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COMMUNICATIONS TO THE EDITOR
phase was thought, without proof of composition, to be (PN),. We have found that the crystalline phase produced under the conditions described, and under other conditions, is P3N5. The solid phosphorus nitrides produced by use of electrical discharges are amorphous to X-rays and usually are heterogeneous in appearance. They decompose a t 800 to 900’ in a vacuum or in ammonia or nitrogen. The volatile products are mainly the elements; under suitable conditions, a homogeneous amorphous withtherefrom. an N : P atomic ratio of unity can be nitride condensed The properties of the residual solid depend upon the starting material and its treatment. Effects of several treatments on various amorphous nitrides are shown in Table I. The heterogeneous amorphous mixtures (products of arc synthesis) invariably yielded crystalline residues with the Same X-ray pattern, and the intensity of the pattern increased as the :p ratio approached 1.66. The conversion of amorphous to crystalline phase was accompanied by an expansion.
1-01, 79
the same crystal phase. The mononitride also reacted with nitrogen. Neither of these materids, however, changed in N :P ratio or yielded a crystalline phase when heated in a vacuum. It was concluded that the crystalline phase was Pax5. The interplanar spacings, d, and the relative intensities, I , are shown in Table IT. E. 0. HUPFMAN DIVISIONOF CHEMICAL DEVELOPMENT GRADY TARBU~TON TENNESSEE VALLEY AUTHORITY GLEXN5’. ELMORE ASTHONYJ , S M I T I I m‘lLSoN ALA. MARYGRIFFINROUNTREE RECEIVED FEBRUARY 21, 1957 PHOSPHORYLATION COUPLED WITH PYRIDINE NUCLEOTIDE OXIDATION‘
Sir:
we are reporting herewith mitochondrial phosphorylations in beef heart and honey bee thoracic preparations. These phosphorylations are coupled to oxidation of triphosphopyridine nucleotide (TPNH), and also depend upon an enzyme or enzymes present in the “soluble” fraction of the TABLE I cells, ;.e., the portion not sedimented by centrifugREACTIONS OF AMORPHOUS KITRIDESIN VACCUM, .%MMONIA ing up to 105,000 X g. A N D NITROGEN AT 800 TO 900’ Table I records the oxidation and phosphorylaInitial material (amorphous) Residual product atomic Atomic tion observed when T P N H is the substrate. Reratio Appearance0 h’: P ratio N:P Crystallinityb sults are similar with catalytic amounts of T P N in the glucose-6-P04 or 6-phosphogluconate oxidaIn vacuum, 10-3 to 10-4 mm. tion systems. It is evident that the “soluble” Homogeneous 0.98 0.98 Absent fraction, which contains the pentose cycle coni1.48 1.48 Sbsent plex, does not possess the necessary terminal elecHeterogeneous 1.25 1.60 Strong tron transport enzymes to promote T P N H and 1.25 1.65 Very strong pentose cycle oxidations. On the other hand, both In ammonia, 1 atm. the “soluble” fraction and the mitochondria are Homogeneous 0.99 1.62 Very weak required for oxidative phosphorylation, in contrast 1.48 1.58 Very weak to Krebs cycle oxidations where the mitochondria Heterogeneous 1.22 1.27 Very weak alone can suffice. Freshly prepared sarcosomes 1.22 1.61 Weak from honey bee thoraces also oxidize T P N H with 1.22 1.63 Strong concomitant phosphorylation, but do not require 1.22 1.66 Very strong a “soluble’) fraction. The P/O ratios in the honey bee are low (0.2) both with T P N H and Krebs cycle In nitrogen, 1 atm. intermediates because of an active ATPase in the Homogeneous 0.99 1.46 Weak preparations. 