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[148] Carbonic anhydrase (plant and animal)
836
RESPIRATORY ENZYMES
[148]
[148] C a r b o n i c A n h y d r a s e ( P l a n t a n d A n i m a l ) By E. RoY WAYGOOD
CO2 ~- HsO ~- H2CO3~ H+ ~- HCO3When COs is dissolved in water it is slowly hydrated to carbonic acid which spontaneously ionizes according to the above equation. Hydration occurs in the pH range 6.5 to I0.0, whereas the reverse dehydration takes place in the pH range 5.5 to 7.5.1 This reversible reaction is catalyzed by many oxy-acid buffers, 2 including phosphate, cacodylate, Veronal, chromate, borate, selenite, and also by the enzyme carbonic anhydrase which catalyzes both phases of the reaction equally.', Assay Methods
Principle. There are two main methods for the determination of carbonic anhydrase activity. 1. Manometric Method. Activity may be determined by measuring the increased rate of COs output when carbonic acid, supplied in the form of a bicarbonate solution, is dehydrated by shaking with a buffer (usually phosphate, pH 6.6 to 6.8) and enzyme. The hydration of carbon dioxide is measured by the increased rate of COs uptake when gaseous COs is shaken with a buffer (usually Veronal, pH 8.0) and enzyme. 2. Colorimetric Method. When a solution of COs is mixed with an alkaline buffer, the pH drops rapidly, owing to the reaction COs-}OH---+ HC03- (above pH 8.0) accompanied and followed by the hydration of COs (below pH 10.0). The decrease in time taken for the buffer containing enzyme to drop to a specified pH is used as a measure of catalytic rate. The lower pH value is determined by a sharp change in the color of an appropriate indicator when the buffering capacity of the solution changes markedly. Procedure. (1) Manometric Procedure. Clark and Perrin 4 have suggested that Meldrum and Roughton's boat-manometric method 5 should be retained provisionally as the standard procedure for measuring enzyme activity. The procedure with elegant refinements which afford a more sensitive means of studying the kinetics of the enzyme under a 1F. J. W. Roughton and V. H. Booth, Biochem. J. 40, 309, 319 (1946); F. J. W. Roughton, Harvey Lectures, Series 39, p. 96 (1943-4). F. J. W. Roughton and V. H. Booth, Biochem. J. 32, 2049 (1938). 3 M. Kiese and A. B. Hastings, J. Biol. Chem. 132, 281 (1940). 4 A. M. Clark and D. D. Perrin, Biochem. J. 48, 495 (1945). 5 N. U. Meldrum and F. J. W. Roughton, J. Physiol. (London) 80, 113 (1933).
[148]
CARBONIC ANHYDRASE (PLANT AND ANIMAL)
837
wider range of conditions 1,2 has been described in considerable detail elsewhere. ~-7 However, for purely routine studies on the distribution or purification of the enzyme and the effects of inhibitors and activators, the use of the W a r b u r g a p p a r a t u s is recommended, since it comprises one of the standard items of equipment available in m a n y laboratories and suffers from no more serious disadvantages than does the b o a t apparatus. Nevertheless, for the accurate interpretation of d a t a derived from a n y manometric procedure measuring carbonic anhydrase activity, the operator m u s t be fully aware of the limitations imposed on the method b y the reaction system. These have been discussed in detail b y R o u g h t o n et al. 1,2,8 and b y Clark and Perrin. 4 Although a n u m b e r of investigators 9-1~ have described reliable methods using the W a r b u r g technique, the one described below, developed by K r e b s and Roughton, 14 is recommended because of its simplicity and the use of a low concentration of phosphate which becomes increasingly inhibitory toward the enzyme at high concentrations. KREBS-t~OUGHTONWARBURGTECHNIQUE. For experiments at 0 °, 2 ml. of 0.1 M N a 2 H P 0 4 and 0.1 M KH2PO4 in the proportion 3.2 are placed in the main c o m p a r t m e n t of a W a r b u r g flask with 0.2 ml. of water or enzyme. One milliliter of 0.1 M N a H C 0 3 is placed in the side arm. At zero time, after equilibration, the two solutions are mixed, shaken at 120 to 180 oscillations per minute, and pressure changes recorded at 30-second intervals over a period of 5 minutes or more. T h e pressure change due to the control (nonenzymically catalyzed reaction) as well as the reaction catalyzed b y small amounts of the enzyme is a linear function of time until a b o u t one-third (ca. 80 mm.) of the final pressure change has been attained. F o r experiments at 15 ° and above, 1 ml. of phosphate buffer and 1 ml. of 0.05 M N a H C 0 3 are used. DEFINITION OF UNIT AND SPECIFIC ACTIVITY. Pressure changes are converted to microliters of C02 b y the use of flask constants (kco,) in the conventional manner. ~ The increased C02 evolved in 30 seconds (i.e., 6 H. Van Goor, Enzyrnologia 13, 73 (1948). 7 F. J. W. Roughton and A. M. Clark, in "The Enzymes" (J. B. Sumner and K. Myrbtick, ed.), Vol. 1, Part 2, p. 1250, Academic Press, New York, 1951. s F. J. W. Roughton, J. Biol. Chem. 141, 129 (1941). 9 M. Leiner and G. Leiner, Biochem. Z. 311, 119 (1942). 10 W. C. Stadie, B. C. Riggs, and N. Haugaard, J. Biol. Chem. 161, 175 (1945). n M. D. Altschule and H. D. Lewis, J. Biol. Chem. 180, 657 (1949). 12E. R. Waygood and K. A. Clendenning, Can. J. Research C28, 673 (1950) ; Science 113, 177 (1951). 13R. U. Byerrum and E. H. Lucas, Plant Physiol. 27, 111 (1952). 14H. A. Krebs and F. J. W. Roughton, Biochem. J. 43, 550 (1948). 15W. W. Umbreit, R. H. Burris, and J. F. Stauffer, "Manometric Techniques and Tissue Metabolism," Burgess Publishing Co., Minneapolis, 1949.
