Aug. 20, 1956
FORMATION OF COPPER COMPLEXES DURINGTYROSINASE-CATALYZED OXIDATION 4 1%
A n d . Calcd. for CI8HlR?;O3.C1O4: C , 54.61; H , 4.58; C1, 8.96; 3 OCHa,23.52. Found: C, 54.59; H , 4.60; C1, 8.77; OCHI, 23.49. Action of Heat on Ga1anthine.--A sublimation tube was packed n i t h 12 mg. of galanthine and heated to 280' (3 mm.). A fluorescent oil sublimed along with much unchanged galanthine. The total solid mas dissolved in benzene, and the solution was chromatographed on a short column of aluminum oxide. .I fluorescent band ITAS eluted e a i l y n ith benzene and 50Yo benzene -ethyl acetate. The
infrared spectrum of the crude product (2-3 mg.) was nearly identical with that of synthetic 11. It exhibited the same action during melting point determination as authentic 11' and, therefore, mas converted t o the corresponding 7-phcnanthridone by air a t 150" and sublimed a t 200' (0.25 mm.). The m.p. of the crude product, 265-270" (reported' 272274"), was not depressed by admixture with authentic phenanthridone.' The infrared spectrum ( K B r ) also \vas identical with that of authentic phenanthridone.' BETHESDA 14, NARYLAXD
[ C O ~ T R I B U T IFROM O N THE SOUTHERN UTILIZATIOX RESEARCH BRANCH, AGRICULTURAL RESEARCH SERVICE, UNITEDSTAr s s DEPARTMENT O F AGRICULTURE]
Formation of Copper Complexes During Tyrosinase-catalyzed Oxidations BY
JETT
c. XRT€lUR, JR.,
AND
T. A. MCLEhlORE
RECEIVEDDECEMBER 27. 1955 The formation of Cu complexes with products of catechol, IiydroTuinone, chlorogenic and caffeic acids, during tyrosinasecatalyzed oxidation, was determined, using radioactive Cu64as a tracer and a n ion-exchange method. The most stable Cu complexes formed were with o-quinone, with their stability decreasing with the introduction of groups on the initial aromatic ring of the compounds investigated. The maximum amount of Cu was complexed with o-quinone a t the time of its formation. If the polymerization of the o-quinone mas ailou-ed to proceed prior t o the addition of C u + + , a smaller amount of metal was bound.
Introduction An investigation of the formation of Cu complexes with products of tyrosinase-catalyzed reactions could indicate some differences in the mechanism of oxidation of different substrates, particularly o-dihydric and @-dihydric phenolic donors. The determination of the formation of Cu complexes using ion-exchange methods,',? radioactive Cu64and catalytic quantities of tyrosinase oxidizing the substrates would offer a new approach to this problem. It is the purpose of this report to present experimental data which indicate formation of Cu complexes with o-quinones. Experimental
mc.ig. About 0.64 g. of Cu was dissolved in 5 ml. of concentrated "OB, then 3 ml. of concentrated H2S01 were added and the mixture was heatcd until SO, was evolved. The acid C U ~ ~ solution S O ~ was diluted with Cu-free distilled water t o yield a stock solution containing 3-4 pg. of Cu per ml. The other chemicals used were C.P. or reagent grade.
