COMMUNICATIONS TO THE EDITOR
Feb. 5, 1955
in thick pentagonal and hexagonal plates which melt in the capillary tube at 230-234' (cor.) with ~ f 2' (c 0.5 in pyridecomposition, [ a ]-163 dine). The values obtained on analysis indicate the empirical formula CazH~sOaNz (578.6) (calcd. C, 66.42; H, 6.62; 0, 22.12; N, 4.84. Found: C, 66.43; H, 6.51; 0, 22.14; N, 4.91). The molecular weight determined by potentiometric titration with 0.1 N HCl was 579. The hydrochloride crystallizes from watery acetone in thin, rectangular plates, m.p. 247-253' (cor.) with decomposition ; C32H~OsN2.HCl(calcd. C, 62.48; H, 6.39; 0, 20.81; C1, 5.76. Found: C, 62.09; H, 6.15; 0, 20.93; C1, 5.86). I n Keller's color reaction with glacial acetic acid containing ferric chloride, and concentrated sulfuric acid, canescine yields the same brownish-violet stain as yohimbine. On alkaline hydrolysis, canescine yields an equivalent of trimethoxybenzoic acid, C10H1206 (212.2) (calcd.: C, 56.60; H, 5.70; 0, 37.70. Found: C, 57.13; H, 5.90; 0, 37.03), m.p. and m.p. when mixed with a genuine sample of trimethoxybenzoic acid were 170" (cor.) If canescine is boiled with sodium methylate in methanol, canescinic acid methyl ester and trimethoxybenzoic acid methyl ester are obtained. Canescinic acid methyl ester has not yet been obtained in crystalline form; CzpH~04Nz(384.5) (calcd. C, 68.72; H, 7.34; 0, 16.65. Found: c , 68.02; H, 7.41; 0, 16.93), [a]"D -80 2' (c 0.5 in pyridine). The ultraviolet absorption spectrum of canescine in ethanol is composed of the chromophores of the unsubstituted indole system and of trimethoxybenzoic acid; Xmax 218 (log e 4.79), Am, 272 (log e 4-26))hmax 290 (log E 4.07). From these data and from biogenetic considerations it may be concluded, with a high degree of probability, that canescine has the structure of a 11-desmethoxyreserpine (formula I).
*
I OCHi
I
Investigations have shown that canescine possesses pharmacological properties similar to those of reserpine; above all, i t produces a marked and prolonged fall in blood pressure.* The methoxy group in position 11 of the reserpine molecule, which is absent in canescine, therefore, does not seem to be necessary for the pharmacodynamic actions typical of these substances. RESEARCH LABORATORIES SANDOZ, INC. BASLE,SWITZERLAND RECENEDJANUARY 5,1955
A. STOLL A. HOFMANN
(8) We are grateful to Prof. E. Rothlin, Director of the Pharmacological Laboratory, Sandoz, Inc., Bade, for supplying us with the preliminary pharmacological findings.