838 ~
RESPIRATORY
ENZYMES
[148]
over-all rate - nonenzymic rate) in the region of 60 to 80 mm. of pressure can be used to calculate Qco, on a nitrogen or other appropriate basis. In the case of labile highly purified enzymes (see later), or when the effect of activators or inhibitors is being studied, it m a y be more accurate to express the enzymic activity as the difference between the true initial unimolecular velocity constants (k, - k,) of the enzymically and the nonenzymically catalyzed reactions, respectively, according to the methods of Mitchell et al. '8 and Clark and Perrin. 4,~7 F r o m the equation a of the first-order reaction kt = 2.303 log a - x' log (a - x) is plotted against t and the initial slope times 2.303 gives the value of the velocity constant. PRECAUTIONS. Phosphate has a strong catalytic effect on the dehydration of carbonic acid, ~ and the Q10 = 2.9 for this reaction differs markedly from the Q~0 = 1.4 for the enzymically catalyzed reaction (over-all r a t e - non-enzymic rate). ~8 Accordingly, in order to work within the range of strict proportionality between activity and enzyme concentration, the apparatus is limited to the use of enzyme concentrations which increase the nonenzymic activity up to sixfold at 0 o and twofold at 38 ° . If measurements are made at higher concentrations of enzyme, the reaction is limited b y the diffusion of COs and should be corrected b y applying the theoretical concepts and experimental treatments of Roughton. 8 According to Roughton el al., ~,7,8 in most cases, when diffusion is limiting the apparent rate m a y be corrected b y applyR,~Ro
ing the formula R - R~ - R~ where R0 is the apparent rate and Rm is the maximum observable rate in the presence of a large a m o u n t of enzyme. R~ is a function of liquid volume and the dimensions of the flask. As yet it has only been determined for the boat apparatus. When highly purified enzymes from blood are used, a proportion m a y be inactivated b y impurities or adsorption. Such enzyme preparations give a sigmoid relationship between activity and enzyme concentration and furthermore are subject to progressive inactivation during shaking. These errors m a y be overcome b y observing strict precautions in cleanliness of glassware, acid washing, use of doubly distilled water, or b y stabilizing the enzyme with 0.05 % peptone.l.4.~9 Or, on the other hand, since these errors result in deviations from the first-order reaction, during the latter part of the reaction time, the initial slope of the plot log (a -- x) le C. A. Mitchell, U. C. Pozzani, and R. W. Fessenden, J. Biol. Chem. 160, 283 (1945). 17A. M. Clark, Nature 168, 562 (1949). ~8F. J. W. Roughton, J. Physiol. (London) 107, 12P (1948). 19D. A. Scott and J. R. Mendive, J. Biol. Chem. la9, 661 (1941); 140, 445 (1941).
[148]
CARBONm ANHYDRASE (PLANT AND ANIMAL)
839
vs. t will give a true value for the velocity constant (k~) independent of the inactivation of the enzyme. 4,~7 Clark 4,~7 has made use of this method to distinguish between stabilizations and activations. By calculating the percentage decrease (D) in the value of k, during the first 100 seconds of reaction, the sensitivity of the preparation may be determined. In a similar way, the degree of stabilization (S) is calculated as 100(1 - d/d ~) where d and d ~are the values of D in the presence and the absence of stabilizing or activating agents, respectively. 2. Co!orimeoric Method. Colorimetrie techniques are usefu) for rapid tests but have a restricted range regarding the conditions under which enzyme activity can be investigated. Owing to the relatively large inhibitions of the enzyme by CO~= in the original method of Brinkman 2° and Philpot and Philpot, 2~ a more reliable technique using Veronal buffer has been developed by Roughton and Booth s and is being used successfully in other laboratories ~,s3 (C. A. Mawson, personal communication). CO2-VERONAL INDICATORMETHOD. Veronal buffer (3 ml. of 0.022 M Veronal in 0.022 M Na salt, pH 7.95), three drops of bromothymol blue, and 2.3 ml. of distilled water (or 0.3 ml. of enzyme and 2.0 ml. of water) are mixed in a 15-ml. stoppered weighing bottle and placed in ice water for 15 minutes. Five milliliters of ice-cold water saturated with COs (0.071 M) is added anaerobically from a long nozzled all-glass syringe. The time is observed for the pH to drop to 6.3, determined with the aid of a bromothymol standard at this pH. The solutions are mixed in less than 1 second without bubbling or loss of COs. The control time averages 90 seconds, compared to about 64 seconds in the presence of 14.3 p.p.m, of a crude chloroform preparation (Step 2, p. 841). DEFINITION OF UNIT. The enzyme unit E.U. -
to - -
t
t
, where t and
to
are time of reaction in the presence and the absence of catalyst, respectively. Since the velocity constant of the uncatalyzed reaction (k~) calculated from the experimental data was found to be 0.0022 mole/1./sec., which agrees well with the accepted value of 0.0021 at 0 °, the validity of the method is established. Accordingly, Roughton and Booth ~ calculated the rate of enzymic hydration of COs in moles per liter per second as follows, allowing a period of 1 second for mixing. R
--
Ro
Ro
to - -
t-
t
1
2~ R. Brinkman, J. Physiol. (London) 80, 171 (1933). 21 F. J. Philpot and J. St. L. Phflpot, Biochem. J. 80, 2191 (1936). 22 K. M. Wilbur and W. G. Anderson, J. Biol. Chem. 176, 147 (1948). .,3 E. R. Trethewie and A. J. Day, Australian J. Exptl. Biol. Med. Sc/. 27, 429 (1949).
840
RESPIRATORY ENZYMES
[148]
where R and R0 are the rates in the presence and the absence of catalyst, respectively. Ro = k~ [average COs] Then Rate = R - R 0 -
((tt 0- - t 1) ) ]c, [average COs]
since k~ = 0.00225 mole/k/see, and [average COs] = 0.0307 mole under the experimental conditions. Therefore, Rate of enzymic hydration of COs
_ (to -- t)
(t - 1) 6.91 X 10-5 mole/1./sec.
The figures for the all-liquid method are approximately 70 % of those expected from manometric data. The discrepancy of 30% may be put down to the inhibitory action of the indicator and experimental error. Wilbur and Anderson 22 have used an automatic syringe to introduce the COs, and the pH changes are measured electrometrically. RAPID-FLOW COLORIMETmCM~.THOD. All methods of assay heretofore discussed are subject to a mixing error and are restricted to the use of low concentration of enzyme owing to diffusion limitations. Accordingly, the development of the Hartridge-Roughton rapid-flow technique, which minimizes these errors and photoelectrically records the color change of a pH indicator, opens up a wider range of conditions under which the enzyme can be studied. Clark and Perrin 4 have used the rapid-mix, quickstop method of Chance. 24 A saturated solution of COs is rapidly mixed at room temperature with Veronal buffer, pH 8.6 and pK 8.0, in a modified Millikan 25 microapparatus. The flow through a capillary tube is stopped in milliseconds, and in the presence of phenol red the pH of the solution is recorded continuously as a function of time. Much higher temperatures and enzyme concentrations may be used in this technique. U s e of C a r b o n i c A n h y d r a s e as a B i o c h e m i c a l T o o l
In decarboxylation or other reactions where gaseous COs is produced, a question of some significance arises as to whether CO2 or HCOa- is the primary product. In systems rapidly producing COs into the gaseous phase, carbonic anhydrase will slow down the rate owing to the accelerated hydration of the COs. On the other hand, it is inferred that if HCO3is the primary product the rate would be initially accelerated. Krebs and Roughton ~4have shown that COs is the primary product of yeast carbox2~B. Chance, J. Franklin Inst. 229, 455 (1940). 25G. A. Millikan, Proc. Roy. Soc. (London) A155, 277 (1936).
