OXIDASE AND PEROXIDASE REACTIONS IN THE PRESENCE OF DIHYDROXYMALEIC ACID*

BY BRI’ITON CHANCE

(From the Johnson Research Foundation, University of Pennsylvania, Philadephia, Pennsylvania)

(Received for publication, November 19, 1951)

The remarkable property of peroxidases to act as aerobic oxidases in the presence of dihydroxymaleic acid has led to the view that peroxidase acts as an oxidase by means of a ferric-ferrous cycle of valency change as do the cytochromcs (1). Appropriate instrumentation and experimental condi- tions have now made it possible to measure simultaneously the oxygen, dihydroxymalcic acid, and peroxidase kinetics and to separate the react.ion nearly completely into the oxidase (Equation 1) and pcroxidase (Equation 2) activities that comprise the over-all reaction of Equations 1 and 2.’

HRP 02 + DHii - Mn++ Hz02 + DKA (1)

Hz02 + DHM - HRP-+ 2HzO + DKA (2)

In this paper we shall reinterpret some of the earlier work and there- fore a brief summary follows: Interestingly enough, Ssent-Gyorgyi (2) began the search for a DHM oxidase because DHM, like catechol and succinic acid, forms colored complexes with ferric salts. Later with Bangs (3) hc found such a DHM oxidasc in many plants, especially horseradish. They found t,hat; 1 atom of oxygen is equivalent to 1 molecule of DHM. Banga and Philippot (4) showed the reaction to be activated by Mn++, demonstrated peroxide formation, and concluded that the oxidation product of DHM is the diketo acid, as in Equation. 1 above. At the same time, Theorell and Swedin (5) also found Mn* activation and catalase inhibition and identified DIIM oxidase as a highly active peroxidase. They found that 2 oxygen atoms are equivalent to 1 DHM molecule. In a model system with cytochrome c as a peroxidase they concluded that Mn++ acts as a mediator between DHM and peroxidase-peroxide (see Equation 1). In a second paper (1) they proposed a rather different mechanism: they

* The research was supported in part by a grant from the Division of Iiesearch Grants and Fellowships, National Institutes of Health, United States Public Health Service, and in part by a grant from the Office of Naval Research.

1 DHM = dihydroxymaleic acid, HRP = horseradish peroxidaee, LPO = lacto- peroxidase, CcP = cytochrome c peroxidaae, DKA = diketo acid of DHM.

577

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

578 OXIDASE AND I’EROXIDASE RE.4C’HOSS

concluded that HRP acts directly as an aerobic oxidase by means of a ferric- ferrous cycle of valency change of peroxidase iron. This conclusion is based upon the light-sensitive carbon monoxide inhibition of the oxidase activity (6) and upon the effect of carbon monoxide upon t.he absorption spectrum of HRP in the presence of DHM. Under the experimental con- ditions of this paper WC find no effect of carbon monoxide upon the absorp- tion spectra of HRP, 1~1’0, and CcP in the presence of DHM and find no carbon monoxide inhibition of the Mn++-activated oxidase activity. These results support the mechanism of Equations 1 and 2. We also find, Mn++ to be essential to maximal activity under our experimental conditions. Thus this reaction requires two different heavy metal cat.alysts.

EXPERIMENTAL

Me&&-The apparatus for measuring the rapid chemical reactions in the visible and ultraviolet regions of the spectrum is described else- where (7), as is the polarographic technique for measuring oxygen uptake (8). The apparatus was modified to permit operation at 4”. The DHM was obtained from the Delta Chemical Works and gave a purity of 80 per cent on iodine titration. The values of tie0 and ~~26 are 10 and 1.7 cm.-’ per m&, respectively, at pH 4.0 (see Table I). The HRP prepara- tion was carried out by Dr. A. C. Maehly and Miss Margit Grenholm, who followed the procedure of Theorell (9), and was very nearly pure on the basis of the value D&Dm = 3.0. CcP of about 20 per cent purity was prepared by Mr. T. M. Devlin and Mr. D. Trevethan, who used un- published modifications of the procedure of Abrams et al. (10). Pure J,PO was kindly donated by Dr. B. D. Polis of the Eastern Regional Research Laboratory.

