a rapid procedure for catalase determination in blood and tissue

A Rapid Procedure for Catalase Determination

in Blood and Tissue Samples with the

Van Slyke Manometric Apparatus

John Esben Kirk

A precise and rapid gasometric procedure is described for catalase determination inblood and tissue samples. The enzyme activity assay is performed with the Van Slykemanometric apparatus. In the developed technic, a 45-sec. reaction period is used,and a large liquid-gas interphase is provided with the purpose of preventing theoxygen diffusion from becoming a rate-limiting factor. Under the devised methodo-logical conditions, the rate of oxygen evolution closely follows a parabolic curvewhich permits the calculation of the initial activity velocity of the enzyme. Theoxygen evolvement is directly proportional to the quantity of catalase present in theanalyzed sample. Less than 4 mm. are required for each catalase measurement.

SEVERAL GASOMETRIC METHODS have been used for determination ofcatalase activity by direct measurement of the rate of oxygen evolve-ment resulting from the enzymic decomposition of hydrogen peroxide,

but during the last 40 years, no procedure has been described in whichthe Van Slyke manometric apparatus is employed for the assay ofthis enzyme. Because of the explosive character of the catalase reac-tion and the fast inactivation of the enzyme by the hydrogen peroxidesubstrate, a rapid method is required for reliable estimation of theinitial velocity rate.

A suitable fast method for gasometric catalase determination hasrecently been devised by Greenfield and Price (4); in that procedure,

a special apparatus is employed in which the evolved oxygen is meas-ured by a pressure transducer and a continuous recorder system. Inview of the simplicity and general availability of the Van Slyke mano-

From the Washington University School of Medicine, St. Louis, Mo.The investigation was supported by Grant PHS-891 from the National Institutes of Health,

U. S. Public Health Service.

763

764 KIRK Clinical Chemistry

metric apparatus, it was considered advisable to develop a new meth-od for catalase assay with the use of that equipment.

In the technic described in the present paper special consideration

has been given to the basic features of the catalase assay which haveproved advantageous in the Greenfield and Price method. The re-leased oxygen is measured manometrically after a very short reactiontime and conditions have been selected and arranged which provide alarge surface area for the assay sample in the Van Slyke extractionchamber, thus facilitating the diffusion of oxygen across the gas-liquidinterphase during the reaction period.

It was empirically observed by the author that the rate of oxygenevolvement under the conditions of the developed manometric proce-dure very closely followed a parabolic curve and was directly propor-tional to the amount of purified catalase and quantity of blood and tis-sue homogenate present in the sample. Since the findings were con-sistent and the results were highly reproducible, these observations

permitted the development of a fast and reliable method for determi-

nation of the catalase activity in blood and tissue samples.

Method

A conventional Van Slyke manometric apparatus with a gas extrac-tion chamber calibrated at 0.5, 2.0, 10.0, and 50.0 ml. is used in the cata-lase assay procedure. The general technic for handling the apparatusmust be employed in carrying out the enzyme activity test. It shouldbe pointed out that for special reasons which will be explained later inthis paper the catalase testisperformed with 3.0ml. of solution (S)

and that the pressure readings are made at a volume (a) of 7.0 ml.(Fig. 1).

Reagents

Hydrogen peroxide solution, 1.0 M Prepare by adding 2.9 ml. of30% H202 solution to 22.1 ml. of 0.01 M phosphate buffer, pH 6.9. ThepH of the commercial hydrogen peroxide stock solution should bechecked and its molarity verified by permanganate titration or bymeasurement of the optical density of diluted samples at 240 m.

Phosphate buffer solution, 0.01 M pH 6.9 Prepare according todescription made by Peters and Van Slyke (8).

Caprylic alcohol

7cc Volume

Vol. 9, No. 6, 1963 CATALASE DETERMINATION 765

Fig. 1. Extraction chamber

of Van Slyke manometric ap-

paratus for use in gasometric

catalase method. Gas is shown

at 7.0-mI. volume for pressurereading.

Surfoc.e Levelof Sompte

Level of -

Mercury Meniscu

Procedure

Transfer of Sample to Extraction Chamber

At the beginning of the test when the wash water is drawn off, thechamber fills with mercury. The chamber stop cock is closed, and thecock to the leveling bulb is left open. With a pipet, 0.9 ml. of phosphatebuffer is measured into the cup of the extraction chamber.

