286

BBA 65194

BIOCHIMICA ET BIOPHYSICA ACTA

ACTIVITY OF CATALASE IN THE RED CELL

PETER NICHOLLS

Department of Biochemistry. State University of New York. Buffalo. N. Y. (U.S.A.J

(Received December 24th, 1964)

SUMMARY

The activity of catalase (H202 :H 202 oxidoreductase, EC I.II.I.6) in the horseerythrocyte and its ability to protect haemoglobin against oxidation by peroxide havebeen studied. Both catalase and haemoglobin are shielded by a permeability barrierin the intact cell, presumably the reel-cell membrane. Once H20~ has penetrated thisbarrier it reacts with haemoglobin, oxyhaemoglobin and catalase at the same ratesas when it reacts with the same substances isolated in vitro.

Catalase in the intact red cell is fully active both catalatically and peroxida­tically and shows a normal sensitivity to inhibitors. It is capable of destroying between99.0 and 99.9% of H 202 entering the cell. Under the present conditions the role ofcatalase in protecting haemoglobin from oxidation to methaemoglobin was foundto be much greater than that of glutathione peroxidase.

The consequences of the present findings for the problems of the physiologicalrole and evolution of catalase are discussed.

INTRODUCTION

BINGOLD1 was among the first to claim that the catalase (H202:H202 oxido­reductase, EC LILI.6) present in the erythrocyte protects the haemoglobin fromattack by H202. According to this view added peroxide is removed by Reaction I

catalase2 HzO a--+ Oa + 2 H 20

HbFeH + HzO e--+ HbFeOHH + COHO]

(I)

(2)

and does not bring about Reaction z (HbFe2+ indicates ferrous haemoglobin).KElLIN AND HARTREE2 , however, found that if peroxide is generated slowly andcontinuously by an enzyme system, Reaction 2 took place quite readily, and theyconcluded that "endoerythrocytic catalase does not protect haemoglobin from oxida­tion to methaemoglobin by very low concentrations of H202 formed in a primaryoxidation reaction", They attributed the protection by catalase against oxidationby a great excess of H 20 2 to a shift in the equilibrium of Reaction 3 resulting from theoxygen produced in Reaction I.

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CATALASE ACTIVITY IN RED CELL

HbFe2+ +°2 ;;= HbFc2+02 (3)

It was supposed that only the deoxygenated haemoglobin reacts with peroxide(Reaction 2). In contrast to this, FOULKES AND LEMBERG3 compared the rates ofcholeglobin formation in haemolysates in the presence and absence of a catalase in­hibitor, azide, and found that even with continuously generated peroxide, catalaseactivity markedly decreased choleglobin formation. LEMBERG AND LEGGE4 had con­tinued to emphasize the protective role while KElLIN AND HARTREE5 advocated ametabolic role for the enzyme in the oxidation of a physiological hydrogen donor(Reaction 4).

catalaseH 20 2 + AH2 ----+ A + 2 Hp

CHANCEB attempted to solve the problem of the physiological role of catalasefrom a theoretical angle by showing that in the presence of a fixed amount of hydrogendonor, the relative amounts of peroxide destroyed peroxidatically (Reaction 4) andcatalatically (Reaction I) depend on peroxide concentration. At high peroxideconcentrations Reaction 1 would predominate, at low concentrations Reaction 4would be most important. This theoretical demonstration did not, however, disposeof the three experimental problems:

(a) does catalase even in the absence of a hydrogen donor behave in the sameway towards low as towards high peroxide concentrations?

(b) does endoerythrocytic catalase behave in the same way towards peroxideas does pure catalase or catalase in a haemolysate?

(c) are there hydrogen donors normally present in erythrocytes or haemolysatesthat make it necessary to take Reaction 4 into account in calculating the effect ofperoxides?

According to CHANCE? the answer to the first question is "yes" J and NICHOLLSB

has more recently confirmed in detail that it is possible to analyse the reactionsoccurring at low peroxide concentrations (IO-LIQ-iO M) in terms of those occurringat higher concentrations (10-2-10-4 M) and vice versa. But it is nevertheless possiblethat subtle but physiologically significant differences may have been missed by theseinvestigations.

