I955

A Spectrophotometric Method for the Determinationof Creatine Phosphokinase and Myokinase

By I. T. OLIVERDepartment of Biochenistry, The University, Sheffield

(Received 28 January 1955)

In the past, methods for the assay of creatinephosphokinase have been based on the chemicaldetermination of one or other of the products ofthe reaction:

creatine +ATP =creatine phosphate +ADP,where ATP = adenosine triphosphate and ADP=adenosine diphosphate. The substances mostcommonly determined have been creatine orcreatine phosphate; e.g. Banga (1943), Askonas(1951), Narayanaswami (1952), Ennor & Rosenberg(1954), Kuby, Noda & Lardy (1954) and Chappell &Perry (1954). These methods may not always beconvenient for the determination of creatine phos-phokinase activity in tissue homogenates, since theamount of tissue used in reaction mixtures makesthe introduction of significant amounts of endo-genous substances a distinct possibility. Themethoddescribed here, although not more sensitive thanthe methods based on the colorimetric estimationof creatine, makes it possible to measure enzymicactivities in tissue homogenates and extractsdiluted 2000 to 20 000 times, and to follow con-tinuously the time course of the reaction in a totalvolume of about 4 ml. Under these conditionseffects due to endogenous substances are negligible.The method is based on Kornberg's assay pro-

cedure for ATP (Kornberg, 1950), whereby theformation of ATP from ADP and creatine phos-phate is linked to the reduction of triphospho-pyridine nucleotide (TPN). When the rate-limitingstep in the reaction sequence is that catalysedby creatine phosphokinase, spectrophotometricmeasurement of the rate of reduction ofTPN givesthe rate ofATP formation, and thus the activity ofthe enzyme.The method has also been applied to the deter-

mination of myokinase activity by measuring therate of formation ofATP from ADP. The methodsfor the assay of myokinase due to Kalckar (1943,1947) are laborious. The most sensitive method isprobably that based on the firefly luminescencesystem for the estimation of ATP (Strehler &Totter, 1952), but is not convenient for general use.The coupled activities of myokinase and creatinephosphokinase have been used by Chappell &Perry (1954) to assay myokinase. The present

method has the advantage of convenience overmost of the older methods. The reactions arefollowed continuously in the spectrophotometer,and the rates catalysed by small amounts of tissuecan be measured with ease.An outline of the method has been given else-

where (Oliver, 1954). Complete experimentaldetails are included here.

EXPERIMENTAL

Materials and method&Adenosine tripho8phate. ATP was prepared by the

method of LePage (1949); the chromatographic proceduredescribed by Krebs & Hems (1953) showed that more than90% of the phosphate was present in the form of ATP.

Adenoeine diphosphate. ADP was obtained fromSchwartz Laboratories Inc. and contained less than 1% ofATP when assayed by the chromatographic proceduresabove, or enzymic procedures based on those of Kornberg(1950).

Adenylic acid (AMP). This was obtained from RocheProducts Ltd. Solutions of AMP were brought to pH 7-0with NaOH before use.Sodium creatine phosphate hexahydrate. This was syn-

thesized from creatine by the method of Ennor & Stocken(1948).

Glucose 6-phosphate. This was a gift from Dr T. H. Wilson,Walter Reed Army Medical Center, Washington 12, D.C.

6-Phosphogluconic acid. This was prepared by bromineoxidation of glucose 6-phosphate according to Seigmiller &Horecker (1951). Chromatographic procedures (Oliver,unpublished experiments) showed that the preparation wasfree of glucose 6-phosphate.

Triphosphopyridine nuceotide (TPN). This was preparedby Mr D. H. Williamson after Horecker (private communi-cation). Preparations containing 65-90% TPN were pre-pared by gradient elution from Dowex-l resin and con-tained no diphosphopyridine nucleotide (DPN) or adenylicacid (AMP), since these substances were eluted from thecolumn at an earlier stage. Crude preparations, in whichthe major impurity was AMP, contained about 10% TPNand 25% DPN.

