COMMUNICATIONS TO THE EDITOR
Sept. 20, 1952
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infrared spectrum of films of @-luciferinlacks fine structure but contains strong absorptions a t 3250, 3825, 1680, 1625, and 1510 an.-',which collectively indicate the amide bond as it occurs in peptidesb$' or in cyclic ureide^.^.^ Accordingly, an attempt was made to degrade /?-luciferin by hydrolysis and, although 0.5 iV hydrochloric acid does not attack the molecule appreciably a t looo, de-oxygenated 4 -IT acid slowly degrades i t a t 125" with loss of activity. /?-Luciferindoes not give a ninhydrin test but the product of its hydrolysis contains a number of ninhydrin-positive substances. These have been presumptively identified by two-dimensional paper chromatography as the amino acids: glycine, threonine, proline, lysine, aspartic acid, glutamic acid, and leucine, isoleucine, or phenylalanine. The hydrolysate contains an unidentified ninhydrinpositive substance and a yellow pigment readily separable from the amino acid fraction. In addition, when /?-luciferin is chromatographed on paper with either hydrogen-saturated n-butanol or iamyl alcohol (Rf 0.8 and 0.65, respectively, determined by the position of light-emitting areas after wetting the chromatogram with luciferase) the N-chloroamide test,1° the retention test," and hydrolysis of eluted substance show that the position of luciferin activity coincides with the position of a polypeptide. Our preparations of /?-luciferin contain in addition polypeptide (Rf 0 in both solvents) which does not possess luminescent activity in the presence of luciferase but which may be related to luciferin since it and the active polypeptide ('2) Thiolacetic acid has been reported previously to react readily with amines, B. Pawlewski. Bcr., 31, 661 (1898). have identical amino acid compositions. Only (3) R. A . Boissonnas, H c h . Chim. Acta, 34, 8 i 4 (1951). the active polypeptide is yellow and accordingly (4) J . R. Vaughan and R. L. Osato, Tms J O U R N A L 74, , 676 (1952). belongs to the class of pigmented polypeptides (6) .1similar method has been used t o prepare S-acyl thiol amino hitherto encountered in Actinornyce~.'~J~Such acid esters; T. Wieland, u'. Jchafer and E. Bokelmann, A n n . , K73, 99 ( I95 1J. substances thus occur in higher organisms, and the (6) G. Aliiger, G. E. P. Smith, Jr.. E. L. Carr and H. P. Stevens, bioluminescent reaction between Cypridina luciJ . Org. C'iurn.,14, 962 (1949). ferin and luciferase is a naturally-occurring phase DEPARTMEST OF CHEMISTRY JOHS C. SHEEHAN in the metabolism of these compounds.
methods, we have prepared N-acylated thiol amino acids and have found them to be active acylating agents2 for amines and amino acid derivatives under mild conditions. Interaction of phenaceturic acid, triethylamine and ethyl chlorocarbonateas4in methylene chloride solution at -10") followed by treatment with excess hydrogen sulfide, led to a 72% yield of thiolphenaceturic acid,j in. p. 116.5-118.0" (dec.). Anal. Calcd. for CloH&02S: C, 3 i . 3 9 ; H, 5.30; h', 6.69. Fourid: C, 5 i . 4 5 ; H, 5.38; N, 6.73. -1 solution of thiophenaceturic acid and aniline in 50-50 ethanol-phosphate buffer of pH 7.3 (0.1 M) deposited 785% of phenaceturanilide, m.p. l63-1Mo, in 18 hours a t room temperature. Phthaloylthioglycine was prepared by a similar procedure in 61% yield, imp. 116.5-118.0". Anal. Calcd. ior C10H7N03S: C, 54.29; H, 3.19; N, 6.33. Found: C, 54.49; H, 3.33; N, 6.36. Treatment of phthaloylglycyl chloride with sodium hydrosulfide in dimethylformamide solution also afforded phthaloylthioglycine in good yield. A solution in methylene chloride of phthaloylthiolglycine, glycine methyl ester hydrochloride and triethylamine reacted a t room temperature to give phthaloylglycylglycine methyl ester. The addition of iodine-potassium iodide6 to an aqueous solution (0-5") of phthaloylthiolglycine and glycine methyl ester hydrochloride containing excess sodium bicarbonate produced an inmediate precipitate of the peptide derivative.
