THE COMPOSITION OF CATALASE-PEROXIDE COMPLEXES *

BY BRITTON CHANCEt

(From the Biochemical Department of the Medical Nobel Institute, Stockholm, Sweden)

(Received for publication, October 30, 1948)

Recently the long sought intermediate compound of catalase and hydro- gen peroxide has been identified, and its spectrum and reaction kinetics have been measured (1, 2). The properties of this complex are not, how- ever, those of a classical enzyme-substrate compound of Michaelis and Menten. One very unusual property of this complex is that not all of the four catalase hematins of erythrocytes are bound to peroxide, even when saturation with peroxide is demonstrated spectrophotometrically. It is the purpose of this investigation to study this property of the catalase- hydrogen peroxide complex in some detail and to obtain a quantitative measure of the number of peroxide molecules bound to the catalase mole- cule.

Several independent experimental methods indicate this property of the complex in a qualitative manner. First, titration of catalase with hydro- gen peroxide gives molar amounts of this complex in excess of the molar amount of peroxide added when the calculation is based upon the com- bination of peroxide with all the catalase hematins. Second, the non- competitive inhibition of catalase activity by cyanide (3) indicates that not all catalase hematins are combined with peroxide under the conditions of the test for catalase activity. Third, the spectrum of catalase hydrogen peroxide is very unusual. The difference of extinction coefficient between catalase and this catalase-hydrogen peroxide complex is rather small, 40 cm.-’ X rn6 at 405 rnp, which is about equal to the difference between the extinction coefficients of a four-hematin horse erythrocyte catalase (380 cm.-’ X mr@) and a three-hematin horse liver catalase (340 cm.-’ X mh@) (1).

On the other hand, the change of extinction coefficient between catalase and the unstable primary catalase-alkyl hydrogen peroxide complexes is large (180 cm.-’ X rnM-l at 405 m,u for horse erythrocyte catalase) (2). A possible explanation of the greater change of extinction coefficient in the latter reaction may be that more alkyl hydrogen peroxide groups than hydrogen peroxide groups attach to catalase hematins. In fact, on the assumption that the change of extinction coefficient per peroxide group

* This is Paper 2 of a series on catalases and peroxidases. t John Simon Guggenheim Memorial Fellow (194648). Present address, Johnson

Research Foundation, University of Pennsylvania, Philadelphia. 1311

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1312 COMPOSITION OF CATALASE PEROXIDES

bound to hematin iron is the same for alkyl hydrogen peroxides and for hydrogen peroxide, the spectral data suggest that 4 times as many alkyl hydrogen groups as hydrogen peroxide groups attach to catalase; i.e., four alkyl hydrogen groups per erythrocyte catalase molecule, but only one hydrogen peroxide group per catalase molecule.

It is of importance to obtain further proof of such a difference in the nature of these compounds, because it may lay the foundation for an explanation of the tremendous difference in the reactivities of catalase towards hydrogen peroxide compared with alkyl hydrogen peroxides. Ca- talase decomposes hydrogen peroxide into oxygen and water with a tre- mendous velocity (in 0.1 M hydrogen peroxide the turnover number is 3.5 X 10’ X 0.1 = 3.5 X lo6 times per second (Bonnichsen, Chance, and Theorell (4))), while catalase scarcely decomposes the alkyl hydrogen peroxides at an appreciable velocity in the absence of alcohols (a turnover number of about 0.02 times per second).

Previous methods of determining the number of peroxide groups at- tached to hematin (for example, those used by Keilin and Ha.rtree (5) in the studies of the methemoglobin-hydrogen peroxide and ethyl hydrogen peroxide complexes) are completely unsuitable here, since not only is the catalase-hydrogen peroxide complex fairly unstable, but a considerable amount of hydrogen peroxide is decomposed during its formation (1).

Even the rapid spectrophotometric titration used in the study of the primary peroxidase-hydrogen peroxide complex (6) is unsatisfactory here, since the mechanism of hydrogen peroxide decomposition must be com- pletely formulated in order to use such titration data in a calculation of the number of hydrogen peroxide groups bound to catalase hematin. It is desirable to use a purely chemical test for unbound hematin groups in catalase-peroxide complexes which requires no postulates of enzymatic mechanism.

If a catalase-peroxide complex is mixed, for example, with cyanide, which reacts very rapidly with free catalase hematins (3), the initial amount of the catalase cyanide compound formed relative to that formed from free catalase is a direct measure of the number of the free catalase hematin groups in the catalase-peroxide complex. The amount of cata- lase-cyanide complex is readily estimated spectrophotometrically. Some of the characteristics of the catalase-cyanide reaction are now reviewed to determine whether cyanide is a suitable reactant.

Reactions of Catalase with Cyanide or Peroxide-The chemical method for determining the number of free hematin groups in a catalase-peroxide complex requires that cyanide and peroxide compete for the same place on the iron atoms of catalase. There is, however, no direct evidence of com- petition between hydrogen peroxide and cyanide in the inhibition of the

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B. CHANCE 1313

destruction of hydrogen peroxide (3), although competitive inhibition can be completely demonstrated for peroxidase (7). In this paper, direct spectroscopic evidence is presented that cyanide can displace hydrogen peroxide from catalase hematin (see Fig. 2), and therefore cyanide is a suitable substance in this respect. Thus the lack of competitive inhi- bition of catalase is caused by the peculiar mechanism of the catalase- hydrogen peroxide reaction and not by the fact that cyanide and peroxide combine at different parts of the hematin group.

