Kato, Masaru

Yoshida, Zenichi

English

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RIGHT:URL:CITATION:AUTHOR(S):ISSUE DATE:TITLE:Studies on Melanin Formation inAnimals (II) : The Acceleration ofTyrosine Oxidation by Copper-Ammine ComplexKato, Masaru; Yoshida, ZenichiKato, Masaru ...[et al]. Studies on Melanin Formation in Animals (II) : The Acceleration ofTyrosine Oxidation by Copper-Ammine Complex. Memoirs of the College of Science,University of Kyoto. Series B 1957, 24(3): 171-1781957-12-25http://hdl.handle.net/2433/258501       MEMOIRS OF THE COLLEGE OF SCIENCE, UNIVERSITY OF l<YOTO, SERIES B,                   Vol. XXIV, No. 3 (Biology), Article 2, 1957            Studies on Melanin Formation in Animals             II. The Acceleration of Tyrosine exidation by                       Copper-Ammine Complex*                                   By             Masaru KATo & Zen-icki YosmDA     Zoological Institute, College of Science, Department ef !ndustrial Chemistry,            University of Kyote Faculty of EngineeriRg,                                              University of Kyoto                           (Received Oct. 30, 1957)    Concerning the mammalian melanin formation, it has been stated by LERNER(1953) that the oxidation of tyrosine catalyzed by the copper enzyme, tyrosinase, isaccelerated 2n the presence of 3, 4-dihydroxyphenyla}aniRe, dopa, but that tyrosinasecan not act on tyrosine in the absence of dopa. In his studies oÅí non-enzymaticoxidation of tyrosine, FosTER (1950) pointed out that tyrosine could be oxidized inthe presence of both cuprlc ions and a trace of dopa. Henceforth, it has beenaccepted by many workers in this field that the presence of a "threshold concen-tration" of dopa is indispensable for the reaction from tyrosine to dopa• •    In our previous paper, lt was reported that the ethylenediamine, a chelatingsubstance, acted according to its concentration inhibitively or acceleratively uponthe non-enzymatic oxidation of monephenol when it was cata!yzed by copper-ammine,and that the different action was due to both (1) higher stability constant betweenchelating substance and Cu(ii) portion of the complex than the stability constantbetween substrate and the CuCii), and (2) amount of the chelating substanceagainst the copper complex. From the resuks obtalned, we have proposed a plausi-ble scheme of tyrosinase reaction. The mechanism in the accelerating autoxidationof tyrosine caused by the presence of a trace of dopa will be discussed in this paper.    Here we wish to thank Professor Dr. M. IcHiKAwA for his help for the prepa-ra.tion ef the manuscript. Our thanks are also due to Dr. Y. FuJiMuRA of theResearch Institute for Food for his generous supply of oxidized form of ascorbicacid. We are also indebted to Professor Dr. S. TANAKA of .the Biochemical Institutein the College of Science, Mr. S. KrTAoKA of the Biochemicai Laboratory in theCollege of Agriculture, for their valuable suggestions.                              Experimental .                          Material and Methods    Accelerating eff ect of cateckol, dopa ana ascorbic acid upon the autoscidation* Aided by a grant for ScientifiÅë Research from the Ministry of Education.'172 Masaru KATo andZen-ichi YOS}IIDAof tyrosine catalszed bN cof)Per-ammine: The main part of each manometrical vesse}contained i.5 ml of phosphate buffer (pH 10), 1 ml of O.i M copper-ammine and 1 mlef O.04M l-tyrosine. The centre well coritained 0.3ml of i095 KOH. The side armcontained 0.5ml of such sDlution as O.O025M catechol, O.OO05M l-dopa or O.O05Mascorbic acid. These solutions were replaced by distilled water in their blank te$t•The gas space ef the vessel was fil}ed with air. The cock was closed after thevessel reached the thermal equilibrium, and then the solution of the materials wastipped from the side arm into the main part. The vessei was shaken for a giveRperiod at a rate of about 110 strokes per minute at 350C, and the volume ef oxygen-uptake in the buret was read at appropriate intervals.    