Resdls

Anuerobit Reaction of HRP, DHM, and H202--In view of the specificity of HRP for the enediol group, the finding of a rapid peroxidatic reaction probably giving the diketo acid is to be expected (Equation 3).

COOH COOH

I I C-OH

HrOz + ( HRP (f=”

C-OH A + 2&O (3)

c=o

I I COOH COOH

Such a rapid reaction is demonstrated by the traces of Fig. 1, A which result from mixing anaerobic HRP with anaerobic H20z and excess DHM. The replacement of the spent solutions of a previous experiment causes the

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

B. CHANCE 579

abrupt downward deflection of the trace at 325 rnp (an increase of optical density). The zero order disappearance of DHM is indicated by the linear rise of the trace. At 425 rnp, the formation and disappearance of the peroxidase-peroxide Complex II are recorded. The nature of the kinetics suggests that the reaction of peroxidase-peroxide Complex II with the electron donor is the rate-determining step and the value of its second

Anaerobic Aerobic Peroxidase Oxidase Action Action

A 6 FIG. 1. Rapid spectrophotometric measurements of the kinetics of DHM disap-

pearance measured at 325 rnr and the appearance and disappearance of the peroxidase complex formed directly from Hz02 under anaerobic conditions in A and from DHM under aerobic conditions in B. The reactions were carried out in rapid flow appara- tus with 0.17 cm. optical path. An increase of optical density is indicated by an upward deflection at 424 rnr and by a downward deflection at 3% rnp. In A, 2 mM anaerobic DHM plus II202 are mixed with 1.64 PM anaerobic HRP. In B, 2 mM anaerobic DHM plus 24 PM Mn++ are mixed with I.64 MM HRl’ in aerated buffer. pH 4.0, 0.1 M acetate buffer. The optical density change of 0.020 at 32.5 rnr corresponds to 70 PM DHM. 4’. (Experiment 821a.)

order velocity constant is about 2 X 1W M+ X sec.-l at pH 4, similar to the value for the enediol ascorbic acid (11). As peroxide is formed from the oxidase reaction, as described below, this peroxidatic reaction will occur as a part of the aerobic oxidase reaction.

Aerobic Reaction of HRP and DHM-Similarities are obvious in the records of Fig. 1, A and B. A peroxidase complex is seen to appear and to disappear in synchronism with the initiation and termination of the over-all activity, and a similar rate of disappearance of DHM is seen in the ter- minal phases of both reactions. The value of the velocity constant for

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

580 OXIDASE AND PEROXIDASE REACTIONS

the slow phase of the oxidese activity agrees to within the experimental error with the value calculated from the anaerobic peroxidase reaction, 1.9 and 2.2 X 104 M-I X sec.-‘, respectively, being obtained in a particular case (Experiment 821a).

In the aerobic reaction, however, there appears a xapid initial disap- pearance of DHM at many times the rate of the slower reaction. These two phases of the aerobic reaction are of roughly equal amplitude and, in the course of the paper, will be shown to represent the reactions of Equa- tions 1 and 2, According to Equation 1, the conversion of O2 to 11202

_ Free Peroxidose(O6yM)

Platinum Microelectrode

Time after-flow stops 0 (sec.)

FIG. 2. A correlation of the rapid disappearance of oxygen and DHM. The ex- perimental conditions are generally as in Fig. 1, B except that 1.2q HRP are mixed with the reactants and the platinum micro electrode polarized at -0.3 volt is in- serted into the capillary observation tube of the flow apparatus. The platinum micro electrode trace goes off scale during the flow of liquid in the observation tube and the point marked 190 JIM 02 is calibrated in a eeparate experiment (Experiment 821b).

causes the rapid disappearance of DHM and then the anaerobic peroxi- datic reaction of Hz02 and DHM causes the slower reaction of Equation 2 to occur at the same rate as in Fig. 1, A.