Results The effect of substrate on tyrosinase oxidation a t $H 6.7 and 25' with C U ~ ~ equivalent SO~ to 3.84 p g . of Cu'j4added at zero time is shown in Fig. 1. The tyrosinase preparation has o-dihydric phenolase activity; however, hydroquinone can be oxidized by the enzyme on addition of a trace of catechol as a mediator. Using about 20 p g . of enzyme per test, the data indicate that initially the mechanism of oxidation of catechol and hydroquinone containing T h e activity of the tyrosinase was measured innnometrically (Warburg apparatus) a t 25" by determining the rate a trace of catechol approximates zero order. of oxygen uptake. The flasks, about 17-18 ml., contained Oxidation was also determined a t PH 3.6 and 7.8 two side arms and a center well. The enzyme and Cu65O0, a t 25'. Very little deviation from the data shown solutions were usually put iii separate side arms. The center well contained a filter paper wick wetted with 0.2 in Fig. 1 for pH G.7 was noted. ml. of 1OyoKOH to absorb COUwhich might be released. The effect of pH and substrate on the amount of Phosphate buffer (0.3 ml. of 0.5 d l ) and substrate solution Cu ion complexed is shown in Table I. Cu6'S04, (optimum amount) previously determined were placed in the maiii reservoir of the flask, suflicient Cu-free distilled water being added to bring the total liquid volume to 3 ml. During the meisurements of the rate of oxidation of different substrates by tyrosinase, CuecS04solution was added. .Ifter a predetermined time, cation exchange material, 0.2 g. of phosphorylated cotton f a b r i ~ was , ~ added to remove the copper ions from the solution. The Cu64 complexed was determined by drying a n aliquot of the solution (which had been extracted with cation-exchange material by shaking the material with the solution for 1 hr.) and measuring its radioactivity in a gas flow counter. The use of this method to determine formation of metal complexes has been outlined by Schubert and Richter.1J The mushroom tyrosinase used was a commercial preparation. About 0.1 ml. of the preparation as received, about 0.5 mg. of enzyme preparation, was diluted with Cu-free water to 25 ml. One-ml. portions of this diluted preparation, about 20 pg. of enzyme, were used per test. The C U (half-life ~ ~ 12.88 hr.; 6-0.57, 0.65; 7-1.34 tilev.) was obtained :is Cu wire, iiiitinl specific activity about 300 ( 1 ) J. Schubert, J . Phys. Coiioid Chein., 5 2 , 340 (194.8). 12) J . Schubert a n d J. W. Richter, ibid., 52, 330 (1948); J O U K N A I . , 7 0 , 4259 (1948). ( 3 ) J . D. Guthrie, I i i d . E n g . Chcin., 44, 2187 (19.52).
Trrrs
TABLE I EFFECT OF p H
AND
SUBSTRP.TE O N C u + + COMPLEXED~ C u_ _complexed, __ ~. + +
Substrate
+
Hydroquinone trace catechol Catechol Chlorogenic acid Caffeic acid Protocatechuic acid Hydroquinone
pgL-
~
p H 5.6
pH 6 7
$13 7 . 8
0.04 .77 .22 ,40
0.19 1.01 0.08 . 23
0.50
.oo
..
.90
.38 . 22 00
.06 .(Jo .oo a ;2ge of diluted enzyme, 24 hr., estimated Cu content of tyrosinase used per test, 0.04 pg.; values reported are in excess of controls.
equivalent to 3.84 p g . of Cu, was added to the enzyme-substrate solution a t zero time. After GO minutes, cation-exchange cotton fabric was added. -Inaliquot of the extracted solution was analyzetl for radioactive Cue4, and, after appropriate corrections for decay time, the p g . of Cu associated with
415-4
J.
L
c. ARTHUR, JR.,
AND
T. h. MCLEMORE
A
TABLE I1 EFFECT OF pH, DESATURATION OF TYROSINASE AND TIMEOF ADDITIONOF Cu64 O N C u + + COMPLEXED~ Age of diluted enzyme
-a F a
3 2 W
0
>
X
0
'7s
complexed (measurements a t the end of 60 minutes) decreased with denaturation of the enzyme.
180
W' Y
1701.
0 2 Uptake a t 1 min., PI.
p H 5.6
pH 6 . 7
C U added ~ ~ 23 Fresh 10 24 hr. Cu64added 17 Fresh a Substrate catechol; values trols.
Cu
+ +
pH 5 6
Complexed, fig.
p H 6.7.-
at 0.0 hr. 34 1.88
2.45 1.01 10 0.77 at 0.5 hr. 0.23 18 1.03 reported are in excess of con-
When Cu6*S04was added after 0.5 hr. of oxidation, the amount of Cu complexed was less than when the Cu was added a t zero time. It is also seen that the effect of the presence of C u + + a t zero time was to increase the oxidation.