~IASSACHUSETTS ISSTITUTEOF TECHNOLOGY CAMBRIDGE 39, MASSACHUSETTS DAVIDA . JOHNSOS RECEIVED AL-GUST4, 1952
THE BETA-LUCIFERIN O F CYPRIDINA'
Sir: The production of light by the marine ostracod crustacean, Cypridinu hilgendorfii, is a result of interaction between oxygen, the enzyme luciferase, and either of two amorphous substances of unknown nature which have been designated a- and ,5-lu~iferin.~* We ~ ' ~wish to report evidence that these luciferins are chromopolypeptides. The most highly purified preparations of the luciferins are oxygen-sensitive orange-yellow resins from which no bioluminescent substance can be sublimed and from which it has not been possible to obtain crystalline fraction^.^ At 6.5' in high vacuum, a-luciferin is converted to B-luciferin; the transformation is reversed in dilute acid. The (1) This investigation was supported by a grant from the Research Corporation, New York. (2) E. N. Harvey, "Living Light," Princeton University Press, Princeton, N. J., 1940; "Bioluminescence," Academic Press, New York, N. Y. (3) R. S. Anderson, J . Gcn. Physiol., 19, 301 (1935). (4) H. S. Mason and E. F. Davis, J . Biol. Chcm., 197, 41 (1952).
(i) .\. Elliot and E. J . Ambrose, .Vnfirue, 166, 921 (1950). ( 6 ) I . Yf. Klotz and P. Griswold, Science, 109, 309 (1949). ( 7 ) 1. 37. Klotz, P. Griswold and I)..\I. Gruen, THISJOURXAL, 71, 161.i (1949). (81 E . K. Blout and pl. J. Fields, J . Biol. Chenr., 178, 335 (1949). (9) E. R. Blout and hf. J. Fields, THISJ O ~ N A L ,72, 479 (1950). (10) K , X . Rydon and P. W. G. Smith, Y a l u r c , 169, 982 (1952). (11) F. A. Robinson, K. L. .4.Fehr and W. Dickinson, Biochem. J . , 61, 298 (1952). (12) S.-4.Waksman and AI. Tishler, J . B i d . Chem., 142, 519 (1942). (13) H. Lehr and J. Berger, Arch. Biochem., 23, 503 (1949).
DEPARTMENT O F BIOLOGY PRITCETOS UNIVERSITS PRINCETON, Iz'. J. RECEIVED .&UGcST 14, 1952
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11. S. MAWN
LIPOIC ACIDICON JUGASE
Sir : Lipothiamide pyrophosphate (LTPP), the amide of lipoic acid (LA) and thiamin pyrophosphate (TPP), is required for the oxidative decarboxylation of pyruvate and a-ketoglutarateby cell-free extracts of an Escherichia coli mutant.'= It has now been demonstrated that cell-free extracts of wild-type E. coli contain an enzyme, lipoic acid conjugase, (1) (a) L. J. Reed and B. G. DeBusk, THIS J O U R N A L74, , 3984 (1952); (b) 74, 3457 (1952).
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which forms LTPP from T P P and bound LA. 'The loss of this enzyme is apparently the critical change resulting from mutation of the parent strain. This conclusion is based upon studies of pyruvate oxidatTon systems, resolved atid partially purified by the method of Korkes, e/ d . , ? from the wild a t i d iiiutant strains of this organisni. Pyruvate dismutation requires transacetylase, lactic dehydrogenase, orthophosphate, diphosphopyridine nucleotide, coenzyme A, TPP, and two enzyme fractions from the wild strain (designated - 1 ~ 7 and Bw)' (Table I ) . However, the enzyme fraction Hw and the cofactor T P P cat1 be replaced by a single substance, LTPP, indicating that the basic pyruvate oxidase system, which is activated by the coenzyme LTPP, is present only in fraction -1. The apooxidase fraction iron1 the riiutaiit strain, A l f , can likewise be activated either by LTPP, or by TPP plus Bw, but rteither apooxidase fraction is activated by TPP plus B L ~the , fraction from the mutant corresponding to fraction BIV of the wild strain.
