FACTORS AFFECTING THE ACTIVITY OF CARBONIC ANHYDRASE

BY MANFRED KIESE AND A. BAIRD HASTINGS

(From the Department of Biological Chemistry, Harvard Medical School, Boston)

(Received for publication, October 14, 1939)

The activity of several hydrolytic enzymes, e.g. papain, urease, phosphatase, can be altered by reducing or oxidizing substances. Some observations suggest that the effects of oxidizing and reducing agents on the hydrolytic and synthetic activities of the enzymes are different. For example, Waldschmidt-Leitz, Scharikova, and Schaffner (1933) report that the hydrolysis of glycerophosphate by kidney phosphatase is inhibited by sulfide and by cysteine, although these substances do not change the synthetic activity of the enzyme. Kayashima (1938) reports that the hydrolytic activity of liver esterase is increased by reduc- tion and decreased by oxidation, while the synthetic activity is increased by oxidation and decreased by reduction. Lens protein and succinic dehydrogenase were used to reduce the enzyme and aeration in the presence of copper sulfate to oxidize it. These observations are not consistent with the classical definition of a catalyst.

In undertaking a study of the modification of the lytic and synthetic properties of an enzyme, it seemed desirable to choose one in which both properties could be studied with the same degree of precision. Carbonic anhydrase meets these require- ments admirably because both the hydration of CO2 and the dehydration of HzC03 can be accurately measured.

The experiments to be presented herein are concerned with (1) the further purification of the enzyme, carbonic anhydrase, (2) the effect of various oxidizing and reducing agents on the enzyme, (3) the effect of pH, and (4) the reexamination of the effect of other substances reported to be inhibiting agents, such as CO, cyanide, and sulfide (Meldrum and Roughton, 1934).

281

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282 Activity of Carbonic Anhydrase

Since the papers of Meldrum and Roughton (1934) and Stadie and O’Brien (1933) deal extensively with the properties of car- bonic anhydrase, they will not be reviewed again here.

Measurement of Activity of Enzyme-The activity of the enzyme was measured by the effect on the rate of hydration of CO2 and dehydration of H&Z03 in 0.2 M phosphate buffer solution, pH 7.0. Hydration and dehydration rates were measured manometrically with an apparatus which is described in the preceding paper (Kiese and Hastings, 1940). Initial CO2 pressures from 100 to 250 mm. of Hg were used. Hydration and dehydration were usually studied consecutively on the same solution. After the hydration had come to equilibrium, the shaking of the reaction vessel was st.opped, the gas space rapidly evacuated, and after the shaker was started again, the rate of dehydration was meas- ured. In many experiments, the rate of dehydration was not separately measured, but only the rate of hydration and the equilibrium pressure. From these data, any effect upon the rate of dehydration could be detected. Since the amount and concentration of buffer and the initial CO2 pressure were kept constant, the equilibrium pressure depended upon the ratio of the velocity of the hydration and dehydration. Therefore, if a catalyst increased the speed of hydration and the reaction came to the same equilibrium as the uncatalyzed reaction, the dehydra- t.ion must have been influenced by the catalyst to the same extent as the hydration.

The concentration of the enzyme which increased the reaction rate by 100 per cent was chosen as the measure of catalytic ac- tivity. The half time to the completion of the reaction was usually used as the measure of the reaction rate. The procedure followed is apparent from inspection of the curves given in Figs. 2, 3, and 5. The equilibrium pressure corresponding to zero time was found by extrapolation. All experiments were carried out at 5.0°, and were repeated two or more times.

Preparation of Carbonic Anhydrase

The enzyme was prepared from washed beef erythrocytes. In the first step, the removal of hemoglobin from the solution of hemolyzed cells, the procedure of Meldrum and Roughton and of Stadie and O’Brien was followed. To the washed erythro-

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M. Kiese and A. B. Hastings 283

cytes were added water, alcohol, and chloroform, each in the amounts equal to half the erythrocyte volume. The mixture was shaken for some minutes, and centrifuged after several h The supernatant fluid containing the enzyme was then dia against running water to remove the alcohol and chloroform. A repeated fractional precipitation of the solution with am- monium sulfate was then carried out. From a 1 per cent solution of the crude material, the highest concentration of enzyme was precipitated between 70 and 85 per cent saturation with am- monium sulfate. The final fraction was electrodialyzed. The whole procedure was carried out at 5”. The enzyme prepared in this way will be referred to as Preparation A. 0.35 y of this preparation in 10 ml. of phosphate, pH 7.35, increased the speed of hydration by 100 per cent. The activity of this enzyme prepa- ration may be compared with that of Meldrum and Roughton. Their preparation doubled the dehydration rate, at 15’, in a con- centration (by weight) of 1:7,000,000 while our Preparation A had Ohe same activity at 5” in a concentration of 1: 30,000,OOO.