[148]
CARBONIC ANHYDRASE (PLANT AND ANIMAL)
841
ylase and the urease reaction. Hansl and Waygood 26 have confirmed their findings and in addition have shown that C02 is the primary product of the plant pyruvic, glutamic, oxalacetic, and a-ketoglutaric decarboxylation systems. More recently, Conway and O'Malley ~7 have concluded from their theoretical and experimental treatments that HC03- may be produced concomitantly with CO2 in the latter two reactions. Source and Purification of the Enzyme Animal. Mammalian red blood cells are the richest source. Among other tissues, the pancreas, gastric mucosa, and kidney contain comparable amounts. The enzyme is absent from plasma and other body fluids with certain exceptions. 5,6 Many methods of purification have been described in the literature (see Van Goor 6) culminating in a crystalline preparation of ammonium carbonic anhydrase by Scott and Fisher. 2s In general, however, investigators have used one or all of three stages of purification, either lysed red cells or the crude chloroform preparation of Meldrum and Roughton 1,5 or highly purified enzymes prepared by the particular method of the investigator. Since many laboratories have successfully used the highly purified preparations of Keilin and Mann 29 and because of its relative simplicity the details of their procedure and those of the crude preparations are described. Step 1. Lysed Red Cells. The red blood cells of defibrinated ox blood are centrifuged from the serum and washed three times with an equal volume of 0.9 % NaC1. The washed cells are hemolyzed with an equal or half-volume of distilled water. Step 2. Crude Chloroform Preparation.1 To 10 ml. of lysed red cells are quickly added 8 ml. of 40 % ethanol and 4 ml. of chloroform. The mixture is stirred in a centrifuge tube for 3 minutes to a thin sludge and allowed to stand for 20 minutes. After centrifugation for 10 minutes at 3500 r.p.m., a three-phase system is formed consisting of a supernatant layer of enzyme solution, a central layer of denatured protein, and a bottom layer of chloroform. Step 3. Method I I of Keilin and Mann. 29 Dialysis of Alcohol-Chloroform Extract. The filtered enzyme solution is dialyzed for 24 hours against running water. Step 4. Total Precipitation with Ammonium Sulfate. The fluid is saturated with (NH4)2S04, and the precipitate after filtering on a Btichner 36 N. 37 E. 3s D. 39 D.
Hansl and E. R. Waygood, Can. J. Botany 30, 306 (1952). J. Conway and E. O'Malley, Biochem. J. 54, 154 (1953). A. Scott and A. M. Fisher, J. Biol. Chem. 144, 371 (1942) ; Nature 153, 711 (1944). Keilin and T. Mann, Biochem. J. 34, 1163 (1940).
842
RESPIRATORY ENZYMES
[148]
funnel is dissolved in a small a m o u n t of water, dialyzed against running tap water and centrifuged. Step 5. Fractional Precipitation with Ammonium Sulfate. T h e supern a t a n t fluid is saturated 4 5 % with respect to (NH4)2S04 and filtered. The filtrate is completely saturated with (NH4)2SO4 and, after filtering, the precipitate is dissolved in water and dialyzed. Step 6. Purification with Alumina C~ Gel. T h e solution is treated with three successive 5-ml. ( = 100 mg.) portions of alumina C~ gel at p H 6.8 and centrifuged, the cakes being discarded each time. Step 7. Fractional Precipitation with Ammonium Sulfate. The above solution is made 50 % saturated with (NH4)2SO4 and, after filtering, completely saturated. The precipitate is dissolved in water and dialyzed against distilled water until free from salt. SUMMARY OF PURIFICATION PROCEDURE a
Step
Total volume, ml.
Absolute E.U.
1 2-3 4 5 6-7
2000 2250 110 85 50
4,000,0002 1,488,000 1,100,000 765,000 500,000
M1./E.U.
Specific activity, E.U./mg.
Zn, %
Recovery, %
0.0005 0.0015 0.0001 0.0001 0.0001
14.3 384 588 830-1000 2220
0.C024 --0.15-0.17 0.33
-37 27 19.5 12
Method II [D. Keilin and T. Mann, Biochem. J. 34, 1163 (1940)]. b Calculated from Step 1, Method I [D. Keflin and T. Mann, Biochem. J. 34, 1163 (1940)]. Plant. Bradfield 8° and Waygood and Clendenning TM found the enzyme to be ~ocalized only in the cytoplasm of leaf tissue. The enzyme is absent from root tissue2 ° Sirois and Waygood (unpublished) recommend spinach (Spinacea oleracea) as a source of the enzyme, since it has the highest specific activity of all plants tested, b u t m a n y other sources are available, n o t a b l y leaves of New Zealand spinach (Tetragonia expansa), nast u r t i u m (Tropaeolum majus), lamb's quarters (Chenopodium album), and beet (Beta vulgaris). 12.13,30.31 Sibly and Wood 3~ have purified the plant enzyme b y the m e t h o d of Keilin and Mann, b u t the percentage recovery was low. We have found t h a t it is essential to protect the plant enzyme with cysteine at all stages during purification, and the following procedure is recommended. Midribs are removed from spinach leaves, and 200 g. is ground in a m o r t a r with 50 ml. of cysteine (final concentration 0.005 M). The brei is 30j. R. G. Bradfield, Nature 159, 467 (1947). sz p. M. Sibly and J. G. Wood, Australian J. Sci. Research B4, 500 (1951).
[148]
CARBONIC ANHYDRASE (PLANT AND ANIMAL)
843
pressed through nylon, and the crude juice is centrifuged for 20 minutes at 15,000 X g. The supernatant fluid (200 ml., A = 15 E.U./mg.) is shaken with 75 g. of Ca3(P04)2 and centrifuged. The cake is eluted with 0.2 M phosphate, pH 6.6, in 0.005 M cysteine. After an initial precipitation with 30 % alcohol, the enzyme is totally precipitated at a concentration of 75% ethanol. The white powder is completely soluble in water. The solution, when dialyzed against 0.005 M cysteine and lyophilized, yields a preparation containing 300 E.U./mg. and 0.05 % Zn with no other metals. The enzyme unit is that defined by Waygood and Clendenning. 12
Properties Specificity. Carbonic anhydrase is specific for the reaction CO2 + H20 ,~- HsCO3. It is doubtful if it catalyzes the reaction COs + O H - --* HC03- which predominates above pH 11.0 and is significant between pH 8 and 10.1,~,3,7 Since the latter occurs as a primary reaction succeeded by, or accompanying, the enzyme-catalyzed reaction, it may introduce errors in some colorimetric methods. There are numerous processes which are limited by the reaction COs + H20 ~---H~CO~, and their rates may be increased by the addition of enzyme, e.g., the deposition and solution of CaCO3, and the dissociation of carbamate into C02. 5,7 Stability. Crude chloroform preparations of the animal enzyme are stable for long periods in the dry state but are progressively inactivated in solution. Purer preparations are less stable. Plant carbonic anhydrase is stable in the dried form but deteriorates rapidly in solution. It is also much less stable during dialysis than the animal enzyme. Cysteine (0.005 to 0.01 M) protects the plant enzyme in solution and during dialysis. 30 Nature of the Enzymes. Carbonic anhydrase is a Zn-protein compound. ~9,32 Keilin and Mann's preparation ~9 and others contained 0.033% Zn, whereas Scott and Fisher's crystalline preparation 28 contained 0.02% Zn and had a molecular weight of 30,00023 The latter value corresponds to 1 atom of Zn per molecule. Our partially purified preparations of the plant enzyme contain 0.05 % Zn with no other metals. Sibly and Wood81 calculated ~hat their preparations of the plant enzyme contained 0.056 % Zn. The failure of dialysis to remove zinc from either animal 29 or plant carbonic anhydrase (Sirois and Waygood, unpublished) and the fact that Tupper et al. 34 have shown that Zn 6~ ions in the medium do not exchange a2 D. Keflin and T. Mann, Nature 144, 442 (1939). 33 M. L. Peterman and N~. ¥. ttakala, J. Biol. Chem, 14§, 701 (1942). 34 R. Tupper, R. W. E. Watts, and A. Wormalls, Bioehem. J. 50, 49 (1952).