The sample to be analyzed (hemolysate of 1:100 aqueous dilution, ortissue homogenate of suitable concentration) is drawn into a 100-l.Linderstr#{216}m-Lang constriction pipet and is added to the phosphatebuffer in the cup. During the addition, the pipet is held in a verticalposition with its point placed under the surface of the buffer. The

transfer of the sample from the pipet is made slowly, the last part be-ing ejected with the use of a rubber bulb. To insure quantitative trans-fer, the Linderstr#{216}m-Lang pipet is rinsed by drawing phosphatebuffer from the cup to the 100-1.J. constriction mark with subsequentdelivery of the pipet content to the cup sample. The 1.0-ml. volume inthe cup is then introduced into the chamber without admitting any air.

Another 0.9 ml. of phosphate buffer is measured into the cup and

similarly run into the chamber. This second portion of phosphate


Table 1. FACTORS FOR CALCULATION OF AMOUNT (sL.) OF OXYGEN EVOLVED

Temp.#{176} Factor

15 8.845

16 8.811

17 8.777

18 8.743

19 8.710

20 8.677

21 8.644

22 8.611

23 8.57924 8.54725 8.516

26 8.484

27 8.453

28 8.423

29 8.392

30 8.360

31 8.33132 8.302

33 8.27134 8.242

35 8.213

Factors were calculated for conditions of S = 3.0 ml. and a = 7.0 ml.

gen evolved per second (Equation 1), micromoles of oxygen evolvedper second (Equation 2), or micromoles of hydrogen peroxide decom-posed per second (Equation 3):

(1)

(2)22.4 Vt

k = PXIX2 (

22.4 Vt

where P = observed pressure of oxygen evolved, expressed in milli-meters mercury and f = factor listed in Table 1.

The enzymic velocity values calculated from the above equationsshould subsequently be corrected for the temperature at which the testwas conducted to convert activities to those exhibited by catalase at25#{176}.This correction is made by use of the factors recorded in Table 2.

As an example, a diluted human blood sample equivalent to 1 /Ll. ofwhole undilutel blood after 45 see. of shaking at a temperature of 29#{176}produced an 88.5mm. increase in pressure (cOrrectedfor blank value).


Fig. 1. Extraction chamber

of Van Slyke manometric ap-paratus for use in gasometric

catalase method. Gas is shown C I

at 7.0-ml. volume for pressure OS 0icc Volume

reading.

Surface Levelof Sample

Level of

Mercury Mmiscus

Procedure

Transfer of Sample to Extraction Chamber

At the beginning of the test when the wash water is drawn off, thechamber fills with mercury. The chamber stop cock is closed, and thecock to the leveling bulb is left open. With a pipet, 0.9 ml. of phosphatebuffer is measured into the cup of the extraction chamber.

The sample to be analyzed (hemolysate of 1:100 aqueous dilution, ortissue homogenate of suitable concentration) is drawn into a 100-.J.Linderstr#{216}m-Lang constriction pipet and is added to the phosphatebuffer in the cup. During the addition, the pipet is held in a verticalposition with its point placed under the surface of the buffer. The

transfer of the sample from the pipet is made slowly, the last part be-ing ejected with the use of a rubber bulb. To insure quantitative trans-fer, the Linderstr#{216}m-Lang pipet is rinsed by drawing phosphatebuffer from the cup to the 100-pJ. constriction mark with subsequentdelivery of the pipet content to the cup sample. The 1.0-ml. volume inthe cup is then introduced into the chamber without admitting any air.

Another 0.9 ml. of phosphate buffer is measured into the cup andsimilarly run into the chamber. This second portion of phosphate


buffer serves to wash into the chamber residuals of the sample whichmay adhere to the bottom of the cup. With a medicine dropper, fourdrops of caprylic alcohol are then placed in the cup, and a sufficientpart of this reagent is admitted to the chamber to bring the mercurymeniscus to the 2.0-mi. mark. A few drops of mercury are then addedto the small amount of caprylic alcohol remaining in the cup, and withthe chamber stop cock closed, the sample is ready for receipt of thehydrogen peroxide reagent.

Addition of Hydrogen Peroxide Reagent

The addition of the hydrogen peroxide reagent to the sample in theextraction chamber is made with a 1.0-mi. Ostwald-Van Slyke pipetequipped with stop cock and a rubber tip. Between use, this pipet isconveniently kept standing in a tall, narrow beaker placed immediate-ly to the right of the Van Slyke apparatus.

