TIIE JOURNAL OP BIOLOGICAL CI~EMISTRY Vol. 243, No. 18, Issue of September 25, pp. 4832-4841, 1968

Printed in U.S.A.

Purification and Properties of Horse Erythrocyte

Carbonic Anhydrases”

(Received for publication, May 9, 19 8)

ANNA J. FURTH~

From the Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138

SUMMARY

Two distinct components of carbonic anhydrase (EC 4.2.1.1) have been purified from horse red cells. In the isolation procedure described, the large excess of hemoglobin was removed by precipitation in ammonium sulfate, and the two carbonic anhydrase proteins were then separated by DEAE-Sephadex chromatography. This separation was facilitated by the remarkably high isoelectric point of the basic component, which appeared to have a net positive charge even at pH 10.0.

This basic component, which was designated enzyme C, constituted only 36% of the total carbonic anhydrase protein but had a high specific activity; with the use of the Wilbur- Anderson assay for CO2 hydration, this was in the order of 40,000 units per mg. The major protein, designated enzyme B, had a much lower specific activity, in the order of 4,000 units per mg.

The extinction coefficients and sZO,ur values of the purified B and C proteins were measured, as well as the K, and V,,, values for their hydrolysis of p-nitrophenyl acetate. The amino acid compositions and the optical rotatory dispersion spectra are also reported.

The two components of horse carbonic anhydrase de- scribed here give a further example of the unusual type of polymorphism already seen with primate carbonic anhydrase. This suggests that it may be a characteristic of the enzyme from the blood of a variety of mammalian species. The salient features seem to be firstly, the presence of two chemi- cally distinct proteins catalyzing the same reaction at differ- ent rates, and secondly, the much greater abundance of the less active form.

The isolation of two horse enzymes brings to seven the number of carbonic anhydrases now studied in a highly puri- fied state. These are classified as either high specific ac- tivity forms (human, rhesus monkey, and horse C enzymes,

* This research was supported by Grant HE-03169-10 from the United Sta.tes Public IIealth Service and Grants GB-1255 and GB- 58% from the National Science Foundation.

: Present address, Department of Pharmacology, University of Oxford, Engla.nd. Reprints may be requested either from the author at her present address or from Dr. J. T. IGdsall at the laboratory where the work was done.

bovine B enzyme) or low specific activity forms (human, rhesus monkey, and horse B enzymes), and the proteins in each category are then compared in terms of amino acid com- position, kinetics, and opticalrotatory dispersion.

Carbonic anhydrase (EC 4.2.1.1, COZ + HpO s H+ + HCO,) has been obtained in highly purified form from human (l), rhesus monkey (a), and bovine red cells (3, 4). A partial purification of the enzyme from other primate bloods has been achieved by Tashian (5) and by Tashian, Riggs, and Yu (6), while Tappan, Jacey, and Boyden (7) have report,ed a chromatographic separation of two guinea pig carbonic anhydrases.

All the highly purified enzymes have a molecular weight of approximately 30,000 and contain 1 zinc atom per molecule. Although the major activity of the enzyme is the reversible hydration of COZ, it has recently been shown to catalyze the hydrolysis of esters (5, 8-11) and the hydration of aldehydes (12).

In most of the species studied, carbonic anhydrase shows polymorphism of an unusual type. There are two forms of the enzyme varying widely in specific activity, and these are present in unequal amounts, the less reactive form being the more abundant. The difference in activity is very striking. In primates, for example, the C component is some 3 times more active than the B component. Presumably, such a marked dif- ference in activity between two apparently similar proteins is re- lated to underlying differences in structure at the active site. A more detailed comparison of high and low specific activity forms would obviously be of interest, but so far only the primate enzymes are available in a sufficient state of purity. The bo- vine enzyme has also been highly purified, and has two com- ponents, but these are almost identical in composition, and both have the same high specific activity (4). There have been re- ports of another low specific activity bovine enzyme (13), but this does not seem t,o be present t.o any great. extent.

The present study of horse carbonic anhydrase establishes another example of a polymorphism similar to that shown by the primate enzymes. Two components of very different specific activity were isolated. These could be clearly distinguished by

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their composition and, here again, the low specific activity form was the more abundant.

The same pattern appeared consistently, even with a variety of purification procedures. The best. method took advantage of the low solubility of horse hemoglobin, which could be removed by precipit,ation with ammonium sulfate. The resulting crude carbonic anhydrase was then separated into two components by ion exchange chromatography. Some of the physical and enzymatic properties of these purified proteins were then studied, and the two components were compared with the corresponding high or low specific activity forms of other species.

