotein moiety of reduced succinate dehydrogenase (EC 1.3.99.I) are not readily accessible to water and hence do not equilibrate with protons of the aqueous phase. A simiiar failure of protons on reduced flavins of certain flavoproteins to equilibrate with aqueous phase protons has been observed in the microsomal DPNH-cytochrome b5 reductase (EC 1.6.2.2) system by DRYSDALE, et al. 4. The authors wish to express their appreciation to Dr. E. RACKER and Dr. E, J E N N E R for many helpful suggestions°
Central Research Department* Experimental Station E. I, du Pont de Nemours and Company Wilmington, Dd. (U.S.A.) I 2 3 4
I). W. W. G.
R. A. G-OLDSBY P. G. HEYTLER
E. GREEN, A d v ~ . Enzymol., (I959) 78. C. SCHNEIDER AND G. I-t. t~2OaEBOOM, dr. Biol. Chem., I83 (I95o) 123. W. KIELLY AND J. Pt. BRONK, dr. Biol. Chem., 23 ° (I958) 521. R. DRYSDALE, M. J. SPIEGEL AND P. STRITTMATTER, dr. Biol. Chem., 236 (I96~) 2323.
Received August 26th, I964 * C o n t r i b u t i o n No. IOlO
Biochim. Biophys. Acfa, 97 (I965) I 6 2 - I 6 4
SHORT COMMUNICATTONS
4 P- JOLLY;S, C. ALA!S AND 7- JOLLY;S, ArC]~. Biochem. Bioptzys., 98 (~962) 56. 5 P- ,~OLL~S, C. ALAIS AND J. JOLLieS, Biochim. Biophys. Aeta, 69 (I963) 5z-t. 6 J. m. NORTHRUP, M. KUNITZ AND R. ~-VJ[.HERRIOTT, Crystalline Enzymes, C o l u m b i a U n i v e r s i t y Press, N e w York, I948, 2 n d ed. 7 W. HABERMANN, H. MATTE~I~E~MER, H. SKY-PEcK AND H. SINOHARA, Chimia, t 5 (t96~ } 339. 8 A. G. MACI
Received August 2zst, I964 Biochim. Biophys. Acta, 97 (I965) I 5 9 - I 6 2
sc 23 074
The radiochemical determination of succinate oxidation Present methods of estimating succinate oxidation depend on measurement of oxygen uptake via polarographic or manometric techniques. The method reported here combines the advantages of convenience and sensitivity afforded b y polarography with the potential for para!1el, simultaneous measurement provided b y manometry. When succinic acid is labeled with tritium in the 2 or 3 position, oxidation causes tritium to appear as tritiated water which can be sensitively assayed b y scintiliation counting. Since this method specifically measures the dehydrogenation of succinic acid, it m a y be used to quantitate succinate oxidation not only b y tile succinate oxidase of mitochondria but also b y D P N + during the energy-linked reversal of electron transport or b y the photosynthetic systems of certain bacteria. The determination is performed in the following manner: tritiated succinic acid (47 m e / m m o ! e ; New England Nuclear Corp.) was dissolved in distilled water and neutralized to p H 8-Io. This solution was placed on a x × Io cm DEAE-Sephadex A-25 coarse or medium anion-exchange column (CI- form) and 2o-25 ml of distilled water were run through the column. This treatment removed contaminants from the [aH]succinate which did not bind to the anion-exchange resin. After elution of the [3H]succinic acid with ! M KC1, solutions of convenient specific activity were prepared b y dilution of the radioactive soIution with carrier succinic acid. The determination of succinate oxidation was carried out b y incubation of [3H]succinic acid with mitochondria for io-2o rain. The reaction was stopped b y the addition of perchloric acid to a ievel of 2 %. The control or zero time vessel contained mitochondria treated with perchloric acid or sodium cyanide prior to the introduction of [3H]succinic acid. After the reaction was terminated, an aliquot of the supernatant solution (o.2-o.5 ml) was placed on a DEAE-Sephadex column which was then Muted with two successive 5-mi portions of water. An aIiquot of the combined eluate was assayed for 3H b y liquid scintillation counting. An internal standard was employed to permit a determination of absoIute tritium content. Mitoehondria were prepared from beef heart after GREEXa or rat liver according to SCt~m'EIDZR AND HOGEBOOM2, while rat-liver-submitochondrial particles were prepared according to the method of KIzzzY a~,~I) BRoxK a. Biochgm. Biophys. Actor, 97 (I965) x 6 2 - I 6 4
BIOCI-IIMICA ET BIOPHYSICA ACTA
163
Table I shows that the numbers of/~moles of succinate oxidized calculated from the specific activity of the succinate multiplied by 4 determines the number of/~atoms of oxygen utilized (see p. 5)TABLE I RADIOCHEMICAL AND MANOMETRIC DETERMINATION OF P / O RATIO E a c h s o d i u m vessel c o n t a i n e d 5.2 m g b e e f - h e a r t - m i t o c h o n d r i a l p r o t e i n , I O O / , m o l e s s o d i u m succ i n a t e (148 ooo d e s i n t e g r a t i o n s / m i n / # m o l e ) , IOO m M KC1, 16 miV[ KH~PO4, 0. 5 mlVi A D P , 45 K M u n i t s h e x o t d l l a s e (E C2.7.I.I) ( S i g m a t y p e III), i o o mlVf glucose, 5 ° m M Tris a n d 5 m M MgCI~. T h e r e a c t i o n w a s r u n a t p H 7.4 for 23 r a i n a t 3 °0 i n a v o l u m e of 2.3 m h
i 2
Net desintegrations/min as ~H~O*
#atoms oxygen (manometric)
#atoms oxygen (tritium assay)
PO~ esterified
395 ooo 402 ooo
9.8 11. 4
lO.7 lO.9
18. 4 17. 3
* N e t d e s i n t e g r a t i o n s / m i n is o b t a i n e d b y s u b t r a c t i n g d e s i n f e g r a t i o n s / m i l l zero t i m e f r o m t o t a l d e s i n t e g r a t i o n s / m i I 1 for i a n d 2.