Direct evidence that cyanide forms a ferric iron covalent compound with catalase is furnished by the magnetic measurements of Theorell and Agner (8). From direct studies of the reaction of catalase and cyanide (3) and from analogy with studies of methemoglobin cyanide (9), it is clear that cyanide ion combines with catalase iron. Magnetic studies (8) further indicate that cyanide ion combines only with intact catalase hematins and not with those partially or completely converted into bile pigment; a three-hematin horse liver catalase molecule binds only 3 cyanide ions.

Methods

Since the catalase-hydrogen peroxide complex begins to decompose several tenths of a second after its formation (see Chance (l), especially Fig. 3), the rapid flow technique (10) is essential.

The ideal form of the rapid flow method for this purpose is the two- mixer technique of Hartridge and Roughton (ll), in which the unstable catalase peroxide is formed in one mixing chamber and, at any short time later, is mixed with cyanide ion in a second mixing chamber. However, completely satisfactory results may be obtained with a single mixing chamber in the following general cases. (1) Catalase is mixed with perox- ide and cyanide simultaneously, but the peroxide reaction is complete before the cyanide reaction has progressed appreciably. (2) The catalase- peroxide complex is stable enough to permit premixing in a test-tube, followed by reaction with cyanide in the rapid mixing chamber.

The first method is applicable to the catalase-hydrogen peroxide com- plex and the second to the catalase-alkyl hydrogen peroxide complexes. The kinetic and equilibrium data for the catalase-cyanide and catalase- peroxide complexes specify the relative cyanide and peroxide concentra- tions for a successful experiment. The following equations define the reaction velocity constants involved in the kinetics and equilibrium. They are written for the specific case of a three-hematin horse liver catalase and a catalase-hydrogen peroxide complex in which only one of the three hematins is bound. Equation 1 is similar to that given previously, except that the value of n = I is used here (I), and the heme-linked hydroxyl group of catalase is indicated (12). The definition of the velocity con-

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1314 COMPOSITION OF CATALASE PEROXIDES

stants and the equation for the reaction of catalase and cyanide were given previously (3). It is assumed that the catalase hematins react independently.

h Cat(OH)s + Hz02 ----

kz Cat(OH)z(OOH) + HgO (1)

Cat(OHMOOH) + 2HCN kg

F---L Cat(CN)z(OOH) + 2H20 Ice

(2)

The initial hydrogen peroxide concentration is x0, and the initial cyanide concentration is &. The values of kl and ks are 3 X 10’ MY’ sec.-’ (1) and 9 X lo5 M-l sec.-’ (3) respectively. The value of ks is very slightly af- fected by whether or not peroxide is already bound to a catalase hematin. The apparent dissociation constant for Equation 2 is 4 X lo+ M (3). The apparent dissociation constant for Equation 1 is about 1 X 10”

M (1). Method l-If the composition of the complex of Equation 2 is to be de-

termined to an accuracy of 3 per cent, the velocity of formation of the hy- drogen peroxide complex (kro) must be at least 30 times greater than the velocity of formation of the cyanide complex (k&J; otherwise, cyanide might compete with hydrogen peroxide.

k~po;3*>30; .‘. 20 P io 6 (3)

Thus the initial hydrogen peroxide concentration need be only slightly greater than the cyanide concentration.

Since catalase must be saturated with hydrogen peroxide, the initial hydrogen peroxide concentration must be larger than lo* M.

The initial cyanide concentration should be large enough to give a read- ily measurable amount of the catalase cyanide compound. In this par- ticular method, it has been convenient to use an initial cyanide concen- tration which gives about 0.5 to 0.75 saturation of catalase with cyanide. When the catalase concentration is comparable to the cyanide concentra- tion, there are appreciable differences in the concentration of uncombined cyanide, depending upon whether the reaction is with free catalase or with the catalase-hydrogen peroxide complex, and appropriate corrections to the concenkation of catalase cyanide are calculated in the “Appendix.”

Method %-The catalase-alkyl hydrogen peroxide complexes are stable for about a minute, and therefore the complexes, preformed in a test-tube, are mixed with cyanide in the rapid flow apparatus.

Spectroscopic Considerations-The primary catalase-peroxide complexes and catalase have an isosbestic point (the same extinction) at 435 rnp,

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B. CHANCE 1315

and therefore the concentration of the catalase-cyanide complex is meas- ured at this wave-length.

Choice of Catalases-If hydrogen peroxide is bound to only one catalase hematin, a 25 per cent decrease in the amount of catalase cyanide com- pound formed on reaction of cyanide with the catalase-hydrogen peroxide complex would be expected with a four-hematin erythrocyte catalase and a 100 per cent decrease would be expected with a one-hematin catalase, in accordance with Equation 2 above. Thus the method is most sensitive with low hematin catalases. Although low hematin catalases may be prepared by the methods of Lemberg and Legge (13), the methods for determination of the bile pigment content require large corrections. The

FIG. 1. The kinetics of the formation of catalase cyanide in the absence (left- hand) and in the presence (right-hand) of hydrogen peroxide. 3.4 pM of hematin iron horse liver catalase, pH 6.5, 0.01 M phosphate buffer (Experiment 82b).

hematin content of horse liver catalase prepared according to the methods of Agner (14) and Bonnichsen (15) has been determined by several meth- ods, including splitting experiments, magnetic titrations (8), and spectro- photometric tests (3, 15), and appears to be rather close to three intact hematins. However, experiments with the four-hematin catalase of horse erythrocytes and with an especially prepared low hematin catalase are described. The catalases used in these experiments were purified by Dr. R. K. Bonnichsen, to whom many thanks are due.