844
RESPIRATORY ENZYMES
[148]
with the zinc moiety of the enzyme from blood indicates that the metal is firmly entrenched in the protein molecule. The evidence that we have been able to provide in favor of the view that plant carbonic anhydrase is a Zn-protein is as follows. The Zn content of the purified enzyme preparation increases proportionately with increasing specific activity during prolonged dialysis against cysteine. No other metals are present, but the possibility that Zn is present in a nonspecific protein is not excluded. Inhibition by cyanide and azide (see later) is also indicative of the metalloprotein nature of the enzyme in plants. The kinetics of the enzyme from blood have been studied in detail by Roughtou and Booth 1 using the refined boat method. Byerrum and Lucas 1~ give some kinetic data for the enzyme from plants. Since it is doubtful if all the necessary precautions were observed in their experiments, the results await confirmation. Effect of Enzyme Concentration. Provided that diffusion is not a limiting factor, there is a linear relationship between activity and concentration of crude preparations of the enzyme. When diffusion is limiting, the apparent rate may be corrected by Roughton's formula (see above). In the case of highly purified preparations a dilution effect may occur, giving a sigmoid relationship between activity and enzyme concentration. As pointed out earlier, this may be overcome by stabilizing the enzyme. Effect of Substrate Concentration. The value of the Michaelis constant (Kin) for the crude chloroform preparation is 0.009 M C02 (+0.001 M) at 0°. 1 The value is independent of pH, although Kiese ~5 has reported values at 1° of 0.0012 M at pH 7.4, rising to 0.0022 M at pH 9.3 for a highly purified enzyme. It is not certain whether Kiese took all the necessary precautions. Calculations from Leiner's data for a stabilized highly purified enzyme gives K~ = 0.0075 M + 20%. T M Turnover Number. The rapidity of the reaction is shown by calculations from the data of Roughton et al. 1,~ which gives a maximum turnover number of the order of 9.6 X 107 at 0 ° and pH 7.3 for the enzyme from blood. The highest turnover number previously recorded is 5 X 106 for catalase. The value of 1.8 × 108 for the velocity constant of combination of enzyme and substrate is also indicative of the rapid reaction and is of the same order as that for catalase. Effect of pH. The activity of the enzyme from blood is at a minimum at pH 6.5 and gradually increases to fivefold the activity at pH 10.0. There is evidence that the activity is increased below pH 6.0.1 The isoelectric point is at pH 5.3. 3~ 35M. Kiese, Biochem. Z. 307, 400 (1941). 36M. Leiner, Biochem. Z. 315, 31 (1943).
[148]
CARBONIC ANHYDRASE (PLANT AND ANIMAL)
845
Effect of Temperature. Recently corrected values give Q10 = 2.9 for the nonenzymic rate and Q10 - 1.4 for the enzymic rate (over-all rate nonenzymic rate). TMA plot of the log activity against l I T shows a linear relationship. Inhibitors. Heavy metal poisons--e.g., cyanide, azide, and sulfide-inhibit the activity of carbonic anhydrase from blood to the extent of 50% at concentrations ranging from 10`4 to 10-e M. 7 Whereas the activity of the plant enzyme is strongly inhibited by azide (70 to 90 % at 10-3 M), 12,3° cyanide, according to Bradfield 3° and Sibly and Wood, 31 only inhibits at much higher concentrations (65 to 75 % at 10-3 M; no inhibition at 10-3 M). However, Waygood and Clendenning ~2 have reported 50 to 75% inhibition of the activity of a crude and dialyzed enzyme preparation from Tradescantia fluminensis by 10-3 M HCN. These values have been confirmed by Sirois and Waygood (unpublished) using stabilized semipurified preparations from spinach leaves. The activity of a crude spinach preparation is inhibited 50% by 2.4 X 10-3 M HCN, whereas the activity of the purified enzyme is inhibited 50% by 1.25 X 10-3 M HCN. Nevertheless, the relative insensitivity of the plant enzyme to cyanide, compared with the enzyme from blood, constitutes one of the more important differences between the two enzymes. The markedly inhibitory effect of low concentrations of sulfanilamide and related substances containing the --S02NH2 group has been extensively investigated. 37-4° N-Substituted sulfonamides are ineffective.89 Thiophene-2-sulfonamide and p-sulfonamidobenzoic acid are eight to twelve times as effective as sulfanilamide which inhibits the activity of the enzyme from animals 50% at 10-7 M. 3s,39 A new and more specific inhibitor and less toxic drug, Diamox 6063 (2-acetylamino-l,3,4-thiadiazole-5-sulfonamide) has recently been reported. 4° In contrast, Bradfield a° and Sibly and Wood ~ found that sulfanilamide at a concentration of 10-5 M caused little or no inhibition of the activity of the plant enzyme from two different sources, thus indicating another important difference between the animal and the plant enzyme. The inhibitory effect of anions on the activity of the enzyme from blood in decreasing order I - > NO3- > Br- > C1- > acetate > SO4--. The effect of C1- is especially interesting, since it appears to cause inactivation by forming an inactive complex with the enzyme. The divalent ion COs-- also causes inhibition of enzyme activity, especially at high pH values.
37T. Mann and D. Keilin, Nature 146, 164 (1940). as H. W. Davenport, J. Biol. Chem. 168, 567 (1945). 39H. A. Krebs, Biochem. J. 48, 525 (1948). 40T. H. Maren, Trans. N. Y. Acad. Sci. 16, 53 (1952).
846
RESPIRATORY ENZYMES
[148]
Activators, Stabilizers and Protectors. Claims of activating substances reported in the literature are now suspect, owing to the questionability of the methods used. Clark and Perrin 4 have shown convincingly that apparent activation of the enzyme by certain substances, e.g., boiled horse plasma and glutathione, is due to the restoration of the activity of purified enzymes, lost by adsorption or the effect of impurities. No activation occurs when the enzymes are stabilized by 0.05 % peptone; thus these substances are acting as stabilizers. Indeed the stabilizing power of plasma obscured a weak inhibitory effect.4 Scott and Mendive 19 report a number of stabilizers of which horse serum (1:40) and 0.05% peptone are the most effective. We have used 0.025% gelatin to stabilize the plant enzyme. One of the most important differences between plant and animal carbonic anhydrase is the dependency of the former on free - - S H groups. Thus Bradfield 3° discovered that cysteine efficiently protected the plant carbonic anhydrase while standing or during dialysis. Furthermore, the enzyme was completely inhibited by 5 X 10-4 M p-mercuriochlorobenzoate, which has little effect on animal carbonic anhydrase. Sibly and Wood 31 have confirmed these findings and also demonstrated complete inhibition of activity by 5 X 10-3 M arsenite, another mercaptide-forming compound. The inhibition caused by both these substances could be reversed by cysteine o r glutathione. Iodoacetate does not cause inhibition, but the presence of sulfhydryl groups on plant carbonic anhydrase has been confirmed polarographically. 3~ The important papers of the Japanese workers, Kondo et al., 4~ on the kinetics of spinach carbonic anhydrase were not available to the author at the time of writing. They have reported the isolation of a metal-free electrophoretically homogeneous protein, stabilized by 0.1 M NaC1 having carbonic anhydrase activity. 41 K. Kondo, H. Chiba, and H. Kawai, Bull. Research Inst. Food Sci. Kyoto Univ. 8, 17-27 (1952); 13, 1-59 (1954). (English abstracts.)