EXPERIMENTAL PROCEDURE

Preparation of Hemolysate

Most of the horse red cells were from whole blood, which we were able to obtain by the kindness of the Massachusetts Public Health Department, Biological Laboratories, Boston. The blood was collected in 0.1 M EDTA at neutral pH to prevent coagulation, and the cells were centrifuged down and washed in 0.9% NaCl as described by Rickli et al. (14).

Some preparations were also made from the blood clots re- maining after serum had been removed for preparation of anti- toxins. In this procedure whole blood is allowed to clot at 4” over a period of 8 days, during which time the serum is slowly extruded. The clots are usually discarded, but we found that most of the red cells could be recovered from them still unlysed by squeezing the clots in cheesecloth. The extruded fluid was centrifuged at 2000 rpm for 20 min. The cells collected in the precipitate and were washed once in 0.97, NaCl. The carbonic anhydrase activity was normal, and the enzyme extracted from these clots appeared identical with that from fresh blood.

The packed cells were lysed by the addition of twice their volume of distilled water, and were dialyzed against distilled water overnight. This gave a hemolysate with a hemoglobin concentration of 10 g/100 ml.

Removal of Hemoglobin

Method 8-Batchwise adsorption on DEAE-Sephadex was the method used. The resin and the hemolysate were separately equilibrated with 0.05 M Tris-0.012 M HCl buffer at pH 8.7 (I), and a mixture consisting of 1 ml of packed resin for every 130 mg of hemoglobin was stirred overnight. The resin was removed by vacuum filtration, and the filtrate was concentrated as described below.

Method B-Denaturation with ethanol and chloroform was used. This method differed only slightly from the procedure described in Reference 14. The proportions used were 700 ml of hemolysate to 125 ml of ethanol and 150 ml of chloroform, and t,he procedure was modified by the addition of 50 ml of 1 M

phosphate buffer at pH 6.8 immediately before the addition of chloroform. This raised the ionic strength and reduced the amount of carbonic anhydrase lost by adsorption to denatured hemoglobin. The hemoglobin precipitate was washed in 0.1 M

phosphate buffer, and the combined washing and supernat,ant were then dialyzed against distilled water to remove traces of organic solvent.

Method C-The third method was removal of hemoglobin by precipitationin 50y0 saturated ammonium sulfate at 25”. One liter of hemolysate was adjusted to pH 6.8 (from pH 7.1) with

12 ml of 0.5 M HCI. Solid ammonium sulfate, 264 g, was added, and the temperature of the preparation was readjusted to 25” after the salt had dissolved. Precipitated hemoglobin was removed by centrifugation at 25” and 8000 rpm for 20 min. The resulting supernatant had a hemoglobin concentration of 0.3 g 100 ml and contained only 3% of the original hemoglobin. The carbonic anhydrase recovery was 67y0, and the yield could be further improved by washing the hemoglobin precipitate with half-saturated ammonium sulfate.

Concentration of Protein Solutions with Use

of Solid Ammonium Sulfate

The large volumes of dilute protein solution resulting from each of the above procedures could be rapidly reduced by use of solid ammonium sulfat,e, if the pH were first adjusted to 7 with concent,rated phosphate buffer. This was part.icularly important for solutions previously at high pH to prevent the release of free ammonia.

The protein solutions were poured into Visking cellophane dialysis sacs (diameter l+ inches) and the sacs were then placed in solid ammonium su1fat.e. Protein inside the sacs precipitated in the resulting saturated ammonium sulfate solution and could be collected by centrifugation for 40 min at 10,000 rpm.

DEAE-Sephadex Chromatography

This was performed on the crude material from which hemo- globin had been partially removed by one of the methods described. The quantity of hemoglobin present therefore varied, but samples containing up to 12 g total protein could be satisfactorily handled t’o yield a complete separation of B and C components, each free from hemoglobin. With such large amounts, adsorption was best achieved by keeping the concentra- tion of hemoglobin in the starting material below 4 g/100 ml, and by applying the sample to the column at a low flow rate.

