SHORT COMMUNICATIONS 351

as high as i . i (see Fig. I), which is comparable to the ratio of ~.55 oi~'reduced/i~'°xidizedrn/~/~28o nl/,~ obtained

for a typical cytochrome c*. The b-type cytochrome was reduced by lactate in the presence of baker 's yeast

lactic dehydrogenase e 9 under anaerobic conditions, but not under aerobic conditions. The enzymic reduction of the cytochrome was measured spectrophotometrically in vacuo in a Thunberg tube modified for spectrophotometry (Fig. 3). When air was introduced into the tube, the reduced cytochrome was immediately oxidized. The autoxidation of the cytochrome was not inhibited by IO -2 M KCN. Neither KCN nor CO caused any modification in the absorption bands as judged from the observation made with a microspectroscope. The biological function of the cytochrome, for which we propose the name "cytochrome b-56I, 554 (Sclerotinia libertiana) ''1°, is a mat ter for further investigation.

This investigation was supported in part by research grant (No. RG-587 I) from the National Institutes of Health, U.S. Public Health Service.

Department of Biology, Faculty of Science, University of Osaka, Nakanoshima, Osaka (Japan)

TATEO YAMANAKA

TAKEKAZU HORIO

KAZUO OKUNUKI

1 j . YAMASHITA, T. HIGASHI, T. YAMANAKA, M. ~IOZAKI, H. MIZUSHIMA, t~. ~/IATSUBARA, T. HORIO AND K. OKUNUKI, Nature, 179 (1957) 959-

2 I. SEKUZU AND K. OKUNUKI, d r. Bioehem., 43 (1956) lO7. 3 G. H/JBSCHER AND M. KIESE, Naturwissenseha]ten, 39 (1952) 524 • a G. t{/3BSCHER, M. KIESE AND R. NICOLAS, Bioehem. Z., 325 (1954) 223, 299. 5 g . SATOMURA, S. 0 I AND A. SAWADA, Bull. Agr. Chem. Soc. Japan, 22 (1958) I94.

S. J. BACH, M. DIXON AND L. G. ZERFAS, Bioehem. J., 4 ° (1946) 229. 7 C. A. APPLEBY AND R. K. MORTON, Nature, 173 (1954) 749. 8 T. YAMANAKA, T. ]-IORIO AND K. OKUNUI(I, J. Bioehem., 45 (1958) 291. 9 j . YAMASHITA, T. HORIO AND K. OI~IJNUKI, ,[. Bioehem., 45 (1958) 707 •

10 F. EGAMI, M. ISHIMOTO, T. MORI, Y. OGIJRA, I(. OKUNUI~I AND R. SATO, J. Bioehem., 44 (1957) 619.

Received November 24th, 1959

• I t should be r e m a r k e d t h a t no c-type cy tochrome has ever been e x t r a c t e d from th i s fungus.

Biochim. Biophys. Acta, 4 ° (196o) 349-351

The condensation of catechols with ethylenediamine

I t has been shown by HARLEY-MASON AND LAIRD 1 that the main product of the reac- tion of ethylenediamine with adrenaline is dihydro-3-hydroxy-I-methylpyrrolo- (4,5-g)-quinoxaline (I), while the reaction with catechol yields 1,2,3,4-tetrahydro- 1,4,5,8-tetraaza-anthracene (II) HARLEY-MAsoN (personal communication) further

~ N \ / ~ - . CHO H # N - - . / - ~ / N H \ c H 2

N ~- ~/ N / 2 % N / ~ \ N H / C H 2 I

CH 3 I II

found that the main product formed from noradrenaline is identical with that formed

Biochim. Biophys. Acla, 4 ° (196o) 351-353

3 5 2 SHORT COMMUNICATIONS

from catechol and tha t the elimination of the/~-ethanolamine side chain of noradre.- naline occurring during this reaction depends on the presence of the j1-hydr.o5 ,:,1 group.

Several paper-chromatographic studies of the reaction product~ obtained fl-om adrenaline and noradrenaline have been reported. They indicated the prese~ce o;; a var iety of compounds. Thus BURN AND FIELD s %und two fluore:qcent deri,, adves (-~ adrenaline and two of noradrenaline, while ~f o~:~<; Asia) FIsc}{E~? :: described the for-- mation of three derivatives from adrenaline and two from noradrenaline. According to YAGI AND NAC.,ATSn ~ the condensation product of adrenaline ::;epm:at:e~ i ;~to two spots of which the fainter one is identical with the main product oi ;~oradrenaHr~e. NADEAU AND JOLY ;5, finally, detected no less than ten different compone~_-,~:> derived from adrenaIine and about the same number derived from noradle;nai~r~e.

In at least some of these experiments concentrations of cateci~o]ami~es arid etk.yl- enediamine were employed which diverged great]y from those specithed for the fluorimetric es~;imation e' and which might therefore have been more fa.';ourabJe for the occurrence of side reactiol?s. 5Iioreover, the possibility cam>of be e:<luded tha*: the pr imary condensation product underwent decomposition e~ther d u r b g !.he pre- l iminary processing or during chromatograp]?y itse]f.