RESPIRATORY ENZYMES
[148]
[148] C a r b o n i c A n h y d r a s e ( P l a n t a n d A n i m a l ) By E. RoY WAYGOOD
CO2 ~- HsO ~- H2CO3~ H+ ~- HCO3When COs is dissolved in water it is slowly hydrated to carbonic acid which spontaneously ionizes according to the above equation. Hydration occurs in the pH range 6.5 to I0.0, whereas the reverse dehydration takes place in the pH range 5.5 to 7.5.1 This reversible reaction is catalyzed by many oxy-acid buffers, 2 including phosphate, cacodylate, Veronal, chromate, borate, selenite, and also by the enzyme carbonic anhydrase which catalyzes both phases of the reaction equally.', Assay Methods
Principle. There are two main methods for the determination of carbonic anhydrase activity. 1. Manometric Method. Activity may be determined by measuring the increased rate of COs output when carbonic acid, supplied in the form of a bicarbonate solution, is dehydrated by shaking with a buffer (usually phosphate, pH 6.6 to 6.8) and enzyme. The hydration of carbon dioxide is measured by the increased rate of COs uptake when gaseous COs is shaken with a buffer (usually Veronal, pH 8.0) and enzyme. 2. Colorimetric Method. When a solution of COs is mixed with an alkaline buffer, the pH drops rapidly, owing to the reaction COs-}OH---+ HC03- (above pH 8.0) accompanied and followed by the hydration of COs (below pH 10.0). The decrease in time taken for the buffer containing enzyme to drop to a specified pH is used as a measure of catalytic rate. The lower pH value is determined by a sharp change in the color of an appropriate indicator when the buffering capacity of the solution changes markedly. Procedure. (1) Manometric Procedure. Clark and Perrin 4 have suggested that Meldrum and Roughton's boat-manometric method 5 should be retained provisionally as the standard procedure for measuring enzyme activity. The procedure with elegant refinements which afford a more sensitive means of studying the kinetics of the enzyme under a 1F. J. W. Roughton and V. H. Booth, Biochem. J. 40, 309, 319 (1946); F. J. W. Roughton, Harvey Lectures, Series 39, p. 96 (1943-4). F. J. W. Roughton and V. H. Booth, Biochem. J. 32, 2049 (1938). 3 M. Kiese and A. B. Hastings, J. Biol. Chem. 132, 281 (1940). 4 A. M. Clark and D. D. Perrin, Biochem. J. 48, 495 (1945). 5 N. U. Meldrum and F. J. W. Roughton, J. Physiol. (London) 80, 113 (1933).
[148]
CARBONIC ANHYDRASE (PLANT AND ANIMAL)
837
wider range of conditions 1,2 has been described in considerable detail elsewhere. ~-7 However, for purely routine studies on the distribution or purification of the enzyme and the effects of inhibitors and activators, the use of the W a r b u r g a p p a r a t u s is recommended, since it comprises one of the standard items of equipment available in m a n y laboratories and suffers from no more serious disadvantages than does the b o a t apparatus. Nevertheless, for the accurate interpretation of d a t a derived from a n y manometric procedure measuring carbonic anhydrase activity, the operator m u s t be fully aware of the limitations imposed on the method b y the reaction system. These have been discussed in detail b y R o u g h t o n et al. 1,2,8 and b y Clark and Perrin. 4 Although a n u m b e r of investigators 9-1~ have described reliable methods using the W a r b u r g technique, the one described below, developed by K r e b s and Roughton, 14 is recommended because of its simplicity and the use of a low concentration of phosphate which becomes increasingly inhibitory toward the enzyme at high concentrations. KREBS-t~OUGHTONWARBURGTECHNIQUE. For experiments at 0 °, 2 ml. of 0.1 M N a 2 H P 0 4 and 0.1 M KH2PO4 in the proportion 3.2 are placed in the main c o m p a r t m e n t of a W a r b u r g flask with 0.2 ml. of water or enzyme. One milliliter of 0.1 M N a H C 0 3 is placed in the side arm. At zero time, after equilibration, the two solutions are mixed, shaken at 120 to 180 oscillations per minute, and pressure changes recorded at 30-second intervals over a period of 5 minutes or more. T h e pressure change due to the control (nonenzymically catalyzed reaction) as well as the reaction catalyzed b y small amounts of the enzyme is a linear function of time until a b o u t one-third (ca. 80 mm.) of the final pressure change has been attained. F o r experiments at 15 ° and above, 1 ml. of phosphate buffer and 1 ml. of 0.05 M N a H C 0 3 are used. DEFINITION OF UNIT AND SPECIFIC ACTIVITY. Pressure changes are converted to microliters of C02 b y the use of flask constants (kco,) in the conventional manner. ~ The increased C02 evolved in 30 seconds (i.e., 6 H. Van Goor, Enzyrnologia 13, 73 (1948). 7 F. J. W. Roughton and A. M. Clark, in "The Enzymes" (J. B. Sumner and K. Myrbtick, ed.), Vol. 1, Part 2, p. 1250, Academic Press, New York, 1951. s F. J. W. Roughton, J. Biol. Chem. 141, 129 (1941). 9 M. Leiner and G. Leiner, Biochem. Z. 311, 119 (1942). 10 W. C. Stadie, B. C. Riggs, and N. Haugaard, J. Biol. Chem. 161, 175 (1945). n M. D. Altschule and H. D. Lewis, J. Biol. Chem. 180, 657 (1949). 12E. R. Waygood and K. A. Clendenning, Can. J. Research C28, 673 (1950) ; Science 113, 177 (1951). 13R. U. Byerrum and E. H. Lucas, Plant Physiol. 27, 111 (1952). 14H. A. Krebs and F. J. W. Roughton, Biochem. J. 43, 550 (1948). 15W. W. Umbreit, R. H. Burris, and J. F. Stauffer, "Manometric Techniques and Tissue Metabolism," Burgess Publishing Co., Minneapolis, 1949.
838 ~
RESPIRATORY
ENZYMES
[148]
over-all rate - nonenzymic rate) in the region of 60 to 80 mm. of pressure can be used to calculate Qco, on a nitrogen or other appropriate basis. In the case of labile highly purified enzymes (see later), or when the effect of activators or inhibitors is being studied, it m a y be more accurate to express the enzymic activity as the difference between the true initial unimolecular velocity constants (k, - k,) of the enzymically and the nonenzymically catalyzed reactions, respectively, according to the methods of Mitchell et al. '8 and Clark and Perrin. 4,~7 F r o m the equation a of the first-order reaction kt = 2.303 log a - x' log (a - x) is plotted against t and the initial slope times 2.303 gives the value of the velocity constant. PRECAUTIONS. Phosphate has a strong catalytic effect on the dehydration of carbonic acid, ~ and the Q10 = 2.9 for this reaction differs markedly from the Q~0 = 1.4 for the enzymically catalyzed reaction (over-all r a t e - non-enzymic rate). ~8 Accordingly, in order to work within the range of strict proportionality between activity and enzyme concentration, the apparatus is limited to the use of enzyme concentrations which increase the nonenzymic activity up to sixfold at 0 o and twofold at 38 ° . If measurements are made at higher concentrations of enzyme, the reaction is limited b y the diffusion of COs and should be corrected b y applying the theoretical concepts and experimental treatments of Roughton. 8 According to Roughton el al., ~,7,8 in most cases, when diffusion is limiting the apparent rate m a y be corrected b y applyR,~Ro
ing the formula R - R~ - R~ where R0 is the apparent rate and Rm is the maximum observable rate in the presence of a large a m o u n t of enzyme. R~ is a function of liquid volume and the dimensions of the flask. As yet it has only been determined for the boat apparatus. When highly purified enzymes from blood are used, a proportion m a y be inactivated b y impurities or adsorption. Such enzyme preparations give a sigmoid relationship between activity and enzyme concentration and furthermore are subject to progressive inactivation during shaking. These errors m a y be overcome b y observing strict precautions in cleanliness of glassware, acid washing, use of doubly distilled water, or b y stabilizing the enzyme with 0.05 % peptone.l.4.~9 Or, on the other hand, since these errors result in deviations from the first-order reaction, during the latter part of the reaction time, the initial slope of the plot log (a -- x) le C. A. Mitchell, U. C. Pozzani, and R. W. Fessenden, J. Biol. Chem. 160, 283 (1945). 17A. M. Clark, Nature 168, 562 (1949). ~8F. J. W. Roughton, J. Physiol. (London) 107, 12P (1948). 19D. A. Scott and J. R. Mendive, J. Biol. Chem. la9, 661 (1941); 140, 445 (1941).
