SOME FACTORS INFLUENCING THE ACTIVITY OF PEROXIDASE”

BY R. W. GETCHELL AND JAMES H. WALTON

(From the Laboratory of General Chemistry, University of Wisconsin, Madison)

(Received for publication, November 20, 1930)

Of the reactions in which catalysis is due to enzymes, those in- volving oxidation and reduction have received far less attention than have the hydrolytic type. The following investigation was undertaken in order to determine some of the factors which influ- ence the reactions of the peroxidases.

Preparation of the Enzyme

An important source and preparation method are reported from the Munich laboratories of WillsGtter (l-3) and his associates who have prepared a highly purified peroxidase from horseradish. The methods of Willstatter, with certain modifications, were em- ployed in the present investigation.

About 14 pounds of horseradish roots were cleaned, sliced thin, and self-dialyzed in running water for 8 days. The slices darkened and, in confirmation of WillstSter’s statement, those which de- veloped a deep brown color throughout the slice gave the best yields. After dialysis the slices were freed from water; the enzyme was “set” with oxalic acid; the solid was finely ground, and the enzyme was extracted with barium hydroxide. The enzyme solution was partially purified by treatment with carbon dioxide and with nine-tenbhs its volume of absolute alcohol and the solution rapidly concentrated below 35” in vacua from 1400 cc. to

* The experimental data in t.his paper are taken from a thesis submitted by R. W. Getchell in partial fulfilment of the requirements for the degree of Doctor of Philosophy at the University of Wisconsin, 1930..

This investigation was made possible by a grant from the University Research Fund, Dean Charles S. Slichter, Chairman.

419

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420 Peroxidase Activity

about 75 CC. The residual yellow liquid, after clarification in a centrifuge, was treated at 10” with 5 volumes of absolute alcohol, resulting in precipitation of the easily filtrable enzyme. It was further purified with dilute sulfuric acid, twice reprecipitated with absolute alcohol, and an aqueous stock solution prepared. This final solution was kept at a low temperature and largely diluted for use.

Composition of the EnLyme Material

The solid enzyme material was of a light cream color and con- tained 5.8 per cent ash. Its response was negative to the usual protein tests, except for a weak xanthoproteic reaction. The ash contained iron but gave a negative test for manganese by the Marshall persulfate method. A purpurogallin number of 205 was obtained when the enzyme was evaluated according to Willstgtter’s method (1).

Effect of Hydrogen Ion Concentration

For establishing the various hydrogen ion conditions, investiga- tors have used organic acids and their salts, mineral acids, am- monia, and the alkali hydroxides, carbonates, and phosphates. In a very extended study of malt amylase, Sherman (4) employed combinations of phosphoric acid, its sodium salts, sodium carbonate and sodium hydroxide. These substances were selected for the present study. Their solutions were prepared from the purest samples obtainable. For enzyme solutions, the stock sample was largely diluted to produce a concentration which would form a convenient amount of product. Resublimed pyrogallol was dis- solved in redistilled water to a concent,ration of 20 gm. per 100 cc. A fresh solution was prepared daily. Merck’s superoxol (30 per cent hydrogen peroxide) was distilled in zlacuo, analyzed, and diluted to a 2 per cent solution. For the color comparison solu- tion, pure purpurogallin was prepared by the method of Perkin and Steven (5). A standard solution of suita.ble color intensity was secured by employing a concentration in ether of 50 mg. per liter. Since it was not feasible to use an ether solution in actual measurements, the color was duplicated by a 0.034 per cent aqueous solution of potassium dichromat,e. The latter solution constituted the color standard. Pure, iron-free, 10 per cent sulfuric acid was

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R. W. Getchell and J. H. Walton

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421

FIG. 1. Chamber for peroxidase reaction in alkaline solutions. R rep- resents the reaction bottle; A and B, tubes for introducing nitrogen; T, Y-tube for connecting to nitrogen supply; P, 0, and N, screw clamps for closing system after disconnecting from nitrogen supply; E, capsule contain- ing enzyme; C, sliding wire support for capsule E; G! capsule containing pyrogallol; W, wire for lowering capsule G; H, hydrogen ion reagent in measured volume of water; K, capillary outlet to relieve pressure during nitrogen charging; and S, stopper supporting tube system.