A column measuring 4 x 50 cm was packed with DEAE-Sepha- dex A-50 resin, which had been equilibrated in 0.05 M Tris-0.012 M KC1 buffer at pH 8.7, as described in Reference 1. All samples were dialyzed extensively against starting buffer, and then applied to the column at a flow rate of 25 ml per hour. The column was then eluted with the starting buffer at a flow rate of 70 ml per hour. The unretarded C enzyme was collected in the first 200 ml, and t,he column was washed with a further 500 ml of the starting buffer before the second buffer, 0.05 M Tris-0.012 M HCl-0.02 M NaCl at pH 8.7, was applied to collect the B enzyme.

For storage, the pH of the two fractions was reduced to 7.0 with 1 M KaH2P04. The solutions were concentrated with solid ammonium sulfate, and the precipitated proteins were stored in the resulting saturated ammonium sulfate solutions.

Sulfoethyl-Sephadex Chromatography

This was performed with the bead form of the resin in 0.1 M phosphate buffer at pH 6.3.

The resin was allowed to swell in the buffer, was washed several times to remove fines, and then was equilibrated with fresh buffer until the pH and ionic strength of the supernatant over the resin remained constant. A column, 1.6 X 50 cm, was packed at 5” and equilibrated for 24 hours under a pressure head of 40 cm.

The sample, containing 20 to 25 mg of protein, was concen- trated to about 3 ml by vacuum dialysis, and dialyzed exten

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4834 Horse Erythrocyte Carbonic Anhydrases Vol. 243, No. 18

sively against starting buffer before being applied to the column. The column was eluted at a flow rate of 13 ml per hour, and frac- tions were collected every 12 min.

Protein Concentration

Protein concentration was measured by the absorbance at 280 rnp, taking A:F0 values of 13.6 for enzyme B and 13.4 for enzyme C. These figures were calculated after the amino acid composition had been determined, by the method described in Reference 14. The absorbance of a protein solution was read, and the molar concentration of protein present was then deter- mined, with the aid of the amino acid analyzer, by measuring the amount of basic amino acids released on hydrolysis. Nineteen moles of lysine were then known to be equivalent to 1 mole of both B and C proteins, so that molecular extinction coefficients could then be calculated. These were converted to A:TO values, taking the molecular weight as 29,007 for B and 27,918 for C (see Table III below).

Assay of Carbonic Anhydrase and Esterase Activity

The carbonic anhydrase activity was determined by the Wilbur-Anderson method, as described in Reference 14.

The esterase activity was determined, with p-nitrophenyl acetate as substrate, at 25”, in 0.01 M diethylmalonic acid buffer, containing 0.67 y0 acetone by volume (1, 11). The velocity was calculated as the moles of substrate hydrolyzed per mole of en zyme per min. The amount of p-nitrophenol released was cal- culated from the absorbance at 348 mp, where the increment in extinction coefficient due to ester hydrolysis is 5.0 X lo3 M-’

cm-’ (11). The K, an d V,,, values were calculated from a plot of S/v against S, with the method of least mean squares. Here S, the initial substrate concentration, was varied in the range of 0.25 t,o 1.25 mM.

Starch Gel Electrophoresis

This was performed at pH 8.6 as in Reference 14 with 0.2 M

NaOH-0.2 M boric acid for the electrode vessel, 0.02 M NaOH- 0.02 M boric acid-O.04 M NaCl for the bridge vessel, and 0.02 M

NaOH-0.02 M boric acid for the gel. The potential gradient was 14.8 volts per cm and the current, 8.0 ma.

For runs at pH 10.0, the pH was raised by addition of 0.2 M

NaOH, or 0.02 M NaOH where appropriate. For runs at pH 5.5 to 6.5, 0.1 M phosphate buffer was used for the electrode vessel and 0.01 M phosphate for the gel. The bridge vessel was omitted.

Amino Acid Analyses

These were performed by the method of Spackman, Stein, and Moore (15). The internal standards, 0.5 mrvr P-thienylala- nine and a-amino-/3-guanidopropionic acid, were added to the 0.2 M citrate buffer at pH 2 in order to correlate analyses on the long and short columns (16). Duplicate samples were hydro- lyzed for periods of 24, 48, and 72 hours. Values for ammonia, serine, and threonine were obtained by extrapolation to zero time, and valine values were taken from the sample hydrolyzed for 72 hours. Cysteine was determined as cysteic acid, after performic acid oxidation (17). Tryptophan was determined spectrophotometrically by the methods of Goodwin and Morton (18) and Bencze and Schmid (19).

