THE NATURE OF THE LUCIFERIN-LUCIFERASE SYSTEM - Journal

J. Am. Chem. Soc. , 1944, 66 (5), pp 848–848. DOI: 10.1021/ja01233a515. Publication Date: May 1944. ACS Legacy Archive. Cite this:J. Am. Chem. Soc. ...
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Vol. 66

(Carboethoxymethy1)-(B-carboethoxyethyl)sul- to 59,430 calories as compared to 57,340 calories fide, obtained by the reaction of thioglycolic ester available by direct oxidation of two hydrogen with acrylic ester in the presence of a small amount atoms on glucose, the substrate known to inof piperidine, was cyclized by means of sodium crease tremendously the luminescence of washed metal in benzene suspension. The product, luminous bacteria. thiophan-3-on-4-carboxylic ester,2 C’IHI~O~S, We have now observed that a concentrated soluboiled at 96’ at 4 mm. and gave a phenylhydra- tion of luciferase, after prolonged dialysis with zone, C13HleN2O2S,m. p. 100-101°3 from aqueous several hundred volumes of distilled water, and ethanol (Calcd. : C, 59.1; H, 6.1 ; N, 10.6. Found: storage for some eighteen months in a refrigerator, C, 59.1; H, 6.0; N, 10.7), and a semicarbazone, will luminesce with oxygen, following reduction CsH18N3O3S,m. p. 176’ (Calcd.: N, 18.2. Found: by (1) Na&04, (2) reduced Coenzyme I solution N, 18.1). The ester, on acid hydrolysis, gave thio- (partially reduced by pure hydrogen and platiphanone-3, C4HaOS,b. p. 83-85’ at 29 mm., dark- nized platinum, then added anaerobically to luciens on standing, semicarbazone, CsHgNsOS, m. p. ferase), (3) reduced riboflavin (similarly treated), (4) washed cells of E. coli plus glucose, and ( 5 ) 196’ dec., dinitrophenylhydrazone, CIOHION~O~S, m.p. 179Odec. (Calcd.: N, 19.8. Found: N, 19.7). growing culture of E. coli.a A portion of the 2-Methylthiophan-3-on-4-carboxylicester, luciferase solution gave luminescence repeatedly CeHlzOsS, b. p. 93-95’ at 4.5 mm., was obtained on successive additions of an excess of hydroby cyclization of (a-carboethoxyethyl) (P-carbo- sulfite then oxygen, but finally ceased. Luminesethoxyethyl) sulfide. On hydrolysis it gave 2- cence continuous for some hours resulted when methylthiophanone-3, CsHsOS, b. p. 82’ at 28 hydrogen, plus slight oxygen impurity, was passed mm., unstable in air, semicarbazone, CdLNsOS, through so’me luciferase solution containing platim. p. 185-186’, dinitrophenylhydrazone, CUHW nized asbestos. Oxidized coenzyme, oxidized NdOdS, m. p. 161-162’ (Calcd.: C, 44.7; 13, 4.1. riboflavin, ferrocyanide, or glucose without bacteria, as expected, did not reduce the luciferase. Found: C, 44.6; H, 4.2). X consideration of the above facts indicates that Circumstances made necessary the termination of our studies on synthetic thiophanes in August, the luminescent system consists. of a pyridine 1942. It is intended to resume these unfinished iiucleotide plus flavoprotein. According to the absorption spectrum, “luciferin” apparently coninvestigations after the war. tains both Coenzyme (I or’II) and a flavine pros(2) The assigned formula is based on analogy; see Prill and Mcthetic group, the former component providing a Elvain, THISJOURNAL, 66, 1235 (1933). (3) Karrer and Schmid (ref. 1, p. 127) obtained two phenyl- reductant, and the latter, after loose combination hydrazones, m. p. 141.5-142.5O and m. p. 167’; posslble explanations with its specific protein, comprising molecules to account for this discrepancy cannot be investigated at this time. excitable by oxidation. Some of the excited moleGATESAND CRELLIN LABORATORIES OF CHEMISTRY cules radiate and are not destroyed, but others, CALIFORNIA INSTITUTE OF TECHNOLOGY EDWINR. BUCHMAN failing to radiate axe destroyed by their absorbed PASADENA 4,C.4LIPORXIA HARRYCOHEN energy. This phenomenon causes the 9rreversible reaction” of luminescence. Such destntcRECEIVED APRIL21, 1944 tion of non-radiating excited molecules is responsible for the familiar degradation of riboflavin in THE NATURE OF TEE LUCIFERIN-LUCIFEUSE solution in the light, and also phthalhydrazides SYSTEM during luminescent oxidation. Sir: Thus, in luminous bacteria, light emission preIn the luminescent system extracted from sumably occurs when flavoprotein, reduced by Cyfiridinu, the substrate, luciferin, is dialyzable, hydrogen from suitable substrates (e. g., glucose) while the enzyme, luciferase, is not.‘ Reduced via the dehydrogenase-coenzyme system, is oxiluciferin undergoes reversible dark oxidation by dized directly by oxygen. The effects of cyanide oxygen or ferricyanide, and apparently irreversible on oxygen consumption and luminescence, respecluminescent oxidation by luciferase plus oxygen. tively, indicate that most of the hydrogen proLike dihydrocoenzyme, with an absorption maxi- ceeds step-wise, by electron transfer, through the mum at 3400 A., considerably purified, reduced cytochrome-heme system to oxygen. With chloroluciferin solutions have an absorption peak near phyll substituted for the related heme molecule, 3200 A. On adding oxygen this absorption di- the same system of catalysts operating in the reminishes, while a new peak at 4300 k. appears, verse direction would lead to photosynthesis. In shifting quickly to 4700 A+,then disappearing.? luminescence two hydrogens are oxidized for the In presence of oxygen, aqueous luciferin is un- quantum emitted, while in photosynthesis single stable. The absorption at 4700 A. resembles that hydrogens are made available. of certain flavoproteins, and practically coincides PRINCETON UNIVERSITY FRANKH. JOHNSON with the luminescence maximum at 4750 A. The PRINCETON, S . J. HENRYEYRINC energy of this luminescent transition corresponds RECEIVED APRIL17, 1944 (1) Harvey, “Living Light,” Princeton IJniversity Press, Princeton, N. J., 1941. (2) Chase, J . B i d . Chcm.. 160, 433 (1943).

(3) Doctors Chase, Schlenk and Kunitr kindly supplied experimental materials.