Hexokinase and glucose 6-phosphate dehydrogenase. Thesewere obtained together in a crude preparation of yeasthexokinase similar to that described by Slater (1953). Theactivity of glucose 6-phosphate dehydrogenase in the pre-paration was increased by taking the protein fraction pre-cipitated from aqueous extracts of baker's yeast by 50-75% saturation with ammonium sulphate. After dialysis

116

CREATINE PHOSPHOKINASE AND MYOKINASEto remove ammonium sulphate, it has been found con-venient to freeze-dry the enzyme preparation, and to storeit in the refrigerator.

Glucose 6-phosphate dehydrogenase activity. This wasassayed by following the rate of reduction of TPN in thespectrophotometer at 340 m,u. when glucose 6-phosphatewas added to a reaction mixture consisting of 0-05Mbarbitone buffer pH 8-6, 001M-MgCl4, 0O1m-KCl, 104MTPN, and the yeast preparation which had- previouslybeen incubated until the optical density became steady.

Hexokinase activity. This could not be determinedquantitatively in such a system, since hexokinase is moreactive than glucose 6-phosphate dehydrogenase in theyeast preparation. Activities less than maximum weremeasured by following the rate of reduction of TPN in thespectrophotometer at 340 mjA. when ATP (final concentra-tion 0.001 m) was added to a reaction mixture containing0-05M barbitone buffer pH 8-6, 0-01M-MgCl,, 0O1m-KCl,0OO1M glucose, 10-4M TPN, and the yeast preparationwhich had previously been incubated until the opticaldensity became steady.Spectrophotoer. A Unicam spectrophotometer model

SP 500 was used for the determination of optical density at340 mIL. Cuvettes (1 cm.) were used and readings weremade against a blank of barbitone buffer. The temperatureof the cuvettes was controlled by a water-jacketed cellcompartment made by Mr G. Fletcher according toCampbell & Simpson (1953).

Syringe. An Agla micrometer syringe (BurroughsWellcome and Co.), fitted with an elongated glass tip bentthrough a right angle, was used to deliver volumes up to50 pl. of the muscle homogenates to the reaction cuvettes.

Muscle homogenates. These were prepared from the hind-leg muscles of male albino rats immediately after killing bya blow on the head. The muscle was minced in a coldmincer, a sample weighed and then treated for 3 min. in achilled Waring Blendor (micro cup) with 6 vol. of ice-cold0-1M-KCI. The homogenates were strained through gauzeto remove coarse particles and ligaments and stored at 00until required. For assay, samples were diluted tenfoldwith ice-cold 0s1M-KC1 immediately before use. If it wasnecessary to retain a preparation overnight, it was storedat -15°.

Buffer system. In using barbitone buffer at pH 8-6 wehave followed the work of Banga (1943). At this pHhexokinase is stable and of high activity.

Principle of the method

Use is made of the assay procedure for ATP (Kornberg,1950) in which ATP reacts with glucose in the presence ofMg2+ and hexokinase, forming glucose 6-phosphate (Eqn. 1),which is then oxidized by glucose 6-phosphate dehydro-genase with simultaneous reduction of TPN (Eqn. 2).

Glucose + ATP-*glucose 6-phosphate +ADP, (1)

glucose 6-phosphate +TPN-+6-phosphogluconicacid +TPNH2. (2)

The rate of reduction of TPN is measured by observing therate of change of optical density in the spectrophotometerat 340 mIA. (Fig. 1). Although the yeast preparationcontaining the hexokinase and glucose 6-phosphate de-hydrogenase is very impure, it is free from several enzymeswhose presence would be disadvantageous. Fig. 2 shows

that two of such enzymes, glucose dehydrogenase and 6-phosphogluconic acid dehydrogenase, are completelyabsent. The system is completely specific for TPN, and itis therefore unnecessary, except where other considerationsarise, to use purified preparations of TPN. In practicecrude preparations containing about 10% of TPN and25% of DPN have often been used in these studies.