Composition of Catalase Hydrogen Peroxide Intermediate--Fig. 1 shows on the left the reaction of catalase and cyanide as recorded spectropho- tometrically in the rapid flow apparatus (16). The top tracing represents flow velocity and its downward deflection indicates the moment at which

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1316 COMPOSITION OF CATALASE PEROXIDES

the flow is started. At that time, previously formed catalase cyanide is washed out of the capillary observation tube by the discharge of the syringes and is replaced by thoroughly mixed catalase and cyanide. The flow velocities employed correspond to such short times after mixing that the reaction of catalase and cyanide does not proceed to a measurable extent. Thus a solution of free catalase is flowing rapidly down the observation tube. The spectrophotometer records the abrupt decrease of optical density caused by the replacement of catalase cyanide by free catalase. But when the flow stops, the reaction of catalase and cyanide proceeds, and an increase of optical density equivalent to an upward oscillograph deflection of 105 mm. is measured from Fig. 1. This deflec- tion is termed D1.

In the right-hand record, the same reaction is repeated, but 10 pM of hydrogen peroxide are mixed with the cyanide solution previous to filling the syringes of the rapid flow apparatus. On initiating the flow catalase is now mixed with both cyanide and hydrogen peroxide. The catalase- hydrogen peroxide complex forms completely at the values of flow velocity used, but causes no deflection of the tracing because the measurement was made at 435 rnp, an isosbestic point between the Soret bands of catalase and catalase hydrogen peroxide (1). Thus a solution of the catalase- hydrogen peroxide complex at its saturation value is flowing rapidly down the observation tube, and, as already mentioned, has the same optical density as free catalase. When the flow stops, the reaction of cyanide with catalase hematins not bound to hydrogen peroxide proceeds with the same velocity as in t.he left-hand section, but gives an upward deflection of only 75 mm. This deflection is termed Dz. Catalase hydrogen perox- ide is unstable and decomposes in about a minute (see Fig. 2 or Chance (1)). As this decomposition occurs, cyanide combines with the freed catalase hematin, as indicated by the slowly rising tracing at the end of the right-hand record of Fig. 1. Finally cyanide binds all three hematins and should give the full deflection of 105 mm. measured in the left-hand record (Dl). That this reaction actually occurs is verified by the deflec- tion of the right-hand record before the flow is started. This deflection was recorded several minutes after a previous identical experiment, after the completion of the decomposition of catalase hydrogen peroxide and the consequent combination of cyanide with all the catalase hematins. The change of optikal density from catalase cyanide formed in this way to the catalase-hydrogen peroxide complex (which has the same optical density as free catalase at 435 mp) is equivalent to an oscillograph deflcc- tion of 105 mm. and is identical to the deflection obtained in the lcft- hand section of Fig. 1. Thus the formation of catalase hydrogen peroxide reduces the deflection from 105 to 75 mm.

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B. CHANCE 1317

If all the hematin groups were bound to peroxide, the deflection would have been reduced to zero (see Fig. 3). If only one of the three hematins were bound to peroxide, the deflection would be reduced by 33 per cent; i.e., from 105 to 70 mm. In fact, these data have been calculated by the formula 1 - (Dz/D1)100, which gives the percentage reduction in the amount of catalase cyanide formed from catalase hydrogen peroxide com- pared with that formed from free catalase.

In Fig. 1, the value of 1 - (DZ/D1)lOO is 28 per cent. The value expected if hydrogen peroxide occupied only one of the three horse liver catalase hematins is actually 27 per cent, not 33 per cent.’ Thus the ex-

TABLE I Variation of 1 - (D2/D1) with Cyanide Concentration

3.4 PM of hem&in iron horse liver cat&se, 10 PM of hydrogen peroxide; X = 435 rnp; 0.01 M phosphate buffer, pH 6.5 (Experiment 82b).

Initial cyanide, pu

l-- (DJD,)lCO.. .

TABLE II Variation of 1 - (DJDI) with Hydrogen Peroxide Concentration

3.4 PM of hem&in iron horse liver catalase, 4 JAM of cyanide, X 435 rn& 0.01 M phos- phate buffer, pH 6.5 (Experiment 84).

Initial hydrogen peroxide, px

1 - (D~/D~)lOO.. . . . . . .

perimental figure and the calculated figure verify that about 1 hydrogen peroxide molecule is bound per catalase molecule.

Experiments over a wide range of cyanide and peroxide concentrations are given in Tables I and II, and the results are expressed again by the factor 1 - (DZ/D1)lOO. In Table I, the values of 1 - (D&) for low cyanide concentrations are considerably higher than the theory requires, but the error in recording the small concentration of catalase cyanide is large. At 8 PM of cyanide and above, the limitations of Equation 3 are exceeded and a decrease of 1 - (DJDJ is seen; here cyanide is competing

1 A calculation shows that the decrease in the amount of cyanide compound to be expected is 27 per cent instead of 33 per cent for a liver catalase with one hematin bound, since appreciably more free cyanide is present in thii case (see “Appendix”).

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1318 COMPOSITION OF CATALASE PEROXIDES

with hydrogen peroxide for catalase hematin. In accordance with the data of Table I and in agreement with Equation 3, 4 PM of cyanide are chosen for the experiments shown in Table II, and the effect of a variation of hydrogen peroxide concentration is studied. The values of 1 - (02/01) for the larger values of hydrogen peroxide concentration are probably accurate and may be compared with the expected value of 27 per cent for a three-hematin catalase.’ The average experimental value (34 per cent) indicates that the catalase-hydrogen peroxide compound consists of about 1 molecule of hydrogen peroxide per catalase molecule.