The 1.OM 11202 reagent is drawn into the pipet which is then insertedinto the cup with the rubber tip pressed against the bottom. The pipetcock isopened, and ifheld in correctposition,there willbe no flowfromthe pipet. The admission of the 1.0 ml. of hydrogen peroxide solution

to the chamber is made by means of the chamber stop cock. Tmmediate-ly before the opening of that stop cock, a stop watch is started; if anelectrical stop watch operated by a foot switch is employed, the start-ing of the watch can be made at the same second the chamber stop cockis opened. When the 1.0 ml. of hydrogen peroxide reagent has been

transferred through the mercury seal into the extraction chamber, thechamber stop cock is closed, and the Ostwald-Van Slyke pipet is with-drawn and replaced in the beaker for storage. The bore of the chamberstop cock is then sealed with the mercury remaining in the cup. Be-tween 9 and 11 sec. are required for the delivery of the hydrogen perox-ide reagent to the chamber.

Reaction Period

By means of the leveling bulb, the mercury in the chamber is lowered

smoothly until the mercury has fallen to the 10.0-mi. mark. The stopcock leading to the leveling bulb is closed and is subsequently used for

adjustment of the mercury meniscus exactly to the 10.0-mi. mark (Fig.1). This adjustment must have been accomplished by the time a 20-sec. reading is registered on the stop watch. The initial pressure (p0)exerted by the sample is quickly read on the manometer and the shak-ing of the chamber is immediately started. The shaking is continued


for 45 sec. at a rate of 250 rpm (the shaking is started when the stopwatch shows a 20-sec. reading and is stopped at 65 sec.).

During the period of shaking, the cock is occasionally opened with asmooth gradual motion to admit mercury from the leveling bulb tokeep the mercury meniscus close to the 10.0-ml. mark. The retainment

of the mercury meniscus in this narrow part of the extraction chamberassures a small area of contact between the hydrogen peroxide-con-taining sample and the mercury, and provides a large surface area be-tween the sample and the gas phase in the chamber.

When the 45-sec. shaking period has been reached (when the watchshows the total time of 65 sec.), the shaking of the chamber is stopped,and the mercury meniscus is exactly adjusted to the 10.0-mi. mark(Fig. 1). The pressure reading (p,) is immediately made on the ma-

nometer and the temperature is recorded.The stop cock leading to the leveling bulb is opened and the sample

is removed from the chamber by suction. The cup and the extractionchamber are then washed twice with 10-15 ml. of distilled water.

A complete catalase test including the measuring of the sample andreagents into the pipets and the washing of the chamber can be made in

less than 4 mm.

Blank Value

A blank value (c) isdetermined by performing a testwith 1.9ml. of

phosphate buffer, 4 drops of caprylic alcohol, and 1.0 ml. of 1.0 lvi H202reagent. The blank value recorded for the 45-sec. test is usually about

6 mm. The mean time required for a blank determination is 31/4 mm.

Calculationof CatalaseActivity

The pressure of oxygen evolved in the 45-sec. reaction period is P =

p1 - p0 - c, where c is the pressure exerted by the blank.The P value is converted to microliters of oxygen (at 0#{176}and 760 mm.

Hg) by multiplication with the appropriate factor listed in Table 1.

Since the catalase reaction rate as recorded in the present manometricprocedure strictly follows a parabolic curve, the initial velocity con-

stant (k) is calculated on the basis of the following formula:

Vt

where q = quantity of oxygen evolved and t = time in seconds.

The initial reaction values may be expressed as microliters of oxy-


Table 1. FACTORS FOR CALCULATION OF AMOUNT (ILL.) OF OXYor..i EVOLVED

Temp.#{176} Factor

15 8.845

16 8.811

17 8.777

18 8.743

19 8.710

20 8.677

21 8.644

22 8.611

23 8.57924 8.54725 8.516

26 8.484

27 8.453

28 8.423

29 8.39230 8.360

31 8.331

32 8.30233 8.271

34 8.242

35 8.213

Factors were calculated for conditions of S = 3.0 ml. and a = 7.0 ml.

gen evolved per second (Equation 1), micromoles of oxygen evolvedper second (Equation 2), or micromoles of hydrogen peroxide decom-posed per second (Equation 3):

(1)Vt

(2)22.4 Vt

k = PXfX2

22.4 Vt

where P = observed pressure of oxygen evolved, expressed in milli-meters mercury and f = factor listed in Table 1.