0-7F2i 0-61- f1

0-5F

0-40 n1cvv

0-2.24)1

-. 0-10n1 -L I I

~0 5 10 151 t + Time (min.)1 2 3

20

Fig. 1. Hexokinase and glucose 6-phosphate dehydro-genase in yeast enzyme preparation. At zero time bothreaction mixtures consist of 0-05M barbitone bufferpH 8-6, 0-01 M-MgCl,, 0-1M-KCl and 10-4M TPN (finalconcentrations). At time (1), 0-1 ml. of 'couplingenzyme' containing 1 mg. dry wt. was added to bothmixtures I and II with stirring. At time (2), 0-2 ml. of0-5m glucose added to both, with stirring, bringing finalconcentration to 0-02M. At time (3), 0-1 ml. of 0-02Mglucose 6-phosphate added to I, and 0-1 ml. of 0-02MATP added to II with stirring. Final concentrations ofthese two substrates was 0-OO1M.

In these coupled reactions, in order that the final rate ofreduction ofTPN shall be equal to the rate of formation ofATP, the activities of hexokinase and glucose 6-phosphatedehydrogenase must be in excess of the activity of theATP-forming reaction, i.e. the latter reaction must be therate-determining step. This situation can be arranged bytrial. Reaction mixtures containing buffer, TPN, glucose,ADP, creatine phosphate and increasing amounts of the' coupling enzyme' (yeast preparation containing hexo-kinase and glucose 6-phosphate dehydrogenase) were usedto measure the rate of ATP formation catalysed by aconstant amount of a highly diluted muscle homogenate.Under these conditions, ATP formed by creatine phospho-kinase and myokinase is detected. The first level of additionto give a maximum rate was used in subsequent quanti-tative experiments. In our experiments 4 mg. of the driedpreparation of the 'coupling enzyme' gave quantitativeassay of creatine phosphokinase or myokinase activity in0-20 p. of a muscle homogenate of dilution one part in 60,i.e. 0-0-33 mg. wet weight of muscle. Under these condi-tions the activity of an enzyme which catalyses the forma-tion of ATP is taken as shown by the initial steady rate ofreduction of TPN, which can be obtained graphically fromthe rate curves of optical density versus time. Using theextinction coefficients for reduced TPN given by Horecker

117Vol. 6I

I. T. OLIVER& Kornberg (1948), i.e. 6-22 x 106 cm.2/mole, activities canbe expressed in terms of micro-moles of ATP formed perunit time, since each molecule of ATP formed gives rise toone molecule of reduced TPN. This relation holds only ifthe enzyme preparation is free of 6-phosphogluconic aciddehydrogenase, and glucose 6-phosphate is the only sub-stance effective for the reduction of TPN. Our system hasbeen tested in this respect.

06 r

ATP will also arise from ADP by the activity ofmyokinase (Eqn. 4).

Creatine phosphate+ADP =creatine+ATP, (3)2ADP=ATP+AMP. (4)

This effect has been overcome by adding excess ofAMP to the reaction mixture. In Fig. 3 is seen theeffect of AMP on the rate of reduction of TPN ina reaction mixture containing TPN, the 'couplingenzyme', glucose, ADP and a muscle homogenate.

0-5E

X' 0-4

._

c.O 0-2

0-1

F :. 0-5EX 0-4(6

co

0-3C

1

0-- 0-2

II

- 4,

5t t2 3

10Time (min.)

Fig. 2. Yeast enzyme preparation. At zero time reactionmixtures consist of 0-05x barbitone buffer pH 8-6,0-01lmMgCl2 and 0-IM-KC. Mixture I contains 10-4MDPN and II contains 10-4M TPN. At time (1), 0-1 ml.of 'coupling enzyme' added to both with stirring. Attime (2), 0-2 ml. of 0- m glucose added to both withstiring. At time (3), 01 ml. of 0-02m glucose 6-phos-phate added to I, and 0- ml. of0-02x 6-phosphogluconicacid added to II with stirring. The DPN contained noTPN.