Ultracentrifuge Runs

These were performed on a Spinco model E analytical ultra- centrifuge. The partial specific volumes needed to determine the ~~0,~ values were calculated from the amino acid composition cw

Optical Rotatory Dispersion

Measurements of optical rotatory dispersion were performed on the Cary model 60 recording spectropolarimeter (21). The protein samples were dialyzed for 24 hours against 0.02 M phos- phate buffer at pH 7. The [m’] values were calculated by taking a mean residue weight of 109 for enzyme B and 110 for enzyme C.

RESULTS

Nomenclature-The more basic of the two components, which was unretarded on DEAE-Sephadex, was designated enzyme C, and the more acidic, which was eluted only on raising the ionic strength, was designated enzyme B. The reasons for using this terminology are explained in the “Discussion.”

Preparation of Crude Carbonic Anhydrase-A major problem in the purification of erythrocyte carbonic anhydrase is the re- moval of hemoglobin, which is present in about loo-fold excess over the enzymes. Since the recovery of carbonic anhydrase is always less than lOO%, there is a danger that one component may be overlooked, particularly if this is of low specific activity. This was especially true of horse carbonic anhydrase B which, as it later appeared, accounted for only 15y0 of the total activit’y. Hence, an enzyme yield of 80% during the removal of hemoglo- bin could consist of 94% recovery of component C and zero re- covery of component 13. Further, the C enzyme has a very high isoelectric point (see below), so that on ion exchange chromatog- raphy it separates from hemoglobin much more readily than the B enzyme. For these reasons a variety of approaches was used in preparing the crude enzyme. The results of these are com- pared in Table I.

For removal of hemoglobin, the most efficient methods were those using ethanol and chloroform, or ammonium sulfate. The use of DEAE-Sephadex to remove hemoglobin by adsorpt.ion was as successful as with the human enzymes (I), but in order to minimize the danger of preferential loss of one component, the amount of resin added here was kept low, so that only 607, of the hemoglobin was removed at this stage.

The relative proportions of B and C components were the same, regardless of the purification procedure used, and most of the work described below employed the ammonium sulfate method.

Separation of B and C Enzymes on DEAE-Sephadex-The crude carbonic anhydrase still contained considerable quantities of hemoglobin, whatever the method of preparation. It was pos- sible to remove this and to separate the B and C forms on a single run, with the use of a stepwise elution as described under “Experimental Procedure.” The highly basic C enzyme emerged in the void volume of the column, while the B enzyme was ad- sorbed to the resin and could be eluted by adding 0.02 M sodium chloride to the Tris-chloride buffer. Under these conditions the hemoglobin still remained firmly bound to the resin.

A typical elution diagram is shown in Fig. 1. It was not possible to calculate recoveries in terms of protein, since the large amounts of hemoglobin in the starting material obscured

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the absorbance at 280 mp, but the total protein emerging from the column could be computed from Asso readings on the eluate, with an A:2 value of 13.4 for C and 13.6 for B. The results of a typical run, given in Table II, show that B is the major eompo- nent in terms of protein. The relative proportions of the two components were independent of the method used for removal of hemoglobin (see Table I), so that there seems to have been no preferential loss of one component during any of t,he three pro- cedures. However, during the DEAE-Sephadex chromatog- raphy itself, there may be disproportionate recovery of the C enzyme, since none of this very basic component is likely to be adsorbed at pH 8.7. There was up to 90% recovery of the total activity applied to the column, but still higher yields could be obt#ained by washing the resin in buffer at high ionic strengths. The eluate then contained large quantities of hemoglobin, and this fraction was usually discarded. If all the activity still unac- counted for were due to loss of B component only, then the proportion of this in the starting material could be as high as 24yo in terms of activity, and much more in terms of protein.

The specific activities of the two components differed by a factor of nearly 10. This difference was found consistently, even though the absolute values varied somewhat. The reason for this variation was not clear. It did not appear to be related to the enzyme source (whole blood or blood clots) or to the method used for the removal of hemoglobin, although the ethanol- chloroform method tended to give highest specific activities at this stage.

The variation in specific activity made no difference in the pattern seen on starch gel electrophoresis at pH 8.6 (Fig. 2). The major component of the crude C enzyme was a highly basic protein, shown here as Band 1. Bands 2 and S represent small am0unt.s of two less basic fractions which often appeared with the C enzyme. The B enzyme was apparently homogeneous (Band 4) and contained no trace of the hemoglobin (Band 6) visible in the starting material.

Further Purification of Carbonic Anhydrase C-To obtain a homogeneous preparation of the C enzyme, it was necessary to remove the two minor C components (Bands d and S of Fig. 2). This was achieved by applying the crude C enzyme from DEAE- Sephadex chromatography to a column of sulfoethyl-Sephadex at pH 6.3.