In order to show that there is a rapid disappearance of oxygen cor- responding to the rapid disappearance of DHM, the enzyme concentra- tion is decreased to permit a clear recording of the platinum micro electrode data and the result is shown in Fig. 2. Nearly all the oxygen disappears in the fast reaction. The slower tail on the platinum micro electrode trace is not caused by the oxidaae reaction; a similar effect is observed in the anaerobic reaction of Fig. 1, A and is caused by the disappearance of DHM. (Quantitatively, 76 per cent of this slow effect is explained in this manner.) A much more accurate correspondence of the fast DHM dis-

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

B. CHANCE 581

appearance with the oxygen uptake is obtained at lower enzyme concen- trations, at which the sensit,ivity of our apparatus unfortunately is inade- quate to regist,er the kinetics of the peroxidase complex.

Stoiehiometry of Peroxidase and Oxidase ReactionsTwice as much DHM will be consumed in the aerobic reactions (Equations 1 and 2) as in the anaerobic peroxidatic reaction (Equation 2). We have first confirmed the stoichiometry of the peroxidatic reaction in which the amount of DHM disappearing in the presence of Hz02 on the basis of spectrophotometric

TABLE I

Determination of Molecular Extinction Coegicients and Purity for Dihydrozymaleic Acid

(Delta, Sample 1.) (Experiments 82Oa, 820b, and 827.) The values of l are in cm.-’ X mK*. ~11 4.0, 0.1 M acetate buffer.

x e (calculated on a t (from anaerobic weight basis) B& titration) t (from It titration) Average l

-__- - ~- ~.-~.

m

290 7.1 10.6 9.50 325 1.22 1.81 / lY.7 1.63

~__

TABLE II

Effect of Oxygen Concentration upon Stoichiometry of Reaction The values of e arc in cm.-* X rnaT1 (Experiments 784a, 814e, 820).

-

Initial 02, PM. . . . . . . 24 48 120 Apparent ~315 = AD,~~/[O~]. . . . . . 5.9 4.1 3.2 True ~a*~*. . . . . 1.7 1.7 1.7

Stoichiometry, ap~r~~~6ms. . . . . . . . . . . . . . 3.6 2.4 1.9

* See Table I.

measurements at 325 rnp is compared with the iodine titration value for DHM, and confirmation of the 1:l stoichiometric value is obtained as shown in Table I. Table II shows the results of a study of the amount of DHM oxidized in the oxidase reaction and reveals a peculiar effect; the [DHM]/[02] value increases at low [O,]. However, in the region of our experiments, the stoichiometric value approximates that required by Equa- tions 1 and 2. In the oxidase reaction that proceeds in the absence of Mn++ at higher temperatures, the [DHM]/[Oz] value is about 1.6 (Experiment 527). Such a variation of the stoichiometry of the reaction is attributed to unstable reaction products that react with DHM at low oxygen concen- trations and with oxygen at high oxygen concentrations and higher tem- peratures. It is also possible that oxidation products of DHM may act

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

582 OXIDASE AND PEROXIDASE REACTIONS

as electron donors. These results indicate the complexity of the chemistry of DHM oxidation and the need for its further study.

Kinetics of Fast Oxidase Reaction-Fig. 3, A and B, shows that the fast reaction is much more sensitive to Mn++ and oxygen concentration than the slow reaction. Again we identify the fast oxidase reaction with Equa- tion 1 and the slow peroxidatic reaction with Equation 2. Maximal oxi-

z Manganous !Sulfate(yM) A

Initial

Rate

FKL 3. The effect of the concentration of Mn *, 02, and Hz02 upon the oxiduse activity. In A, 1.64 PM HRP in aerated buffer are mixed with 1.54 mM DHM plus various [Mn++] in anaerobic buffer. The ordinates represent the slope of the oscillo- graph traces which show DHM disappearance. The unite are in milIimcters per second. In B, 1.64 PM HRP in buffers of various [O,] are mixed with 1.76 rnM DHM

plus 24 PM Mn++ in anaerobic buffer. The ordinates are the same as in A. In C, 1.64 PM HRP in aerated buffer are mixed with 1.54 mu DHM plus various [lIzOr] in anaerobic buffer. pH 4.0, 0.1 M acetate buffers, 4“. (Experiments 823, 824, and 825.)