[148]
CARBONm ANHYDRASE (PLANT AND ANIMAL)
839
vs. t will give a true value for the velocity constant (k~) independent of the inactivation of the enzyme. 4,~7 Clark 4,~7 has made use of this method to distinguish between stabilizations and activations. By calculating the percentage decrease (D) in the value of k, during the first 100 seconds of reaction, the sensitivity of the preparation may be determined. In a similar way, the degree of stabilization (S) is calculated as 100(1 - d/d ~) where d and d ~are the values of D in the presence and the absence of stabilizing or activating agents, respectively. 2. Co!orimeoric Method. Colorimetrie techniques are usefu) for rapid tests but have a restricted range regarding the conditions under which enzyme activity can be investigated. Owing to the relatively large inhibitions of the enzyme by CO~= in the original method of Brinkman 2° and Philpot and Philpot, 2~ a more reliable technique using Veronal buffer has been developed by Roughton and Booth s and is being used successfully in other laboratories ~,s3 (C. A. Mawson, personal communication). CO2-VERONAL INDICATORMETHOD. Veronal buffer (3 ml. of 0.022 M Veronal in 0.022 M Na salt, pH 7.95), three drops of bromothymol blue, and 2.3 ml. of distilled water (or 0.3 ml. of enzyme and 2.0 ml. of water) are mixed in a 15-ml. stoppered weighing bottle and placed in ice water for 15 minutes. Five milliliters of ice-cold water saturated with COs (0.071 M) is added anaerobically from a long nozzled all-glass syringe. The time is observed for the pH to drop to 6.3, determined with the aid of a bromothymol standard at this pH. The solutions are mixed in less than 1 second without bubbling or loss of COs. The control time averages 90 seconds, compared to about 64 seconds in the presence of 14.3 p.p.m, of a crude chloroform preparation (Step 2, p. 841). DEFINITION OF UNIT. The enzyme unit E.U. -
to - -
t
t
, where t and
to
are time of reaction in the presence and the absence of catalyst, respectively. Since the velocity constant of the uncatalyzed reaction (k~) calculated from the experimental data was found to be 0.0022 mole/1./sec., which agrees well with the accepted value of 0.0021 at 0 °, the validity of the method is established. Accordingly, Roughton and Booth ~ calculated the rate of enzymic hydration of COs in moles per liter per second as follows, allowing a period of 1 second for mixing. R
--
Ro
Ro
to - -
t-
t
1
2~ R. Brinkman, J. Physiol. (London) 80, 171 (1933). 21 F. J. Philpot and J. St. L. Phflpot, Biochem. J. 80, 2191 (1936). 22 K. M. Wilbur and W. G. Anderson, J. Biol. Chem. 176, 147 (1948). .,3 E. R. Trethewie and A. J. Day, Australian J. Exptl. Biol. Med. Sc/. 27, 429 (1949).
840
RESPIRATORY ENZYMES
[148]
where R and R0 are the rates in the presence and the absence of catalyst, respectively. Ro = k~ [average COs] Then Rate = R - R 0 -
((tt 0- - t 1) ) ]c, [average COs]
since k~ = 0.00225 mole/k/see, and [average COs] = 0.0307 mole under the experimental conditions. Therefore, Rate of enzymic hydration of COs
_ (to -- t)
(t - 1) 6.91 X 10-5 mole/1./sec.
The figures for the all-liquid method are approximately 70 % of those expected from manometric data. The discrepancy of 30% may be put down to the inhibitory action of the indicator and experimental error. Wilbur and Anderson 22 have used an automatic syringe to introduce the COs, and the pH changes are measured electrometrically. RAPID-FLOW COLORIMETmCM~.THOD. All methods of assay heretofore discussed are subject to a mixing error and are restricted to the use of low concentration of enzyme owing to diffusion limitations. Accordingly, the development of the Hartridge-Roughton rapid-flow technique, which minimizes these errors and photoelectrically records the color change of a pH indicator, opens up a wider range of conditions under which the enzyme can be studied. Clark and Perrin 4 have used the rapid-mix, quickstop method of Chance. 24 A saturated solution of COs is rapidly mixed at room temperature with Veronal buffer, pH 8.6 and pK 8.0, in a modified Millikan 25 microapparatus. The flow through a capillary tube is stopped in milliseconds, and in the presence of phenol red the pH of the solution is recorded continuously as a function of time. Much higher temperatures and enzyme concentrations may be used in this technique. U s e of C a r b o n i c A n h y d r a s e as a B i o c h e m i c a l T o o l
In decarboxylation or other reactions where gaseous COs is produced, a question of some significance arises as to whether CO2 or HCOa- is the primary product. In systems rapidly producing COs into the gaseous phase, carbonic anhydrase will slow down the rate owing to the accelerated hydration of the COs. On the other hand, it is inferred that if HCO3is the primary product the rate would be initially accelerated. Krebs and Roughton ~4have shown that COs is the primary product of yeast carbox2~B. Chance, J. Franklin Inst. 229, 455 (1940). 25G. A. Millikan, Proc. Roy. Soc. (London) A155, 277 (1936).