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422 Peroxidase Activity

employed. The ether and diluting water were redistilled for use. For acid solutions, measurements were made in an atmosphere of air; for basic solutions, a nitrogen atmosphere was very success- fully employed.

TABLE I

Efect of Hydrogen Ion Concentration on Activit~~ of Perozidase

Hydrogen ion reagent hncentrstion

in reaction mixture

HaPOd. ............................ “ ............................. ‘I .............................

NaHsPOa. .........................

dd ml7.

0.00045 1.1 0.00030 1.1 0.00015 1.5 0.040 2.3

NaHzPOa. ......................... 0.012 HaPOd. ............................ 0.00008

NaH2P04 .......................... 0.006 HaPOd. ............................ 0.000065

NaHzPOa .......................... “ ..........................

None ............................ NanHPOa ..........................

0.0025 0.0005

0.001

NatHPOa. ......................... 0.005 Na2C03. ........................... 0.00024

NatHPOa. ......................... 0.005 Na2C03. ........................... 0.00084

NaOH ............................. Na2COa. ........................... NaOH. ............................

“ ............................. ‘I ............................. “ .............................

0.001 8.7 8.0 0.0016 8.4 8.3 0.0015 5.2 8.4 0.002 4.5 8.5 0.006 4.4 9.0 0.010 0.0 10.6

Purpurogallin PH

3.3 3.4 3.7 4.4

2.5 4.4

3.4 4.4

3.6 4.8 3.7 5.1

5.5 6.0 8.2 7.3

8.1 7.5

9.0 7.8

All reactants were measured directly from the ice bath. Into the measuring flask were introduced 5 cc. of pyrogallol solution, 1 cc. of hydrogen peroxide, and the desired volume of hydrogen ion reagent; the whole was diluted with water to 99 cc. After

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R. W. Getchell and J. H. Walton 423

thorough mixing the solution was cooled to 0” in the reaction flask and 1 cc. of the enzyme was added in a small capsule. The reac- tion was allowed to proceed for exactly 12 minutes, then arrested by the addition of 5 cc. of sulfuric acid. The purpurogallin was next extracted in a separatory funnel with four portions of ether and the combined extracts made up to 50 cc. A sample of this extract was calorimetrically compared with the standard in a series of ten readings per determinat.ion.

For reactions in a basic solution, the above process was modified slightly (Fig. 1). The hydrogen ion reagent, with the measured volume of water (H), was introduced into the 500 cc. wide mouth reaction bottle (R), which was fitted with a 4-hole rubber stopper (8). The enzyme solution was placed in the capsule (E), sup- ported in a loop of light copper wire attached to a heavy wire (C) which could be raised and lowered through an air-tight rubber collar in the stopper. The pyrogallol was contained in a larger capsule (G). The system was charged with purified nitrogen through the Y-tube T and tubes A and B, the pressure being relieved through capillary K. After the oxygen was entirely swept out, the apparatus was disconnected, the contents of the capsule (G) were emptied by raising tube B, the system was cooled to 0”, and the enzyme was finally introduced by lowering capsule E. After the reaction had proceeded for exactly 12 minutes, sulfuric acid was introduced through tube B and the product was extracted with ether and measured as described before.

As a met,hod of determining hydrogen ion concentrations, so- lutions were prepared duplicat,ing those mentioned above but wit,hout the 1 cc. of enzyme solution. The presence of the enzyme, wit,h the resulting oxidation of pyrogallol, produced constantly changing values. It was determined, however, in the more strongly acid solutions where enzyme activity is low, that the presence of the purified enzyme solution did not appreciably modify the hydrogen ion concentration. The quinhydrone elec- trode was used for the acid region and the hydrogen electrode for the basic solutions.

In Table I the activity of the peroxidase is expressed in terms of mg. of purpurogallin formed for each concentration of hydrogen ions. On account, of the catalysis of hydrogen peroxide by bases, it was necessary to run blank determinations on the basic

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424 Peroxidase Activity

solutions in order to measure t.he amount of purpurogallin so produced. Such corrections are included in the data of Table I. Blank determinations on the acid mixtures gave negative results. The phosphate ions are not without influence on the reaction rate, but their effect is minor. This is borne out by the work of Smir- now (6) who observed that phosphates exerted but little influence on crude peroxidase of wheat. No hydrogen ion reagent could be selected for these measurements which would not produce some effect of its own. It would not be permissible, however, to assume that the effects of the enzyme and reagent are additive and correct the data accordingly. The 12 minute reaction time was selected as best under the conditions of experimentation. A shorter period introduced too high a percent,age of error and a much

TABLE II

Effect of Enzyme Concentration on Yield of Product

pH 4.8.