The enzyme was purified further by a procedure which we shall call fractional denaturation. The enzyme was dissolved in a slightly acid buffer and sodium bicarbonate added. A vigorous foaming ensued which denatured and precipitated protein. If the amount of bicarbonate was too large, practically all the protein in solution was denatured, including the enzyme. But if the foaming was not excessive and the protein precipitation incom- plete, the enzyme remaining in solution was in a relatively purer state. The optimal amount of bicarbonate had to be determined by preliminary experiment. The enzyme so obtained was still slightly yellowish in color, but less so than before treatment. The colored substance could be adsorbed on an old aluminum hydroxide B (WillstSitter and Kraut, 1924) without loss of enzyme. Continuing these two procedures, fractional denaturation and adsorption, gave a colorless enzyme preparation whose purity was increased 3 to 5 times, compared with that of Preparation A.

Some Chemical Properties of the Enzyme

The enzyme lost very little activity if kept in 0.5 per cent solu- tion at 3” for several weeks. However, in 0.001 per cent solution at 3”, the activity decreased 50 per cent in 3 to 6 days.

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Activity of Carbonic Anhydrase

The iron content of several preparations was estimated by the method of Willstatter (1920). After a single ammonium sulfate precipitation, the enzyme preparations contained 0.02 to 0.05 per cent iron. With repeated fractional precipitation with am- monium sulfate, the iron content decreased. Preparation A con- tained 0.008 per cent iron, Further purification did not further decrease the concentration of iron.

Since this work was completed, Keilin and Mann (1939) have reported the important observation that the activity of their carbonic anhydrase preparations is paralleled by the zinc content of the preparations, their best material containing 0.31 to 0.34 per cent zinc. Iron, copper, manganese, and magnesium were absent from their purest preparations. Unfortunately, no ob- servations were made on the zinc content of our preparations.

No phosphorus was detected in our enzyme preparations. In view of the error of the method employed and the amount of material used, this would indicate that the phosphorus content was less than 0.01 per cent.

The mobility of the enzyme in the Tiselius cataphoresis ap- paratus (1937) has been determined in several experiments.’ (Purification of the enzyme by this procedure has, so far, been unsuccessful.) The enzyme preparation was dissolved in acetate buffer at 0.1 ionic strength, concentration approximately 1 per cent. In solutions of pH 3.7, 4.3, 4.5, and 4.7, only one strong boundary was found to migrate symmetrically to the cathode. The enzyme activity always accompanied this boundary. In a solution of pH 5.3, the protein solution no longer appeared to be homogeneous. One boundary appeared to move to the cathode and another one to the anode. Both fractions contained active enzyme material. From the speed of migration of the protein in solutions of varying pH, the isoelectric point of the enzyme in acetate buffer was estimated to be approximately pH 5.

E$ect of Oxidation and Reduction

It was found that a number of oxidizing agents inhibit the activity of carbonic anhydrase. In Table I, the concentrations

1 The experiments were carried out in the Department of Physical Chemistry, Harvard Medical School, through the courtesy of Dr. Edwin J. Cohn and Dr. Ronald M. Ferry.

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M. Kiese and A. B. Hastings 285

of the oxidizing agents which inhibited activity of the enzyme by 50 per cent are given. Some relation between oxidizing in- tensity and effectiveness as inhibitors is evident. Perchlorate and periodate, which are stronger oxidizing agents than chlorate, were stronger inhibitors. Chlorate was stronger than bromate,

TABLE I

Inhibition of Carbonic Anhydrase by Different Oxidizing Agents

The enzyme was dissolved in 0.2 M phosphate, pH 7.0.

Substance added Concentration which inhibited ensyme activity 50 per cent

Permanganate Iodine Periodate. Perchlorate. . Chlorate....... Persulfate. Bromate

5 x IO-6 5 x 10-S 1 x 10-S 2 x 10-a 4.5 x 10-a I x 10-Z 2 x 10-Z

100.