Competition between Hydrogen Peroxide and Cyanide for Catalase Hema- tin-According to the experiments just described, catalase hematins not bound to hydrogen peroxide in the catalase-hydrogen peroxide complex can combine readily with cyanide. The reaction of these free hematins is shown by the records of Fig. 1 and by the data of Table II to be inde-

OPTICAL DENSITY

INCREMENT AT 417 mu

+-FREE CATALASE

CATALASE

+HYDROGEN PEROXIDE

HYDROGEN PEROXIDE phi 20 20 CYANIDE- )rtvl 0 200 ETHANOL-MM 100 IO0

FIG. 2. The effect of cyanide upon the kinetics of the catalase hydrogen peroxide compound measured at the isosbestic point for catalase and catalase cyanide (Ex- periment 86). 3.4 PM of hematin iron horse liver catalase.

pendent of hydrogen peroxide concentration, a non-competitive reaction. These free hematins also participate in the destruction of hydrogen perox- ide (3) and, therefore, the inhibition of catalase activity by cyanide ion is non-competitive, as is shown by the data of von Euler and Josephson, Stern, and Keilin and Hartree, in which roughly the same degrees of cy- anide inhibition of catalase were obtained in spite of large variations in the initial hydrogen peroxide concentration (for a summary, see Chance (3), Table V).

On the other hand, we can now show that the catalase hematin bound to hydrogen peroxide in the catalase-hydrogen peroxide complex is involved in competitive inhibition with cyanide. Table I shows how increasing cyanide concentrations can nearly completely prevent the formation of the catalase-hydrogen peroxide complex. Table II shows how increasing initial hydrogen peroxide concentrations can displace cyanide from the catalase-hydrogen peroxide complex. And finally, Fig. 2 shows directly

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B. CHANCE 1319

the decrease in the concentration of the catalase-hydrogen peroxide com- plex caused by cyanide competing with hydrogen peroxide. This record was obtained at the isosbestic point for catalase and catalase cyanide (417 mp), and at this wave-length the kinetics of catalase hydrogen per- oxide are recorded without interference from the kinetics of catalase cy- anide. The left-hand record shows the usual kinetic curve for the rapid formation (sharp drop), and slow disappearance (slow exponential rise) of catalase hydrogen peroxide (see Chance (1)). The right-hand tracing clearly shows that 200 PM of cyanide have “captured” 82 per cent of the catalase hematin bound by hydrogen peroxide in the left-hand record. But the dissociation constant of catalase cyanide (4 X lo+ M) indicates that nearly 100 per cent of the catalase hematin should have been bound by cyanide if no peroxide were present. The dissociation constant of catalase cyanide calculated from Fig. 2 is KI = 44 X lo6 M. Thus hydrogen peroxide and cyanide exhibit competition in this record.

The theoretical increase of the catalase cyanide dissociation constant may be calculated for the usual formula KI’ = Kr[l + (zo/K,J]; this is the usual formula for simple competitive inhibition (7), where ~0 = 20 X 10” M hydrogen peroxide (initially) and Km = 0.6 X 10” M (see

Chance (1); the value of Km is about half that of the catalase molarity). KI’ is calculated to be 1 X 10m4 M. Thus 65 per cent of the catalase hem&in is calculated to be bound by cyanide, and 82 per cent is actually indicated by the right-hand side of Fig. 2.

These simple calculations are in error because the hydrogen peroxide concentration at the time the cyanide ion combines with catalase is less than the initial peroxide concentration, owing to the destruction of perox- ide by catalase. This reaction is, of course, partially inhibited by cyanide. Thus the calculated value of KI’ should be considerably less than 1 X low4 M and, therefore, in better agreement with the experimental value.

A further proof of the competition between cyanide and peroxide is furnished by the data of Tables I and II, which are further analyzed in Tables III and IV. The data of Table I represent the titration of the catalase-hydrogen peroxide complex with cyanide. Since cyanide ion is competing with the peroxide molecule for the catalase hematin, the dis- sociation constant is no longer 4 X 10T6 M but is calculated in Table III to be K: = 16 X lo-” M in the range in which the data are most reliable. Thus there is no doubt of competitive inhibition, for the values in Table III are about 3 times greater than the dissociation constant of the catalase cyanide in the absence of peroxide. However, the decomposition of hy- drogen peroxide causes less than the amount of competition calculated ac- cording to the equation above: KI’ = 5 X 10m5 M.

The data of Table II represent the titration of catalase cyanide with hydrogen peroxide, and the dissociation constant (K’) of the peroxide

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1320 COMPOSITION OF CATALASE PEROXIDES

molecule competing with the cyanide ion for the catalase hematin is calcu- lated in Table IV in the range in which the data are most reliable. K’ is found to be 2 X lo+ M compared with the calculated value of 1.2 X lo+ (K’ = K (1 + [CN]/KJ). Here K’ is greater than the calculated value. No explanation of this discrepancy can be given at present.

Thus the competition between cyanide and peroxide for catalase hematin involved in the catalase-hydrogen peroxide complex is clearly demon-

TABLE III Competition between Cyanide and Hydrogen Peroxide for Catahee Hematin, Calcu-

lated from Table I 10 PM of peroxide.