Accele7ating gff e ct of exidized foywtsof catechol, dopa a•nd ascorbic acid uPonthe autoxidation of ,tyrosi•ne catai.Nxecl bycoPPer-ammine: The main part of eachmanometrical vessel contained 1.5ml ofphosphate buffer (pH10), lml of O.i.Mcopper-ammine and 0.5 ml of such soltitionas O.O025M catechol, O.O05M dopa ore.O05M oxidized form of ascorbic acid.The side arm contained lml of O.04Ml-tyrosine. Other eonditiens are the sameas previous. After shaking at an afore-said rate for 40 minutes in the case withcateÅëhol, and for 80 minutes in the casewith dopa, when the rate of oxygen-uptakebecame constant (zero), the cock wasopened, and then the tyrosine solution wftcstipped from the side arm into the mainpart. (The constant oxygen-uptake inthese cases means the formation of theexidized forms of the chelating sub-stances.) The volume of oxygen-uptake wasthen estimated successively at appyopriateintervais. In blank test the tyrosine solu-tion was replaced by distilled water. Inthe case with the oxidized form of ascor-bic acid (prepared by Dr. Y. FuyMuRA)the experimenta} procedures and conditionswere the same as in the case of ascorbicacid.A"'svoMsimatdi&itosll8017e160150t40lool2G110looboee70oosc40scco10  to 20 oo 4e se ee 70 ep"etl leq 11e lor).          --. time (min.)Fig. I. Accelerating effect of catechol  on autoxidation of tyrosine catalyzed  by copper-ammine complex (pH 10,  350C).Al B', C' and D': oxygen-uptake in the  system of catechol involving various  concentrations of o-benzoquinone and  copper-ammme.A, B, C and D: oxygen-uptake in each  system of the catechol, tyrosine and copper-ammine; A corresponds to A' and so on.E: oxygen-iiptake in the system of tyro- sine and copper-ammine.Studies on Melanin Formation in Animals. II 173                                   Resuks    AcceZerating effec't of cateel•iol iepon tl•ie autoecidation of tyrosine cataZyzed bycePPer-ammiRe; As a preliminary experiment the autoxidation of the catecholcatalyzed by copper-ammine was examinedin the presence of the various concen-tratioRs oÅí o-benzoquinone. The resuits are indicated by AC B', C' and D' in Fig• 1•The figure indicates that the rates of autoxidatien of these solutions decrease in theorder, A'>B'>C'>D', and that the concentrations of o-benzoquinone before the aut-oxidation increase in the reversed order: A'<B'<C'<D'.    Next, the rates oÅí oxygen consuraption in the system of copper-arnmine, tyrosineand catechol were measured, and the results obtained are shown by A, B, C and Din Fig• 1. The rates of tyrosine autoxidation in each system'catalyzed by copper-catechol (1:1) complex increase in accordance with the decrease of o-benzoquinoneconcentration or with the increase of cateehol cQncentration. These results revealthat the accelerated autoxidation of tyrosine may be dependent upon the concen-tration of catechol involved in the system, but not so much on the concentration ofo-benzoquinone. The autoxidation of tyrosine catalyzed by copper-ammine complexis iRdicated by E in Figs. i, 2 and 3..tAgEvo•Igttrp8gegTl8ei70leolse,l40l30120]ioltX)cjOso7eeese4e3e201() v  IO 20 3e 40 Se 60 70 Se 90 lco 110 12e 13e 14e 150 160 17e ISO l90 2(X)                   -" time (min.