[148]
CARBONIC ANHYDRASE (PLANT AND ANIMAL)
841
ylase and the urease reaction. Hansl and Waygood 26 have confirmed their findings and in addition have shown that C02 is the primary product of the plant pyruvic, glutamic, oxalacetic, and a-ketoglutaric decarboxylation systems. More recently, Conway and O'Malley ~7 have concluded from their theoretical and experimental treatments that HC03- may be produced concomitantly with CO2 in the latter two reactions. Source and Purification of the Enzyme Animal. Mammalian red blood cells are the richest source. Among other tissues, the pancreas, gastric mucosa, and kidney contain comparable amounts. The enzyme is absent from plasma and other body fluids with certain exceptions. 5,6 Many methods of purification have been described in the literature (see Van Goor 6) culminating in a crystalline preparation of ammonium carbonic anhydrase by Scott and Fisher. 2s In general, however, investigators have used one or all of three stages of purification, either lysed red cells or the crude chloroform preparation of Meldrum and Roughton 1,5 or highly purified enzymes prepared by the particular method of the investigator. Since many laboratories have successfully used the highly purified preparations of Keilin and Mann 29 and because of its relative simplicity the details of their procedure and those of the crude preparations are described. Step 1. Lysed Red Cells. The red blood cells of defibrinated ox blood are centrifuged from the serum and washed three times with an equal volume of 0.9 % NaC1. The washed cells are hemolyzed with an equal or half-volume of distilled water. Step 2. Crude Chloroform Preparation.1 To 10 ml. of lysed red cells are quickly added 8 ml. of 40 % ethanol and 4 ml. of chloroform. The mixture is stirred in a centrifuge tube for 3 minutes to a thin sludge and allowed to stand for 20 minutes. After centrifugation for 10 minutes at 3500 r.p.m., a three-phase system is formed consisting of a supernatant layer of enzyme solution, a central layer of denatured protein, and a bottom layer of chloroform. Step 3. Method I I of Keilin and Mann. 29 Dialysis of Alcohol-Chloroform Extract. The filtered enzyme solution is dialyzed for 24 hours against running water. Step 4. Total Precipitation with Ammonium Sulfate. The fluid is saturated with (NH4)2S04, and the precipitate after filtering on a Btichner 36 N. 37 E. 3s D. 39 D.
Hansl and E. R. Waygood, Can. J. Botany 30, 306 (1952). J. Conway and E. O'Malley, Biochem. J. 54, 154 (1953). A. Scott and A. M. Fisher, J. Biol. Chem. 144, 371 (1942) ; Nature 153, 711 (1944). Keilin and T. Mann, Biochem. J. 34, 1163 (1940).
842
RESPIRATORY ENZYMES
[148]
funnel is dissolved in a small a m o u n t of water, dialyzed against running tap water and centrifuged. Step 5. Fractional Precipitation with Ammonium Sulfate. T h e supern a t a n t fluid is saturated 4 5 % with respect to (NH4)2S04 and filtered. The filtrate is completely saturated with (NH4)2SO4 and, after filtering, the precipitate is dissolved in water and dialyzed. Step 6. Purification with Alumina C~ Gel. T h e solution is treated with three successive 5-ml. ( = 100 mg.) portions of alumina C~ gel at p H 6.8 and centrifuged, the cakes being discarded each time. Step 7. Fractional Precipitation with Ammonium Sulfate. The above solution is made 50 % saturated with (NH4)2SO4 and, after filtering, completely saturated. The precipitate is dissolved in water and dialyzed against distilled water until free from salt. SUMMARY OF PURIFICATION PROCEDURE a
Step
Total volume, ml.
Absolute E.U.
1 2-3 4 5 6-7
2000 2250 110 85 50
4,000,0002 1,488,000 1,100,000 765,000 500,000
M1./E.U.
Specific activity, E.U./mg.
Zn, %
Recovery, %
0.0005 0.0015 0.0001 0.0001 0.0001
14.3 384 588 830-1000 2220
0.C024 --0.15-0.17 0.33
-37 27 19.5 12
Method II [D. Keilin and T. Mann, Biochem. J. 34, 1163 (1940)]. b Calculated from Step 1, Method I [D. Keflin and T. Mann, Biochem. J. 34, 1163 (1940)]. Plant. Bradfield 8° and Waygood and Clendenning TM found the enzyme to be ~ocalized only in the cytoplasm of leaf tissue. The enzyme is absent from root tissue2 ° Sirois and Waygood (unpublished) recommend spinach (Spinacea oleracea) as a source of the enzyme, since it has the highest specific activity of all plants tested, b u t m a n y other sources are available, n o t a b l y leaves of New Zealand spinach (Tetragonia expansa), nast u r t i u m (Tropaeolum majus), lamb's quarters (Chenopodium album), and beet (Beta vulgaris). 12.13,30.31 Sibly and Wood 3~ have purified the plant enzyme b y the m e t h o d of Keilin and Mann, b u t the percentage recovery was low. We have found t h a t it is essential to protect the plant enzyme with cysteine at all stages during purification, and the following procedure is recommended. Midribs are removed from spinach leaves, and 200 g. is ground in a m o r t a r with 50 ml. of cysteine (final concentration 0.005 M). The brei is 30j. R. G. Bradfield, Nature 159, 467 (1947). sz p. M. Sibly and J. G. Wood, Australian J. Sci. Research B4, 500 (1951).
[148]
CARBONIC ANHYDRASE (PLANT AND ANIMAL)
843
pressed through nylon, and the crude juice is centrifuged for 20 minutes at 15,000 X g. The supernatant fluid (200 ml., A = 15 E.U./mg.) is shaken with 75 g. of Ca3(P04)2 and centrifuged. The cake is eluted with 0.2 M phosphate, pH 6.6, in 0.005 M cysteine. After an initial precipitation with 30 % alcohol, the enzyme is totally precipitated at a concentration of 75% ethanol. The white powder is completely soluble in water. The solution, when dialyzed against 0.005 M cysteine and lyophilized, yields a preparation containing 300 E.U./mg. and 0.05 % Zn with no other metals. The enzyme unit is that defined by Waygood and Clendenning. 12
Properties Specificity. Carbonic anhydrase is specific for the reaction CO2 + H20 ,~- HsCO3. It is doubtful if it catalyzes the reaction COs + O H - --* HC03- which predominates above pH 11.0 and is significant between pH 8 and 10.1,~,3,7 Since the latter occurs as a primary reaction succeeded by, or accompanying, the enzyme-catalyzed reaction, it may introduce errors in some colorimetric methods. There are numerous processes which are limited by the reaction COs + H20 ~---H~CO~, and their rates may be increased by the addition of enzyme, e.g., the deposition and solution of CaCO3, and the dissociation of carbamate into C02. 5,7 Stability. Crude chloroform preparations of the animal enzyme are stable for long periods in the dry state but are progressively inactivated in solution. Purer preparations are less stable. Plant carbonic anhydrase is stable in the dried form but deteriorates rapidly in solution. It is also much less stable during dialysis than the animal enzyme. Cysteine (0.005 to 0.01 M) protects the plant enzyme in solution and during dialysis. 30 Nature of the Enzymes. Carbonic anhydrase is a Zn-protein compound. ~9,32 Keilin and Mann's preparation ~9 and others contained 0.033% Zn, whereas Scott and Fisher's crystalline preparation 28 contained 0.02% Zn and had a molecular weight of 30,00023 The latter value corresponds to 1 atom of Zn per molecule. Our partially purified preparations of the plant enzyme contain 0.05 % Zn with no other metals. Sibly and Wood81 calculated ~hat their preparations of the plant enzyme contained 0.056 % Zn. The failure of dialysis to remove zinc from either animal 29 or plant carbonic anhydrase (Sirois and Waygood, unpublished) and the fact that Tupper et al. 34 have shown that Zn 6~ ions in the medium do not exchange a2 D. Keflin and T. Mann, Nature 144, 442 (1939). 33 M. L. Peterman and N~. ¥. ttakala, J. Biol. Chem, 14§, 701 (1942). 34 R. Tupper, R. W. E. Watts, and A. Wormalls, Bioehem. J. 50, 49 (1952).
