1006
`
`( Chem. P hann. Bull.)
`I t( 5 )1006-1010(19'11)
`
`Vol. 19 ( 1971)
`
`UDC 547. 466.8. 03.04: 546.56.04
`
`Manometric Study of the Copper-Catalyzed Oxidation of Cysteine
`
`Ax1RA HANAKI and HIROKO K AMIDE
`
`National lnslilute of Radiological Sciences•>
`
`(Received December 8, 1970)
`
`The copper-catalyzed oxidation of cysteine was followed from the consumption
`of oxygen using the conventional Warburg method and from the formation of hydrogen
`peroxide using the spectrophotometric method. The rate of oxygen uptake varied with
`pH and showed a maximum at pH 7.2. Hydrogen peroxide was produced progressively
`during the oxidation, which indicated the possibility of the successive four equivalent
`reduction of oxygen via hydrogen peroxide to water. The amounts of oxygen uptake
`and of hydrogen peroxide formed decreased with the increase of pH. From the peroxide
`formation and the oxygen consumption, the mode of the utilization of molecular oxygen
`was discussed. In the low p H region, the two equivalent redox-reaction, in which oxygen
`is reduced to hydrogen peroxide, may be predominant. As pH increases, hydrogen
`peroxide formed is utilized for the reoxidation of copper(!) ion, and thereupon the
`successivefour equivalent redox- reaction becomes predominant.
`
`Recently, an enzyme catalyzing the oxidation of 2-mercaptoethylamine to hypotaurine
`was found to contain copper, iron and zinc ions. 2 > Copper and iron ions in this enzyme might
`mediate the electron transfer between the substrate and oxygen molecules. The metal ion
`is turned over, during the course of the reaction, between the high- and low-valence states,
`and thereupon electron is transfered from the substrate to the high valence metal ion and from
`the low valence metal to oxygen molecule. The sulfhydryl group is autoxidized to disulfide,
`In the disulfide formation, the tum-over of the metal catalyst
`sulfinic and sulfonic acids.
`is probably coupled either with the two equivalent reduction of oxygen to hydrogen peroxide
`or with the four equivalnt reduction of oxygen to water. The present work was undertaken
`to investigate the copper-catalyzed oxidation of cysteine in order to elucidate some of the
`features of the biological oxidation.
`In some copper-containing enzymes, i.e., galactose oxidase3 > and diamine oxidase,4> the
`tum-over of copper is coupled with the two equivalent reduction of oxygen and hydrogen
`peroxide is accumulated as the final product from oxygen. In other enzymes, i.e., laccase5>
`and ascorbic acid oxidase,6> the turn-over is coupled with the four equivalent reduction of
`oxygen and water is the final product.
`In the latter enzymes, since hydrogen peroxide is not
`utilized efficiently for the oxidation, it is considered that oxygen is reduced directly to water
`but not via hydrogen peroxide, i.e., the simultaneous four equivalent reduction.71
`In the
`<:hemical oxidation, oxygen may be reduced via hydrogen peroxide to water. The present
`paper dealt with the utilization of the molecular oxygen in the copper-catalyzed oxidation.
`The determination of the rate was done by measuring oxygen consumption.
`
`1) Location: Anagawa-4, Chiba.
`2) D. Cavallini, S. Dupr~. R. Scandurra, M.T. Graziani and F. Cotta-Ramusino, European j. Biochem.,
`4, 209 (1968).
`3) J.A.D. Cooper, W. Smith, M. Bacila and H. Medina,]. Biel. Chem., 234, 445 (1959).
`4 ) S.Y. Levine and S. Kaufman, J. Biol. Chem., 236, 2043 (1961) .
`.5) B.G. Malmstrom, "Oxidases and Re.lated Systems," ed. by T.E. K ing, H .S. Mason, l\1. Morrison,
`J. Wiley and Sons, Inc., New York, 1965, p. 207.
`,6) G.K. Stark and C.R. Dawson, " The Enzymes", ed. by P. Boyer, H.A. Lardy, K. l\lyrback, Academic
`Press, Inc., New York, 1963, p. 297.
`7) E. Frieden, S. Osaki, and H. Kobayashi, J. Gen. Physiol., 49, 213 (1965).
`
`Eton Ex. 1058
`1 of 5
`
`