The results of KElLIN AND HARTREE2 , together with some observations oncatalase in yeast cells", have suggested that intracellular catalase may indeed havedifferent properties from catalase in free solution, including a lower activity. AlthoughCLAYTON1owas able to explain all such differences in bacterial catalase activity in termsof a single diffusion barrier, such an explanation does not seem to account for the otherdata2,9 .Moreover, COHEN AND HOCHSTEINll have performedexperiments whichindirect­ly indicate a low catalase activity in the red cell towards slowly generated peroxide.

The presence of catalase hydrogen donors in mammalian systems has beenwidely reported. Peroxidative activity has been observed in liver slices12 and inisolated rnitochondria'". In the latter case, a possible role in maintaining mitochon­drial structure is indicated>, MARGOLIASH AND SCHEJTER15 have adduced indirectevidence for peroxidative activity of erythrocyte catalase towards a donor presentphysiologically.

It was to throw light on some of these questions, by carrying out a quantitativestudy of the reactions of H202 with haemoglobin and catalase in the erythrocyte,that the following experiments were done.

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288

METHODS AND MATERIALS

P. NICHOLLS

Horse blood was obtained fresh from the slaughter hous e, and suspensions ofcorpuscles were prepared by washing twice with an equal volume of isotonic salineand centrifuging. Such corpuscles could be stored at 4° for several da ys without losingtheir characteristic permeability to hydrogen peroxide (see RESULTS) .

Barcroft differential manometers were used for measurement of cat alaticactivity and for experiments in "coupled oxidatiori'". A Hilger Uvispek spectro­photometer with a glass prism was emplo yed in the spectrophotometric studies. Purecatalase was prepared from horse liver as previously described-e. Notatin (glucoseoxidase) was from a sample prepared by Boot s' Pure Drug CO.

H:P2 solutions were prepared by suitable dilutions from "roo volume" AnalaRsolution. Sodium azide was obtained from BDH Ltd. 3-Amino-I .2,4-triazole was a giftfrom Professor D. ApPLEMAN to Professor D. KElLIN. GSH was obtained from L.Light and Co. Other reagents were of analytical or equivalent grade.

RESULTS

The catalatic reaction within the erythrocyteUsing the horse red blood corpuscles washed free of plasma, resuspended in

isotonic saline to the original volume and st ored at 4°, it is possible to measure thecatalase activity manometrically with both intact and lysed cells. The presence of0 .15 M NaCl at pH 7-4 has no appreciable influ ence on the reacti on rate. In bothsyst ems the rate of oxygen evolu tion is proportional t o the number of cells and toperoxide concentration (5-20 mM), and the ratio ofthe rates in the two systems (I lL)remains constant (E qn. 5) :

Catalatic activity oj intact cells-=-.,.....,.~--:-:---;-=--':--:---:--:- = 0.3 = I ILCatalatic acti vity oj haemolysate

(5)

(Actual values varied from 0.23 to 0.3X). For such horse cells, the concentrat ionratio, in terms of haematin, [catalase]j[haemoglobin] was found to be 3 ' 10-4•

assuming an average value for th e velocity constant of Reaction I to be 4' 106 M-1sec- 1.A closely similar concent ra tion ratio seems to occur in human red cells.

Cells treated with isotonic nitrite and washed with saline (methaemoglobincorpuscles) also showed a catalatic activity proportional to [H202Jand cell concentra­tion , but the absolute rates were lower and sometimes increased during the reaction(especially with intact cells), and the ratio IlL approached La. We assume that cat a­lase in "methaemoglobin cells" is in the same state as in normal cells, but that thenitrite treatment leaves residual amounts of nitric oxide (a catalase inhibitor withvery high affinity) and increases the permeability and spontaneous lysis of the cells.