>Fig. 2. Accelerating effect of dopa on autoxidation of tyrosin;  catalyzed by copper-ammine complex (pff 10, 350C).A and B: oxygen-uptake in the system of depa (B; centained dopaquinene), tyrosine and copper-ammine.A' and B'; oxygen-uptake in the system of dopa (B'; contained dopaquinone) and copper-ammine.E: oxygen-uptake in the system of tyrosine and copper-arnmine,174 Ma$aru KATo andZen-ichi YOSHIDAA{'EvoteV.?5gegAIAsEuyortf""?5erg3ISO17016G150140lsoma110loocoee?eoose 4eoune 10 •o   10 ac lse 40 50 co 7e se se lcrJ 11e 12e lsu           - tjme (min.)Fig. 3. Accelerating effect of ascorbic acid  on oxidation of tyrosine catalyzed by  copper-ammine complex (pH IO, 350C).A: oxygen-uptake in the system oÅí ascorbic  acid, tyrosine and copper-ammine.A': oxygen-uptake in the system of a$corbic  acid and cepper-ammine.E: exygen-uptake in the system of tyrosine  and copper-ammine.looooeoroeese4eseab10• o    ACCeieratiiZg effeCt of doPa uPonthe aUtOXidatiOn of tYrOSine Catai.YXedby coPPer-ammine: A' and B' in Fig. 2show the autox!dation (catalyzed bycopper-ammine complex) of a freshlyprepared co!ourless dopa and a brownco}oured dopa which was exposed toair foy three days at room temperature.The order of A'>B' is parallel to theerder in the case of catechoi, i.e., itis general that oxygen-uptake of thereduced form is greater than that ofthe oxidized form.    A and B in-1•Fig. 2 indicate theaccelerating effect of the colourlessfresh dopa and brown coioured dopaupon the autoxidation of tyrosine re-spective!y. From the Åëomparisofi of A                                .and B, the former is obviously superiorin effectiveness to the latter.    ACCeLerating effeCt of aSCOrbiCaCid UPOn the aUtOXidatiOn of tYQSiUeCatalYZed bY COPPer-aMMine: Autoxi-dation in the composite system oftyrosine, ascorbic acid and copper-ammine complex, and the autoxidationof ascorbic acid catalyzed by copper-   le oo se 4o e le oo so 40 se ee 70 se 9(} loo 11e lee lsu                  -> time (min.)Fig. 4. Acc61erating effeCt of oxidized form of catechol  upon the autoxidation of tyrosine catalyzed by copper-  ammine complex (pH 10, 350C).A:oxygen-uptake in the system of catechol and           . copper-ammme.B:oxygen-uptake in the system of tyrosine, cop- per-ammine and oxidized form of catechol.C:oxygen-uptake in the system of tyrosine and           .  copper-ammlne.D:oxygen-uptake in thett system of copper-ammine  and oxidized forrn of  catechol.M: showing the time when  the cock was epened ancl tyrosine was added.Studies on Melanin Formation in Animals. II l75ammine comp}ex are given by A and A' in Fig. 3. E in the same figure repre-sents the autoxidation of tyrosine catalyzed by copper-ammine complex. From thecomparison of these three lines the accelerating effect is c!ear of the reducingagent on the autoxidation of tyrosine.    Accelerating effect of oxidized form of cketating substances uPon the aute.xi-dation of tyrosine catalvxed bs coPPer-ammine; As is shown by A in Fig. 4, the rateb&Evort:v.?ggegtooeoro60se4eseeele o- ,- time (min.)Fig. 5. Accelerating effeÅët of oxidized form of dopa upon the autoxidation  of tyrosine catalyzed by copper-ammine complex (pH IO, 350C).A: oxygen-uptake in the system of dopa and copper-ammine.B: exygen-uptake in the system of tyrosine, copper-ammine and oxidized  form of dopa.C: oxygen-uptake in the system of tyrosine and copper-ammine.D: oxygen-uptake in the systern of copper-ammine and oxidized form of dopa.