Fig. 3. shows the results of a typical experiment, in which 90% of the enzymatic activity was recovered. Most of the active protein was retarded by the resin, and the minor components were eluted first. The different peaks could be identified with the electrophoretic components of the crude enzyme by com- bining the eluate to give Fractions II, III, and IV, as shown, and running aliquots on electrophoresis.

A typical gel electrophoretic pattern with the three C com- ponents is shown in Fig. 4. The slightly retarded Fraction II corresponded mainly to Band S of the crude preparation, while Fraction III cont.ained both Bands 1 and 6. These two latter components were incompletely separated on the column; the protein which yielded Band 2 emerged as a shoulder preceding the major protein in Fraction IV. Although neither of the minor components was electrophoretically pure, a homogeneous prepa- ration of the major C component could be obtained by this method, if the latter part of the main peak (designated Fraction IV) was collected as shown in Fig. 3. This fraction had the highest specific activity (up to 40,000 units per mg of protein) and

TABLE I Composition of crude carbonic anhydrase after removal of hemoglobin

by three different methods

The last two columns give the proportions of B and C enzymes present in each of the carbonic anhydrase preparations and were obtained after chromatography (see Fig. 1) of the crude prepara-

tions. The percentages are in terms of activities.

Method

% % DEAE-Sephadex ............. 60 75 16 84 Ethanol CHCI,. ............. 99.7 67 15 85 Ammonium sulfate ........... 98 77 15 85

/ 1

behaved as a homogeneous protein in the ultracentrifuge. The significance of the two minor C constituents is discussed later, and all further studies on the C enzyme refer to the homogeneous fast moving Band 1 isolated in Fraction IV.

Peak I, the fraction which was not retarded on sulfoethyl- Sephadex, is not shown on the gel. It normally contained only protein with no carbonic anhydrase activity, but in this case there were considerable quantities of enzyme B, since the DEAE- Sephadex separation had been incomplete.

Further Purification of Carbonic Anhydrase B-The specific activity of this component, after its isolation by DEAE-Sephadex chromatography, was only 4000 units per mg. Since this is rather less than that of other carbonic anhydrases, several attempts at further purification were made, even though at pH 8.6 the protein appeared electrophoretically homogeneous (Fig. 2).

Electrophoresis at pH 5.5, 6.0, and 6.5 again showed a homo- geneous protein, although at pH 5.5 the direction of migration was reversed. There was very little mobility at pH 6.0, which suggests that this is in the region of the isoelectric point. With the use of sulfoethyl-Sephadex chromatography with low ionic strength and a pH slightly above this (pH 6.5 in 0.01 M phosphate buffer, p = 0.015), it was possible to cause slight retardation of the protein. But here, as in all the chromatographic systems tried, there was only one major protein peak and no significant change in specific activity. The same single band was still seen on electrophoresis, and it must be concluded that the B enzyme, as separated from crude carbonic anhydrase by DEAE-Sephadex chromatography, is essentially a homogeneous protein.

Ultracentrifuge Data-The homogeneity of the two purified proteins was tested by sedimentation velocity runs. A single peak was obtained with each component. Partial specific volumes were found by calculation from amino acid composition (20) to be 0.725 for B and 0.732 for C, and with the use of these figures, the ~~0,~ values were 2.74 S for B (at 11.6 mg per ml) and 2.71 S for C (at 6.2 mg per ml).

Ultraviolet Absorption-The molar extinction coefficient, EM, at 280 rnp is important as a working standard to give some meas- ure of protein concentration. If the amino acid composition of the protein is known, eM can be calculated directly as described under “Experimental Procedure,” by measuring the Azso of a protein solution, hydrolyzing it, and then determining the molar concentration on the amino acid analyzer. The respective e,$#

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4836 Horse Erythrocyte Carbonic Anhydrases Vol. 243, No. 18

Ml of elude

FIG. 1. Elution of horse carbonic anhydrases B and C from DEAE-Sephadex. O-O, absorbance at 280 mr. A-A, carbonic anhydrase activity as determined by the Wilbur-Ander- son procedure. The eluting buffer was 0.05 M Tris-0.012 M HCl, pH 8.7, for enzyme C, and 0.05 Tris-0.012 M HCI-0.02 M NaCI, pH 8.7, for enzyme B.