I Initial cyanide concentration, PM

8

Measured y. catallase hematin bound by cyanide, (33 - (1 - (Da/D1) 100)/33)

39

(y)i==( i-l)i=Ki,dfXl@ 12

TABLE IV

20 50 200

55 76 91

10 16 20

Competition between Cyanide and Hydrogen Peroxide for Catalase Hem&in Calcu- lated from Table II

4 JLM of cyanide.

I Initial hydrogen peroxide concentration, px

1 2 4 s 20 -----

Measured To catalaee hematin bound 26 50 56 35 85 by hydrogen peroxide, (1 - (Dn/D1) loo)/34

Calculated equilibrium constant, 2.0 1.4 2.7 0.8 4.0

[&OS] = K’, Y X 10”

strated. Nevertheless, the inhibition of the destruction of hydrogen peroxide by catalase is non-competitive, because catalase hematins not involved in this complex are required for the destruction of hydrogen peroxide.

Composition of Catalase Ethyl Hydrogen Peroxide Compound-Experi- mental results obtained by use of Method 2 are shown in Fig. 3. Records A and B are directly comparable with those of Fig. 1. The first reaction (A) is simply a calibration of the amount of catalase cyanide that forms

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B. CHANCE 1321

from all three hematins. In the second record (B), ethyl hydrogen per- oxide lvas first added to catalase in a test-tube, and the mixture was sucked up into the syringe of the rapid flow apparatus and there, 0.3 minute after mixing in the test-tube, Tvas mixed Tvith cyanide in the capillary. This record contrasts sharply Tvith the corresponding record of Fig. 1; no appreciable evidence of the rapid cyanide reaction is obtained, but only of a slo~v reaction at about the rate at which the catalase-ethyl hydrogen peroxide complex is breaking doJvn. The remaining records confirm this; after 1.5 minutes, an appreciable amount of catalase hematin is free to react rapidly Tvith cyanide, and, after 3 minutes, nearly all the catalase

FIG. 3. The kinetics of the formation of catalase cyanide in the absence of ethyl hydrogen peroxide (A) and during the decomposition of the catalase-ethyl hydrogen peroxide complex (at 0.4, 1.5, 2.5, and 3 minutes). In record A, catalase is mixed with cyanide in the rapid flow apparatus. In records B to E, the catalase ethyl hydrogen peroxide is preformed and is then mixed with cyanide in the rapid flow apparatus. 3.4 ,UM of hematin iron horse liver catalsse, 10 PnI of cyanide, 67 MnI of ethyl hydrogen peroxide (Experiment Did).

hematin is free. This record is taken to indicate that ethyl hydrogen peroxide can bind all three of the catalase hematins of horse liver. Table V sho\vs the variation of 1 - (Dz/D1) with ethyl hydrogen peroxide con- centration. The equilibrium constant for catalase ethyl hydrogen per- oxide as calculated from the data of Table V appears to be too large (17), but a considerable portion of the substrate was decomposed before Dz was measured.

The composition of catalase methyl hydrogen peroxide can be assumed to be the same as that of the ethyl hydrogen peroxide complex, since their Soret bands are very similar (18).

Simultaneous Reactions of Hydrogen Peroxide and Ethyl Hydrogen Perox- ide with Catalase Hematin-Strong evidence for the accuracy of these

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1322 COMPOSITION OF CATALrlSE PEROXIDES

conclusions is afforded by using ethyl hydrogen peroxide instead of cy- anide in the reaction of catalase and hydrogen peroxide. Since ethyl hydrogen peroxide combines more slowly than cyanide (2, 17), a somewhat greater concentration of the former is required.

These measurements were made at 410 rnp, at which the extinction co- efficients of the catalase peroxide compounds are large and, per mole of hematin iron bound, are very nearly equal (18). Thus these data record the sum of the concentrations of catalase hydrogen peroxide and catalase ethyl hydrogen peroxide.

A typical experiment is shown in Fig. 4. In record A, the saturation value for catalase ethyl hydrogen peroxide is recorded on a rapid time scale (DA = 70 mm.) on the bottom line and on a slow time scale directly above. In record B, the saturation value (DB = 45 mm.) and life time of the in- termediate compound are decreased by the addition of 400 pM of ethyl alcohol so that the succeeding records can be completed more rapidly (17).

TABLE V

EJect of Ethyl H:~ldrogen Peroxide Concentration upon Percentage of Catalase Hema- tins Free to React with Cyanide

3.4 PII of hematin iron horse liver catalasc, 10 PM of cyanide; 0.01 ~4 phosphate buffer, pII 6.5; X = 435 rnp (Experiment 9ld).

Etl~yll~ydrogen peroxide,/m

In record C, the saturation value (DC = 21 mm.) and the kinetics of cat alase hydrogen peroxide are recorded. This compound forms very rapidly, nearly completely at the fastest flow velocity. Thus there is only a very small change of its concentration iv-hen the flow stops. The cor- responding record (above) on a slow time scale shows that alcohol has also accelerated the decomposition of this intermediate (cl. Fig. 2, left-hand section).

If now both hydrogen peroxide and ethyl hydrogen peroxide react simul- taneously with catalase hematin, as in record E, the hydrogen peroxide compound forms first, as indicated by the 21 mm. rapid drop of the oscillo- graph tracing as the flow starts.2 After the flow stops, the slower reaction

2 This record depicts what would happen if some hydrogen peroxide were acci- dentally present in the ethyl hydrogen peroxide. That in excess of the enzyme molarity would he rapidly decomposed a.nd the remainder would react exactly as shown in record E. Indications of such a sharp step have not been noticed in the kinetics of formation of ethyl hydrogen peroxide compounds.