-M: showing the time when the cock was opened and tyrosine was added.b-EvoM.aFSgegTlcoseeoroeose4eseee10 e- > time (min.)B:oxygen-uptake in the system of  oxidized form of ascorbic aÅëid, tyro-  sine and copper-ammine.C:oxygen-uptake in the $ystem of tyrosine and cQpper-ammine.D:oxygen-uptake in .the system of copper-ammine and oxidized form of ascorbic acid.Fig. 6. Accelerating effect of exidized forrr} of ascorbic acid upon the autoxidation of tyresine catalyzed by copper-ammine com- plex (pH 10, 350C).176 Masaru KAToand Zen-ichi YOSI{MAof oxygen-uptal<e due to the autoxidation of catechol reaches constant (zero) afterabout 40 minutes as in the case of A', Bi, C' and D' in Fig. !. This rneans thatthe catechol becomes converted into the oxidized form. The rate ef exygen-uptakeof the oxidized catechol thenceforth can be presented by D in Fig. 4. C in the samefigure represents the rate of oxygen-uptake of tyrosine catalyzed by copper-amminea}one. When the two materials are mixed, the rate of oxygen-uptake is foundremarkably enhanced synergistically, as is shown by B (tyrosine, exidized form ofcatechol and copper-ammine).    The similar results have been obtained in the experiments using both oxidizedform of dopa and ascorbic acid as shown in Figs. 5 and 6 respectively. From theseresults, it is clear that the autoxidation of tyrosine is accelerated by the oxidizedform of chelating substances such as catechol, dopa and ascorbic acid. But, thisacceleyating effect is more marked when the reduced form of each substance is used•                         Discussion and Sumrr!ary   It has been pointed out by LERNER and FosTER that dopa is converted into dopa-quinone when it is participating in the accelerating oxidation of tyrosine (1, 2).Their view has been supported also in our previous paper (3, 4) in which we haveconcluded that this acceleratien caused by a small amount of chelating sttbstancewould be due to the special property of a metal complex which increases thestabiiity constant between tyrosine and Cu(ii). Butin the present experiments withcatechol, dopa and ascorbic acid the autoxidation of tyrosine was more acceleratedthan in the experiment with the oxidized form of these substances, as is clear!yunderstandable from the comparison ef Figs. 1, 2 and 3 and Figs. 4, 5 and 6. ThisR// h-o- x/ Rf/Xx,,-ox / rR//1-ox /   - + cu(m •-> vl cu(m •-, L l cua)  xx]-o- /Å~ K/Lo/X LV_o."' xx  dopa Tyrosinase (I) (II)     R //X-ox /02 _t.. T ,.,i R //Å~_o ... Jl)yrosine+o2> cu(I) Y ne > cttal) .--.-......------------->       xv-,o,., X-02H xv,o., X C?IZS;k:.i:pg:e     02 Tyrosine Tyrosine 02--02 Å~/ X/ Å~/+02 X/-ww--- > Cu(i) imrmum> Cu(iD -> Cu(i) >                                                   Cu(I)      / X. -•zzf i X -TfY,r.O,Si:ai,,l/ Å~ / X      (VII) (VIII) (T(b•) (Ix) (x)         Fig. 7. Reaction mechanism of tyrosine oxidation            presence of small amount of dopa. (R:,_,R (l)g.(III)     Tyrosine  Å~/   Cu(D  /x]   (VI) _ -02-    accelerated by theCH2•CH•Nff,•COOH)Å~/  /Cu(I)  Å~ /Cu(Ir) Å~(I)]Studies on Melanin Formation in Animals. II 177 evidence indicates that the form of dopa, for instance, when participating in the accelerating reaction, would be dopasemiquinone as shown in Fig• 7.     The complex (II) in the Fig. 7 would be produced at first by the coordinative binding between dopa and Cu(ii) portion of copper complex of tyrosinase (I). After one electron is transported from dopa to Cu(ii) portion, the corr}plex (II) will be converted into the unstable dopasemiquinone-Cu(i) complex (III). Cu(D portion of the complex (III) will be attacked by oxygen molecule dissolved in the system to produce the complex (IV) and to set an •Oif ion free. The }atter is formed from oxygen in the complex (IV) by addition of one electron from Cu(i) portion. The position occupied by this oxygen is coordinated by tyrosine as is shown in the complex (V). In the complex (V), one electron is transported from dopasemiquinone to Cu(ii) portion, and the complex (V) separates into a dopaquinone and tyrosine- Cu(i) complex (VI). The dopaquinone will be consumed as mentioned by FosTER for melanin formation as soon as it is produced. From the equation,                        K _ [dopaquinone-Cu(ii)]                          - [dopaquinone][Cu(ii)itherefore, higher concentration of dopaquinone-Cu(ii) complex will not be expectedusually in these reaction systems.     