In all experiments it was found necessary to incubate the'coupling enzyme' with the substrates for about 5 in.before starting the reaction to be studied. During thispreliminary incubation some reduction of TPN takes place(see Fig. 1) which is allowed to come to completion beforethe required reaction is started. This effect is due to tracesof ATP or glucose 6-phosphate in the reagents, and also toa substrate in the yeast preparation which has not beenfully identified.

RESULTS

Measurement of creatine phosphokinase activityThe work for which this assay procedure has beendeveloped has been concerned with enzymicactivities in homogenates or tissue extracts.Accordingly, the isolation of a single phosphatetransfer reaction for study has been one of theproblems. The reaction catalysed by creatinephosphokinase (Eqn. 3) can be measured in terms ofthe production of ATP, but in tissue homogenates

D

X0

I ~ ~ ~ ~ ~ ~~~ ,. I5 10 15t t Time (min.)2 3

Fig. 3. Effect of AMP on myokinase. At zero time reac-tion mixtures consist of 0-05M barbitone buffer pH 8-6,001M-gl,, 0-lm-KCI, 0-001m ADP, 0-02m glucoseand 10-4m TPN. Mixture I contains in addition, 001MAMP. At time (1), 0-4 ml. of 'coupling enzyme' (con-taining 4 mg. dry wt.) was added to both with stirring.At time (2), 10pl4. of a muscle homogenate diluted 1 in60 with 0 lm-KCl were added to both with stirrng. Attime (3), 0-2 ml. of 0-2m AMP was added to II, bringingfinal concentration to 001 mx.

When the concentration of AMP is ten times thatof ADP, the myokinase activity is completelyinhibited. This makes it possible to distinguishbetween the ATP-forming reactions of creatinephosphokinase and myokinase. The measurementof creatine phosphokinase activity in the presenceof myokinase then becomes possible. In practice,a control from which residual myokinase activitycan be determined is always included in assaysof creatine phosphokinase. The control contains,besides TPN, glucose and the 'coupling enzyme',only AMP and ADP in the ratio of 10 to 1 (seeFig. 4). Errors caused by myokinase activity underthe conditions of assay for creatine phosphokinasecan thus be evaluated, but in fact these are rarelysignificant.The effect of high concentrations ofAMP on the

activity of creatine phosphokinase was tested. Inexperiments in which the ADP concentrationremained constant at 0-OO1M and the AMP con-centration was increased from 0-0025 to 0:015m, theactivity of creatine phosphokinase did not changeappreciably until the highest concentration ofAMP

118 I955

- 0 &..*-

CREATINE PHOSPHOKINASE AND MYOKINASE

06 r

,c L*U'bE5t 0~4

! 03C4.

X 02._

O0 0-2

r

Table 3. Effect of ADP on activity ofcreatine phosphokinase

Reaction mixture otherwise as in Table 1. AMP 0O01 M.

4-

1

Conen. ofADP(mM)051-02-0

2

5 10Time (min.)

15

Fig. 4. Determination of creatine phosphokinase. At zerotime the reaction mixtures consist of 0 05m barbitonebuffer pH 8.6, 001M-MgCI,, 0-1M-KCI, 002m glucose,0-001m ADP, 0O01m AMP and 10-M4 TPN. Mixture IIcontains also creatine phosphate, 0-01M. At time (1),0 4 ml. of 'coupling enzyme' (containing 4 mg. dry wt.)is added to both mixtures. At time (2), 10,. of a musclehomogenate diluted 1 in 60 with 0.1 M-KCI are added toboth I and II with mixing. Curve I is the control fromwhich residual myokinase activity can be determined.The tangent to the linear portion of Curve II gives therate of optical density change which is proportional tothe ratio of reduction of TPN. This allows the evaluationof the rate of ATP formation, which is defined as theactivity of the enzyme.