The enzymic velocity values calculated from the above equationsshould subsequently be corrected for the temperature at which the testwas conducted to convert activities to those exhibited by catalase at25#{176}.This correction is made by use of the factors recorded in Table 2.

As an example, a diluted human blood sample equivalent to 1 l. ofwhole undilutel blood after 45 sec. of shaking at a temperature of 29#{176}

produced an 88.5 mm. increase in pressure (corrected for blank value).


Table 2. FACTORS FOR CONVERSION OF BLOOD CATALASE ACTIVITIES OBSERVED AT VARIOUS

TEMPEEATURES TO THOSE EXHIBITED AT 25#{176}

Temp.#{176} Factor

15 1.1374

16 1.1292

17 1.1200

18 1.1096

19 1.0980

20 1.083921 1.071122 1.054223 1.038924 1.019825 1.0000

26 0.9773

27 0.9558

28 0.9301

29 0.903530 0.8758

31 0.851332 0.8231

33 0.797134 0.7680

35 0.7404

According to Equation 1, this corresponds to:

88.5 X 8.392 742.7= = 110.7 pi. 02/sec. in initial reaction

v’45 6.708

The values, expressed as micromoles of oxygen evolved per second(Equation 2) and micromoles of hydrogen peroxide decomposed persecond (Equation 3) are, respectively, 4.94 and 9.88. A conversion ofthese figures to enzymic activity at 25#{176}(Table 2) yields values of 92.9l. of oxygen evolved per microliter undiluted blood per second; 4.15M of oxygen evolved per microliter undiluted blood per second; and8.30 p.MI of hydrogen peroxide decomposed per microliter undilutedblood per second.

If desired,the shaking reaction period may be changed from 45 sec.

(V45 = 6.708) to 30 sec. (v’3O 5.477) or 60 sec. (V60 = 7.746).

Cleaning the Chamber with Chromic-Sulfuric Acid Solution

When the manometric apparatus is used for catalase activity deter-

minations, it is occasionally necessary to clean the extraction chamber


with a chromic-sulfuric acid solution as described by Peters and VanSlyke (8) ; because of the importance of the pH of the reaction mixturein the catalase determination, the acidity of the final wash water shouldbe checked after this cleaning. After removal of the chromic-sulf uricacid solution, a satisfactory pH (between 6.3 and 7.0) was achieved

when the chamber was washed successively with three portions of wa-ter, one portion of 0.5 N NaOH, and three additional portions of water,

the volume of each washing solution being between 25 and 50 ml.

Experimental

Catalase Reaction Rate

For determination of the correlation between time of reaction andquantity of oxygen evolved,experiments were conducted with samplesof human hemolysates and liver homogenates, and with commercial

catalase preparations. The oxygen evolution was recorded after vari-

ous times of shaking of the chamber; the reaction periods chosen were

15,30, 45, 60, 90, and 120 sec. of shaking. Since it is not possible to makeaccurate readings on the manometer during the shaking of the extrac-tion chamber, the tests were made successively on equal aliquots of thesame catalase-containing samples. Duplicate analyses were per-formed in all the studies and usually showed very close agreement.

The constructed graphs regularly displayed a shape indicating a

parabolic curve. In order to prove the parabolic reaction rate, thequantities of oxygen evolved were plotted against /t. In all the ex-periments this plotting resulted in a straight line passing through theorigin (the origin representing the time at which the shaking was

started). These findings verify the parabolic nature of the catalasereaction rate under the present conditions of assay (3). At the varioustimes of reaction, a close correlation was found between the amount of

enzyme present and the recorded activity values; as an example, thevalues observed for a blood sample are presented in Fig. 2. Similarproportionality was encountered in assays of freshly prepared solu-tions of purified catalase samples (Fig. 3).

Relationship between Shaking Rate of Chamber and Catalase Reaction

A shaking rate of 250 rpm of the driving wheel was chosen for the

manometric catalase procedure because in the determinations of bloodand tissue samples, nearly identical activities were observed with 240,250, and 260 shakes per minute. Slightly lower values were recorded at210 rpm, and a markedly reduced rate of oxygen evolution was found

P (Pienure)

in mm.Hg. 200 Cu. mm.


without shaking of the chamber (15% of that observed with 250 shakes

per minute).At the selected 250 rpm, approximately similar catalase activity

values were noted for the 45-sec. shaking period when the shaking of

Fig. 2. Correlation betweentime of reaction (shaking pe-riod), volume of blood used, andquantity of oxygen evolved. The

manometric readings are ex-

pressed in mm. Hg corrected for

blank values. A 1:100 dilutedhuman blood sample was used in

test.