The tyrosine-Cu(ii) complex (VIII) will be Xformed from the O,-tyrosine-Cu(i) cemplex (VII) which is produced through the interaction between a molecular oxygen and Cu(i) porticn in complex (Vl) (5). By transfering one electron from tyrosine to Cu(ii) portion in the complex (VIII), a tyrosine free radical (TÅë•), precuysor of dopa, and Cu(i) complex (IX) will be produced, and the Cu(i) comp}ex (X) will be returned to the initial form, the complex (I), through the interaction between a molecular oxygen and Cu(i) portion in complex (X).     Under these consideration, it is safe to say that an important meaning of the presence of asmall amount of dopa for the autoxidation of tyrosine is in the Åíorma- tion of an unstab}e intermediate metal complex of dopasemiquinone.     In the oxidation from tyrosine to dopa catalyzed by tyrosinase, an acce}erating mechanism by dopaquinone has been suggested by LERNER. He speculated also that the catalytic form of copper pertion of tyrosinase is Cu(i) form in his scheme. But usually, the Cu(ii) form of tyrosinase must be more suitable ior transfering the electron from tyrosine te copper portion of tyrosinase. For the electron-transÅíer from tyrosine to Ctt(ii) portioR of the complex, it wil! be required that the electron density of Cu(ii) portion of the complex must be as smal} as possible. The electron distribution wouid be considered to be smaller in Cu(ii) portion of dopaquinone complex. Namely, it would be that dopaquinone has higher oxidation potential than does dopasemiquinone.    Therefore, the participatien of dopaquinone complex in the autoxidatien of tyrosine shou].d be easier than that of dopasemiquinone complex. Nevertheless, the results obtained in the present experimepts were opposite against our expectation. Unfortu- nately we can not understand the reason for it. l78 Masaru I<ATo and Zen-ichi Yos}imA   A possible scheme of enzyme action due to the dopasemiquinone complex isrepresented in Fig. 8. And the reactlon in the acceierating oxidaÅíion of tyrosinewill be given in the cyclic form as is shown in Fig. 9•                                                                                                                                                                                                         Fig. 8. Scheme of enzyme action.                         R: CH2-CH•NH2•COOH                ,.=.f/seL}/di...,f,SpminZtrie        xif,z,z:r ::;z,. nt 027sopa`rv``''!"ac OX} tm)          (TtpD1.2.3,4.5.      7si,t`i))leg.--g">-rss,,.,cit.:paer)su) eX4LJiAy)           o2- `eliiYx ._.._> gspattute         7pme-":l.Sili?pmx<iu.,aufkZl'kaem:,imne?'crpteqavaac-',ma"e`"'                                         Popapm                  Fig. 9. Cycle of tyrosinase action.                      RefereneesLERNER, A. B. (1953) Advances in Enzymology, 14, 73.Fos7'ER, M. (1950) Proc. Natl. Acad. Sci. U. S., 36, 606.KATo, M. & Z. YosmDA (1957) Biological Science (Tol<yo, japan). 9, 6.KATo, M. & Z. YosmDA (in press).MAsoN, H. S. (1957) Science, 125, 1185.

Studies on Melanin Formation in Animals (II) : The Acceleration of Tyrosine Oxidation by Copper-Ammine Complex

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Studies on Melanin Formation in Animals (II) : The Acceleration of Tyrosine Oxidation by Copper-Ammine Complex

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