TABLE II Separation of crude carbonic anhydrase into B and C components

by DEAE-Sephadex chromatography

Percentages of total protein and total activity were calculated as a percentage of the combined B and C eluates. Specific activity was expressed as Wilbur-Anderson units per mg of protein.

component

Percentage of total protein. . 64 36 Percentage of total activity.. 15 85 Specific activity. 3,600 34,800

B C

values were then 3.95 x lo4 M-I cm-i for B and 3.73 x lo4 M+ cm+ for C.

Molar absorptivity can also be calculated by Wetlaufer’s method (22) be assuming a contribution of 5500 from each tryptophan and 1340 from each tyrosine residue. The eM values found in this way (cdl calculated) should be lower than the ex- perimentally observed value, because of hyperchromism of the chromophores when they are embedded in the intact protein, but the ratios of EM observed to EM calculated were only 1.03 for both proteins.

iimino Acid Composition-Table III gives the amino acid composition of the two purified proteins. The lefthand column shows the average values obtained by taking phenylalanine as 11.0 residues per molecule. This gave the best near-integral values, and the calculated minimum molecular weights, assuming 1 atom of zinc per molecule, were then 29,007 for B and 27,918 for C. These amino acid compositions, of course, do not repre- sent exact or final values for the more abundant residues.

Tryptophan values were estimated spectrophotometrically by the method of Goodwin and Morton (18). Some calculations were also made by the Bencze and Schmid method (19). For the B enzyme, both methods gave a tyrosine to tryptophan ratio of 1.58, but with the C enzyme the ratio obtained was 1.35 by the Goodwin and Morton method and 0.76 by the Bencze and Schmid method. The former figure gives 5 tryptophans per molecule, and a calculated EM value for the C enzyme of 3.71 x 104 M-' cm*. This is in good agreement with the experi-

mental value of 3.73 X lo4 M-l cm-r, whereas the Bencze and Schmid ratio would give 9 tryptophans per molecule and a cal-

culated EM value of 5.9 X lo4 M-I cm-l. Similar discrepancies between these two spectrophotometric methods have been found before (23), and it appears that where the apparent tyrosine to, tryptophan ratio is less than unity, there is a tendency for the tryptophan content to be overestimated with the use of the, Bencze and Schmid method.

An interesting feature of the amino acid composition of the B protein is the presence of 2 cysteine residues. Up to now, no more than a single -SH group has been found in any carbonic anhydrase protein, so that the question of disulfide bond forma- tion has not arisen. When the B enzyme was treated with 0.2 M p-mercaptoethylamine, there was no change in activity, which suggests that both cysteines are fully reduced in t’he native enzyme.

Zsoelectric Point-In testing the homogeneity of the B enzyme, starch gel electrophoresis was performed at pH 5.5, 6.0, and 6.5. At pH 6.0 the protein had very little mobility, while at pH 5.5. it traveled toward the cathode and at pH 6.5 toward the anode. The isoelectric point is therefore close to pH 6, which is in the same region as pH 5.7 for the human B enzyme (14).

In contrast, the horse C enzyme was far more basic than other carbonic anhydrase proteins. At pH 8.7 it migrated rapidly toward the cathode, ahead of’bther basic proteins such as lyso- zyme, which has an isoelectric point of 10.8 (24). There was little change in mobility when the pH of t,he gel was raised to 10.0, which is to be expected if only lysine residues (pK 10.5) are titrating in this region. Once the amino acid composition of the protein is known, the reason for its very basic character becomes

3 3 3

4 4

5 5

0 + FIG. 2. Starch gel electrophoresis of fractions obtained from

DEAE-Sephadex chromatography of crude hemolysate. B and C are samples from the two carbonic anhydrase peaks similarly labeled in Fig. 1. HEM. is the crude hemolysate applied to the column, and contains at least five electrophoretic components. Band 1 is the major component of carbonic anhydrase C; Bands 2 and 3 are minor components of enzyme C and may be artifacts (see text) ; Band 4 is carbonic anhydrase B; Band 5 is hemoglobin. The electrophoresis was run in borate buffer at pH 8.6.

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clear. The large number of amide groups (Table III) must result in the masking of all but 21 of the total 53 p- and y- carboxyl groups. With the use of the pK values for the free amino acids taken from Reference 20, arginine and lysine should -give a total of 23.7 positive charges at pH 10.0. If this is bal- anced by only 21 ionized carboxyl groups, there will still be a net positive charge of 2.7 at pH 10. Hence, the amino acid composition alone appears sufficient to explain the unusually basic character of this protein.