90.

z 00.

t z 70.

: 5 60.

“z 50. z d 40.

2 30.

20.

IO. i

1 i 4 b A io ia 14 i6 I'0 2'0 2'2 24 is Zb

CONCENTRRTION OF IODlNE-mO16w10's/liter

6’1o. 1. The inhibition of carbonic anhydrase by iodine. Enzyme Preparation A in 0.2 M phosphate buffer, pH 7.0.

and iodate, 0.02 mole per liter, did not show any inhibitory effect. The inhibition produced by different concentrations of iodine, one of the strongest inhibitors, is shown in Fig. 1. In all experi- ments, a concentration of enzyme Preparation A, which, in the absence of inhibitors, increased the rate of hydration 4 times, was used. Control experiments with oxidizing agents but without

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Activity of Carbonic Anhydrase

enzyme had no influence upon the reaction. In all cases, the inhibition was found to be reversible by certain reducing agents when the contact between the oxidizing agent and the enzyme did not exceed 10 minutes. Reducing agents such as ascorbic acid, cysteine, or hydroquinone, restored the activity of the enzyme completely after inhibition by the oxidizing agent. When ascorbic acid was used, pH change was avoided by addition of an equivalent amount of alkali. The pH was checked with the glass electrode. In the concentrations used, ascorbic acid did not, of it,self, affect the hydration of CO2 to a measurable extent.

In Fig. 2, a typical example of the reversible inhibition by iodine is given. The curves of comparable experiments with per- manganate are essentially the same as those presented for iodine.

Several experiments were carried out with each oxidizing and reducing agent studied. The results of all comparable experi- ments were identical. The activity of the enzyme could be only partially restored after 2 hours contact with KMn04. With iodine, however, the inactivation was completely reversible even after 4 hours. Potassium ferricyanide, 0.01 M, did not inhibit the enzyme; nor did porphyrindin, a dye with a rat.her high oxidation-reduction potential (E’o = +0.57 volt at pH 7.0) (Kuhn and Desnuelle, 1938), inhibit the enzyme in a concentra- tion of 0.002 mole per liter.

Unfortunately, the effect of oxygen and hydrogen with Pt or I’d as catalyst on the activity of the enzyme could not be studied, as was done with phosphatase (Kiese and Hastings, 1938). Car- bonic anhydrase was completely inactive in the presence of platinized or palladium asbestos, or colloidal palladium. Ap- parently, the enzyme was adsorbed on the catalysts, since filtrat’es of solutions to which these catalysts were added were inactive.

From the data in Fig. 2, it can be seen that in both the active and inactive state, the influence of the enzyme on hydration and on dehydration was the same. This was also true for incomplete inhibition of the enzyme by different concentrations of oxidizing agent, as illustrated in Fig. 3, when the enzyme was inhibited by different concentrations of permanganate. The hydration rate and the equilibrium pressure were measured. Within the limits of accuracy of the method, the same equilibrium was reached in the catalyzed and uncatalyzed reaction. It may also be men-

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M. Kiese and A. B. Hastings 287

HYDRF4TION

*-

0

k 60.

20.

f

DEHYDRRTION

10

FIG. 2. Reversibility of iodine inhibition of carbonic anhydrase by ascorbic acid. Enzyme Preparation A in 0.2 M phosphate buffer, pH 7.0. Hydration and dehydration were measured in the same solution. Arrows designate times of half hydration and half dehydration. X indicates un-

catalyzed reaction; l , carbonic anhydrase alone; 0, carbonic anhydrase inhibited by iodine (0.5 X 10-B mole per liter); A, carbonic anhydrase + iodine (0.5 X 10-a mole per liter) + ascorbic acid (0.01 mole per liter).

FIG. 3. Partial inhibition of carbonic anhydrase by KMn04. Enzyme Preparation A in 0.2 M phosphate buffer, pH 7.0. X indicates uncatalyzed reaction; l , carbonic anhydrase alone; 0, carbonic anhydrase inhibited by KMn04 in four different concentrations. The horizontal line inter- sects the curve at the points of half hydration.

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288 Activity of Carbonic Anhydrase

tioned that the same result was obtained when the enzyme was inhibited by sulfide or cyanide.

It may be concluded, Oherefore, that, our experiments give no evidence that the hydration function of carbonic anhydrase can be inhibited to a greater or less extent than the dehydration function.