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B. CHANCE 1323

of catalase and ethyl hydrogen peroxide gets under way, but finds one of the catalase hematins already bound to hydrogen peroxide. Thus the extent of the slow reaction (DE) is only 27 mm. as compared with Dg = 65 mm. in record B. As the record above on a slow time scale shows, the reaction proceeds as in record B; the hydrogen peroxide compound soon breaks down, and all three hematins are available for ethyl hydrogen

FIG. 4. The kinetics of the formation (lower section) and the course of the reaction (upper section) of catalase and ethyl hydrogen peroxide in the absence (A, B) and in the presence of hydrogen peroxide (D, E). 3.4 PM of hematin iron horse liver catalase; X = 410 rnp, pH 6.5, 0.01 M phosphate (Experiment 91a). The optical density changes are as follows: D A, from free catalase to saturated catalase ethyl hydrogen peroxide; Da, from free catalase to 0.65 saturated catalase ethyl hydrogen peroxide; DC, from free catalase to saturated catalase hydrogen peroxide; DD and DE, from saturated catalase hydrogen peroxide to 0.65 saturated catalase ethyl hydrogen peroxide.

peroxide. There is no indication in this slow record of the conversion from the catalase-hydrogen peroxide complex to the catalase-ethyl hydrogen peroxide complex, since the extinction coefficients per hematin are very nearly equal.

Record D, made just a few seconds after a previous injection, demon- strates the great kinetic difference between the two catalase peroxides. Since the capillary had just been filled with reactants before record D was

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1324 COMPOSITION OF CATALASE PEROXIDES

started, the initial base-line corresponds to a mixture of the two catalase peroxides at a little less than their saturation values. Rapid flow of fresh reactants prevents the formation of the ethyl hydrogen peroxide com- pound but not the hydrogen peroxide compound. Thus the tracing does not rise to the optical density of free catalase, only to that of catalase hydrogen peroxide. After the flow stops, the ethyl hydrogen peroxide reaction proceeds, and the subsequent portions of record D duplics te those of record E.

A simple calculation gives the number of hematins bound by hydrogen peroxide, since all these can be bound by ethyl hydrogen peroxide.

TABLE VI

Composition of Catalase-Hydrogen Peroxide in Liver Catalase

Hematin compound i ,Density mcrement

CatH,O,, DC ............................... Cat(C2H500H)2, DEDA/DB. ............... Cat(C,H,OOH),, DA ........................

mm.

21 42 70

Ratio of den- Expected city increments ratio

1.0 1.0 2.0 2.0 3.3 3.0

TABLE VII

Composition of Catalase-Hydrogen Peroxide in Krythrocyte Catalase

Density increment

mm.

CatHzOz, DC.. . 23 1.0 1.0 Cat(CzH500H)3, DEDJDe.. 63 2.7 3.0 Cat(CPH500H)4, DA.. 92 4.0 4.0

The ratio 1 - (Dz/DB) = 1 - (27/45) = 40 per cent as compared with the expected value of 33 per cent. The data are summarized in Table VI, and the value of DE is corrected for partial saturation (DA/DB) to give 42 mm.

The extinction coefficients of these compounds per mole of hematin iron are nearly equal (18); the data confirm the conclusions from the cyanide experiments (Table II).

In experiments in which a 1.35 PM horse erythrocyte catalase having four intact hematins was used, the data of Table VII have been obtained by a repetition of Fig. 4. The value of 1 - (DE/DB) = 31 per cent com- pared with the expected value of 25 per cent. The data are, however, in agreement with those obtained by using the three-hematin liver catalase,

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B. CHANCE 1325

and therefore show that the composition of catalase hydrogen per- oxide is unrelated to the bile pigment content of the catalase.

Furthermore, no change in the composition of the catalase-hydrogen peroxide complex in a liver or an erythrocyte preparation is indicated by the change in optical density at 410 rnp on formation of the complex: 23 mm. for 1.35 PM of erythrocyte catalase and 20 mm. for 1.17 FM of liver catalase.

Composition of Catalase Hydrogen Peroxide in High Biliverdin Catalase -Bonnichsen has prepared some catalases of high biliverdin content (19). One of these preparations was tested by these methods in order to detect with greater sensitivity the effect of a variation of the number of hematins

418 sec. f+- I , I

I I

1 I

CATALASE I I I I”

tI=380cm”xmMJ~

CATAASE- METHYL HYDROGEN

PEROXIDE -

FIG. 5. The composition of catalaae hydrogen peroxide formed in the presence of the notatin system. 0.95 PM of horse blood catakse, 0.04 MM of notatin added at A, 6.7 1~ of glucose added at B, 10 PM of methyl hydrogen peroxide added at C and at D; A = 405 w, pH 7.0, 0.01 M phosphate buffer (Experiment 251).

present in the catalase preparation upon the composition of catalase hydrogen peroxide.

This catalase, when compared to a solution of erythrocyte catalase of equal optical density at 405 rnp gives 36 per cent less cyanide compound at 435 rnp and 39 per cent less methyl hydrogen peroxide compound at 405 rnp (18) and may be considered to have about 2.7 hematins. On the basis of a cyanide-hydrogen peroxide test similar to that in Table II, the value of 1 - (QJDJ is found to be 38 per cent, and on the basis of a hydrogen peroxide-methyl hydrogen peroxide test according to the method just described, the value of 1 - (DE/De) is 39 per cent.