Enzymatic Activity-Both human and rhesus monkey carbonic -anhydrases consist of two components with widely different

I / I I 1 I ( I (

0.71 i7

2 0.6

3

‘r 8 04

c 2

05 L 0.3 b B 0.2 Q

0.1

0 0 40 80 120 160 200 240

MI. of elude

FIG. 3. Sulfoethyl- Sephadex chromatography of carbonic anhydrase C. O-O, absorbance at 280 rnp. A-A, car- bonic anhydrase activity as determined by the Wilbur-Anderson procedure. The eluting buffer was 0.1 M phosphate at pH 6.3, and the starting mat.erial was a sample of carbonic anhydrase C prepared by DEAE-Sephadex chromatography similar to that of Fig. 1. Vertical lines indicate the pooling of eluted fractions. Fraction I consisted mainly of Enzyme B which had been incom- pletely separated in the previous step; Fractions II and III con- tained minor components of the C enzyme, Fraction III being heterogeneous (see Fig. 4); Fract,ion IV gave a homogeneous preparation of the major component of the C enzyme.

(a> II III m

FIG. 4. Diagram of starch gel electrophoresis of fract,ions ob- tained after sulfoethyl-Sephadex chromatography of carbonic anhydrase C. Fractions II to IV refer to the pooled fractions indicated in the elution diagram of Fig. 3. Sample a was the ma- terial applied to the column, and contained the same Bands 1 to 4 described under Fig. 2. The electrophoresis was run in borate buffer at pH 8.6.

TABLE III Amino acid composition of horse carbonic anhydrase B and C Hydrolysis was carried out for periods of 24, 48, and 72 hours.

Data were calculated on the assumption that the proteins both contained 11 phenylalanine residues per molecule. The figures are the average of three independent determinations on each protein. The calculated molecular weight equals the sum of the amino acid residues plus 1 zinc plus 1 HzO.

Lys .................

His .................. Arg ................. Amide NH,. ......... Asp ................. Glu ................. Gly ................. Ala. ................. Val .................. Leu ................ Ile .................. Pro ........... Ser. ................. Thr ................ cys ..................

I Met ................. Phe ................. Tyr ................ Trp .................

Total no. of residues. Molecular weight. ..

18.5 9.7 5.2

(22.4) 32.3 25.4 23.1 14.9 19.8 21.4

9.2 17.5 27.9 12.1

2.5

19 10

(2:) 32 25 23 15

20 21 9

18 28 12

2

2.0 2

11.0 I1 8.0 8 5.2 5

29,

265 7

Carbonic anhydrase B residues per molecule

Nearest integer

I -

Carbonic anhydrase C residues per molecule

Found

19.1

12.0 9.0

(32.0)

26.9 25.9 23.2

16.9 18.7 21.6

6.9 15.7 18.1 12.0

1.3

1.4 11.0 7.0 5.1

27,91:

Nearest integer

19 12

(392) 27 26 23

17

19 22

7

16 18 12

1

1 11

7 5

252

s

specific activities, and the horse enzyme shows the same features. When the COz activity was measured by the Wilbur-Anderson method, there was some variation in absolute values, the specific activity being in the order of 40,000 units per mg of protein for the C enzyme and 4,000 units per mg for the B enzyme. The ratio of activities was always constant,, the C enzyme being 10 times more active than the B enzyme under these conditions.

A more reliable method of assay makes use of the weak esterase activity of carbonic anhydrase, which occurs in primate and bovine enzymes, even after extensive purification, and which, like the COZ activity, is strongly inhibited by acetazolamide. Al- though this is probably not the primary function of the enzyme, it seemed reasonable to use the reaction to compare the activities of horse B and C enzymes in a more quantitative way. Assays were performed as described under “Experimental Procedure.” The esterase activity was considerable, being in the same range as that of other carbonic anhydrases. Again, there was a IO-fold difference in the activities of B and C components, a typical result being 5.2 and 51.5 moles of substrate hydrolyzed per mole of enzyme per min, respectively (pH 7.6 and 1 mM substrate con- centration). As with the other carbonic anhydrases, this activity could be distinguished from that of other esterases by its marked susceptibility to inhibition by acetazolamide, a potent inhibitor of the COZ activity. At an inhibitor concentration of 50 FM, there was no discernible ester hydrolysis.