Fig. 3. Catalase activities ex

hibited by purified preparation

recorded at Various enzyme con-centrations under standard con-

ditions of manometric method

(100 ul. of solution employed Is

equal to 5.7 tug. of eatalase). Re-action time (shaking period), 45

see.

P1 Pressure)in mm. Hg.

100

0 00 200 300 400 500Cu. mm. Cotolose Solution

the chamber was started either 20 or 25 sec. after the initiation of thehydrogen peroxide addition to the sample. The choice of a 45-sec. shak-ing period, from 25 to 70 sec. instead of the described period startingat 20 sec. and discontinued at 65 sec., may be preferable to analysts whoare not routinely trained in the handling of the Van Slyke apparatus.

Influence of Hydrogen Peroxide Concentration on Catalase Reaction Rate

The catalase activities of blood and tissue samples were found to benearly independent of the 11202 concentration within the range of 0.625


to 1.25 M (final concentrations 0.208 to 0.417 lvi). Somewhat lowercatalase values were noted with the use of 0.3125 and 2.50 lvi 11202 re-

agent solutions.Under the technical conditions described in the present paper most

of the hydrogen peroxide substrate remains undecomposed during thetest even at the highest catalase reaction rates measurable by themanometric procedure. The evolution of oxygen exerting a 400-mm.Hg pressure is equivalent to a decomposition of 304 /.LM of hydrogenperoxide which represents only 30.4% of the 1000’M of 11202 (0.1 ml.

of 1.0 M 11200) added to the catalase-containing sample in the extrac-tion chamber.

Effect of Temperature on Catalase Activity Rate

A thorough investigation was made of the effect of temperature onhuman blood catalase activity as determined by the present mano-metric method. Five human blood samples were included in the study.The catalase activities of these samples were measured over the rangeof 15-35#{176}.The control of the reaction temperature was accomplishedthrough continuous circulation of water with a special constant tem-perature through the chamber jacket. Large needles were inserted

through the upper and lower rubber stoppers of the jacket, and rubbertubing was attached to the heads of the needles. The circulating waterwas admitted to the jacket through the lower needle.

When a change in the temperature was made, the extraction cham-ber was left filled with mercury for 20 mm. to assure a more uniformtemperature level in the apparatus. After each test, the sample tem-perature was immediately measured.

The phosphate buffer solution employed in the tests was maintainedin a water bath at the same temperature; hemolysate samples werekept in a refrigerater at 4#{176}between assays. The experiments werestarted at 35#{176},and the temperature was then gradually lowered to 15#{176}.In order to verify that no measurable degradation of the blood cata-lase had occurred during the long experimental period, the tempera-ture was finally raised to the original level of 35#{176}.Essentially similarvalues were recorded in the initial and final experiments conducted atthe same temperature.

The effect of the temperature on the blood catalase activity of thefive samples studied was found to be quite similar. Since the tempera-ture curve is not linear, the observed graph is presented in Fig. 4 andthe mean Q,and Q10values are listed in Table 3.

15 7 9 21 23 25 27 29Temperature(‘C)


% of Activityat 25C115

Ito

Fig. 4. Percentage values ofoxygen evolved (expressed in ml.at 0#{176},760 mm. Hg) by catalase 00

in human blood samples at vari-

ous temperatures, as compared 95

with activities recorded at 25#{176}.90

85

Table 3. Qo AND Qio VALUES FOR BLOOD CATALASE REACTION AS DETERMINED BY MANOMETRIC

PROCEDURE

Temp. rang #{128}0 SE. (.10 SE.

15-20 1.048 0.010

20-25 1.086 0.01025-30 1.143 0.01230-35 1.192 0.01115-25 1.139 0.025

20-30 1.240 0.021

25-35 . 1.345 0.022

Velocity Values Observed for Commercial Catalase Preparations

On the basis of the initial velocity rate determined with the mano-metric method, the turnover number for the enzyme in a purified cata-lase powder* prepared from beef liver was found to be 5,120,000. Thequantity of enzyme present in the catalase solution used in the test was

confirmed through measurement of the optical density of tile stock so-lution at 280 m in a spectrophotometert and by assay according to theprocedure of Beers and Sizer (1); the observed enzyme content of thebeef liver catalase powder agreed closely with that reported by theSigma Chemical Co. (Lot C41B-085). An essentially similar turnover

number of 5,180,000 was found for a catalase preparation obtainedfrom Worthington Biochemical Corp., Freehold, N. J. (Lot 5553-78).The high turnover number for purified catalase recorded with thepresent manometric technic indicates the validity of that procedure.