daae activity is obtained with 50 PM Mn * for the particular experimental conditions, a value which is 20 times the peroxidase activity, corresponding2

1 This value of k, correaponde to &o, = 73,ooO 4. per hour per mg. for 1 mM DHM at 0” and at the higher DHM concentrations used by Theorell and Swedin (5 IILM) a value of &a = 370,ooO would be expected. This gives an oxidaae activity per metal atom of the came magnitude aa that of ascorbic acid oxidaae: the value of turnover number of the DHM oxidsee is measured to be 400 get.-’ for 1 mM DHM at 0”, and Dawson (12) gives 683 sec.-l per Cu tF atom for optimal conditions with ascorbic acid oxidaae at room temperature. It haa not been possible to reproduce the value of&o,= 2,400,ooO ~1. per hour per mg. found earlier by Theorell and Swedin (1).

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

B. CHANCE 583

to k, = 4 x lo6 M-’ x sec.-r. Half maximal oxidase activity requires 70 PM O2 for these conditions. Hz02 in the absence of Mn* gives rise to some oxidase action, as shown in Fig. 3, C, but the rate is one-tenth the maximal rate with Mn++ only (Fig. 3, A). At 4”, the free enzyme gives very little oxidase activity without Hz02 addition, as shown by the ordinate intercept of Fig. 3, C. Thus the peroxideperoxide complex is a much

3.5 OJM)

FIG. 4, A AND B. The effect of a reduction of the peroxide concentration caused by catalase or hydroquinone upon the oxidase activity of HRP. 1.4 FM HRP, 500 ELM DHM, 0.1 M acetate buffer, pH = 4.7, 2.5”. (Experiments 26% and 2&k.)

c FIO. 4, C. The effect of cyanide upon the fast and slow phases of the disappear-

ance of DHM. 1.64 JLM HRP plus various [HCN] in aerated buffer are mixed with 1.14 rnx DHM plue 24 PM Mn* in anaerobic buffer. The ordinates are defined in Fig. 3, A. At 170 PM HCN the fast reaction still haa a rate of 1.2 PM per second. pH 4.0,O.l M acetate buffer, 4’. (Experiment 822.)

better oxidase catalyst than the free enzyme. The experiment of Fig. 3, A is imperfect in this respect; the oxidase rate with zero [Mn++] is usually about 3 per cent of that with optimal [Mn++].

Swedin and Theorell have already shown that HzOz is necessary for the oxidase activity by demonstrating catalase inhibition (1) ; our quanti- tative results are shown in Fig. 4, A. Similar inhibition ehould also be caused by addition of an electron donor in addition to DHM that would accelerate the reaction of Equation 2 and thereby reduce the peroxide concentration. Hydroquinone does just this, as shown by Fig. 4, B.

Carbon monoxide causes no measurable inhibition of the oxidase activity

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

584 OXIDASE AND PEROXIDASE REACTIONS

of the Mn++-activated syst.em (Experiment 7&k), although cyanide in- hibits both the oxidase and peroxidatic reactions, as shown by Fig. 4, C. The cyanide inhibition of the latter reaction is expected (13) ; the inhibition of the former is in accord with our observation that the peroxidase-peroxide complex is a better catalyst than the free enzyme. A large excess of cyanide does not stop the Mn++-activated reaction; at 170 I.~M of cyanide 10 per cent of the oxidase activity remains.