That the low activity of intact cells is due to a permeability barrier as in bac­teria-? was shown by incubating the cells in isotonic sodium hypophosphite, a ratherspecific and almost irr eversible catalase inhibitorw. The activity decreased progres­sively over a period of days when measured after washing and resuspending the cellsin NaCl solution. After 5 days, the activity was found to be approx. 3%of the initialactivity, and I lL = I.O. This suggests that the intracellular catalase is in fact asactive as when in free solution and that the diminished app arent activity is due neither

B ioohim, B iophy s. A cta, 99 (rg65) 286 - 297

CATALASE ACTIVIT Y IN RED CEL L 289

to the presence of reversibly inactivated enzyme" nor to an unfavorable, e.g. highlyviscous, intracellular environment.

If kd is the diffusion constant (moles cm-a sec- I M-l) for H 20a across the red­cell membrane, then we have Eqns, 6-8

kdA(H .O.]o = hdA( H P .1t + k/ V(cat] [H 20 .) j

[HP .li /(H P 2)o = I II.

(IlL ) (VIA ) k/[cat )h« = 1 - (IlL )

(6)

(7)

(8)

where I lL = 0.3 (pH 7.4, 20°), according to Eqn. 5, VIA is the ratio of volume toarea of the erythrocyte (45/70' ro - 4 cmF7, k/ = 4' 1 0 6 M-I sec- I, and the intracellularcat alase concentration is 5.6 ,uM. The diffusion constant k« has the value 6· 10-4 ernsec", higher than the values found for most non-electrolytes (but close to that formethanol), and lower than the values for gases-",

R eactions of added peroxid e with erythrocyte haemoglobinRaOa m ay react wit h haemoglobin in at least four ways (Eqns. 9 -12 ; Eqn. 10

is formally equivalent to Eqn. 2).

H bFeH O. + H 20 . --+ H bFeH + OH - + O2 + [OHO] (9)

H bFe2+ + H20. --+ HbFe3+H20 + OH - + [OHO] (10)

H bFeHH20 + H .O. --+H bF eOH + I-l.O+ + [OHO] (II)

Hb(Fe2+ or F e2+) + H 20. --+ CholeHb (FeS+ or Fe2+) (12)

Table I gives the rate s and sto ichiometries observed for Reactions 9-II inhaemolysates and intact cells when catalase activity is inhibited by azide. Reaction 9was followed spectrophotometrically at 577 mil, Reaction 10 at 555 m,u under Na,Reaction II at 580 m,u. Reaction 1 2 was not studied ; although both FOULKES AND

L EMBE RG3 and MILLSI9 used choleglobin formation to det ermine catalase protectiveaction , as neither the form of haemoglobin nor the immediate oxidising agent involved

TABL E I

REA CTION RATES FOR SEVERAL FO RMS OF HAEMOGLOBIN WITH H.O.

p H 7.4 phosphate, 0.I5M NaCJ, 20 °, Hb from 40 to 90/tM, H 20 . from 20ftM upwards, 4mMazide, experiments with H b FeHO. and HbFest in air, experiments with HbFe2+ under N s­I lL is the ratio of t he acti vit y in whole cell suspensions compared to that of the ha emolys at e.

Haem oglobin Oxidationsubstrate product

Oxidation ratehaemolvsate(M- 's;C- ')

iJ(HbJI(H.O. J IlLstoichiometry

HbFeH O. H bFe' +(N. - ) 20 ( 12-25) 1.0HbFe2+H.O HbFe·+(N.-) 300 (280-420) 0.9-0.95 0.7HbFe2+(N. -) H bF eOH 60 0 (450-800)' ~0.3" 0.25

• Calculated for fre e HbF eH ,

•• P eroxide compo und is u nstable.

Biochim, B iopbys . A cta, 99 (1965) 286- 297

P. NICHOLLS

is known, and as the reaction does not show the straight-forward kinetic behaviorof Reactions 9-II, it was not considered useful for quantitative study.