Discussion The determination of the CU"+ complexed is based primarily on the fact that the quantity of a cation bound to a definite amount of cation exchanger a t equilibrium is proportional to the conE centration of free ions in the solution.',? If meassurements are made in the absence and presence of 0 IO 20 30 40 50 60 complexing agents, then a t equilibrium the total TIME, MINUTES. metal species in solution, free metal ions and comFig. 1.-Effect of substrate on tyrosinase oxidation a t plexed metal, is proportional to metal on the cation PH 6.7, 25': A, hydroquinone 3 mg., catechol 0.4 mg.; exchanger.* Therefore, under given experimental B, catechol 20 mg.; C, chlorogenic acid 3 mg.; D, caffeic conditions, an increase in the amount of Cu remaining in the solution is directly proportional to the acid 3 mg.; E, protocatechuic acid 3 mg.; F, hydroquinone formation of Cu complexes. 3 mg. The data indicate that the most stable Cu coniplex formed, in the cases investigated, were with othe enzyme, substrate and/or oxidation product was calculated. Controls, consisting of buffered quinone. o-Quinones of chlorogenic and caffeic solution of the chemicals and of buffered solution acids also formed Cu complexes; however, the containing the enzyme in absence of substrates, formation constants were apparently less than for were also run. Residual radioactivity in the ex- o-quinone. Protocatechuic acid and hydroquinone tracted solutions in these cases indicated less than were poor substrates for this tyrosinase preparatibn. Although hydroquinone, containing a trace of 0.09 p.p.m. of CUT+,equivalent to about 0.15 pg. catechol, is a good substrate for this tyrosinase (see of Cu in the solution. The copper content of tyrosinase is about 0.27,,, Fig. l), the Cu complex formed is less stable than and for 20 pg. of enzyme only about 0.04 pg. of Cu the Cu-o-quinone complex and is also more dependent on pH. The mechanism of the oxidation is present. of hydroquinone, using catechol as a mediator, has I t was observed that in the case of catechol subbeen proposed as (I) the catalyzed oxidation of strate, the largest amount of Cu was complexed. catechol, ( 2 ) the chemical reaction of o-quinone For the o-dihydric phenols of chlorogenic, caffeic and hydroquinone to yield catechol and hydroxyand protocatechuic acids, the addition of other groups to the aromatic ring affected the apparent hydroquinone and then ( 3 ) catalyzed oxidation ~f activity of the enzyme and the amount of Cu com; the former to o-quinone and of the latter to hydroxy-o-q~inone.~ plexed. The data in Table I would indicate that Cu coinIn the case of hydroquinone, a 9-dihydric phenol, the amount of Cu complexed was very low. Hydro- plexes of the oxidation products of catechol and quinone containing a trace of catechol was a very hydroquinone have significantly different formation active substrate (see Fig. 1); the amount of Cu constants. If the proposed mechanism of oxidation of hydroquinone is accepted, it would appear that complexed was significant. the presence of the OH group of hydroxy-0-quinone The effect of denaturation of the diluted tyrosinase by aging and the time of addition of C U ~ ~ S is O a~ determining factor in the formation constants of on the amount of the Cu complexed is shown in its Cu complexes. Another possibility is that hy(4) A. E. Martell and M . Calvin. "Chemistry of t h e M e t a l Chelatc Table 11. The decrease in 0 9 uptake in the first Prentice-Hall, Inc., New York, N. Y , 1953. minute is a measure of the denaturation of the Compounds," (J) J R f . Nelson and C. R. n a w s o n , A i i v n i i r ~ rin E N Z Y W O, !4 , $ I ! ] enzyme. I t is observed that the amount of Cu (1 $144).