Enzyme concentration I

Purpurogallin formed

mg. m!?.

0.14 0.2 0.28 1.4 0.42 2.5 0.57 3.1 0.85 6.7 1.70 13.2

longer time unnecessarily prolonged the measurements. In most of the reaction mixtures a typical yellow color was developed during the 12 minute period, but varying in shade with the reaction of the solution. The ether extract was yellow and the aqueous extraction residue was colorless or pale pink. 65 cc. of ether, added in four portions as described, proved to be the most efficient practice for the complete extraction of the purpurogallin. Nearly 20 cc. were miscible with the water, leaving the total volume of the extracts a little less than 50 cc. Duplicate determinations usually gave excellent checks, the calorimeter average readings varying by not more than ho.2 mm. With a view to extending the length of the reading day, various illuminating devices were tested with the calorimeter. In no case would artificial lighting afford

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R. W. Getchell and J. H. Walton 425

the reading accuracy attainable with north sky light. The point of inactivity in the basic region is, by extrapolation on a curve, too far into the basic field. Inasmuch as this relatively high concen- tration of base resulted in the formation of other colored products, an accurate determinat,ion of the exact point of inactivation was not, possible.

Effect of Concentration of the Enzyme

Although enzymatic concentration has no effect on the final reaction equilibrium, it does influence reaction velocities. In order to study this factor in relation to peroxidase, various concen- irations of the enzyme were evaluated by the method already described, all other factors being maintained constant. The result,s are given in Table II. Since we possess no generally accept’ed explanation of the mechanics of enzymatic oxidations, a discussion of the effect of the concentration of a given enzyme can do no more than establish t,he velocity values for the system under consideration. An explanation advanced by Willstatter and Weber (7) assumes, for peroxidase, the formation of two addition compounds, via. RHO. OH and RH, 0: 0, the one active and the other inactive. At any rate, preponderance of opinion seems to favor the view that the substrate is oxidized by activated oxygen formed through union of t,he enzyme wit,h the peroxide.

E$ect of Concentration of Substrate

This relationship has been most widely studied in connec- tion with the hydrolysis of sucrose by the invertase of yeast. For that enzyme Nelson and Schubert (8) report a maximum yield at a 5 per cent subst,rate concentration. For the dsta of peroxidase recorded in this paper, concentrations of pyrogallol were employed ranging from 0.2 to 5 gm. per 100 cc. The enzyme was added to the solutions containing this subst#rate and hydrogen peroxide, and evaluated as usual. In Fig. 2 are shown graphs of the results.

The decreased activity at low concentrations is probably a mass action effect. It is not due to lack of sufficient pyrogallol to pro- duce maximum yield. The falling off of the curve from the point of maximum activity can best be explained as a reduction in t,he active surface of the enzyme, brought about by the formation of an

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Peroxidase Activity

addition compound with the large excess of the pyrogallol. The hydrogen ion concentrat’ion of all of the solutions was so nearly constant (pH 6.1 A= 0.1) that this factor played very little part in the variations of enzymatic activity. The concentration for maximum activity is that which was employed throughout this research.

FIG. 2. Effect of concentration of substrate on activity of peroxidase

E$ect of Salts

The presence of salts in all enzymatic processes of the animal organism renders the st)udy of their influence of especial interest. Solutions of pure salts, whose concentrations in each reaction mixt.ure are indicated in the data, were added to the pyrogallol- hydrogen peroxide mixture; the enzyme was introduced and the reaction followed as described before. For the first series, several concentrations of each salt, were used. Blanks were run on each sample and if the salt alone was thus shown to catalyze the rcac-

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R. W. Getchell and J. H. Walton 427

tion, its effect is indicated in Table III under “Activity of salt alone.” When the enzyme was used with a catalytically active salt, it could not be assumed that their combined effect was addi-