Table 1. Effect ofAMP on activityof creatine pho8phokinase

Reaction mixture consists of 'coupling enzyme' in0*05M barbitone buffer pH 8.6, 0-01M-MgCl, 01m-KCl,0.02M glucose, 0001m ADP, OOlm creatine phosphate,10-4M TPN, and 10pl. of muscle homogenate containingabout 20 mg. wet wt./ml.

Concn. ofAMP(mM)

2-55.01015

Activity of creatine phosphokinase(pmoles ATP/hr./mg. wet wt.)

-~A-(i (ii) ORi)4-855-17 6-20 2-75- 6.00 2.64- *518

Table 2. Effect of creatine pho8phate on

activity of creatine pho8phokinase

Reaction mixture otherwise as in Table 1. AMP OOlm.

Activity of creatineConcn. of phosphokinase

creatine phosphate (pmoles ATP/hr./mg.(mM) wet wt.)10 7.5520 7.5530 7-6140 7-90

I-

_45s 30

co

v5

0

'

C

0

Fe C

Activity of creatine phosphokinase(pmoles ATP/hr./mg. wet wt.)

A

(i (ii) (iii)8-25 5.38 5-809 90 5.448-72 4.40 5-62

5 10Volume of muscle homogenate (Il.)

15

Fig. 5. Concentration of creatine phosphokinase andactivity. The data were obtained from measurementsof the linear rate of reduction of TPN, catalysed byincreasing amounts of muscle homogenate. Reactionmixtures as in Fig. 1 with the addition of OOlX AMP,001m creatine phosphate, 0O001M ADP and 0-02Mglucose. Activity expressed as ,umoles ATP produced/hr.

was reached, when a slight decrease in activity wasseen. Accordingly the highest concentration ofAMP used was 001m (see Table 1).When the concentration of creatine phosphate

was raised through the range 001-004M in foursteps, only a small increase in activity could bemeasured, and the concentration usually used, inorder to minimize the amount ofcreatine phosphaterequired, was therefore OOlm (see Table 2).When the concentration ofADP was varied from

00005 to 0-002m no consistent increase in theactivity of creatine phosphokinase in a musclehomogenate could be measured, and the usualconcentration of ADP in the system was 0001M(see Table 3).The above concentrations of ADP, AMP and

creatine phosphate were used in experiments toestablish the validity of the assay method. InFig. 5 the activity of creatine phosphokinase in a

Vol. 6I 119

! _-

2-55

muscle homogenate is plotted against the relativeconcentration of the enzyme. The activities calcu-lated from the initial steady rate of reduction ofTPN (see Fig. 4) are directly proportional torelative concentration. In a further experiment theactivity of creatine phosphokinase was determinedin two identical samples of a muscle homogenateby the spectrophotometric method, and an inde-pendent method previously used in this laboratory.This method depends on the analysis of creatinephosphate in a reaction mixture after its separationon a paper chromatogram. An alkaline solventdescribed by Hanes & Isherwood (1949) consistingof a mixture of n-propanol, concentrated ammoniaand ethylenediaminetetraacetic acid, has been usedby the author for the separation of creatine phos-phate from adenosine phosphates, inorganicphosphate, triose and hexose phosphates in tissueextracts. This technique is easily applicable toenzyme reaction mixtures after deproteinizationwith trichloroacetic acid and neutralization of theextracts, and in this way creatine phosphate wasisolated for analysis from a reaction mixture similarto those used in the other method. The rate ofbreakdown of creatine phosphate determined byphosphate analysis on spots cut from the chromato-gram according to Eggleston & Hems (1952) thengave the activity of creatine phosphokinase. Thismethod is very much less sensitive than thespectrophotometric method and in order to adjustconditions so that the decomposition of creatinephosphate could be detected analytically, the' coupling enzyme' was added to the reactionmixture together with glucose. This has the effectof maintaining the starting concentration of ADP,which in turn extends the time of breakdown ofcreatine phosphate and also the degree of decom-position. It is then possible to follow the reaction,using concentrations of ADP and creatine phos-phate identicalwith those inthe spectrophotometricsystem. Results of a typical experiment gave theactivity of creatine phosphokinase at 250 to be 2-85by the spectrophotometric method and 2-33 by theanalytical procedure (imoles ATP formed/hr./mg.wet weight of muscle). Since the analytical pro-cedure is less accurate than the spectrophotometricmethod, these values represent fair agreement.