Thus the composition of the catalase hydrogen peroxide is again shown

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1326 COMPOSITION OF CATALASE PEROXIDE

to be unaffected by a rather large change of bile pigment content, from 0 to 1.3 catalase hematins.

Composition of Catalase Hydrogen Peroxide Formed in Presence of Nota- tin, Glucose, and Oxygen-The formation of catalase hydrogen peroxide I from hydrogen peroxide produced by the notatin system has been reported (2). Since a continuous supply of hydrogen peroxide is available, the complex is stable for several minutes, and the number of free catalase hematins is simply determined by the addition of excess alkyl hydrogen peroxide, as in Fig. 5.

In these tests, a recording spectrophotometer equipped with a 1.3 cm. cuvette was used. The formation of catalase hydrogen peroxide is shown at B and gives a deflection of 8.5 scale divisions on Fig. 5. As soon as a steady state is reached, an excess of methyl hydrogen peroxide is added, and the total density increment is 28.5 scale divisions. Thus 8.5/28.5 or 30 per cent of the four catalase hematins is bound in the hydrogen peroxide complex.

These reactions provide a simple method for determining the number of intact hematins in a catalase containing biliverdin. If the ordinary spec- trophotometer is used, about 30 times stronger catalase is required, and some increase in the notatin concentration may be necessary to insure saturation of the catalase-hydrogen peroxide complex. Catalase solutions should be dialyzed thoroughly before this test to remove any alcohol, which would lower the saturation of the catalase-hydrogen peroxide com- plex owing to the peroxidatic reaction of this complex with ethanol.

DISCUSSION

The preliminary conclusion drawn from previous studies of catalase hydrogen peroxide is substantiated by these studies: the compound con- sists of very nearly 1 peroxide molecule per catalase molecule. In four determinations by two independent methods, the amount of hydrogen peroxide exceeded 1 per catalase molecule by 26, 21, 23, and 20 per cent (34/27, 40/33,31/25, and 30/25) for horse erythrocyte and liver catalases. The inherent error of the method would allow a discrepancy of about 12 per cent in these values.3 The average of these data gives the compo- sition of the catalase hydrogen peroxide as 1.2 f 0.1 peroxide-bound hematins.

* The error of these methods is usually between 2 and 4 per cent of the total deflec- tion measured. Since the deflection due to the catalase-hydrogen peroxide complex is only one-third to one-fourth of the total deflection, the error is correspondingly greater. These errors are due to inadequate gain of the light intensity control, irregularities in the relative delivery from the syringes, and, in the case of horse liver catalase, errors in the determination of the number of intact hematins.

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B. CHANCE 1327

‘There are two interpretations of the result. The first is that the cata- lase hematins are identical in all respects and are completely independent. In this case, each one of the four catalase hematin groups is slightly over 0.25 saturated with hydrogen peroxide. The cause of the partial satura- tion of the enzyme-substrate complex is readily explained by the “purely kinetic” theory of catalase activity (2) ; catalase activity involves the con- secutive reactions of hydrogen peroxide molecules with catalase and with catalase hydrogen peroxide, and the relative values of the velocity con- stants of the first and second reactions determine the saturation of the catalase-hydrogen peroxide complex in the steady state (2). This ex- planation, however, requires modifications to account for the constant composition of the catalase-hydrogen peroxide complex in catalases con- taining bile pigment. According to this theory, the measured compo- sition of the complex would decrease with a decrease in the number of intact hematin groups; a 2.7 hematin catalase should have only 0.8 perox- ide-bound hematin per catalase molecule, in contrast with the experimental data. Explanations for this discrepancy between the theory and the ex- periments can be obtained by postulating interaction bettveen pairs of catalase hematins (L. Pauling, personal communication). But no evi- dence of such interaction has yet been found in the reaction of catalase with cyanide (3) or with methyl hydrogen peroxide (17).

The second interpretation is that the composition of the catalase- hydrogen peroxide complex is not a statistical effect but is due to “special properties” of the catalase-hydrogen peroxide complex. After the com- bination of hydrogen peroxide with any one of the catalase hematins, the catalase molecule acquires “special properties,” which permit the rapid destruction of hydrogen peroxide at the free catalase hematins. Accord- ing to this simple theory, the composition of the catalase-hydrogen perox- ide complex would be exactly 1 peroxide molecule per catalase molecule and would be independent of the bile pigment content of the catalase mole- cule. The latter effect is in accord with these experiments, but the former conflicts with the composition of catalase hydrogen peroxide of 1.2 perox- ide molecules per catalase molecule. Also there are no “special proper- ties” of the catalase-alkyl hydrogen peroxide complexes (17).

The composition of the catalase-hydrogen peroxide complex is remark- ably independent of the peroxide concentration: no significant change of its spectrum was obtained in 4 mM of hydrogen peroxide (1) or in the presence of the very dilute hydrogen peroxide (-lo+’ M) generated by notatin, glucose, and oxygen (2), a total range of about lo6 in hydrogen peroxide concentration.

That the composition of the catalase-hydrogen peroxide complex is a consequence of the “catalatic” reaction (the rapid breakdown of hydrogen

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1328 COMPOSITION OF CATALASE PEROXIDES

peroxide by catalase) is clearly shown by comparison with the composition of the catalase-alkyl hydrogen peroxide complexes in which all the intact catalase hematin groups are bound to alkyl hydrogen peroxide and no “catalatic” reaction is observed; the breakdown of dilute alkyl hydrogen peroxide solutions by catalase in the absence of alcohols is very slow (2).