In the Mn++-free system, we can demonstrate some CO inhibition. The oxidase activity is so low under these conditions (1.3 mM of DHM, 4”, pH 4.0) that the rate of production of H202 from the oxidase system is

500540580 500 540 580 520 560 600 NyJ) Ahyd Xmyd

FIG. 5. The visible bands of the compounds formed by HRP, Ccl’, and LPO in the presence of H202 and DHM. For HRP, 200 PY DHM, 5.1 pM horseradish peroxidase, pH 4.7,0.1 M acetate buffer. For CcP, 200 pM DHM or 70 PH H& plus 5.45 PX cyto- chrome c peroxidase, pH 4.7, 0.1 M acetate buffer. For LPO, 200 PM DHM or 140 PM Hz02, 3.2 PM lactoperoxidase, pH 4.7, 0.1 M acetat.e buffer. Complex III for HRP is from Keilin and Hartree (15) (RS-3).

accurately and sensitively measured directly from the rate of formation of the peroxidase complex. The half time of 30 seconds for the latter reaction is doubled in the presence of 75 per cent CO, presumably because CO-sensitive iron impurities are catalyzing the oxidase effect in the absence of Mn++ (Experiment 783b).

Nature of Peroxidase Complex Formed in Presence of DHiW-In the presence of low concentrations of peroxide and an electron donor, one would expect to find peroxidase in the form of Complex II (14). But Swedin and Theorell observed visually the absorption bands of Complex III and, in the presence of DHM only, we confirm their results in a quan- titative manner, as shown by Fig. 5, C, where there is illustrated a close correspondence of the visible absorption bands of Complex III formed from

8 These spectral data were obtained by Mr. T. M. Devlin by using a rapid record- ing spectrophotometer constructed by Dr. C. C. Yang.

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

B. CHANCE 585

a large excess of Hz02 (set Keilin and Hartree (15)) or from DHM. The addition of DHM to LPO also gives the tertiary complex, but only the secondary complex is obtained in the case of CcP, for which no tertiary complex is known.

Similar agreement is obtained in the region of the Soret bands of these three enzymes, and the experimental results are shown in Fig. 6. In the case of HRP the spectrum of Complex III formed from excess Hz02 repre- sent.s a correction of the spectrum obtained earlier ((16) and unpublished data).

In summary, DHM reacts with these peroxidases as does an excess of II202; somehow DHM causes the transition from Complex II to III to

so. O4 ’ ’ ’ ’ 360 420 4k3 Npp) Nrnp) aby)

FIG. 6.. The Sorct bands of the compounds formed by HRP, CcP, and LPO in the presence of HZ02 and DHM. For LPO, 300 PM DHM or 200 /AM Hz02 plus 0.32 PM lactoperoxidase, pH 4.7, 0.01 M acetate buffer. For Ccl’, 63 ~NI DHM or 2 ,AX H202 plus 0.85 PM cytochrome c peroxidase, pH 5.5, 0.01 M Pod’. For HRP, 300 PM DHM plus 1.6 PM horseradish peroxidase, pH 4.7,0.1 M acetate buffer (RS-2). (The Sorct band of Complex III is from unpublished data of the author.)

occur at much lower average concentrations of oxidase-generated peroxide than peroxide in solution, but the oxidase-generated peroxide may achieve a high local concentration at the hematin iron group of peroxidase.

Since the identity of the complex formed in the presence of DHM is established as a secondary complex for CcP and a tertiary complex for LPO and HRP, and these complexes were not previously found to react with carbon monoxide, it is hard to explain how Theorell and Swedin could obtain ferroperoxidase-CO by bubbling CO through the solution of HRP and DHM. Our results for CcP, LPO, and HRP are given in Fig. 7, A, B, and C. In none of the experiments were we able to obtain the CO bands without the addition of hydrosulfite, as would be expected if the complexes formed from DHM are ferric peroxide complexes. With HRP and DHM an intensification of the spectrum is observed upon ad-

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

586 OXIDASE AND PEROXIDABE REACTIONS

dition of CO. This effect could have been mistaken for the formation of the CO compound by the earlier workers.