Mea8urement of myokina8e activityThe reaction catalysed by myokinase

2ADP=ATP +AMPgives rise to ATP, and the activity of this enzymecan thus be measured by a method similar to thatdescribed for creatine phosphokinase. When theformation ofATP is linked to the reduction ofTPNin the presence of glucose, hexokinase and glucose6-phosphate dehydrogenase, the reaction proceeds

I955to the right, and since each molecule of ATP givesrise to one molecule of reduced TPN and one mole-cule of ADP, the activity can be calculated.In this system purified TPN was used, since the

impure samples of TPN contained adenylic acid asa major impurity. This AMP may inhibit the ATP-forming reaction and so give rise to spuriously lowvalues for activity. Accordingly, in a systemsimilar to that used for the assay of creatinephosphokinase, purified TPN, the 'couplingenzyme', glucose and ADP form the reactionmixture necessary to assay myokinase activity.Working in such a system, with the 'couplingenzyme' added in excess, the effect of ADP con-centration on the activity of myokinase in a musclehomogenate was studied. Raising the concentra-tion ofADP from 0.001 to 0-004m did not increasethe activity measured and in fact a slight decreasewas noticed (see Table 4). Accordingly, all assays

Table 4. Effect of ADP on activity of myokina8e

No AMP or creatine phosphate. Reaction mixtureotherwise as in Table 1.

Activity of myokinaseConcn. of ADP (.moles ATP/hr./mg.

(mM) wet wt.)1.0 9-032-0 8-603 0 812

2 6-00:i-

4,o

.'

o 24.b_6

o5 10

Volume of muscle homogenate (jl.)Fig. 6. Concentration of myokinase and activity. The data

were obtained from measurements of the linear rate ofreduction of TPN catalysed by increasing amounts ofmuscle homogenate. Reaction mixtures as in Fig. 1 withthe addition of0001M ADP and 0-02M glucose. Activityexpressed as jumoles ATP produced/hr.

120 I. T. OLIVER

CREATINE PHOSPHOKINASE AND MYOKINASE

were carried out at a concentration of 0-001M ADP.At 0-001M ADP assays of myokinase in differentamounts of the same muscle homogenate gave astraight line when plotted against relative concen-tration of enzyme (see Fig. 6).

Application to the assay of enzyme in tissueThe method has been applied to the assay of

enzymic activities in various tissues of the rat.With skeletal muscle and heart muscle theactivities were determined in 10-20,1 . of homo-genates diluted one part in 60. The homogenates ofheart muscle in 0- 1m-KCl werepreparedby grindingthe heart with sand in a chilled mortar. Assays onbrain, kidney, liver and spleen were obtained withextracts since the diluted homogenates were tooopaque. The tissue was homogenized in 6 vol. ofice-cold 0- M-KlKC, centrifuged, and the tissuedebris re-extracted twice with 3 vol. of saline. Thecombined supernatants were clarified by centri-fuging in the cold at 19 000 g, and activities deter-mined by the usual methods. Further extraction ofthe tissue residue yielded insignificant amounts ofenzyme (less than 5% of the total activity ob-tained by the above procedure).