Several reducing agents were tested for their effect upon car- bonic anhydrase. Ascorbic acid in a concentration of 0.1 M did not affect the enzyme; 0.05 M cysteine inhibited it slightly, but 0.01 M was without any effect; 0.005 M anthraquinone-/?-sulfonate had a very slight inhibiting effect in both the oxidized and re- duced state; phenosaphranine, 0.008 M, did not affect the enzyme in either the oxidized or reduced state.2

Injluence of Hydrogen Ion Concentration

A study of the relation of pH to carbonic anhydrase activity has been made. In the preceding paper, it was shown that the influence of pH on the catalytic effect of hypobromite and sulfite is quite different.

Meldrum and Roughton (1934) observed that t.he catalytic effect of the enzyme upon the hydration was smaller at pH 10 than at pH 7.6. Booth (1938) studying the effect of a carbonic anhydrase inhibitor in blood presented a curve for the activity of the enzyme in 0.2 M phosphate at pH 6.8 to 8.1. He found that the enzyme activity increased steadily from pH 6.8 to 8.1, being 6-fold larger at 8.1 than at 6.8.

WC have investigated the activity of the enzyme in the pH range from 6.1 to 10.1, employing phosphate, pyrophosphat,e, and carbonate as buffers. Typical results arc given in Table II. The concentration of the enzyme Preparation A, expressed in y per 100 cc., required to double the rate of hydration, has been dctcrmincd at different pH values. At the same pH, the activity of the enzyme was practically the same in both phosphate and

2 It may be mentioned that the dyes, brom-thymol blue and phenol red, inhibited the carbonic anhydrase. Both dyes have been used in colori- metric methods for the determination of carbonic anhydrase (Brinkman, 1934; Philpot and Philpot, 1936). In a concentration of 1OW M, brom- thymol blue inhibited the enzyme activity about 50 per cent. Phenol red, in the same concentration, inhibited it to an extent of 10 to 25 per cent.

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M. Kiese and A. B. Hastings 289

pyrophosphate. The optimal activity for the total hydration appears to be approximately pH 8.

The hydration of COZ proceeds in two ways: COZ + Hz0 + H&03, and COZ + OH- G HC03-. At present, we cannot de- cide whether the carbonic anhydrase catalyzes both reactions. Some enzymes have different optimal pH values, depending upon the concentration of substrate. Within the limits of substrat’e concentration used in our experiments, no differences in optimal pH values were observed for carbonic anhydrase. Our measure- ments were made with two different substrate concentrations, namely an initial pressure of 100 mm. of Cog, and 230 mm. of

TABLE II

Effect of pH on Over-All Rate of Hydration

PH

6.10 6.90 7.05 7.54 7.60 8.10 8.55 9.0 9.3

10.1

Concentration of en- zyme preparstion re- quired to double rate

of hydration BUfflX

y per 100 cc. .+I

17.5 0.2, phosphate 7.0 0.2, “ 7.0 0.1, pyrophosphate 3.7 0.2, phosphate 4.0 0.1, pyrophosphatc 2.3 0.1, “ 3.3 0.1, “ 5.0 0.1, “ 6.3 0.1, arsenate

18.0 0.1, carbonate

CO2 (all CO2 in the gas phase), but no difference in the pH curves was found.

Carbon Monoxide

Meldrum and Roughton (1934) reported that carbon monoxide produced considerable inhibition of carbonic anhydrase. This inhibition was stronger in the dark than in the light. We rc- peated these experiments with different results.

Carbon monoxide was prepared by dehydration of formic acid by sulfuric acid. The gas was washed by an alkaline solution of hydrosulfite and anthraquinone-@sulfonate, and stored in a reservoir. When drawn from the reservoir, the gas was bubbled

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290 Activity of Carbonic Anhydrase

through the hydrosulfite solution, followed by strong sodium hydroxide to remove traces of sulfide from the CO. For experi- ments in the dark, the reaction chamber was painted black.

The buffer solution with the enzyme was freed from gas by evacuation. The buffer-enzyme mixture was then saturated at a certain CO pressure and kept for different lengths of time. Without shaking, the vessel was then evacuated, the CO replaced by a mixture of CO and CO.+ and the rate of hydration measured as usual.