The rate of H202 removal by Reaction II is of the same order of magnitude asthe rate of the catalatic reaction in the uninhibited erythrocyte (600 X [Hb] \£2

2-IOu X [cat]). The similar values of IlL (Reactions I and II) therefore indicatethat the diffusion barrier to reaction with H202 is the same for both catalase andhaemoglobin. As haemoglobin is essentially "packed in intracellular solution" thisimplies either that catalase is free in solution, or that, if it is segregated, the barrierinvolved is more permeable to H 202 than the erythrocyte membrane itself.

0·40

[1]

1·8

I1-7

§''"r-

j.t~~r.~in

"'"fib

\

1·6

1·5

2 3 4Time (min)

Fig. I. Effect of added H,O, on haemoglobin in intact cells (I) and haemolysate (L). Approx,50,uM haemoglobin (haem concentration) and 15 m/lM catalase are present in each case. CurveL indicates the oxidation of haemoglobin to methaemoglobin in a haernolysa.te, Curve I the oxi­dation with intact cells. Curve I' represents the initial reaction in the intact cells when the extra("induced") oxidation (E) is subtracted. Curves C. (haemolysate) and C1 (cells) are the curvesfor the disappearance of H.O, (H,O. calc. indicates calculated peroxide concentration) in thetwo systems, estimated from separate manometric experiments (k = 4' 106 M-t sec-t). Effectiveprotection in haemolysate -99%. pH 7-4.0.15 M NaCl, 20'; 2.8 ml final volume, I em opticalpath.

Fig. I shows the effect of added peroxide on haemoglobin when the endogenouscatalase is active. The following characteristics of the reaction may be noted:

(a) The initial reaction of peroxide with haemoglobin proceeds independentlyof the presence of active catalase, in both cell and haemolysate.

(b) In the haemolysate, the rate of oxidation of haemoglobin decreases accordingto a kinetic path determined by the disappearance of H 202 catalatically (Hb is actingas an "indicator" of H 20 2) .

(c) The initial rate of reaction with the intact cells is about 1/4 of that in thehaemolysate, although the extent of the initial reaction is similar in the two systems.

(d) The oxidation in the intact cell continues slowly after the calculated timefor peroxide disappearance, and the final extent of methaemoglobin formation istherefore somewhat greater in the intact cells.

Parallel experiments showed that the amount of methaemoglobin formed on

Biochim. Biophys, A eta, 99 (I960) 286-297

CATALASE ACTIVITY IN RED CELL 291

the addition of a given amount of peroxide is independent of the haemolysate concen­tration used. In the presence of 4 mM azide the initial oxidation rate continued tocomplete methaemoglobin formation in both cells and haemolysate (cj. Table I).

The slow final oxidation occurring in intact cells was not an optical artifact,and seems to depend upon (i) the cells being initially intact, for subsequent saponintreatment failed to halt the slow oxidation completely, (ii) the presence of H202 foran appreciable time in the system, as the initial addition to intact cells of an exogenousconcentration of catalase, approx. 5 m,uM, sufficient to induce a peroxide decomposi­tion rate similar to that of the haemolysate, decreased the extent of excess oxidationconsiderably. As the total extent of oxidation in cells and haemolysate should be thesame, an extra feature of the intracellular system must be postulated to account forthe excess "induced" oxidation. Two possibilities exists: the formation of a greateramount of inactive catalase compound II in the intact cells, and the generation of anextra oxidant (e.g. by the reactions of the OR' radical) within the cell.

The "protective" effect of catalase on the initial rapid haemoglobin oxidationin both cell suspension and haerriolysate may be summarized as in Table II. This table

TABLE II

RELATIVE RATES OF REACTION OF HAEMOGLOBIN AND ERYTHROCYTE CATALASE

I mM HaOa at pH 7.4, 20°.