Yol. 74
VERATROBASZNE AND GERALBINE, TWO NEW ALKALOIDS ISOLATED FROM VERATRUM ALBUM'
.Jir
.liter separation of the ester alkaloids, protoveratrine and veralbidine,2 and the alkamines jervirie and rubijervine from the mixture of alkaloids coiitained in Veratrum album, the mother liquor was divided into two fractions, one of which contained the inarkedly basic alkaloids, and the other the weakly basic alkaloids. From the fraction containing the first group an unknown alkaloid could be crystallized out of a solution in ethyl acetate.$ 'This was purified as its hydrochloride which is only slightly soluble in water. Xfter cleavage of the ialt with dilute ammonia the pure base was obtained. This we intend to call veratrobasine. 'The new base crystallizes from methanol in large prisrns, which turn yellow from 270" upward, and melt a t 2X5-288", with decomposition. Its optical D and in rotation in pure alcohol is [ c Y ] ~ ~ -76.6") pyridine [ C Y ~ ~ O D - 1%' %'hen veratrobasine was dissolved in ch-L'( sulfuric acid (2 mg. of the base 111 10 cc. acid) an intensely orange fluorescent 'r.4BL.I:. 1 iolutioii w'is obtained. The solution kept this k'TKUV.4TE I ) I S M L T A T I O S \VITII PYRIFIED E S Z Y M E F K A C color for over 24 hours. Anal. Calcd. for C X H ~ ~ IIOSh C, 74 44; H, !).03; N, 3.62. Found: C, p l l ?roducts i n !rO i i i i i i 7 !.-Hi, 74.3h; H,( j . h l , U..5i; N, 3.86, 3 . G . Acetyl Carbon phosIn possessing only 24 carbon atoms, the new Coin pun en t? dioxide phate Lariat? ~tlkCiloid is iigiiificm tly different from the other x \ v + TPP IJ il 02 :iIl\ariiines $( J f,ir obtained from T brcctrum album mv :{ti :;-I : I ; .itit1 17eriifriiiii viritle, which all have 27 carbon 11 0 Ii 9 An TPP + 1,A .itoui\. Veratrobasiiie has one N-CHj group, the 1.; 4.-1 -1.7 Ax + LTPP first to be found in the 1-eratrum alkaloids: N-CH, A W -t Incubated" F i x 3 t- 'TPP 3 , ,-I ::,:I :i 4 calcd. 3.83, found 3.94. 0.1 lJ,2 li2 An; + Control' The base also contains two actil-r hydrogen A M + TPP (1.1 1.1.1 1.1.1 ,itoiiis: calcd. O.Xh, found 0.51. + B\\- w P :$.ti 3.'j :I.,-) 'l'he infrared nbsorptiori spectrurll shows no 1.7 4.4 -1,s Aw + LTPP hand tyI)ical of betones, the ultraviolet absorption A H + i n c u b a t e d ~i R\r- t T P P :j ;, :I 2 :; -1 spectruin, however, shows ;L definite miximum at Ii I/ ri 1 .I\( + Culirrctl' 2;i2 rlly (log € 2,I-l). 0.1 0.1 11:: .-I,!\ -cn\r 7 - TPIJ Froill the fraction contaiiiing the weakly basic A!\- .+ Ii\l T P P f 1,A I),! 0.1 11:: ,&aloitls, ;1 further new alkaloid could be crystalx u -t H\I + 'TPP 0.1 0.1 1J.1 lired iroiii ethyl acetate. This new alkaloid, which Preseiit a t following irvels: exizyiiie fractioiis, 2.11 nig. we hdVe called gevalbine, crystallizes from aqueous protein; T P P , 1UOy; LA, 1Oy; LTPP, 2 i y of crude syn- acetoiie in large prisms, and from a mixture of ethyl thetic preparationlb; final volume, 2 ml. Supplements :tiit1 acetate and ether ( I : I ) in rectangular plates which experimental conditions as previously described.'* lnmelt a t 221-223" with slight yellowish discoloracubated 90 miri. a t 35O, boiled 10 inin., atid superiiatant :added to A N or A w R \ r incubated :rnd hoilerl prior t o tion. In contrast to the other alkaloids isolated (mnt:tc't with T P P from T'erntrum, geralbine exhibited no measurable iricubation of fractiori Hu- aloiie with 'TPP rotation in pure alcohol, chloroform or pyridine. I'roduces a heat stable product, presumably I,TPP, M'hen dissolved in 84% or pure sulfuric acid 'i which can subsequently activate the apooxidase of light yellow solutiori was obtained which had not either strain ; however, heating fraction Bly prior lost its color after twelve hours. In crystalline to its contact with T P P results in an incubation form geralbine is fairly stable but when dissolved mixture havirig no cooxidase activity. Fractioii in alcohol or chloroform, the solution turns yellow I3Liv must furnish lipoic. acid rorijugase :is well ;IS withiii J iew hours. A m l Calcd. for CZZHuOdY lipoic acid, presumably bound to the coijugase, or C, 70.91, ti, 5.68, N , 4.07 Found: C, 7 6 . i ; ; , less likely t o a contaminating protein, by a union 76.78; H, 9.79, 9.78; N, 3.96, 3.98. Geralbine hydrochloride crystallizes from methnot dissociable by dialysis. anol-ether in fine needles, and melts a t 2iO'. BIOCHEMICAL INSTITUTE AND Anal. Calcd. for CzzHa402NCl: C, 69.55; H, 9.02: LESTERJ. REED D E P A R ~ E NOF T CHEMISRY BETTY G. DEBUSK CI, 9.3.5. Fourld C, 69.96; H, 9.23; CI, 9.35, 9.3; IJN+VERSrfY O F TEXAS, AND ' j
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CLAYTOX FOCSDATION FOR RESEARCII AL'STIN, TEXAS
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