`

`No. 5
`
`1007
`
`Experimental
`
`Material--The Cu(II) solut ion was prepared from copper sheet, 99.999% purity. The copper sheet
`accurately weighed was dissolved in a small amount of cone. HNO3 and then diluted with then twice distilled
`water. This stock solution, standardized with the complexometric titration method if necessary,•> was
`diluted t o a desired concentration with 0.1 N KNO1 . The cysteine solution was prepared just before use
`from commercially available L•cysteine H CI monohydrate. The buffer solutions used were as follows;
`0.02M phosphate, pH 5.9 to 7.1, 0.02M glycylglycine, p H 6.8 to 8.4 and 0.02lt glycine, pH 8.4 to 9.5. The
`ionic strength of all the solutions were adjusted to 0.1 with KNO1 .
`Kinetic Procedw-e--The consumption of oxygen was followed with the conventional Warburg mano(cid:173)
`metric technique at 20°. The main compartment of the reaction vessel contained 2.0 ml of 1.00 x 10-:M
`cysteine, 5.0 ml of the buffer solution and 2.0 ml of 0.1N KNO1, and the side arm contained 1.0 ml of 1.40x
`10-•M copper solution. Routinely, the total volume of the reaction mixtures was 10 ml. Being connected
`with the manometer, the reaction vessel was thermostatted at 20° and the solution was saturated with pure
`oxygen. After 20 min of temperature equilibration, the catalyst was tipped from the side a rm and the reac(cid:173)
`tion was started. The oxygen uptake was read every 1 min thereafter. During the measurement, the vessel
`was shaken mechanically at the rate of 135 oscillations/min. After the uptake of oxygen reached t o a con(cid:173)
`stant level, the reaction was stopped and H 1O1 produced was determined spectrophotometrically with TiCl,.1>
`In another experiment, the formation of H 1O 2 was followed spectrophotometrically. The composition
`of the reaction mixtures was same as the manometric measurement. During the me,t_<mrement, the oxygen
`gas presaturated with 0.1 NKNO3 was bubbled continuously into medium at 100 ml/min.
`
`Result and Discussion
`
`It is postulated that the first step in the metal-cat alyzed oxidation is the formation of
`the metal-substrate complex, which is decomposed subsequently to the low valence metal
`ion and probably the free radical of the substrate.10> The molecular oxygen may play a role
`in the reoxidation of the low valence metal ion, which is catalyt ically inactive, to the high
`valence metal ion. The reoxidation of the low valence metal ion, copper (I) in the present
`case, is expressed as follows:
`• + Hz(),
`2Cu• + 02 + 2H• +==!: 2Cu2
`2Cu• + H,O, + 2tt• += 2Cu2• + 2Hz0
`
`( 1)
`
`(2)
`
`Provided that the reactions (1) and (2) proceed very rapidly, this assumption may be valid,
`the rate of oxygen uptake can be used as the rate of cysteine oxidation .
`The consumption of gaseous oxygen increased linearly in the initial stage. The rate of
`the reaction was determined graphically from the initial linear part of the reaction curve.
`The reaction curve, plotted the oxygen consumption against the reaction time, was shown
`in Fig. 1. The rate of oxygen uptake and the total amount of oxygen uptake, 0.25 to 0.5
`equivalent with respect to the substrate, were appeared to vary with pH. The pH dependence
`of the rate and the amount of oxygen uptake were shown in Fig. 2. Both curves displayed
`maxima near pH 7.
`Molecnl:i.r oxygen is utilized for the reoxidat ion of the catalytically inactive copper (I)
`If hydrogen peroxide produced in the reaction (1) has not an ability to reactivate the
`ion.
`catalyst. the peroxide is the final product of oxygen and the over-all reaction for the cysteine
`oxidation may be shown by the reaction (3);
`2CySH + O, ~ CyS-SCy + Hz02
`
`(3)
`
`8) G. Schwarzenbach, "Die komplexometrische Titration," Ferdinand Enke Verlag, Stuttgart, 1955, p.68.
`9) A. Weissler, Ind. Eng. Chem. Anal. Ed., 17, 695 (1945).
`10) I. Pecht, A. Levitzki and M. Anbar, J. Am. Chem. S oc., 89, 1587 (1967); M. M. Taqui Khan and
`A.E. Martell, J. Am. Chem. Soc., 89, 4176 (1967); A. van Heuvelen and L. Goldstein, ]. Phys. Chem.,
`72,481 (1968); D. Cavallini, C. de Marco and S. Dupre, Arch. Biochem. Biophys., 130, 354 (1969);
`A. Hanaki, Chem. Pharm. Bull. (Tokyo), 17, 1839 (1969).
`
`Eton Ex. 1058
`2 of 5
`
`