1.48 1.35 Absent Against the possibility that the phosphorylaHeterogeneous 1.26 1.56 Medium tions may have been due to glycolysis (from ADPUnder microscope. All crystalline phases gave same myokinase-glucose-hexokinase), additional experiX-ray pattern. ments were carried out using glucose-C14 and in~ ~glucose-G-C’4 Homogeneous amorphous materials with N :p hibitors. By using g l u ~ o s e - 1 - Cor in the presence or absence of iodoacetate it was ratios of 1 and l.5--corresponding to ( p ~ )and , (PzNs),-reacted similarly with ammonia to yield found that the phosphorylation associated with T P N H oxidation was not due to glycolysis. S k i lar P/O ratios were observed during oxidation of TABLE I1 DPNH. This may be due a t least in part to glyX-RAYPOWDER DIFFRACTION PATTERN OF P3Ns“(Cu K, RADIATION‘ colysis. The simultaneous requirement for both the d, d, d, A. I A. I A. I “soluble” and particulate fraction to promote oxi4.56 S 2.02 M\V 1.34 1~ dative phosphorylation with T P N H is observed either with fresh or frozen and thawed prepara4.20 M 1.83 VW 1.29 \I’ 1.71 W 1.21 U‘ tions. While problems of mitochondrial per3.60 VS 1.61 W 1.20 meability to T P N and T P N H remain to be settled, 2.72 MW 2.55 2.43
VI&. Mil’
2’36
””
1.53 1.50
”*‘
M \V
2.27 MW 1.41 W Determined by James P. Smith.
\TIT
1.17 1.12
117
(1) Aided by research grants from t h e American Cancer Society, Oregon Heart Association and National Institutes of Health, U. S. Public Health Service. Published with the approval of the Mono-
“04
vw
graphs Publicativns Committee, Research Paper No. 312, School Science, Department of Chemistry. (2) R. W. Newburgh and V. H. Cheldelin, ibid., 218, 89 (1956).
CJf
COMMUNICATIONS TO THE EDITOR
April 5 , 1957
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TABLEI OXIDATIVE PHOSPHORYLATION BY BEEFHEARTMITOCHONDRIA AND S OLUB LE PREPARATIOX Each vessel contained 27.2 pmoles of phosphate buffer, pH 7.4, containing P32(1187 c.p.m./pmole phosphorus), 20 pmoles of MgC12, 10 pmoles of ADP, 50 pmoles of glucose, 6 mg. of hexokinase; and where indicated, 20 pmoles of pyruvate, 5 Final pmoles of malate, 10 pmoles of TPNH; 0.3 ml. of mitochondria, 0.5 ml. of &,WO enzyme fraction as indicated. volume 3.0 ml., incubated 15 minutes a t 30”. The reaction was stopped by adding 0.2 ml. of 3 M TCA. Inorganic phosphate was measured colorimetrically using a 0.1-ml. aliquot. Differences from the control of 0.3-0.4 optical density units of 0.20 represents 0.27 pmole of P. Esterified phosphorus was measured using the extraction were found, where 40.~. procedure of Cooper and Lehninger.8 The amount of phosphorus esterified agreed by the two methods used. The mitochondria were prepared by the method of Hogeboom and Schneider‘ and used after storing for 24 hours a t -10’. The SX,WO fraction was the supernatant layer obtained after centrifuging a cell homogenate a t O D for three hours a t 25,000 X g, in two volumes of medium containing 0.9% KCl and 0.001 M ethylenediamine tetraacetate (“Versene”). The supernatant fraction was dialyzed against 2 liters of 0.02 M tris buffer, pH 7.4 for 12 hours. I n other experiments not shown, the SIO~,OOO fraction was employed with similar results. patoms oxygen consumed Enzyme added
Mitochondria Mitochondria
+
Sz5,ooo
Mitochondria Mitochondria S25,OOO
+
I
Substrate
Pyruvate SZ&,OOO Pyruvate Pyruvate TPKH S ~ ~ , O O OTPNH TPNH
+ malate + malate + malate
11.8 10.7 0 5.4 5.9 0
I1
14.1 11
...
10.7 9.1
...