82 1
YEAST ALCOHOL DEHYDROGENASE, A ZINC METALLOENZYME
Sir: Crystalline alcohol dehydrogenase (ADH) of yeast' is a zinc metalloenzyme; zinc is an integral and enzymatically functional component of the apoenzyme molecule. Qualitative and quantitative emission spectrography was performed in duplicate,2*a as were microchemical determinations of Protein weights were determined by trichloroacetic acid precipitation6 and separately by measurement of absorbance at X 280 mp. Crystalline preparations from our own and commercial sources uniformly contained large quantities of zinc, lesser and variable quantities of magnesium and insignificant amounts of all other elements. Table I gives typical, quantitative spectrochemical data on twice crystallized preparations of yeast ADH, having high activity. Preparation 1 contained 1660 pg. of zinc per gram of protein, preparation 2 contained 1440 pg. per gram. Fractionation of ADH demonstrated an increase in the zinc:protein ratios in fractions in which the activity: protein ratio was increased. The metal : TABLEI
EMISSION SPECTROGRAPHIC ANALYSISOF LIZED
Element
'TWICE CRYSTALYEASTALCOHOL DEHYDROGENASE
Preparation no. 1 Line/ pg./g. internal yeast standard ADH
Preparation no. 2 pg./g. Line/ internal yeast standard ADH
Zn 3345 1660 Zn3345 1440 Bi 2897 V 3185 a By Na Di165 Copper ethyldithiocarbamate Fe3020 80 Fe4283 81 Iron V 3185 v 4395 Aluminum A13961 79 A1 3961 48 V 3185 V 3185 Mg2776 1180 Magnesium Mg2798 296 Bi 2897 V 3185 Calcium Ca 4318 39 Ca4302 105 Bi 2897 v 4395 Sr 4077 4 Sr 4077 2 Strontium V 4111 v 4395 Ba 4554 11 Barium Ba4554 20 V 3185 v 4395 Mn 2576 2 Manganese Mn 4030 U V 4111 v 4395 Lead Pb 4057 45 Pb 4057 x V 4111 v 4395 Cadmium Cd2268 13 ... x V 3185 Chromium Cr 4254 8 Cr 4254 ?r V 4111 v 4395 S o t detected: x , and also beryllium, cobalt, lithium, molybdenum, nickel, potassium, silver, tin. O Lost. Zinc
(1) E. Racker, J . B i d . Chem., 184, 313 (1950). (2) B. L. Vallee. in preparation for publication. (3) B. L. Vallee and H. Neurath, THISJOURNAL,76, 5006 (1954). (4). B. L. Vallee and J. G. Gibson, 2nd, J . B i d . Chcm., 178, 435 (19481. .~., (5) F. L. Hoch and B. L. Vallee, ibid., 181, 295 (1949). (6) F. L. Hoch and B. L. Vallee, Anal. Chem., P I , 317 (1953).
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COMMUNICATIONS TO THE EDITOR
Vol. 77
protein ratio of all other elements studied decreased Health, Education and Welfare, the Rockefeller with increasing enzyme purification. hnalytical Foundation, the Research Corporation, and the and enzymological data thus far do not indicate Howard Hughes Foundation for Medical Research. that magnesium is an integral functional part of the BIOPHYSICS RESEARCH LABORATORY apoenzyme. Preparations which had as little as 5 OF THE DEPARTMENT OF MEDICINE HARVARD MEDICAL SCHOOL AND BERT L. VALLEE pg. of zinc per gram of protein have full activity. FREDERIC L. HOW PETERBENTBRIGHAM HOSPITAL Microchemical analyses for zinc4z5 in many BOSTON, MASSACHUSETTS crystalline preparations have corroborated the RECEIVED DECEMBER 14, 1954 spectrochemical data indicating the presence of about 0.20y0 of zinc, the exact value being a function of the state of purity of the preparation. A SYNTHETIC COMPOUND WITH CATALASE-LIKE The zinc content of crystalline ADH preparaACTIVITY tions is not reduced below about o.17yOby dialyses Sir: under varying conditions, the choice of which Triethylenetetramine (TETA), HzNCHaCH2hTHindicated the successful maintenance of the zinc- CH&HgNHCHzCHzNHz, forms a quadridentate protein bond against competitive agents. chelate compound with ferric ion. Steric conBoth the rate of appearance of D P N H a t pH siderations show that it is energetically improbable 8.8 in the presence of ethanol, and the disappear- to have the four N atoms and the Ferrr ion in ance of D P N H a t pH 6.5 in the presence of acet- (TETA)-FeIII located in one plane, but that the aldehyde are inhibited strongly, e.g., when the structure with one primary amine N atom above enzyme is preincubated with 1,lO-phenanthroline. and the other below the plane determined by the This inhibition is prevented by