DISCUSSION

In this work the average time taken for a singleassay once the necessary reagents are to hand isabout 20 min. In the course of a routine workingday, assays of creatine phosphokinase and myo-kinase can be carried out on all the principaltissues of a single experimental animal.The sensitivity of the spectrophotometric

method enables the use of highly diluted homo-genates and tissue extracts, and in this way re-actions can be studied free of interfering effectsresulting fron¶ the introduction of unknownamounts and kinds of endogenous substances.Very small amounts of ATP or ADP can functioncatalytically in promoting decomposition of crea-tine phosphate when AMP is added to systemscontaining creatine phosphokinase and myokinase(see Ennor & Rosenberg, 1954). The occurrence ofsmall amounts of ADP in tissue preparations asused by Narayanaswami (1952) probably accountsfor the results obtained by this author, whoobserved creatine phosphate decomposition inbrain homogenates in the presence of AMP as thesole added acceptor. However, using the present

Table 5. Activitiem of creatine pho8phokiname and myokinase in rat tissues

Results stated in ,umoles ATP/hr.fmg. wet wt. Temperature 300. pH 8-6.

Creatine phosphokinase

Rat 3A__________ I__ Myokinase

No 0-01MmTissue Rat 1 Rat 2 cysteine cysteine Rat 1 Rat 2

Skeletal muscle 14-9 18-0 12*1 26*0 18*6 23*3Heart muscle 2-38 2-95 3 50 7*01 5 90 7-40Brain 0-96 1-36 0-87 1-62 1-88 2-03Kidney 0-16 0.01 1*22 1*68Liver 0-02 0-06 0-07 0-38 0 93Spleen 2-10 1-42 0-14 0-22 1*80 5-36

Table 5 presents the results of two typical assayson various tissues of thb rat. Tissues were obtainedfrom a single animal immediately after killing by ablow on the neck.The effect of cysteine on the activity of creatine

phosphokinase in tissue homogenates and extractswas also measured (see also Chappell & Perry,1954). At a concentration of OO1M cysteine, theactivity of creatine phosphokinase is about doublethat measured in its absence (Table 5). In tissuepreparations aged 24-48 hr. at 00, the activity inthe presence of cysteine is about the same as thatin the fresh preparations, but the activity in theabsence of cysteine is lowered 10-20% by ageing.No effect of cysteine on the activity of myo-

kinase in tissue preparations could be demon-strated.

method, it has been shown (Oliver, 1954) that thereis no reaction between creatine phosphate andAMP in muscle homogenates or brain extractswhich were highly diluted, and that reaction oc-eurred only when ADP was added.In the determination of creatine phosphokinase

activity by the present method the reactions aresuch that the concentration of ADP, althoughsmall, is maintained approximately constant overthe time required for the assay. In the assay ofmyokinase, the ADP concentration falls by less than10% over the whole ofthe reaction curve. This doesnot appear to affect the evaluation of reactionvelocities since these curves are usually linear forthe first 3-5 min. after mixing, giving betweensix and ten points on which to calculate thevelocity.

Vol. 6I 121

122 I. T. OLIVER I955A disadvantage of the method is connected with

the activity of ATPase in tissue homogenates. Ifthe activity of this enzyme is high compared withthe ATP-forming enzyme, then the ATP cannot becoupled to the reduction of TPN. The extent towhich the activity of the 'coupling enzyme' can beraised is limited, and so successful competitionbetween the 'coupling enzyme' and the ATPasecannot always be arranged. If too much 'couplingenzyme' is added, then the reduction of TPN dueto an unidentified substance in the preparationmakes the optical density too high to be measuredwith the accuracy required for the determinationof reaction velocities. For this reason it has beenfound advantageous to reduce the ATPase activityof some tissues, e.g. brain (see also Oliver, 1954),liver, kidney and spleen. If the tissues are homo-genized in 0 1 M-KC1 and centrifuged at 19000g for15 min., then much of the ATPase is removed.The rates of reaction catalysed by 0 1-0 5 mg.

wet weight of muscle are usually measured inroutine work, while for other tissues ten times thisamount, i.e. about 5 mg., can be satisfactorilyassayed. The use of such a method for those inter-ested in studies where only small amounts of tissueare available can be appreciated.