Component Rate constant Relative Relative( M-1sec-1) concentration' reaction rate"

Catalase (initial) 9' 106 3' 10-4 r35Catalase plus Compo II

(steady state) 2' 106 3' ro-4 3°Oxyhaemoglobin (in air) .......20 [IJ [rJOxyhaemoglobin (complete)?" ....... 3 1 0.15Reduced haemoglobin 3°0 <1 <ISMethaemoglobin 600 <r <30

• Compared to that of the equilibrium Hb ee HbOj, mixture in air .•• By extrapolation from experiments in presence of increasing amounts of dissolved 0a

(up to r atmosphere O2) ,

also shows that the rate of reaction of "pure" oxyhaemoglobin is considerably slowerthan that observed in air. Hence we have the protection by oxygen observed by KElLIN

AND HARTREE2 and the secondary effect of catalase. But the primary protective actionof catalase (reacting with more than 99% of the added peroxide) is direct, and is infact increased when the haemoglobin is fully combined with oxygen; catalase thendestroys more than 99.9% of the added peroxide. The extent of methaemoglobinformation is thus reduced by a factor of between 100 and 1000 in the presence ofcatalase.

Two extra features of the reaction of haemoglobin with H202 may be noted.Firstly, contrary to the tentative claim of KElLIN AND HARTREE2, Reaction 9, thedirect oxidation of oxyhaemoglobin, proceeds at a finite albeit low rate. Secondly,the rate of reaction of the equilibrium haemoglobin mixture in air is greater thanwould be calculated for an equivalent mixture of "Hb40s" (99%) and "Hb

4" (1%).

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P. NICHOLLS

The observed rate may imply that the rate constant for the reaction of the deoxy­genated haem in "Hb40a" with H 202 (Eqn. ro) is about r700 M-l sec-I (5-6 timesfaster than the haems of Hb4) .

Reactions of enzymically generated peroxide with erythrocyte haemoglobinThe formation of methaemoglobin by slowly generated peroxide is shown in

Fig. 2. A sigmoid reaction curve is observed in each case proceeding to completeformation of methaemoglobin (or its peroxide and azide derivatives). In the haernoly-

0·9

0·8

1'7"-Er-. 06"""''"

0·5

0·4

[L.I)_

0·7

0·2

10 15 20Tim. (min)

Fig. 2. Effect of continuously generated H.O. on haemoglobin in intact cells (I and hINa) andhaemolysate (L and LHNa)' Approx. 50 {1M haemoglobin ane! 15 mflM catalase are present ineach case. Curves I and I HN. show the reaction in intact cells, Curves Lane! LHN. in haemolysates,4 mM azide is present for the reactions of curves I HNa ane! LHN a- pH 7.4, 0.15 M NaCI, zoo, o.z%glucose in air (z50 pM 0.); 2.5 ml final volume, I cm optical path. 1.0 absorbancy unit has beensubtracted from I and I HNa to correct for light scattering.

sate, the initial rate of reaction, 6· ro-4 M-I sec-I, corresponds to a theoretical steadystate H202 concentration of 30 ,uM. This concentration is the same as that calculablefrom Eqn. r3

[HP.J "steady state" = r/k/[catJ

where r is the rate of peroxide generations (5' xo- 7 M' sec-I), k1' is 2' r0 6 M-1sec-l , and[cat] = 8· ro-9 M. The rate of reaction with intact cells is somewhat less than 1/3 thisvalue. The progressive increase in reaction rate may be attributed to the removal ofoxygen from the system to give the more reactive deoxygenated forms of haemoglobin.

Here the protection by catalase is upon the rate and not as above on the extentof reaction. The rapid increase in oxidation rate when catalase is inhibited (Fig. 2) isassociated with a rapid rise in peroxide concentration. The difference between theoxidations in presence and absence of catalase is given by the ratio of peroxideconcentrations as in Eqn, 14

[H.O.] "steady state"[HP.] "catalase inhibited" k,' [cat] t

(14)

where t is the time elapsed (sec). Under the conditions of Fig. 2 the ratio given byEqn, 14 has the value 6 after 3 min, as was found experimentally. If peroxide was

Biochim. Biophys. Acta, 99 (19 65) 286-297

CATALASE ACTIVITY IN RED CELL 293

generated in vivo within the erythrocyte, the much greater local concentration ofcatalase would give a much greater relative protection, above 99% after 6 sec.