Since the catalase-hydrogen peroxide complex forms from an erythrocyte catalase having no bile pigment, there is no reason for supposing that, in liver catalase, a combination with the bile pigment hematins is responsible for the catalase-hydrogen peroxide complex.

The non-competitive inhibition of the destruction of hydrogen peroxide by catalase has led some investigators to conclude that cyanide and perox- ide do not attach to the same place on the catalase hematin (3,20). How- ever, non-competitive inhibition is, in this case, clearly due to the combination of cyanide with the free hematins of the catalase-hydrogen peroxide complex. The fact that catalase activity is inhibited by a com- bination of these hematin groups with cyanide is proof that they are neces- sary for catalase activity.

In these experiments, it has been possible to demonstrate spectroscopi- cally the competition between cyanide and peroxide for catalase hematin. Thus cyanide and peroxide do combine at the same point on catalase hem- atin. Therefore the earlier explanations of the non-competitive inhibi- tion of catalase activity are incorrect and are now unnecessary.

SUMMARY

1. In the catalase-hydrogen peroxide complex, 1.2 f 0.1 hematin groups are occupied by hydrogen peroxide as determined by the reaction of this complex with cyanide or alkyl hydrogen peroxides.

2. The composition of the complex is not appreciably altered by a bile pigment content equivalent to 1.3 hematin groups or by a large variation in the hydrogen peroxide concentration.

3. Hydrogen peroxide is shown to combine with the iron atom of cat- alase hematin by competition between cyanide and hydrogen peroxide.

4. In the catalase-alkyl hydrogen peroxide complexes, all the intact catalase hematins are bound to peroxide.

5. The non-competitive inhibition of catalase activity by cyanide demonstrates that catalase hematins not involved in the catalase-hy- drogen peroxide complex are required for the destruction of hydrogen peroxide.

APPENDIX

Calculation of Change in Amount of Catalase-Cyanide Formed in Pres- ence and Absence of Hydrogen Peroxide--The concentration of free cyanide

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B. CHANCE 1329

is greater in the presence of hydrogen peroxide and a correction to the concentrations of catalase cyanide is calculated as follows:

In the absence of hydrogen peroxide and for a liver catalase (E), the combination with cyanide (I) is represented as in an earlier paper (3). The initial enzyme concentration is e3, and the initial cyanide concentra- tion is io. The concentration of catalase cyanide (EIS) is ~3. Thus the free enzyme concentration is e3 - q3, and the free cyanide concentration is i0 - q3.

ks E-j-31--E&

b

Since the iron atoms of catalase act independently, the equilibrium con- ditions are equivalent to the reaction of a single cyanide ion with a single iron atom. The dissociation constant is to be calculated; therefore, the equations are written in the reciprocal form.

In the presence of enough hydrogen peroxide to convert E into ES, the catalase-hydrogen peroxide complex, Equation 3 is written, in which the symbols have the same meanings as in Equation 1.

ES + 2I \ ks \ ks

ESIa

Ki - (8% - @)(io - d

41

(3)

(4)

Solving Equations 2 and 4 for qa and qz respectively gives

Then .-

!D -b- z/b” - 4c

& - -b’- dm (5)

where b = - (6 + e3 + &), c = e3&; b’ = - (& + e2 + IG’), c’ = e&o. It is here assumed that Kr = Kr’.

BIBLIOGRAPKY

1. Chance, B., Acta. chem. Stand., 1, 236 (1947). 2. Chance, B., Nature, 161,914 (1948).

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1330 COMPOSITION OF CATALASE PEROXIDES

3. Chance, B., J. Biol. Chem., 179, 1299 (1949). 4. Bonnichsen, R. K., Chance, B., and Theorell, H., Acta them. &and., 1, 685 (1947). 5. Keilin, D., and Hartree, E. F., Proc. Roy. Sot. London, Series B, 117,l (1935). 6. Chance, B., J. Biol. Chem., 161, 553 (1943). ‘7. Chance, B., J. Cell. and Comp. Physiol., 22, 33 (1943). 8. Theorell, H., and Agner, K., Ark. Kemi, Mineral. o. Geol., 16 A, NO. 7 (1942). 9. Coryell, C. D., Stitt, F., and Pauling, L., J. Am. Chem. Sot., 69,633 (1937).

10. Roughton, F. J. W., and Millikan, G. A., Proc. Roy. Sot. London, Series A, 166, 258 (1936).

11. Hartridge, H., and Roughton, F. J. W., Cambridge Phil. SOC., 23, 450 (1926). 12. Agner, K., and Theorell, H., Arch. Biochem., 10,321 (1946). 13. Lemberg, R., and Legge, J. W., Biochem. J., 37, 117 (1943). 14. Agner, K., Ark. Kemi, Mineral. o. Geol., 16 A, NO. 6 (1942). 15. Bonnichsen, R. K., Arch. Biochem., 12, 83 (1947). 16. Chance, B., Rev. Scienl. Instruments, 13, 601 (1947). 17. Chance, B., J. Biol. Chem., 179, 1341 (1949). 18. Chance, B., J. Biol. Chem., 179, 1331 (1949). 19. Bonnichsen, R. K., Dissertation, Karolinska Institutets, Stockholm (1948). 20. van Euler, H., and Josephson, K., Ann. Chem., 466,l (1927).

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Britton ChanceCATALASE-PEROXIDE COMPLEXES

THE COMPOSITION OF

1949, 179:1311-1330.J. Biol. Chem. 

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