3208
COMMUNICATIONS TO THE EDITOR
Vol. 84
Crystal Violet (CV) and Malachite Green (RIG), cation radical of TMB. This spectrum recently has as well as 9,p’-methylenebis-(N,N-dimethylaniline)been interpreted in detaiL6 (MBis) all yield N,N,N’,N’-tetramethyl-benzidine Finally, the uncertainty exists that, (a) one (TMB) and its corresponding diquinoid (TRIBOx) molecule of TLSB arises by oxidation of 2 molecules upon oxidation a t platinum and carbon electrodes of dye each losing a K-substituted phenyl, followed in acidic, aqueous buffers. I t can be shown that by chemical coupling reactions, or, (b) that one dye this reaction must take place Yia ejection of an in- molecule loses the central carbon phenyl unit and tegral unit composed of the central carbon atom the remaining two N-substituted phenyl groups attached to a phenyl group. In the case of the couple intra to give TMB. I t a r i be shown that the latter is the case. The peak current due to TMB RfBis, where the central group is merely -CH2--, formaldehyde results. The ethylated dyes Ethyl which arises from AIG, CV or MBis is ca. 1.8 times Violet (EV) and Brilliant Green (BG) which are that obtained from similar oxidation of dimethylanalogous to Crystal Violet and RIalachite Green, aniline. Since it has been shown that 2 moles of respectively, yield the corresponding N,N,N’,N’- dimethylaniline are oxidized to give 1 mole of tetraethylbenzidine. These results are especially TMB, these results can be interpreted to mean that interesting in view of the recent work of Eastman, 1 mole of 1IG, CV or MBis gives 1 mole of T1IB. =Inother interesting facet of the triphenylmethEngelsma and Calvin, who showed CV was formed by oxidation of dimethylaniline with ch1oranil.l ane dye oxidation is that i t occurs in a %step wave. Here the central carbon of the CV cation must I t can be shown that the two waves are due to the originate from a methyl carbon of dimethylaniline. oxidation of the hydrated and non-hydrated forms Our results show the central carbon residue can be of the dye proposed by Cigen.’ I t is only the removed by the relatively mild process of electro- hydrated form which gives rise to the TRSB during chemical oxidation. Thus i t would appear that the anodic oxidation in strong acid medium. il decentral carbon of triphenylmethane dyes (and re- tailed interpretation of the electrochemical results lated compounds) is an unusually facile portion of a will be given soon. rather complex molecule. These results appear to Acknowledgment.-This work was supported be of fundamental interest in complex organic by the Atomic Energy Commission through conoxidation-reduction processes. tract AT( 11-1)-686 and this support is gratefully The cyclic voltammetry and other electrochemi- acknowledged. cal techniques used here were identical with those (6) 2. Galus and R . N. Adams, J . Cltem. Phys., 36, 2814 (19G2). reported in the study of the anodic oxidation of (7) R. Cigen, Acta Chern. Scartd., 12, 1456 (19%). X,N-dimethylaniline. DEPARTMENT OF CHEMISTRY In 1 N sulfuric acid-sodium sulfate medium, CV UNIVERSITY OF KAXSAS Z. GALUS KAXSAS R A L P I I3‘.ADAMS oxidizes a t ca. 0.8 v. vs. s.c.e. Using a 2 v./min. LAWRENCE, triangular wave sweep voltage, no evidence of any RECEIVEI)J U N K 20, 19ti2 0.8 v. is evident on the oxidation a t less than iirst anodic sweep. However, on the second and all subsequent sweeps, an almost reversible oxidaON THE MECHANISM OF THE ENZYMATIC DECARBOXYLATION OF ACETOACETATE. 