Peroxidatic activity of erythrocyte catalaseUsing the "coupled oxidation" method of KEILIN AND HARTREE2,5 with

haemolysates or intact red cells to provide the catalase, and glucose oxidase and glu­cose to generate H 20 2 , no peroxidatic reaction (Eqn. 4) could be detected in the ab­sence of added hydrogen donor. Under the test conditions used, this meant that lessthan S mM of any hydrogen donor as active as ethanol was present in the erythrocytes.In the presence of added ethanol, intact cells and haemolysates were each as activeas purified catalase in promoting its peroxidation, the calculated velocity constantin all cases being close to the theoretical one. In the absence of ethanol, or in itspresence at low haemolysate or cell concentrations, all the generated peroxide (from40 ftg glucose oxidase in 3 ml O.I% glucose, pH 6.0) was decomposed catalatically(Eqn. I).

Under these conditions, whether in the presence or absence of ethanol, haemo­globin destruction occurred. With cells diluted 1:3 (3 mM Hb, I,uM catalase), 3%methaemoglobin formation occurred; with cells diluted I :30 (0.3 roM Hb, O.I ,uMcatalase), 80% methaemoglobin was formed; with cells diluted I:300 (30 ftM Hb,IO mftM catalase), IS % methaemoglobin and 80% cholehaemoglobin and similarproducts were formed. There were no marked differences between cells and haemoly­sates.

The presence of 25 mM aminotriazole in systems containing continuouslygenerated peroxide produced a rapid inhibition of catalase whether in cells or haemo­lysate. No relative protection of intracellular catalase was observed as might havebeen expected in the presence of endogenous donors such as those postulated byMARGOLIASH AND SCHEJTERI5. How then do we account for: The low steady stateamount of the primary catalase peroxide compound-s and the weak catalatic activity-!reported in red cells at very low rates of peroxide addition?

For concentrations of peroxide above 10-8 M the rate of turnover of eachequivalent of catalase peroxide compound (Eqn. IS) exceeds 0.2 sec-I.

6· rag M-1sec-1

Cat FeS+ + HP2 ) Cat Fe8+H202

r.8 . r 07 M-1sec-1Cat FeS+H202 + H 202 > Cat FeS+ + 2 H 20 + O 2

(Isa)

(ISb)

(16)

In the experiments of MARGOLIASH AND SCHEJTERI6and of COHEN AND HOCHSTEINll,

however, the rates of peroxide generation are so low as to give turnover rates forcatalase below 0.02 sec-I. Under these conditions "endogenous" and "entrained"donors-" associated with the catalase molecule itself can decompose the peroxidecompound more rapidly than H 20 2 (Eqn. ISb). Under such conditions there will be adecrease in the amount of peroxide compound present and the apparent catalaticactivityll,15 even though all the peroxide is still reacting with catalase (Eqn. I6).

H 20 ".Cat H 20 2"",",- /"AH 2 [endol", ;;fB"\/ . \1 "I

• I .?

H202)"-Catalase JI''''' A [endO]--/!'·..BH.

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294 P. NICHOLLS

The rate of this reaction will depend upon the effective "concentration" of endogenousdonor AH2JEqn. 16 and hence upon the systems (BH 2) responsible for its regenerationin the cell. These may include glutathione'! and other compounds'" in the erythrocyte.

Relationship bettseen erythrocyte catalase and glutathione peroxidaseMILLS19,20 and COHEN AND HOCHSTEINll, in contrast to the conclusions reached

above, assert that another enzyme, glutathione peroxidase, is responsible for theremoval of low steady states of peroxide (Eqn, 17) even in the presence of fully activecatalase.

