Prrvious reports have suggested that succinic dchydrogenase occurs only in low concentrations in plantR (3) and it has several times been reported completely absent. (1,2). In view of the importance of this enzyme in all schemes of respiration, it secmcd to us worth while to examine its occurrence in plants more thoroughly. With the Thunbrrg technique, using 2,6-dichlorophenol-indophenol as dye, thts presence of the enzyme in oat and pea seedlings was readily demonstrated. Also satisfactdry was o-cresol-indo-2,6-dichlorophenol. Neither methylenc blue nor thionin, under the same conditions, showed any apprcciahlc reduction. Not, only is the selection of dye of c*ritical importance, hut the prrsencat? of small amounts of protein is also necessary. Gelatin, ovalhumin, serum albumin, and tht globulin and albumin from pea seedlings tlave all been found cffectivc at concentrations around 0.05%. A typical experiment is shown in Fig. 1. The enzyme is nonsiderably less stable than that from animal sources, as was found also by Goddard (4) for the preparation from wheat. This fact, tog&her with tjhc experimental points above, douhtless explains its reported absence from many plant. tissues. It is perhaps worth noting that the diaphorase of plants has also been report,ed to be very unstable (6). In the prcscnt, instance it was found that 24 hr. storage at 4°C. in the dark led to virtually complete inactivat,ion (Fig. 1). The method of preparation found most effective u-as differential centrifugation of freshly macerated seedling tissue over 0.4 ,W sucrose in t.he cold. This procedure is essentially that of Hogeboom et al. (5) for the preparation of mitochondria from liver. Its effect,iveness suggested to us that the succinic dehydrogenase of plants may also
Fra. 1. 1: Complete system. 2: Same plus fluoride 10m2 ii/l. 3: Minus protein in Thunberg tube. 4: Stored overnight without protein-buffer preservative. Complete system: 0.0008 M 2,6-dichlorophenol-indophenol (1: 30,000); 0.05 M sodium phosphate, pH 6.8; 0.025 M sodium succinate; 0.1 mg./ml. bovine serum albumin; enzyme isolated from 50 mg. (fresh weight) etiolated 7-day-old pea stems. Final volume: 4.0 ml. Temp. 25°C. Reduction followed on Klett-Summerson colorim-
be present in mitochrondria. blethods found effective in stabilizing the giant mitochondria of insects (7) have been found to increase the stability of the plant succinic dehydrogenase. Best results have been obtained with 1% crystalline serum albumin plus 0.53 M phosphate buffer, pH 7.5, and complete exclusion of oxygen. Such preparations have been kept for 96 hr. without appreciable loss of activity. The enzyme is inhibited 100% by 5 X 10e6 M p-chloromercuribenzoate or by lo-* M phenylmercuric nitrate. It is also inhibited by malonate. Amongst other inhibitors t,ried, an unusual reaction was found with fluoride: 3 X 10m3M NaF produced slight inhibition, and 5O’$c inhibition resulted at about lo-* :\I (Fig. l).’ The explanation of this inhibition requires further study. REFERENCES 1. BERQER, J., AND AVERY, G. S., JR., Am. J. Botany 30, 290 (1943). 2. BONNER, J., AND WILDMAN, S. G., Arch,. Biochem. 10, 497 (1946). 3. DAS, N. B., AND SEN GUPTA, P. K., Ann. Biochem. Exptl. Med. (India) 1, 117 (1941). 4. GODDARD, D. R., Am. J. Botany 31,270 (1944). 5. HOQEBOOM, G. H., SCHNEIDER, W. I?., AND PALLADE, G. E., .J. Biol. Chem. 172, 619 (1948). 6. LOCKHART, E. E., Biochem. J. 33, 613 (1939). 7. WILLIAMS, C. M., private communication, 1951.
tiarvard University, Biological (Cambridge 58, Massachusetts Received May 29, 1951
CARL A. PRICE KENNETH V. THIMANN
r This inhibition by fluoride has been reported also by Slater and W. D. Bonner Biochem. Sot., June 16, 1951) for the enzyme from heart muscle, and is ascribed to a competitive process. (PTOC.