On the basis of the reactioln mechanisms elucidated bv HA~<.~:.-r-3,ia-<)>.r it is to be expected that the condensation product of 3,4-dJhydroxym;ndelic acid with ethylenediamine would b~e identicai with those formed from catechoi and J~oradrena- line, but different ~ronl those of 3,4-dihydroxyphenylacet ic acid and 5,4-dihydroxy- phenylalanine which do not possess, the fi-hydroxyl group i~ ~:he side chain. This assumption is supported by the parti t ion of the condensation p~-oducts between aJ-~ aqueous and an isobutanoi phase: whereas the product from 3,4-dihydr.o:-.yrnandelic acid, like tha t from catechol or noradrenaline, Js almost quant i ta t ively ,:~xtracted into isobutanol, those f~:om 3,4-dihydroxyphenylacetic add and 3,4-dihydrc~×yphe,qyl- alanine remain ]arge]y in the aqueous layer 7,

The possible identi ty of the condensation products of cat:'cno], no~:adreJnaline and 3,4-dihydroxymar:delic acid has now been further investiga~:ed by a comparison of their fluorescence spectra and by paper chromatography.

For the s tudy of the fluorescence spectra o.35 g,g catechol or eq~_~ivaien< quantities of noradrenaline or 3,4-dihydroxymandeiic acid, in zo ml aque.ou~' solution, were condensed with ethv!enedJamine under the conditions described by \Vs;rz?<~aJ~s~En, m; AND ]BONE a, The resulting isobutanol extract was examined in the Amir~co-Bowmar~ Spectrophotofluorometer. The three extracts had identical maxim;~ of acth:atio~ and fluorescence at 4~o and 485 mla., respectively. The fluorescence curves rose sharply from zero at 45 ° m/~ to tlne maxinmm and returned more gradually to zero at 59 ° m;~. After correction for slight differences of intensity the three curves were completely superimposable. In contrast to the slight a symmet ry of these fluorescenec curves, the fluorescence curve of the adrenaline derivative (peak at 5e5 m,~.) wg~s perfect]y symmetrical, which is i~ favour of the assumption that it is caus<'.d ~v a sing/e fluo- rescent product.

For the paper-chromatographic experiments the condensation was carried out under conditions close to tlnose chosen for the fluorimetric assay: a.r, aqueous solution containing 2o/,g in 5 ° ml was incubated with z. 5 ml ~ M ethy]enediamir;e dihydro- chloride and 3.5 m] ethylenediamine at 55 ° for 3o rain. After cooling the mixture was saturated with NaCI and extracted with 3o ml isobutanol. The J'~obuta ~o] extract:

]3ioch~:~< Biop;lys, A<f<y 4o 9960) 35z 35~

SHORT COMMUNICATIONS 353

was evaporated in vacuo to dryness at 3 o0 in a rotary "flash"-evaporator, the residue dissolved in small portions of methanol and concentrated in a test tube in vacuo to about o.3 ml. In view of the reported photosensitivity of some of the condensation products s, amber or red glassware was used throughout. The application of the solution (o.o 5 ml) to the paper (Whatman No. 3) was carried out in red light and the chromatography jars were kept under a darkened hood. Out of ten different solvent systems the best results were obtained with a mixture of isobutanol-ethylenediamine- water (5o : 3 : 20; organic phase) used in the descending direction. As shown in Table I

T A B L E I

PAPER CHROMATOGRAPHY OF ETHYLENEDIAMINE CONDENSATION PRODUCTS

Solvent : isobutanol-ethylenediamine-water (5o : 3 : 2o), descending technique.

Number Substance of spots RF Fluorescence

Adrenaline Noradrenaline

Catechol

Dihydroxymandelic acid

I 0 .30 -0 .34 Yellow 3 (I) o,o 7 Green-yellow

(2) o .15-o .22 Light green (3) o .55 -o .59 Blue-green

2 (I) o. I5-O.22 Light green (2) o .55 -o .59 Blue-green

2 (i) o . 1 5 - o . 2 z Light green (2) 0 .55 -0 .59 Blue-green

the condensation product of adrenaline gave a single spot with intense yellow fluo- rescence. Each of the products formed from catechol, noradrenaline and 3,4-dihydroxy- mandelic acid yielded two indentical spots; a third faint spot with low RF value was observed among the noradrenaline derivatives.

These results suggest that the condensation of ethylenediamine with adrenaline leads to a single fluorescent product and that side reactions are negligible. In the case of catechol, noradrenaline and 3,4-dihydroxymandelic acid two main products which appear to be identical for the three compounds are formed.

Clinical Neuropharmacology Research Center, National Institute of Mental Health, St. Elizabeths Hospital,

Washington, D,C. (U.S.A.) H . W E I L - M A L H E R B E

1 j . HARLEY-MASON AND A. H . LAIRD, Biochem. J., 69 ( i958) 59P. 2 G. P. BURN AND E. O. FIELD, Nature, 178 (1956) 542. 3 j . G. YOUNG AND R. L. FISCHER, Science, 127 (1958) 139o. a K. YAGI AND T. •AGATSU, Nature, 183 (1959) 822. 5 Cr. •ADEAU AND g . P. JOLY, Nature, 182 (1958) 18o.

H . ~VEIL-IVgALHERBE AnD A. D. BONE, Biochem. J., 51 (1952) 311. 7 H . \~VEIL-MALHERBE, Pharmacol. Rev., I I (1959) 278. 8 A. GOLDFIEN AND R. I{ARLER, Science, 127 ( i958) 1292.

Received September I6th, 1959

Biochim. Biophys. Aeta, 4 ° (196o) 351 -353