*Sigma Chemical Company, St. Louis, Mo.

I Model DU, Beckman Instruments Inc., Palo Alto, Calif.


Catalase Activities of Human Blood Specimens and Liver Tissue Homogenates

Catalase activity measurements were performed on 100 humanblood samples obtained from patients at St. Louis Chronic Hospital(average age of subjects, 75 years). Hematocrit and hemoglobin de-terminations were made by conventional methods. Hemolysates of thespecimens were prepared by adding 100 l. of each blood sample to 9.9in!. of redistilled water. The average initial velocity rate calculatedfrom the manometric catalase assays was 115 l. of oxygen evolved permicroliter undiluted blood per second (S.D. of dist., 35); this activity

rate is somewhat higher than those previously reported with gaso-metric technical procedures (2). The average difference between du-plicate measurements of the samples was 2.6%. When expressed permicroliters of red blood cells, the mean oxygen evolution was 286J./sec. (S.D. of dist., 89) ; the corresponding value per milligrams ofhemoglobin was 842l. (S.D. of dist., 265).

For determination of the catalase activity of liver tissue, 10 speci-mens were obtained fresh at autopsy, and 1.0% aqueous homogenateswere prepared with a Kontes Duall type tissue grinder. The mean

catalatic value observed for human liver tissue was 93 jJ. of oxygenevolved per milligram tissue per second. This activity rate is of thesame order of magnitude as that reported by Greenstein et al. (3) for

normal mouse liver.

Discussion

The factors responsible for the oxygen evolution at a rate corre-sponding to a parabolic curve are not known, but the experimentallyestablished consistency of this type of catalase reaction under the em-ployed technical conditions permits an accurate calculation of the ini-

tial activity velocity. Attempts will not be made to correlate this em-pirical finding with the available general information concerning theaspects of catalase kinetics.

It is of interest to note that a calculation of gasometric catalase datareported by Morgulis (7) and Maisin and Pourbaix (6) indicates a

tendency toward a parabolic curve; this was noted by plotting the pub-lished values of oxygen evolvement against the square root of reactiontimes. However, the graphs constructed on the basis of these previ-ously reported catalase activity measurements do not pass directly

through the zero point, possibly due to the fact that the technics usedby those authors differ in many respects from the procedure describedin thispaper.


In the present method, the use of a 3.0 ml. sample volume and themaintenance of the mercury meniscus at the 10.0-ml. mark were chosenwith the purpose of establishing a large liquid-gas interphase area

(Fig. 1). The surface of the sample in the Van Slyke extraction cham-ber is approximately 1.3 sq. cm./ml. of sample volume; this is an evenhigher ratio than that of 0.5 sq.cm./ml. of solutioncalculated on the

basis of the size and shape of the reaction flask used in the method byGreenfield and Price (4). The conditions are very satisfactory for

swirling the sample over the inside of the glass surface in both these

rapid gasometric technics.

References1. Beers, R. F., and Sizer, I. W., J. Biol. Chens. 195, 133 (1952).2. Deutsch, L., and Frank!, J., Ztschr. f. Krebsforsch. 40, 98 (1934).3. Feldman, W. M., Biom4jtheonatics, Griffin, London, 1923, p. 299.4. Greenfleld, H. E., and Price, V. E., J. Biol. Chein.. 209, 355 (1954).5. Greenstein, J. P., Jenrette, W. V., Mider, G. B., and Andervont, H. B., J. Nat. Cancer Inst.

2, 293 (1941-42).6. Maisin, J., and Pourbaix, Y., Compt. Rend. Soc. Biot. 129, 46 (1938).7. Morgu)is, S.,J. Biot. Chem. 47, 341 (1921).8. Peters, J. P., and Van Slyke, D. D., Quantitative CUnical Ohemistry, vol. 2, Williams and

Wilkins, Baltimore, 1932, pp. 236, 816.

a rapid procedure for catalase determination in blood and tissue

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