11‘ tion-reduction system is found a t the lesser anodic potential of ca. 0.55 v. The anodic and cathodic sir: half-peak potentials of this system correspond The decarboxylatioi~ of acetoacetate by the ‘ioif/iiiz2 millivolts with those of TMB-TRIBOx in decarboxylase2 purified from Cl. acetobutylicuna the same medium. previously had been shown to involve obligatory Chemical oxidatiou of triphenylmethane dyes to exchange of the carboiiyl oxygen atom with the ‘I‘MBOx is fairly well e ~ t a b l i s h e d . ~The , ~ most oxygen of the water used as ~ o l v e n t . ~ These recent work of Hanousek and Matrka 011 1 I G is findings suggest3 that a Schiff base formed between probably most definitive.5 To have further proof the enzyme and its substrate may be an active that the compound formed electrochemically was intermediate in the decarboxylation. We have ’I‘hIBOx, CV, -11G and hIBis were oxidized with therefore tried to trap the postulated Schiff base lead peroxide in sulfuric acid and the corresponding intermediate by reduction with borohydride as oxidation products were isolated as perchlorates. has been done with the Schiff bases present i n Solutions of all these compounds showed cyclic other e n z y ~ i i i c ~and - ~ similar s y s t e ~ n s . ~The suc\roltainmetry in complete agreement with the cessful results of these experiments are reported TlIB-TMB Ox oxidation-reduction system formed below. by electro-chemical means only. (1) This work was supiwrted, in part, by N. I. H. grant number Further, these oxidation products were dissolved RG-6687. iii 30% acetone-50yo aqueous buffer of pH 3.8. ( 2 ) G. Hamilt,m and 1 , €I. Westheimer, .I. A m . Chevi..Sot., 81, 2277 These solutions all showed electron paramagnetic (1959). (3) G. Hamilton and I:. H. Il’estheirner, J . Ant. Chem. Soc.. 81, 6332 resonaiice (e.1l.r.) spectra identical with that of the
+
+
+
(1959). (4) E. H. Fischer, A. B. Kent, E. R. Snyder and E . G. Krebs, J. Arn. Chem. Soc., SO, 2906 (1968). ( 5 ) E. Grazi, T . Cheni: and R. T,. Horerkrr, Biochem. nitd BioDhyT. Resrrnvch Conmi., 7, 350 (1Sfi2). (6) R. 1.. Horecker. S . P o n t r e m o l i , C. Rirci and T. Cheng, P u n i (1922). .Yuf,Acnd. Si-i..47, 1919 (1961). (j)V Hanousek and 32. l l a t r k a , Coli. Caeclioslor,. Cheiiz. C o f i ~ i i i . , ( 7 ) R. B. Dempsey and H. N. Christensea, J . Bioi. C/icW., 237, 1113 (1962). 24, I 6 (1959).
( I ) J. W. Eastman, G . Engelsma and M. Calvin, J . A m . Cheiii. Sa:.. 84, 1339 (1962). ( 2 ) 2. Galus and R . N. Adams, .I. A m . C h o n .Snr., 84. 2061 (1982). 0 ) J . Knop, Z . n ~ t n i Chrii!., . 8 5 , 2.7:3 (1931). (41 F. Kehrmann, G , R o y 1 1 , and K a m m , H1.12’. ( ‘ I i i i i i . . I , li:, 6, 16.;
COMMUNICATIONS TO THE EDITOR
Aug. 20, 1962
The enzyme was prepared from CI.acetobutylicurn (NRRL-B-527) obtained from the U. S. Department of Agriculture a t Peoria, Illinois, and was purified according to Hamilton.8 (This purified but uncrystallized enzyme is similar to that obtained e a ~ - l i e rfrom ~ . ~ A.T.C.C. 862, which is unfortunately no longer available.) When the enzyme (100 Mg./ml.) in 0.1 M potassium phosphatecitrate buffer a t pH 7 was treated with 0.025 M sodium borohydride plus 0.025 M acetoacetate a t 0" for 30 minutes, enzymatic activity was irreversibly lost. Enzymatic activity was assayed after exhaustive dialysis by observing the disappearance of the enol absorption band of 0.027 M acetoacetate a t 270 mp in 0.1 M potassium phosphate buffer, pH 5.9 a t 30'. The data are recorded in Table I. TABLE
1
70 activity Sample, treatment prior t o assay
after dialysis
Enzyme 100 100 0.025 M acetoacetate Eiizyme Enzyme 0.025 M BHI90 f. 5 0.025 M acetoacetate 0.025 kf Enzyme BHI25 f 5 Same second treatment with acetoacetate plus BHr12 Same third treatment with acetoacetate plus BH47 Enzyme 0.025 M acetoacetate 2.5 X 10-4 M HCN 84 Enzyme 0.025 Ai' acetoacetate 0.026 B H ~ - 2.5 x 1 0 - ~ X H C N 69 Eiiq-me 0.025 -1f acetoacetate 0.025 M BHa5 X J I acetopyruvate 68
+ + +
+
+
+ + +
+
+ + +