The data of Table II show that when the succinate oxidase system is blocked by cyanide, the amount of tritium appearing as 3H~0 is inhibited by 96 %. Table n also shows significant amounts of 3H20 are not liberated in subsequent DPN+-linked oxidation of Krebs cycle acids derived from succinate oxidation since rotenone effectively blocks DPN+-linked oxidations. In our hands, this method is capable of measuring succinate oxidase activity in as little as 20/~g of mitochondrial protein. TABLE II EFFECT OF CYANIDE AND ROTENONE E a c h vessel c o n t a i n e d 1.2 m g of r a t d i v e r - m i t o c h o n d r i a l p r o t e i n , i o # m o l e s s o d i u m s u c c i n a t e (9.48" lO5 d e s i n t e g r a t i o n s / m i n / / * m o l e ) , xoo m2Vf KC1, 13 m M KH~PO4, o. 5 mM A D P , 45 K M u n i t s h e x o k i n a s e (Sigma t y p e I I I ) , IOO m M glucose, 5o mlVf Tris a n d 5 m M MgC12. T h e d e t e r m i n a t i o n s w e r e c o n d u c t e d a t p H 7-5 for 3o m i n a t a t e m p e r a t u r e of 3 °o i n a v o l u m e of 3.15 ml.
I 2 3 4 5
System
Net desintegrations/min × zo -5
#atoms oxygen used (manomelric)
#moles succinate oxidized (radiochemical)
Mitoehondria Mitochondria M i t o c h o n d r i a + 1.2-1o -5 M r o t e n o n e 1Viitoehondria + 1.2- l o -5 M r o t e n o n e M i t o c h o n d r i a + 2. 4. l o -3 M lqaCiN
6.39 7 .28 7.18 5.75 o.288
2.63 2.63 2.98 2.22 o
2.70 3.08 3.o 4 2.42 o.I2
Table III shows that it is possible to measure phosphate esterification using [a2p] orthophosphate and succinate oxidation utilizing [3H]succinate in the same vessel. Because of differences in the energy of/3 particles emitted from sH and 3~p, radiochemical determination of esterified phosphate may be conducted in the presence of tritium. Manometric calibration has shown that the number of /~moles of succinic acid oxidized by beef-heart or rat-liver mitochondria may be calculated by assuming the Biochim. Biophys. Acta, 97 (1965) I 6 2 - I 6 4
16 4
SHORT COMMUNICATIONS
specific activity to be 0.25 of its measured value. The observation that the e~ective specific activity of the succinic acid is o.25 of the measured activity may be attributed to two factors - statistical elimination of label and isotope effects. TABLE III MEASUREMENT OF P / O RATIOS IN SUBMITOCHONDRIAL PARTICLES USING 3 ~ AND 32p E a c h vessel c o n t a i n e d 0.76 rag r a t - l i v e r - s u b m i t o c h o n d r i a I p r o t e i n , 4 mM s o d i u m s u c c i n a t e (69oooo desintegrations/min/#mole), I6 mM K H 2 P O 4 (1.6. i o a counts/min/~tmoie); o15 m M A D P ; 45 KN[ u n i t s h e x o k i n a s e (Sigma t y p e ][II); Ioo m M glucose; 5 ° mM Tris a n d 5 mM MgCl 2. T h e r e a c t i o n w a s r u n a t p H 7.4 for 2o rain a t 28°.C.
No.
Total desinlegrations/min all20
/~moles /*moles P,i succinate oxidized esleri]bed
P/O
I 2
98 × IOs 117 X IOs
0.57 o.68
0.23 0.20
o.I3o o.I35
The [aH]succinate used in these experiments was randomly labeled in the 2 or 3 position. Thus, statistical elimination of two hydrogens from succinic acid would result in the loss of tritium only hail the time. The additional apparent reduction of the measured specific activity b y a factor of 2 might be due to an isotope effect, not unexpected when one replaces 1H by ~H. When uptake of oxygen by mitochondria is blocked with cyanide, no significant exchange between protons of water and protons at the 2 and 3 positions of succinic acid occurs. This demonstrates that in mitochondria, the protons on the flavoprotein moiety of reduced succinate dehydrogenase (EC 1.3.99.I) are not readily accessible to water and hence do not equilibrate with protons of the aqueous phase. A simiiar failure of protons on reduced flavins of certain flavoproteins to equilibrate with aqueous phase protons has been observed in the microsomal DPNH-cytochrome b5 reductase (EC 1.6.2.2) system by DRYSDALE, et al. 4. The authors wish to express their appreciation to Dr. E. RACKER and Dr. E, J E N N E R for many helpful suggestions°
Central Research Department* Experimental Station E. I, du Pont de Nemours and Company Wilmington, Dd. (U.S.A.) I 2 3 4
I). W. W. G.
R. A. G-OLDSBY P. G. HEYTLER
E. GREEN, A d v ~ . Enzymol., (I959) 78. C. SCHNEIDER AND G. I-t. t~2OaEBOOM, dr. Biol. Chem., I83 (I95o) 123. W. KIELLY AND J. Pt. BRONK, dr. Biol. Chem., 23 ° (I958) 521. R. DRYSDALE, M. J. SPIEGEL AND P. STRITTMATTER, dr. Biol. Chem., 236 (I96~) 2323.
Received August 26th, I964 * C o n t r i b u t i o n No. IOlO
Biochim. Biophys. Acfa, 97 (I965) I 6 2 - I 6 4