TABLE III

Effect of Different Concentrations of Various Salts on Activity of Peroxidase -

Salt

Ferric chloride

Manganese sulfate

Copper sulfate

Mercuric chloride

Sodium chloride

Sodium tungstate

Uranium chloride

Thorium nitrate

Potassium cyanide

hncentration in reaction

mixture

M

0.0000001

0.0000005 0.000001

0.0001 0.001

0.00001 0.0001 0.0005

0.0000001 0.000001 0.00001 0.0001

0.0001 0.001 0.01

0.000001 0.0001

0.000001 0.0001 0.0003

0.0000001 0.000001 0.0001

0.0000001 0.000001 0.00001 0.0001

Activity of salt alone

per cent

0 2.6 3.4

Activity ‘f emw~ and

per cent

100

95 90

0 86 0 74

0 100 2.5 75 5.0 63

0 0 3.3

100 99 68 10

0 92 0 84 0 57

0 Very high

0 0 0

0 0 0

0 0 0 0

89

83 11 0

95 54 0

99 66

2 2

PH

4.8

5.9

4.8

4.9

6.0

5.9

5.7

5.2

6.1 6.8 8.5

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428 Peroxidase Activity

tive. Hence the data for their effect, recorded under “Activity of enzyme and salt,” are not corrected for the influence of the salt alone. In Table III and subsequent tables the term “percentage activity” is used to indicate the catalytic power of the enzyme under t,he designated experimental conditions. The actual weight of the purpurogallin produced could not serve as a basis of corn- parison because the activity of the enzyme did not remain constant throughout the period of the investigation. The percentage activity was computed by dividing the weight of purpurogallin formed under the modified, designated conditions of each experi- ment by the weight formed by the enzyme alone in the absence of such dist’urbing factors (Table III).

A few comments on Table III are pertinent. In a solution of pH 6, with a 0.000001 M concentration of ferric chloride, the salt alone showed no catalyzing effect, but the percentage activit,y of the enzyme in i,ts presence dropped t,o 79.5 per cent. When 68 mg. of the enzyme were mixed with 0.00001 mol of ferric chloride in a volume of 8 cc. and allowed to stand for 30 minutes, the salt was shown to be completely adsorbed from the solution and the activity of the enzyme was reduced 6 per cent’. When measurements were made with a copper sulfate concentration of 0.0001 M but at a pH 5.9, the percentage activity of the enzyme dropped from 75.0 to 22.9. In anot,her study on the influence of copper, samples of the enzyme were mixed with the copper sulfate solution of 0.0001 M concent’ration 5 hours and 12 hours before being subjected to measurement. The results were as follows:

Treatment Percentage activity

Mixed and measured at once. . . . . . . . 75.0 After 5 11~3.. . . . . . . . 71.7

“ 12 ‘( . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37.8

When the 0.0005 M concentration stood in contact with the enzyme for 12 hours, the percentage activity dropped’ from 63 to 27.0. Hence the toxicity of copper sulfate increases with the concentra- tion of the salt, with decreasing acidity of the solution, and with increasing time of preliminary contact of enzyme and salt. Con- t,act of the uranium salt with hydrogen peroxide produced a grad- ual separation of a peruranate (9), hence the effective mass of uranium ions was exceedingly small. This may account for the apparently greater inactivating effects of thorium.

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R. W. Getchell and J. H. Walton

Effect of Anions

429

Soluble fluorides are harmful to ma,ny enzymes. When applied to peroxidase, 0.0001 mol of sodium,fluoride, at pH 6, reduced the percentage activity to 27. In the case of potassium cyanide the poisonous action was even more marked (Table III). Its pH value of 6.8 would, of itself, correspond to high activity, yet the potassium cyanide actually produced almost complete inactivation. The sulfide anion proved highly toxic. When 1 cc. of the enzyme solution was saturated with purified hydrogen sulfide and the latter subsequently removed by aeration, the percentage act.ivit,y wa,s

TABLE IV

E.#ccl of Single Concentrations of Various Salts on Activity of Pwoxidase

Salt Concentration in reaction mixture

Lead nitrate Cobalt chloride Nickel “ Chromous “ Aluminum sulfate Barium chloride Calcium “ Magnesium sulfate Sodium chlorate

“ nitrate ‘I sulfate

Potassium chloride

,M

0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.001 0.0001

Net activity PI-I

per cent 60 5.6 67 6.0 74 6.1 84 4.6 88 4.3 74 6.2 80 6.2 82 6.2 93 6.1 97 6.2 97 6.2 88 6.2 98 6.2

only 16. The enzyme was completely inactivated by treatment with 0.00001 mol of potassium sulfide at pH 7.5. Treatment of the enzyme with the platinum poisons, carbon monoxide and arsine, for 10 minutes reduced the percentage act,ivity to 91.5 and 86.2, respectively.