zinc ions but not two secondary amine N atoms and the FerlI ion by magnesium ions. The inhibition by 1,lO- is stable. I n this chelate ion the four N atoms phenanthroline seems to be competitive with the form four coordination bonds with FelI1 along four coenzyme. of the six octahedral d2sp3hybrid atomic orbitals Based upon a molecular weight of yeast ADH of the latter, the two unoccupied hybrid orbitals of 150,000' the data in Table I give a ratio of 3.3 are directed along the two remaining adjacent and 3.8 moles of zinc t o 1 mole of ADH apoenzyme. octahedral directions. In aqueous solutions these One of our further purified and dialyzed prepara- last two orbitals are presumably used to form two tions contained 1720 pg. of zinc per gram of pro- additional coordination links between Fer1I and tein, corresponding t o 3.9 moles of zinc per mole of two hydroxide ions or a hydroxide ion and a water ADH. A ratio of four moles of zinc to one mole of molecule. In the presence of hydrogen peroxide, ADH apoenzyme seems to be the correct value. either or both of these unidentate ligands may be These data establish yeast ADH as a zinc displaced by the OOH- ions. Each OOH- ion is nietalloenzyme as previously defined.*s9 potentially capable of forming two coordination These findings throw new light upon the inter- links with FeIII, with one electron pair donated action of DPN with ADH. The zinc content of by each 0 atom. But since the 0-0 bond yeast ADH, the participation of zinc in enzyme length is only 1.3 A., the resulting chelate, activity, the kinetics of competitive inhibition and (TETA)-FeII'-OH, is unstable and tends to stretch the strong zinc-protein bonding suggest that the 'oi four molecules of D P N or DPNH involved in yeast ADH activity are bound to the apoenzyme through the 0-0 bond until the second activated complex (TETA)-Fe'T'-OH is formed. Direct splitting of zinc. The hypothesis that other dehydrogenases linked '0 with pyridine nucleotides may similarly prove to be the 0-0bond in an isolated H20,molecule requires metalloenzymes is being tested experimentally. about 35 kcal. of energy per mole. But in the above In this regard it may be suggested that liver complex ion the energy consumed in splitting the XDH, with a molecular weight of 73,000,10 which 0-0 bond is partially compensated by the energy binds two molecules of DPN, does so in similar gained through the formation of more stable FeIII-0 fashion. A41so,the function of the hitherto unexbonds, because the 0 atom and the OH- ion are plained large amounts of zinc in may now be attributed to a high content of ADH, active now separate ligands and can orient themselves for overlapping with the two vacant octain the dehydrogenation or formation of Vitamin AI. maximum hedral atomic orbitals of the FeII'. The second These studies were aided in part by a contract activated complex can then readily react with a between the Office of Naval Research, Department second OOH- ion to yield 0% and OH-. of the Navy, and Harvard University Contract This reasoning was subsequently substantiated No. Nr5ori-07660, and grants-in-aid from the by measurements of the rate of catalytic deconiNational Institute of Health of the Department of position of H202 by (TETA)--FeIT1. In each ( 7 ) J. E. Hayes, Jr., and S. F. Velick, J . B i d . Chem., 207, 225 experiment, a measured volume of about 0.15 .M (1954). H202 solution was mixed with a known amount of ( 8 ) B. L. Vallee, Scientific MonthZy, 62, 368 (1951). (TETA)-FelI1 solution a t zero time. TETA (9) B. L. I'allee, F. L. Hoch, and W. L. Hughes, Jr., Arch. Biochem. Aliquot parts of the reaction mixture were taken B i o p h y s , 48, 347 (1954). (10) H Theorell and R . Bonnichsen, Acta Chem. S c u n d . , 5 , 329 a t short time intervals, mixed with equal volumes (1951) 2.5 N H2S04and titrated with standard ceric of 1 I 1 A 1 I.eiurr xud C. Lriner, h d .?ttlt7., 6 4 , 293 ( l Y 4 4 ) sulfate sulutiorl with o-phenanthroline-Fe" a.; r i l ' i J .If, Bowness, K. A . Murtuii. hf H.Shakir rud A. L Stirbbi, indicator The turnover numbers uf ( T E T A L < j o ~ h ~ Jm ., 5 1 , 5 2 1 119521
+
(