844
RESPIRATORY ENZYMES
[148]
with the zinc moiety of the enzyme from blood indicates that the metal is firmly entrenched in the protein molecule. The evidence that we have been able to provide in favor of the view that plant carbonic anhydrase is a Zn-protein is as follows. The Zn content of the purified enzyme preparation increases proportionately with increasing specific activity during prolonged dialysis against cysteine. No other metals are present, but the possibility that Zn is present in a nonspecific protein is not excluded. Inhibition by cyanide and azide (see later) is also indicative of the metalloprotein nature of the enzyme in plants. The kinetics of the enzyme from blood have been studied in detail by Roughtou and Booth 1 using the refined boat method. Byerrum and Lucas 1~ give some kinetic data for the enzyme from plants. Since it is doubtful if all the necessary precautions were observed in their experiments, the results await confirmation. Effect of Enzyme Concentration. Provided that diffusion is not a limiting factor, there is a linear relationship between activity and concentration of crude preparations of the enzyme. When diffusion is limiting, the apparent rate may be corrected by Roughton's formula (see above). In the case of highly purified preparations a dilution effect may occur, giving a sigmoid relationship between activity and enzyme concentration. As pointed out earlier, this may be overcome by stabilizing the enzyme. Effect of Substrate Concentration. The value of the Michaelis constant (Kin) for the crude chloroform preparation is 0.009 M C02 (+0.001 M) at 0°. 1 The value is independent of pH, although Kiese ~5 has reported values at 1° of 0.0012 M at pH 7.4, rising to 0.0022 M at pH 9.3 for a highly purified enzyme. It is not certain whether Kiese took all the necessary precautions. Calculations from Leiner's data for a stabilized highly purified enzyme gives K~ = 0.0075 M + 20%. T M Turnover Number. The rapidity of the reaction is shown by calculations from the data of Roughton et al. 1,~ which gives a maximum turnover number of the order of 9.6 X 107 at 0 ° and pH 7.3 for the enzyme from blood. The highest turnover number previously recorded is 5 X 106 for catalase. The value of 1.8 × 108 for the velocity constant of combination of enzyme and substrate is also indicative of the rapid reaction and is of the same order as that for catalase. Effect of pH. The activity of the enzyme from blood is at a minimum at pH 6.5 and gradually increases to fivefold the activity at pH 10.0. There is evidence that the activity is increased below pH 6.0.1 The isoelectric point is at pH 5.3. 3~ 35M. Kiese, Biochem. Z. 307, 400 (1941). 36M. Leiner, Biochem. Z. 315, 31 (1943).
[148]
CARBONIC ANHYDRASE (PLANT AND ANIMAL)
845
Effect of Temperature. Recently corrected values give Q10 = 2.9 for the nonenzymic rate and Q10 - 1.4 for the enzymic rate (over-all rate nonenzymic rate). TMA plot of the log activity against l I T shows a linear relationship. Inhibitors. Heavy metal poisons--e.g., cyanide, azide, and sulfide-inhibit the activity of carbonic anhydrase from blood to the extent of 50% at concentrations ranging from 10`4 to 10-e M. 7 Whereas the activity of the plant enzyme is strongly inhibited by azide (70 to 90 % at 10-3 M), 12,3° cyanide, according to Bradfield 3° and Sibly and Wood, 31 only inhibits at much higher concentrations (65 to 75 % at 10-3 M; no inhibition at 10-3 M). However, Waygood and Clendenning ~2 have reported 50 to 75% inhibition of the activity of a crude and dialyzed enzyme preparation from Tradescantia fluminensis by 10-3 M HCN. These values have been confirmed by Sirois and Waygood (unpublished) using stabilized semipurified preparations from spinach leaves. The activity of a crude spinach preparation is inhibited 50% by 2.4 X 10-3 M HCN, whereas the activity of the purified enzyme is inhibited 50% by 1.25 X 10-3 M HCN. Nevertheless, the relative insensitivity of the plant enzyme to cyanide, compared with the enzyme from blood, constitutes one of the more important differences between the two enzymes. The markedly inhibitory effect of low concentrations of sulfanilamide and related substances containing the --S02NH2 group has been extensively investigated. 37-4° N-Substituted sulfonamides are ineffective.89 Thiophene-2-sulfonamide and p-sulfonamidobenzoic acid are eight to twelve times as effective as sulfanilamide which inhibits the activity of the enzyme from animals 50% at 10-7 M. 3s,39 A new and more specific inhibitor and less toxic drug, Diamox 6063 (2-acetylamino-l,3,4-thiadiazole-5-sulfonamide) has recently been reported. 4° In contrast, Bradfield a° and Sibly and Wood ~ found that sulfanilamide at a concentration of 10-5 M caused little or no inhibition of the activity of the plant enzyme from two different sources, thus indicating another important difference between the animal and the plant enzyme. The inhibitory effect of anions on the activity of the enzyme from blood in decreasing order I - > NO3- > Br- > C1- > acetate > SO4--. The effect of C1- is especially interesting, since it appears to cause inactivation by forming an inactive complex with the enzyme. The divalent ion COs-- also causes inhibition of enzyme activity, especially at high pH values.
37T. Mann and D. Keilin, Nature 146, 164 (1940). as H. W. Davenport, J. Biol. Chem. 168, 567 (1945). 39H. A. Krebs, Biochem. J. 48, 525 (1948). 40T. H. Maren, Trans. N. Y. Acad. Sci. 16, 53 (1952).
846
RESPIRATORY ENZYMES
[148]
Activators, Stabilizers and Protectors. Claims of activating substances reported in the literature are now suspect, owing to the questionability of the methods used. Clark and Perrin 4 have shown convincingly that apparent activation of the enzyme by certain substances, e.g., boiled horse plasma and glutathione, is due to the restoration of the activity of purified enzymes, lost by adsorption or the effect of impurities. No activation occurs when the enzymes are stabilized by 0.05 % peptone; thus these substances are acting as stabilizers. Indeed the stabilizing power of plasma obscured a weak inhibitory effect.4 Scott and Mendive 19 report a number of stabilizers of which horse serum (1:40) and 0.05% peptone are the most effective. We have used 0.025% gelatin to stabilize the plant enzyme. One of the most important differences between plant and animal carbonic anhydrase is the dependency of the former on free - - S H groups. Thus Bradfield 3° discovered that cysteine efficiently protected the plant carbonic anhydrase while standing or during dialysis. Furthermore, the enzyme was completely inhibited by 5 X 10-4 M p-mercuriochlorobenzoate, which has little effect on animal carbonic anhydrase. Sibly and Wood 31 have confirmed these findings and also demonstrated complete inhibition of activity by 5 X 10-3 M arsenite, another mercaptide-forming compound. The inhibition caused by both these substances could be reversed by cysteine o r glutathione. Iodoacetate does not cause inhibition, but the presence of sulfhydryl groups on plant carbonic anhydrase has been confirmed polarographically. 3~ The important papers of the Japanese workers, Kondo et al., 4~ on the kinetics of spinach carbonic anhydrase were not available to the author at the time of writing. They have reported the isolation of a metal-free electrophoretically homogeneous protein, stabilized by 0.1 M NaC1 having carbonic anhydrase activity. 41 K. Kondo, H. Chiba, and H. Kawai, Bull. Research Inst. Food Sci. Kyoto Univ. 8, 17-27 (1952); 13, 1-59 (1954). (English abstracts.)