I
23.3 24.4 0 0.6 9.7 0
patoms P esterified Expt.
p/o I1
I
24.8 25.2
2.0 2.3 0 0.1 1.6 0
...
2.0 18.9
...
I1
1.8 2.3
...
0.2 2.1
...
the present findings may suggest an in Vivo cooperation between mitochondria and “soluble” enzymes, that could permit utilization of energy from such systems as the pentose cycle.
between these results and the oxidation products of cyclooctatetraene (111). Reppe and co-workers report4 that neutral permanganate oxidizes I11 to benzoic acid and benzaldehyde, while chromic acid oxidizes i t to benzaldehyde and terephthalic (3) C. Cooper and A. L. Lehninger, J. Biol. Chcm., 219, 489 (1956). acid. It seemed possible therefore that norcara(4) G. H. Hogeboom and W. C. Schneider, { b i d . , 194, 513 (1952). diene-carboxylic acid might be formed as an inter(5) Postdoctoral Fellow of the United States Public Health Service. SCIENCE RESEARCH INSTITUTE VERNON H. CHELDELIN mediate in the oxidation of cyclooctatetraene and, AND DEPARTMENT OF CHEMISTRY CARL WIDMER5 in view of the results described above, tropylium STATE COLLEGE SUDHAJOSHI salts also could be f ~ r m e d . ~ OREGON CORVALLIS, OREGON R. W. NEWBURGH We have now found that appreciable yields RECEIVED NOVEMBER 12, 1956 (5%) of tropylium salts can be isolated from the reaction products following the oxidation of cyclooctatetraene with permanganate in dilute sulfuric OXIDATION OF CYCLOOCTATETRAENE TO THE acidacetone mixtures. To isolate the tropylium TROPYLIUM CATION salt it is necessary t o first extract the other neutral Sir : and acidic products of the reaction, then convert I n a previous paper’ i t was stated that the tro- the water soluble tropylium sulfate to ditropyl pylium cation (I) was oxidized readily by per- ether, which is then extracted and converted to manganate to benzaldehyde. During subsequent tropylium bromide with HBr. This method cerexperiments, designed t o see whether this reaction tainly involves losses and the figure of 5% is the could be used as a specific test for the presence of minimum yield of tropylium sulfate formed in the small amounts of tropylium salts in aqueous solu- reaction. When the oxidation is performed under tion, we have found that norcaradiene-carboxylic neutral or alkaline conditions the yield of tropylacid (11)2is similarly readily oxidized to benzalde- ium salt is negligible. hyde. The tropylium cation probably is formed We consider that the formula scheme shown as an intermediate in the latter reaction for we adequately accounts for the formation of the trohave been able to isolate tropylium salts in yields pylium cation in this reaction. of up to 3070 from the reaction mixture. We have obtained similar result^,^ though with varying yields of tropylium salts, following the oxidation of norcaradiene-carboxylic acid with other oxidants, e.g., ceric salts, Pb(OAc)r, HIQ, Na2S208, I11 IV and we consider that a common two-electron transfer mechanism operates in most of these reactions. Other oxidation products of norcaradiene-carboxylic acid with permanganate are benzoic acid and benzaldehyde. Terephthalic acid also was I1 I obtained when chromic acid in acetic acid wasused as first step is the formation of a glycol which, the oxidant. There appears to be a close parallel in The the acid solution used, gives rise to the ion IV. (1) M. J. S. Dewar and R. Pettit, J . Chem. Soc., 2026 (1956). This then undergoes the normal pinacol-pinacolone (2) The absolute structure of norcaradiene-carboxylic acid is not rearrangement to give the cation V, which is proyet established definitely.: A 1,3,5-cycloheptatriene formulation is used in this paper. (4) w. Reppe, et al.. A n n . , 660, 1 (1948). (3) Full details to be published elsewhere.
(5) W. von E. Doering, e l al., THISJOURNAL, 78, 5448 (1956).