For the second series of salt studies, sahs were selected which proved to be without catalytic effect upon a hydrogen peroxide- pyrogallol mixture at the concentrations employed. Hence their in- fluence upon the catalytic action of the peroxidase would be due to the specific action of the salt itself and to t,he hydrogen ion concen- t,ration which it established. These two influences may not be

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430 Peroxidase Ae tivity

purely additive but it appeared t,hat a closer evaluation of the spe- cific salt effect might be arrived at by considering t.hem as additive. The pH of the solutions containing the neutral salts was 6.2 and in all other cases less than 6.2. Hence the purpurogallin yield at pH 6.2 was taken as the standard, and in the “Net activity” of the enzyme in the presence of the salt (Table IV), correction has been made for any reduction in the activity of the enzyme due to a pH of less than 6.2.

E$ect of Various Organic Compounds

Absolute ethyl alcohol was mixed in varying proportions with the reaction mixture, producing inactivations proportional to its concentration (Table V). It is considered that inhibition by

TABLE 1’

ISfeet oj’ Alcoh~ol on Activity of Peroxidase

Alcohol ratio

5:95 20:80 30:70 50:50

Activity

pel cent

95 76 42 0.5

alcohol is due both to a reduction in the degree of dispersion of the enzyme and to its actual destruction.

The glucoside of horseradish is sinigrin, not obtainable on the market. Peroxidase was unaffected by five other glucosides: amygdalin (0.00001 mol), and digitalin, saponin, salicin, and strophanthin (each 0.00005 mol).

The susceptibility of the enzyme to alkaloids was examined. It was not affected by caffein (21 mg.), atropine (69 mg.), cinchonidine sulfate (48 mg.), quinidine sulfate (8 mg.), strychnine sulfate (17 mg.), and quinine bisulfate (55 mg.). Allowance being made for its hydrogen ion concentration, nicotine (16 mg. or 0.0001 mol) produced an accelerat,ion of 44 per cent. Unless this alkaloid is specific, some undetermined factor must be operative.

A number of organic compounds were selected for st,udy because of their wide distribution in cell contents and plant structure. Glucose (90 mg.), sucrose (34 mg.), and soluble starch (50 mg.)

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R. W. Getchell and J. H. Walton 431

produced no effect. Purified gelatin (32 mg.) and egg albumin (32 mg.) had no pronounced influence but their emulsifying action prevented quantitative evaluation. With allowance for hydrogen ion effects, dextrin (32 mg.) reduced the activity 33 per cent and tannic acid (32 mg.) 55 per cent. Reduction of the active surface of the enzyme by adsorption on the colloidal dextrins could account for their inhibit.ing influence. Since tannic acid is a strong reducing agent it, as well as the pyrogallol, may be an oxygen acceptor in the enzyme catalysis, thus reducing the amount of oxidation product of the latter. The convenient compound formation theory offers another explanation for the inhibiting effect of tannic acid.

E$ect of Physical Factors

Irradiation-l cc. of enzyme, both alone and when contained in the diluting water, was exposed in quartz for 10 minutes to ultra- violet light. The volume of water reduced the destructive action of the rays.

Treatment Percentage activity

Irradiation in 94cc. water.................................. 13.5 “ “ 1 “ “ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0

It is generally observed that ultra-violet light is more potent in enzyme destruction than x-rays, radium, or visible light.

Aeration-The method of aeration consisted of bubbling the gas from the tip of a very fine capillary tube through 1 cc. of the enzyme contained in the capsule. The latter was then introduced into the reaction mixture. In one series filtered air was used, in another, pure oxygen, in a third, carbon dioxide followed by pure oxygen to sweep out the former. In each case the percentage activity was reduced to about 85. An extension of pure oxygen aeration from the 10 minute period to 20 minutes did not modify the value. This effect may be due to the oxidation of some component of the enzyme system or to a reduction in the degree of dispersion of the colloidal enzyme similar to the flocculating effect of gases upon ferric oxide sols, as observed by Stark (10).