`

`1008
`
`Vol. 19 (1971)
`
`-:::- 100
`::i..
`.,
`
`.;,I; .. C.
`Q ..,
`>,
`><
`0
`
`:,
`
`C
`
`.s
`~6
`...:,
`
`C
`
`., ,..,. ..
`g- 4
`., ..,
`>,
`...
`>(
`0 2
`0 .,
`'° ~
`
`150
`
`140 3
`.,
`130 ~
`~ :,
`..,
`120 ~
`>,
`><
`llOO
`
`Time (hr)
`
`Fig. 1. Typical Reaction Curves of the
`Oxidation of Cystcinc
`-0-0-: pH 6.2
`---()-()-: pH 7.4
`_____ , pH 8.4.
`copper ion: 1., ox10-•,.
`
`cysteine: 2.oox10-••
`
`pH
`
`Fig. 2. Effect of pH on Oxygen Uptake
`-0-0-: rate of oxygen uptake
`t;.-t;.-: amount of oxygen uptake
`-
`oopper: 1.40x10-•~
`,~y!.t~n~ : 2.oox 10 -a . .,
`
`In this case, the
`where CySH and CyS-SCy represent cysteine and cystine, respectively.
`rate of oxygen uptake corresponds exactly to the oxidation rate. The rate of oxygen uptake
`is equal to that of peroxide formation. The amount of oxygen consumption will be half equi(cid:173)
`If the reaction (2), as well as (1), contributes to the reacti(cid:173)
`valent with respect to cysteine.
`vation of the catalyst, both the oxygen consumption and the peroxide formation will reduce
`in accordance with the increasing contribution of the step (2). The reactivation of the catalyst
`mentioned above is coupled with the successive four equivalent reduction of oxygen via
`hydrogen peroxide to water. Besides this mechanism, the contribution of the simultaneous
`four equivalent reduction, not via hydrogen peroxide, may be possible.
`
`~
`
`" 3
`' 0
`~ .,
`2
`>< 0 ...
`~
`C ... ...,
`0 ... ..,
`>,
`:r:
`
`10
`
`20
`Time (min )
`
`~o
`
`Fig. 3. Hydrogen Peroxide Formation
`in the Oxidation of Cysteine
`copper: l . .Ox-l0-•11
`cystelne: 2.00xl0-'11 pH 7.4.
`
`(4)
`
`According to this redox- system, hydrogen per(cid:173)
`oxide is never formed in any step of the re(cid:173)
`action. On the contrary, in the mechanism of
`the successive four equivalent redox-reaction,
`hydrogen peroxide should be detected except
`in the extreme case where the rate of the step
`(2) is far rapid as compared with that of (1).
`The formation curve of hydrogen peroxide
`shown in Fig. a indicates the impossibility of
`the simultaneous
`four equivalent redox(cid:173)
`reaction.
`The tot al amounts of oxygen uptake and
`of hydrogen peroxide formed, measured at four
`different pH values and in the definite con(cid:173)
`centration of the substrate, were presented in
`. Table I. The ~aximum value for the oxygen
`uptake was appeared at pH 7 .2. The ratio of
`
`Eton Ex. 1058
`3 of 5
`
`