Both H C N g and a c e t o p y r u ~ a t eare ~~~ strong, ~ reversible inhibitors of the enzyme. The details of the inhibition by acetopyruvate remain unknown,1° but HCN may be presumed to add to the C=N bond of a Schiff base intermediate, in the manner of the addition of HCN in the Strecker synthesis. This interpretation is supported by the observation that HCN inhibition develops with perceptible slowness (e.g., 1 min. a t pH 6) and that the time lag cannot be shortened by preincubation of HCN with either enzyme or acetoacetate; the inhibition reaction occurs only in the simultaneous presence of enzyme, substrate and HCN. Both HCN and acetopyruvate partially protect the enzyme against reduction and irreversible inhibition by borohydride plus acetoacetate. The reduction has been performed with 3-C14acetoacetate (New England Nuclear Corporation). Enzyme (520 p g . ) dissolved in 6 ml. of 0.10 M potassium phosphate-citrate buffer, pH '7, was treated with 0.00445 Jd 3-C14-acetoacetate (1.68 X lo6 disintegrationjmin. pmole) and 0.025 M BHI-. After 30 minutes a t 0 ' the solutions were exhaustively dialyzed a t 0' against 0.05 M potassium phosphate, pH 5.9, lyophilized and counted with a Packard Model 1314X Tri-Carb Scintillation Counter. The results are shown in Table 11. ( 8 ) G . Hamilton, Thesis, Harvard University, 1959. (9) R. Davies, Biochem. J., 36, 582 (1942); 37,230 (1943). (10) R. Coleman, Thesis, Radcliffe College, 1962.
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TABLE I1 Sample
Enzyme Enzyme
+ labelled acetoacetate
+ labelled acetoacetate + BH4-
D P.M./200 pg. enzyme
345 5450
On the assumption that the borohydride reduction was 75% complete, the equivalent weight of the enzyme is about 50,000. A sample of the CI4 labelled enzyme was hydrolyzed for 24 hours a t 110' in 6 N hydrochloric acid in an evacuated, sealed tube and 200 pg. of the hydrolysate subjected to two dimensional paper A radioautograph showed a chromatography." single spot, well removed from both the origin and the solvent fronts. These results indicate that borohydride reduces a compound formed between the enzyme and acetoacetate, and that this reduction forms a bond stable to acid hydrolysis. The available evidence thus supports the validity of the hypothesis3 that the enzymatic decarboxylation of acetoacetate, like the amine catalyzed non-enzymatic decarboxylation,l* proceeds by way of a Schiff base (or Schiff base salt) intermediate. The identification of the group on the enzyme which is reponsible for the formation of the intermediate is actively under investigation. (11) R . R. Redfield, Biochem. et Biophys. A z l a , 10, 344 (1953). (12) K. J. Pederson, J . P h y s . Chem., 38, 559 (1934).
THE JAMBS BRYhXT C O N A N T LABORATORY HARVARD UNIVERSITY IRWINFRIDOVICH CAMBRIDGE, MASSACHUSETTS F. H. WESTHEXMER RECEIVED JULV 5, 1962 THE HIGH TEMPERATURE CONTAINMENT OF SUBSTANCES ABOVE THE MELTING POINT OF THE CONTAINER'
Sir : Present high temperature research is close to the practical limit of solid refractory containers set by their melting points. The highest melting metal is tungsten, melting point, 3643OK., the highest oxide is ThOa, melting point, 3300'K., and the highest melting carbide is TaC, with a melting point 4200'K. A special case is graphite, with a sublimation point of 3800'K. In practice a t our laboratories, these maxima usually cannot be reached due to (1) chemical reaction between container and substance (2) eutectic mixtures, which lower the melting point and (3) thermal shock. While the limit of thermal stability of chemical cornpoundsisaboutSOOO'K., i t w a s d e m o n ~ t r a t e d ~ ~ ~ . ~ recently that the liquid range (i.e., the range from their melting point to their critical point) of some metals extends to 20,000'K. As elementary substances, they cannot decompose, in contrast to chemical compounds. Thus in the case of many liquid metals, experiments could be carried out far This work was supported by the Xational Science Foundation Grant No. NSF-G 18829. A. V. Grosse, R . I. T. U. Repart of Sept. 5, 1960; Paper K O . A.R.S.'s Space Flight Report t o the Nation, N. Y . City, Oct. 9-15, 1961. (3) A. V. Grossc, J . Inorg. A'ucl. Chem., 1 2 , 23 (1961). (4) A. V. GrQsse,I n o u g . Chew,, 1, 436 (1962). (1) under (2) 2159,