OF TETRACYCLINE ANTIBIOTICS ACIDITYCONSTANTS
*4ug. 20, 1956
droquinone is oxidized to p-quinone. Then the differences in Cu, not extracted from the solution by the cation-exchange materials, would indicate a higher formation constant for Cu-o-quinone than for Cu-p-quinone. The apparent effect of denaturation of the tyrosinase on the Cu+" complexed, as shown in Table 11, is probably related to the amount of o-quinone formed rather than change in the enzyme. The ap-
4155
parent effect of time of addition of C U ~ ~ to S Othe ~ oxidizing solution on the amount of Cu-+ complexed is probably due to the polymerization of the o-quinone and the decrease in active binding sites. T h a t is, if the Cuf+ was present a t the time of formation of o-quinone, a greater amount was complexed than if the o-quinone was allowed to begin polymerization prior to addition of the Cu"'S0,. SEIV
ORIAE.4SS,h C I S I A S . 4
[CONTRIBUTIOK FROM THE RESEARCH LABORATORIES O F CHAS. PFIZER & CO., COXVERSE MEMORIAL LABORATORY O F HARVARD UNIVERSITY]
INC., A S U THE
Acidity Constants of the Tetracycline Antibiotics BY C. K. STEPHENS, K. MURAI,K. J. BRUNINGS A N D K. B. WOOD\VARL, RECEIVEDFEBRUARY 20, 1956 The three observed dissociation constants of the antibiotics oxytetracycline, chlorotetracycline and tetracycliiie have beer1 assigned to specific acidic groupings. I n each case. the first dissociation is due t o the tricarbonyl system in ring A , the second to the dimethylammonium function' and the third to the phenolic p-diketone system (C.10, C . l l , C.12). The findings reveal t h a t in neutral solution these antibiotic? exist largely as zwitterions.
The antibiotics oxytetracycline, tetracycline and chlorotetracycline may be illustrated by the general structural formula I.? It will be noted that the molecule I contains several acid groupings of a CH,, R I CH3 OH R?\A7/
-,lo
1111
12
contains three distinct acid groups-the tricarbonylthe ammonium cation B, and the methane system -1, -
CHI
Ki
CH3 . OH : - LzR
1
~
B (+ 1 CHI \yH/
,'1
O H 0 HO 0 I Ia, OxytetracyAine R1 = H,R:, OH Ib,Chlorotetracycline Ri C1, R? H IC,Tetracycline R: = R2 H
~ _ _
-~
_.
~
~.
I1
phenolic diketone system C.3 The problem is thus to associate the 3 , 7 and 9 pKa values with the correct system (X, B or C ) .
TABLE I rather unusual type. This communication is coil,OK,, \7ALUES'z (01; ~ ~ Y D R O C H L O R I D E S ) IS A Q U b O C S ~ O I ~ U T I ( 1 S cerned with the assignment of the observed acidity A T 25' constants of each of the three antibiotics to the OsytetracJ-cline ( I a j 3.27 7 :32 :I. 11 specific functions responsible for the dissociation. Chlortetracycline ( I b ) 3 30 7 11 9 27 From an examination of Table I, it is apparent Tetracycline ( I C ) ;3 30 7 (j8 Y rig that the three antibiotics are closely similar in acidity properties. This, of course, is consistent These are carefully corrected thermodyilxmic pK,'. with their structural relationship. Thus, if we They differ very slightly from previously reported value. assign the p K a ' s in any one of the antibiotics we cf. reference 2 and A . Albert, Suture, 172, 201 (1953). will assume a similar relationship to apply in the Our earliest data pertinent to the assigninent of other two. dissociation constants was derived through etheriThe general formula I , written as an acid salt 11, fication studies on Terramycin. * c M'ith diazoI I JAnoED IS P K C U F ~- Our more recent studies have placed the methane, a lOyoyield of a dimethyl etherza (Cz4H2sand p K , ? in serious jeopardy; see ref d h v e assignments for PK;,, NPOY)was obtained in addition to a larger amount I1 of water-soluble, unstable, amorphous material. ( 2 ) (a1 1' .Z Hochstein, C . I< Stephens. I. I I . Ciinuver. P P. Ilegna, I-n is C o , Inc , iur tetrticycline, the registered trade-mark of Chas Pfizer Achromycin is the tr:irle-mark of American Cyanamirl Co I < r this antibiotic.
1,s) In the C ~ T P, > f s y s t c m C !re \vi11 mnkc n o rt?orL ti, s/)ecuI.itc c ! \ t o which hydruiyl function i i ~i , the C 10 or C 1 2 ---O€I. etc x t u a l l y loses its prr,tim in this strongly chelated yntupini: of iitcims