`

`No. 5
`
`1009
`
`the peroxide formation to the oxygen uptake decreased with the increase of pH. Since
`hydrogen peroxide produced in the step (i) may be partly utilized for the reoxidation of the
`catalytically inactive copper (I) ion, the over-all reaction concerning the reactivation of
`the catalyst may be rewritten as folloWs:
`
`2(l+n)Cu+ + 0 2 + 2(l+n)H+ .=::t 2(l+n)Cu'+ + 2nHs0 + (l-n)H202
`
`(5)
`
`where n, a positive real number less than unity, means the extent of the contribution of the
`step (2) in the reactivation. From the concentration ratio of hydrogen peroxide to oxygen
`uptake, the n value can be estimated:
`n = 1 - (H~,]/[O,]
`
`(6)
`
`It is described statistically that the reactivation of copper (I) ion is the 2 (l+n) equivalent
`oxidation. The result shown in Table I indicates that the peroxide is utilized undoubtedly
`for the reoxidation of copper (I) ion and that the reactivation is inclined to the successive four
`equivalent redox reaction beyond pH 7.
`
`TABLE I. Oxygen Consumption and Hydrogen Peroxide Formation at Various pH's
`
`pH
`
`6.7
`6.9
`7.4
`8.2
`
`H 10 1 formation
`10-<114
`
`0 1 consumption
`µl
`
`[0.J/[CySH]0
`
`4 >
`
`3.16
`3.28
`2.20
`0.31
`
`266
`284
`268
`230
`
`0.28
`0.30
`0.28
`0.24
`
`n
`
`0.43
`0.44
`0.60
`0.94
`
`11) Mole of oxygen consumed for the oxidation of l molar cysteine.
`total coocentratioo ol copper, [Cu].: l.40x 10-•11
`initial 0DDC!f"..ntr;atint\ of c~kiM• [CySHJ.: t .00x l04 x
`
`The peroxide formation and the oxygen uptake were appeared to vary, depending on the
`substrate concentration. Some examples were presented in Table II. Though the real rate
`for the oxidation, d[CySH]/dt, should increase with the substrate concentration in any pH
`region, the rate of oxygen uptake did not always display the concentration dependence as shown
`in Table II: The rate increases with concentration of the substrate at pH 6.9, while it did
`not increase and showed somewhat a constant level at pH 7.4. The peroxide formation did
`not depend regularily upon the substrate concentration. Those irregular relations may come
`from the different participation of the step (2) in the reactivation of the catalyst, because the
`n value indicates that the successive four equivalent redox reaction proceeds effectively as
`
`ThllL£ II. Oxygen Consumption and Hydrogen Peroxide Formation
`
`Cysteine
`10-1M
`
`Rate
`µI/mm
`
`H 10 1 formation
`10--'M
`
`0 1 consumption
`µl
`
`0.50
`1.00
`2.00
`4.00
`0.50
`1.00
`2.00
`4.00
`
`3.93
`6.52
`8.45
`10.73
`9.78
`9.16
`9.50
`9.39
`
`1.83
`2.65
`3.22
`2.63
`1.86
`2.32
`2.20
`0.62
`
`102
`176
`290
`492
`106
`164
`266
`452
`
`pH
`
`6.9
`6.9
`6.9
`6.9
`7.4
`7.4
`7.4
`7.4
`
`n
`
`0.13
`0.28
`0.47
`0 .74
`0.15
`0.32
`0.62
`0.93
`
`total CODcentration of copper:l.40x 10-•11
`
`Eton Ex. 1058
`4 of 5
`
`

`

`1010
`
`" ·- 5 ~
`3
`
`4
`
`7
`
`8
`
`9
`
`pH
`
`Fig. 4. pH Dependence of the Corrected
`Rate of Oxygen Uptake
`experimental details as under Fir. 2
`
`Vol. 19 (1971)
`
`the concentration of the substrate increases.
`The oxidation with hydrogen peroxide would
`depend on the concentration of the substrate.
`The mode of the reaction was thus difler(cid:173)
`ent according to the pH variation. Since the
`total amounts of oxygen uptake vary de(cid:173)
`pending on the pH variation, the apparent rate
`has to be corrected relative to the amount of
`oxygen consumption under the corresponding
`condition. The pH variation of the corrected
`rate was presented in Fig. 4. The maximum
`was appeared at pH 7.3-4. The maximum
`rate in the iron-catalyzed oxidation is ap(cid:173)
`peared at pH 8.11> The reason why the maxi(cid:173)
`mum rate in the copper-catalyzed oxidation
`is appeared in the lower pH region would be
`explained from the thermodynamically strong
`interaction of the metal to the substrate.
`
`Acknowledgement
`
`The authors wish to thank Dr. S. Akaboshi of this institute for helpful advide.
`
`11) J .E. Taylor, J.F. Yan and J. Wang,]. Am. Chem. Soc., 88, 1663 (1966).
`
`Eton Ex. 1058
`5 of 5
`
`