Nature of Complex Thai Forms in Presence of DHM and M&t-Under our conditions for optimal oxidase activity, the characteristic band of Complex III is not observed at its full intensity, as shown by Traces B and C in Fig. 8: as the amount of Mn++ approaches the optimal value, the height of the Soret band falls to that of Complex II ((16) and unpublished data). The presence of Complex II under conditions similar to those for Trace C (Fig. 8) is verified by measurements in the visible region of the spectrum which show that the ratio AQ,~o ,,,,,/AQ,~ mp is 0.8, compared to the

+DHMtCO

500 540 580 500 540 580 Aby) Xy) A(@

Fxo. 7. The effect of carbon monoxide upon the compounds formed by HRP, CcP, and LPO in the presence of DHM, with and without the addition of hydrosulfite. The traces for the compounds formed with DHM are the same as in Fig. 6. The traces labeled +CO are after 2 to 5 minutes of bubbling with carbon monoxide, and the traces labeled +Na&O, are after the addition of about 0.1 mg. of hydrosulfite (RS-1).

value of 1.7 for Complex III and 0.54 for Complex II (15). Thus Com- plex II predominates and Complex III may not be associated with maxi- mal oxidase activity.

At small Mn++ concentrations, the complete release of the ensyme from the complex may not occur when the oxygen concentration falls to zero. Trace D of Fig. 8 shows some complex still present several minutes after the oxygen is used up. At higher Mn++ concentrations, the free enzyme is recovered more rapidly after the oxidase activity is terminated.

The primary peroxidase-peroxide complex is not observed under these experimental conditions and this is characteristic of peroxidase action in a coupled oxidation system: no Complex I is observed to precede the formation of Complex II when peroxide is continuously generated by nota- tin, glucose, and oxygen (personal communication from D. Keilin, and

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

B. CHANCE 587

unpublished experiments of author). The possibility that the Mn++-ac- tivated catalyst involves only the peroxidase protein is eliminated by our failure to obtain any oxidase activity with a peroxidase that was split into heme and protein.

The indirect peroxidatic oxidation of Mn++ to Mn*, as found by Kenten and Mann (17), proceeds at a negligible rate at the low Mn++ used in these studies.

Existence of Mn++ Complexes with DHM and HRP--There is reason to believe that complexes of Mn++ with DHM and HRP exist (1, IS), but we have obtained no measurable shift of the absorption bands of DHM

FIG. 8. Spectra of the complex during rapid oxidase activity. Trace A, 0.5 PM HRP in 0.1 M acetate buffer at 4“. Trace B, HRP plus 0.6 mu DHM and 6.7 m Mu++, and 67 pod Mn* (Trace C). These traces were made as soon ae a steady state was obtained. Trace D was taken 4 minut.es after the termination of the steady state corresponding to Trace B (Experiment 846).

or HRP upon addition of [Mn*] that gives maximal activation of the oxi- dase effect.

Explanation of Reaction Kin&k-A reaction mechanism that exhibits the characteristics of the experimental data follows:

A peroxidatic reaction is

h El + SI - Ed%, (el - PJ (2) (PI)

An oxidase reaction is

Ed% + ~‘5% ! i E, +P,

(PA (a)

ks Et + Sa - Et.‘%, ks Ed31 + Sr - Er + SI (e2 - P2) (l/l (P2) (Pi) (a) (2)

Ei = HRP, El& = Complex II, Si = H202, Sz = DHM, Sa = 02, EZ = oxidase (Mn++ . HRP. Ht02), Ed& = oxidase-oxygen complex. The

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

588 OXIDASE AND PEROXIDASE REACTIONS

concentration of each reactant at any time is indicated by the letter directly underneath the terms of the chemical equations. We have as- sumed that the oxidase has the same concentration as the peroxidase.

In the steady state, the oxygen disappears at a rate lcsapz. The rate of disappearance of DHM, in the presence of oxygen, is the sum of two terms, ksapz and k,apl, corresponding to the fast portions of the records of Fig. 1, B, and Fig. 2. When the oxygen concentration is zero, p2 = 0 and the rate is slower and equal to krap,. The Hz02 concentration is the

difference of two integrals, ~tkpp2dl - ~‘k~apdt. The velocity constant

that we calculate above for the oxidase activity is ka. ks has not been measured but can be approximated from the value of oxygen concentra- tion that gives half maximal activity (-70 PM, see Fig. 3, B); 70 X 1OW = k&k& = lo6 X 0.68 X 10M3/k6; ka 2 lo6 M-’ X sec.-l.