GSHH 202 + 2 GSH ) GSSG + 2 H 20

peroxidase(17)

But at all concentrations of peroxide, catalase, whether free or combined as peroxidecompound, reacts with peroxide at a rate of at least 6.106 M-1sec-1 (Eqn. IS). Calcula­tion of the activity of glutathione peroxidase from MILLS' data-? shows that acorresponding equivalent weight of glutathione peroxidase protein (saturated withrespect to glutathione )removes H Z0 2 at a rate of 8.102 M-1sec1, more slowly thancatalase by a factor of 104 ,

Even if glutathione peroxidase were highly active in vivo, the relative protec­tive effect of catalase should nevertheless be the same. For, as catalase reacts roo timesfaster than haemoglobin (Table II) with any residual peroxide, when it is activehaemoglobin will be oxidized much less extensively than when it is inactive, whateverother complexities occur in the system. There is, however, no direct evidence thatglutathione peroxidase is highly active in vivo; the activity of the isolated enzymein the assay system used in vitro2 0 was only about twice the blank glutathione peroxi­dation rate.

Glutathione may: (i) interact with haemoglobin itself to diminish the rate of itsoxidation by H 20 2 , (ii) provide a source of reducing equivalents for methaemoglobinreductions", (iii) preserve the methaemoglobin reductase enzymes in an active state.In the present experiments, the "starved" cells used probably contained glutathionelargely in the oxidized state. The addition of glucose in the absence of azide tendedto reduce the slow final oxidation occurring in intact cells after the addition ofperoxide, presumably by promoting the intracellular reductase activity. No effectof glucose was observed on the rapid "initial" oxidation of haemoglobin in the pres­ence of azide.

In haemolysates, the addition of glutathione (7 mM) diminished the rate ofoxidation by added peroxide and the initial rate of oxidation by continuously gene­rated peroxide. This direct action must be attributed to a direct interaction betweenglutathione and haemoglobin to reduce the rates of Reactions 9 or 10. The protectionoccurred in both presence and absence of azide, although azide still increased the rateof the protected reaction. The protective effect disappeared before sufficient peroxidehas been produced to oxidise all the glutathione.

It is concluded that both the theoretical considerations and the experimentalresults argue against the possibility of glutathione peroxidase playing a major role inhaemoglobin protection under the present conditions.

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CATALASE ACTIVITY IN RED CELL

DISCUSSION

295

Does catalase playa protective or a metabolic role in the erythrocyte?When the effect of oxygen (Table II) has been taken into account, and the

different ways in which catalase protects haemoglobin from oxidation, by added andby generated peroxide, noted-it reduces the extent of oxidation in the former case,the rate of oxidation in the latter-there remains no fundamental discrepancybetween the results of KElLIN AND HARTREE2 and of FOULKES AND LEMBERG3•

KElLIN AND HARTREE2 correctly noted the greater extent of oxidation by generatedperoxide, and the marked effect of oxygen on the oxidation rate; FOULKES AND

LEMBERG3 correctly noted the increased oxidation of haemoglobin produced whencatalase is inhibited.

The absence of appreciable quantities of hydrogen donors in the erythrocyte(except for the low activity observed in very dilute peroxide-s discussed above, eventhis not being observable in all speciesv) supports the concept that the most likelyrole for catalase here is a protective one. A more powerful theoretical argument maybe advanced in support of this view, however. A metabolic role would be expected tobe a continuous one, the physiologically necessary oxidation of the metabolite AHz(Eqn. 4). But acatalasaemics'" show no metabolic defect. A protective role may beintermittent. In fact it is conceivable that catalase has a protective role and yet isactive only on rare occasions. For example, a calculation based on bioenergetic con­siderations may be made. The requirement for maximal energetic efficiency in main­taining haemoglobin reduced is that the sum of the energies required (over a 4-monthperiod, the erythrocyte lifetime) for the synthesis of catalase ("protective") and forreducing oxidized haemoglobin ("restorative") be a minimum. The energy requiredto synthesize a given amount of catalase 'will be directly proportional to that amount,while the amount of oxidized haemoglobin produced and hence requiring reductionwill be inversely proportional to the amount of catalase present. When a cell contains33 %haemoglobin and 0.04%catalase, as does the horse erythrocyte, assuming thatz ATP molecules are needed per peptide bond in protein synthesis and g ATP mole­cules are equivalent to the one NADPH2 involved in reduction of 2 HbFe3+-+z HbFe2 ,

this amount of catalase would be "evolutionarily optimal" in an environment in­volving an oxidation of haemoglobin with a half-life of about one week (k!:~2IO-R seer").