Dialysis-The purpose of this study was (a) to examine the effect of membranes upon the enzymatic activity and (b) to determine whether the poisoning effect of heavy metal salts, e.g. mercuric chloride, is exerted directly upon the enzyme. For the first study,

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432 Peroxidase Activity

small dialysis sacs of collodion, of parchment, and of viscose were prepared, thoroughly washed, and charged with 1 cc. of the enzyme solution. After dialysis in running distilled water they were rinsed into the reaction capsule and evaluated. The data in Table VI indicate reduced activity with each membrane, increasing with time of contact,. Supplementary determinations also es,tab- lished the following fatits: (1) no enzyme passed through the membranes; (2) percentages of recovery of the enzyme in the transfer process alone were for collodion 100, viscose 100, parch- ment 96.5. Adsorption of the enzyme by the colloidal membranes, thus reducing its active surface, explains the inactivation effects.

In the second study, mixtures of 1 cc. of enzyme and 1 cc. of mercuric chloride (2.7 mg.) were dialyzed in collodion and exam-

TABLE VI

Effect of Dialysis Membranes on Activity of Peroxidase

Collodion Parchment

Viscose

Dialysis

1 day 30 min.

4 hrs. 2 days

30 min. 3 hrs. 2 days

Activity

per cent

39 67 55

7 a7 75 36

ined at the end of 1 and of 2 days. The percentage activity values were at the end of 1 day 20.0, at the end of 2 days 7.7. Other samples so dialyzed showed no mercury ions in the mixt,ure at the end of the 1st day. Reference to Table VI will show that-the mercuric chloride inactivation is higher than the membrane alone could accomplish. Hence the toxic action of mercuric chloridelis, in part at least, exerted directly upon the enzyme.

Temperature Coeficient-Measurements were conducted as usual, with thermostat-controlled baths. Since it was not con- venient to remove samples for test at frequent intervals, the quan- tities of purpurogallin formed in 12 minutes at the different tem- peratures were taken as a measure of the enzymatic activity or velocity. In the 12 minute period the weight of product formed

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R. W. Getchell and J. H. Walton

at 10” was 140 per cent of that formed at O”, and at 20’ the product was 120 per cent of that formed at 10”. Hence the temperat,ure coefficient. was 1.4 in the O-10” range and in the 10-20” interval it was 1.2. A correction for heat decomposition effects upon hydro- gen peroxide is incorporated in the 20” value. At higher tempera- tures, 50”, 40”; and even 30”, thermal decomposition of hydrogen peroxide and of the enzyme rendered quantitative data undepend- able.

SUMMARY

1. Modifications in the methods of preparing and evaluating peroxidase have been developed.

2. The effects on the activity of peroxidase of varying concen- trations of hydrogen ion, substrate, and enzyme have been measured.

3. The activity of peroxidase has been evaluated after subject- ing it to irradiation, to aeration, to different react,ion temperatures, and to several dialysis membranes.

4. Data are reported for the catalytic activity of peroxidase in the presence of a wide variety of inorganic and organic compounds.

BIBLIOGRAPHY

1. Will&litter, R., and Stoll, A., Ann. Chem., 416,21 (1918). 2. Willstiitter, R., Ann. Chem., 422, 47 (1921). 3. WillstLtter, R., and Pollinger, A., Ann. Chem., 430,269 (1923). 4. Sherman, H. C.; Thomas, A. W., and Baldwin, M. E., J. Am. Chem. Sot.,

41, 231 (1919). 5. Perkin, A. G., and Steven, A. B., J. Chem. Sot., 83,192 (1903). 6. Smirnow, A. J., Biochem. Z., 155,l (1925). 7. Willst&tter, R., and Weber, H., Ann. Chem., 449,156 (1926). 8. Nelson, J. M., and Schubert, M. P., J. Am. Chem. Sot., 60,2188 (1928). 9. Price, T. S., Peracids and their salts, London, 107 (1912).

10. Stark, H. M., J. Am. Chem. Sot., 62,273O (1930).

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R. W. Getchell and James H. WaltonACTIVITY OF PEROXIDASE

SOME FACTORS INFLUENCING THE

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