FIG. 4. Activity of carbonic anhydrase in the presence of carbon monox- ide. Enzyme in 0.2 M phosphate, pH 6.9. Curves 1 and 2 represent rates with two different amounts of carbonic anhydrase; 0, without exposure to CO; +, with exposure to 800 mm. of CO for 5.5 hours; X, the uncatalyzed reaction.

The CO pressure was varied from 200 to 1200 mm. of Hg and the time of exposure of the enzyme to CO, before the activity was measured, from 10 minutes to 12 hours.

In a series of ten experiments, we did not observe any evidence of inhibition of the carbonic anhydrase. Fig. 4 shows one ex- ample of these experiments. We have no explanation for the difference between our results and those of Meldrum and Roughton.

In view of the relation between activity and zinc content,

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M. Kiese and A. B. Hastings 291

demonstrated by Keilin and Mann, carbon monoxide might be expected to have no effect on the activity of the enzyme.

Sulfide and Cyanide

Cyanide and sulfide were found by Meldrum and Roughton (1934) and Stadie and O’Brien (1933) to be strong inhibitors of carbonic anhydrase. Our experiments confirm these observations. We found even a higher sensitivity of our enzyme Preparation A to cyanide than Meldrum and Roughton describe. Table III summarizes the experiments on the inhibition by different con- centrations of sulfide and cyanide. The experiments were car-

TABLE III

Inhibition of Carbonic Anhydrase by Cyanide and SulJide

0.2 M phosphate, pH 7.0. Enzyme concentration = amount necessary to accelerate uninhibited rate of hydration 4-fold.

Concentration of cyanide or sulfide

moles x 10s per 1.

0.1 0.2 0.5 1.0 2.0 4.0

10.0 20.0

Inhibition by cyanide Inhibition by sulfide

per cent

22 40 59 70 82 92

100

pet cent

18 43 78 96

ried out with a concentration of enzyme Preparation A which increased the hydration rate 4-fold. The enzyme activity was inhibited 50 per cent by sulfide, 0.23 X 10k5 M, or cyanide, 0.7 X 1O-5 M. Sulfide and cyanide are stronger inhibitors than iodine and permanganate, which required a concentration of 5 X 1O-5 mole per liter to inhibit the activity 50 per cent. Sul- fide and cyanide did not affect the uncatalyzed reaction.

SUMMARY

Experiments on the purification of carbonic anhydrase have been carried out and the properties of the enzyme preparation studied.

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292 Activity of Carbonic Anhydrase

Hydration of CO2 and dehydration of H&OS are influenced by the enzyme equally. The activity of the enzyme may be inhib- ited by a series of oxidizing agents and the activity restored by certain reducing agents. The effect of the inhibitors was shown to be the same on both the hydration and dehydration activity.

The activity of the enzyme in relation to the hydrogen ion concentration was investigated over a range from pH 6.1 to 10.1. The rate of hydration by the enzyme was found to be optimal at pH 8.1.

Carbon monoxide at pressures up to 1200 mm. of Hg did not inhibit the activity of the enzyme. In confirmation of others, we found sulfide and cyanide to bc strong inhibitors of the enzyme.

BIBLIOGRAPHY

Booth, V. H., J. Physiol., 91, 474 (1938). Brinkman, R., J. Physiol., 80, 171 (1934). Kayashima, S., J. Biochem., Japan, 28, 175 (1938). Keilin, D., and Mann, T., Nature, 144, 442 (1939). Kiese, M., and Hastings, A. B., Science, 88, 242 (1938); J. Riol. Chem.,

132, 267 (1940). Kuhn, R., and Desnuelle, P., 2. physiol. Chem., 261, 14 (1938). Meldrum, N. U., and Roughton, F. J.-W., J. Physiol., 80, 113 (1934). Philpot, F. J., and Philpot, J. St. L., Biochem. J., 30, 2191 (1936). Stadie, W. C., and O’Brien, H., J. Biol. Chem., 103, 521 (1933). Tiselius, A., Tr. Fara&q Sot., 33, 524 (1937). Waldschmidt-Leitz, E., Scharikova, A., and Schaffner, A., 2. physiol.

Chem., 214, 75 (1933). Willstatter, R., Ber. them. Ges., 63, 1152 (1920). Willstatter, R., and Kraut, H., Ber. them. Ges., 67, 1082 (1924).

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Manfred Kiese and A. Baird HastingsOF CARBONIC ANHYDRASE

FACTORS AFFECTING THE ACTIVITY

1940, 132:281-292.J. Biol. Chem. 

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