Contrary to certain views 2,ll , sudden formation of peroxide in the blood may bemore likely than continuous formation. Red blood cells have an exceedingly lowrespiratory rate, and are not known to produce peroxides. On the other hand, asudden change in foodstuffs might introduce autoxidisable substances. In a sense theblood is "external" to the organism, and must contain regulatory mechanisms func­tioning sporadically. The plasma y-globulins represent a known analogy.

In this context it may be of interest to note that the Swiss "acatalasaemics"possess an apparently normal catalase in about 0.3% of the usual concentrationw,indicating a change in a control gene rather than the coding gene. Such a activity levelwould give 30-7°% protection of haemoglobin compared to the 99-99·9% usuallyobserved (Table II).

Evolution and function of catalaseFrom the above discussion we conclude that in the horse erythrocyte (and hu-

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296 P. NICHOLLS

man erythrocytes behave similarly): catalase is fully active and just as accessible toperoxide as is haemoglobin; intracellular catalase competes effectively with haemo­globin for peroxide, destroying 99-99.9% of HzOz either added or generated in solu­tion; no hydrogen donors are normally available to decompose the catalase primaryperoxide compound at a rate greater than 0.1 sec-I, the level of activity of the"endogenous" donor; the protective effect of glutathione in starved cells or haemoly­sates therefrom does not involve glutathione peroxidase; the role of erythrocytecatalase, if any, must be protective and its action catalatic (Reaction I).

It is by no means necessary to conclude from this that the "function" of catalaseis to destroy peroxide by Reaction 1. The existence of healthy acatalasaemicse.wmakes it probable that any role in man is marginal, whether catalatic in the blood, orcatalatic and peroxidatic-s in the liver. Different roles may be subserved underdifferent conditions, and the role which is evolutionarily most "primitive" cannot bedetermined in this way.

CALVIN25, in a series of articles on the origin of life, has presented the "evolution"of the catalatic function as a paradigm of chemical evolution. The data he presents,taken from STERN 26, had been used by STERN, who wished to find a chemical modelfor catalase, to illustrate the greater activity and specificity of the enzyme by com­parison with inorganic iron or non-specific iron complexes. But this does not implythat the catalatic function of the enzyme evolved from that of inorganic iron. Theconcepts of "evolution of a morphological structure" and "evolution of a chemicalproperty" are logically different.

Although catalase decomposes peroxide more rapidly than haematin, and hae­matin more rapidly than FeCla solution, (a) under comparable conditions of pH, thecatalatic activity of haematin is not very different from that offree iron 26,27, the mainproblem with haematin being its rapid degradation by peroxide; (b) the catalaticactivity of free iron is low only because it is usually measured at acid pH; by extra­polation, iron, if soluble in alkaline solution, would then be as active as catalaseitself 28 ; (c) the reaction mechanisms in the three cases are different-", the main bio­logical advantage of catalase being that it produces no free radicals-",

It is most likely that both porphyrin structure and catalase activity have"evolved" within a biological nexus containing many proteins and enzymes. Catalase,the need for which probably appeared at a definite time, could have been producedby modification of another haemoprotein with quite a different role and possibly evenlower catalatic activity than iron or haematin alone. It is not possible to discover theoriginal role of catalase by studying its present activities. We must remember the"motley" of the biological world.

In horse-, and probably hurnan-, erythrocytes, catalase seems to have either aprotective catalatic function or none at all. In mammalian liver, it may act peroxida­tically-s. In other systems'" a function has yet to be demonstrated. But the searchfor a general, or original, function is doomed to failure.

ACKNOWLEDGMENTS

The study ofthis problem was initiated by the late Professor D. KElLIN and theinvestigations described were begun in his laboratory at the Molteno Institute in

Biochim, Biophys, Acta, 99 (19 65) 286-297

CATALASE ACTIVITY IN RED CELL

Cambridge, England. I gratefully acknowledge his interest and support up to the timeof his death in February 1963.

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