SUMMARY

1. A rapid spectrophotometric method has beendeveloped for the assay of creatine phosphokinaseand myokinase in tissue extracts and homogenates.The formation ofATP by these enzymes is coupledto the reduction of TPN and the rate of reductiongives a measure of the activities.

2. The sensitivity of the method enables thedetermination of enzymic activities in tissue pre.parations diluted as much as one part in 20 000

corresponding to a quantity of tissue of about300 Ag. wet weight. Under these conditions re-actions due to endogenous substances are negligible.

3. The method has been applied to the measure-ment of creatine phosphokinase and myokinase invarious tissues of the rat.The author wishes to thank Professor H. A. Krebs,

F.R.S., for encouragement and advice, and Mr A. E.Nicholson for technical assistance.

REFERENCES

Askonas, B. A. (1951). Ph.D. Dissertation, CambridgeUniversity.

Banga, I. (1943). Stud. Inst. med. Chem. Univ. Szeged, 3,59.

Campbell, H. & Simpson, J. A. (1953). Chem. & Ind. p. 887.Chappell, J. B. & Perry, S. V. (1954). Biochem. J. 57, 421.Eggleston, L. V. & Hems, R. (1952). Biochem. J. 52, 156.Ennor, A. H. & Rosenberg, H. (1954). Biochem. J. 57, 295.Ennor, A. H. & Stocken, L. A. (1948). Biochem. J. 48, 190.Hanes, C. S. & Isherwood, F. A. (1949). Nature, Lond.,

164, 1107.Horecker, B. L.-& Kornberg, A. (1948). J. biol. Chem. 175,

385.Kalckar, H. M. (1943). J. biol. Chem. 148, 127.Kalckar, H. M. (1947). J. biol. Chem. 167, 467.Kornberg, A. (1950). J. biol. Chem. 182, 779.Krebs, H. A. & Hems, R. (1953). Biochim. biophys. Acta,

12, 172.Kuby, S. A., Noda, L. & Lardy, H. A. (1954). J. biol. Chem.

209, 191.LePage, G. A. (1949). In Manometric Techniques and

Tissue Metabolim. Minneapolis: Burges Publishing Co.Narayanaswami, A. (1952). Biochem. J. 52, 295.Oliver, I. T. (1954). Biochim. biophys. Acta, 14, 587.Seigmiller, J. E. & Horecker, B. L. (1951). J. biol. Chem.

192, 175.Slater, E. C. (1953). Biochem. J. 53, 157.Strehler, B. L. & Totter, J. R. (1952). Arch. Biochem.

Biophys. 40, 28.

Peroxidatic Activity of Catalase

BY H. LASER*Molteno Institute, Univer8ity of Cambridge

(Received 17 February 1955)

The peroxidatic function of catala8e consists in itsability to catalyse the oxidation by hydrogenperoxide of different substances, such as alcohols(Keilin & Hartree, 1936, 1945b), nitrite (Heppel &Porterfield, 1949), and formate (Chance, 1948).This property was first revealed in experiments(Keilin & Hartree, 1936) in which the peroxide was

produced as the result of a' primary enzymicreaction, as for example the oxidation of hypo-xanthine by xanthine oxidase. When this reactionwas allowed to take place in the presence of catalaseand ethanol, the latter underwent oxidation toaldehyde. Attempts to obtain peroxidatic oxidationof ethanol by preformed free H202 in presence ofcatalase were at first unsuccessful (Keilin &Hartree, 1936). Later on, however, very small

* Member of the Scientific Staff, Medical ResearchCouncil.