SUMhL4RY

Under our particular experimental conditions, the following conclusions may be drawn.

1. Under anaerobic conditions, HRP causes the peroxidatic oxidation of DHM ; the secondary peroxidase-peroxide complex reacts with DHM at a second order velocity constant of 2 X 104 M-I X sec.-l at pH = 4 and 4”. ’

2. The aerobic oxidation of DHM by HRP can be made to occur in two phases, the first a rapid oxidase reaction producing H202, the second a peroxidatic reaction similar to that of (1) above. The second order ve- locity constant for the oxidase effect is about 4 X lo6 M-I X sec.-l at pH 4.0 and 4”.

3. The catalyst for the oxidase reaction appears to be a Mn++-activated peroxidase-peroxide complex; much smaller activities are obtained with the peroxide complex alone or with the Mn ++-activated, cyanide-inhibited HRP.

4. In view of the large Mn++ content of some plants, the reaction studied here is more likely to occur under physiological conditions than that studied by Swedin and Theorell (1, 19) in the absence of Mn++.

5. A study of the spectra of the peroxidase complexes formed from Hz02 and DHM alone shows their great similarity and Complex III is formed with HRP and LPO. No conversion of these complexes to the ferro- peroxidase-CO compound could be obtained without the addition of hy- drosulfite. In the presence of Mn ++, DHM, and oxygen, the compound formed is mainly Complex II. This DHM oxidase activity of HRP involves no valence states of iron not found in ordinary peroxidatic re- actions.

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

B. CHANCE 589

BIBLIOGRAPHY

1. Swedin, B., and Theorell, H., Nature, 143, 71 (1940). 2. Szent-Gyiirgyi, A., 2. physiol. Chem., 264, 147 (1938). 3. Banga, I., and Szent-Gyorgyi, A., 2. physiol. Chem., 266, 57 (1938). 4. Banga, I., and Philippot, E., 2. physiol. Chem., 268, 147 (1939). 5. Theorell, H., and Swedin, B., Naturwissenschaften, 27, 95 (1939). 6. Theo&l, II., Mitt. Naturforsch. Ges., Berne, 1, 73 (1944). 7. Chance, B., Rev. Scient. Instruments, 22, 634 (1951). 8. Davies, I’., and Brink,. F., Rev. Scient. Instruments, lS, 524 (1942). 9. Theorell, H., Ark. Kemi, Mineral. 0. Geol., 16 A, No. 2 (1942).

10. Abrams, R., Altschul, A. M., and Hogness, T. R., J. Biol. Chem., 142, 303 (1942). 11. Chance, B., Arch. Biochem., 24, 389 (1949). 12. Dawson, C. R., in McElroy, W. D., and Glass, B., Copper metabolism, Balti-

more, 1 (1950). 13. Chance, B., J. Cell. and Comp. Physiol,, 22.33 (1943). 14. Chance, B., Arch. Biochem., 22, 224 (1949). 15. Keilin, D., and Hartree, E. F., Biochem. J., 49, 88 (1951). 16. Chance, B., rlrch. B&hem., 21, 416 (1949). 17. Kenten, R. II., and Mann, P. J. G., B&hem. J., 46, 67 (1950). 18. Smith, E. L., and Lumry, R., Cold Spring Harbor Symposia Quant. Biol., 14, 168

(1949). 19. Goddard, D. R., and Slater, A. M., Biol. Bull., 93, 227 (1947).

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Britton ChanceDIHYDROXYMALEIC ACID

REACTIONS IN THE PRESENCE OF OXIDASE AND PEROXIDASE

1952, 197:577-589.J. Biol. Chem. 

  http://www.jbc.org/content/197/2/577.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  tml#ref-list-1

http://www.jbc.org/content/197/2/577.citation.full.haccessed free atThis article cites 0 references, 0 of which can be

by guest on May 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from