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4838 Horse Erythrocyte Carbonic Anhydrases Vol. 243, No. 18

The pH profiles were both similar to those found for the hy- drolysis of p-nitrophenyl acetate by the human carbonic anhy- drases (11, 25). The activity rose steeply between pH 6 and 8 and flattened off at higher pH values. The V,,, and K, values at pH 7.6 were found by measuring velocities in the range of sub- strate concentrations between 0.25 and 1.5 mM. Both enzymes obeyed Michaelis-Menten kinetics over this range, and gave good linear plots of S/v against S. The K, values determined from this were 6.1 mM for enzyme B and 9.1 mM for enzyme C. The Vmax/J& values were 20.4 mine1 for B and 525 min-1 for C (E. is the total enzyme concentration).

Optical Rotatory Dispersion-carbonic anhydrases give a very characteristic rotatory dispersion pattern which is greatly in- fluenced by Cotton effects from the aromatic groups. The optical rotatory dispersion is therefore difficult to interpret in terms of known structures such as the a-helix or random coil, but is nonetheless useful in following changes in conformation. The horse proteins have somewhat fewer aromatic residues

TABLE IV

Amino acid composition of high speci$c activity and low speci$c activity carbonic anhydrases

Values are given to the nearest integer. Data for human enzymes are from Guidotti (see Reference 1); for rhesus monkey enzymes from (2); for bovine B enzyme from (4); for horse en- zymes from Table 3. For other data on the human enzymes, in close agreement with those given here, see References 4 and 26. Total basic residues equal the sum of lysine, histidine, and arginine residues. Total carboxylic residues are set eqrial to the sum of the aspartic and glutamic residues, plus one COOH- terminal group, minus the amide ammonia.

Residues per molecule to nearest integer

High specific activity

Lys ............... His ................

Arg ................ Amide NH,. ...... Asp ............... Glu ................ Gly ................ Ala ................ Val ................ Leu. ............... Ile ................. Pro ............... Ser. .............. Thr ................ cr. ...............

Met. .............. Phe ................ Tyr ................ Trp ................

Total residues. ....

Basic. .............

Carboxylic .........

Iumal 1 1 L&x- Bovim C k eY c B

Horse C

luma1 B

blon- .ey B

HOW B

25 12

($1 29 24 22

13 17 27

9 18

19 13

I

24

12 8

19 19 18

11 12 11

i& (3;) (2;)

32 27 31 24 26 22 20 23 16 17 17 19 20 19 17

26 22 20

5 7 9 20 16 17

16 18 30

15 12 14

0 1 I

18

9

7

30 26 22

12 14

24 10

16 18 I1

1

36 22

15 16

16 19 10

17 30

13 1

19

10

(2:) 32

25 23

15 20 21

9 18 28

12 2

1 2 3 1 2 1 2 13 11 11 11 11 10 11

9 7 8 7 8 9 8

7 7 7 5 6 7 5

266

44

33

255

44

263

39

33

252

40

22

259

36

28

256

34

265

34

36 -

Low specific activity

-

200 210 220 230 240 250 260 270 280 290 300 310 Wavelength in my

FIG. 5. Optical rotatory dispersion spectra of horse carbonic anhydrases B and C. O---O, Enzyme B; A--A, Enzyme C. Note the difference in scale on the Zeft and right sides of the figure. The solvent was 0.02 M phosphate buffer at pH 7.0 and 25”.

than other carbonic anhydrases (see Table IV), but in spite of this, the optical rotatory dispersion curves of the two native enzymes shown in Fig. 5 have the characterist,ic carbonic anhydrase features, and show only minor differences with respect to the corresponding curves for primate (2, 21) and bovine enzymes (27).

At high wave lengths the most marked Cotton effect,s are the two beginning with the trough at 297 rnp and peak at 293 rnp, followed by a second trough at 288 rnl.c and peak at 284 mp. Up to this point the B and C curves are very similar. They differ in the fine structure below 280 rnp, and, in particular, an in- teresting feature is the absence of the 265 rnl.s peak in the B curve. This peak seems to be particularly sensitive to changes in conformation, and is the first one to disappear during acid denaturation of human B enzyme (21).

At low wave lengths the optical rotat,ory dispersion patterns of carbonic anhydrases usually show a shallow trough at 222 to 225 rnp which shifts to the 232 mp region, more typical of ac-helices, only after denaturation. Both the native horse enzymes behave characteristically in this respect. The spectrum of the B enzyme has a trough at 223 rnp ([m’] value, - 1,370) ; that of the C enzyme is at 222.5 mp ([m’] value, -1,481). The B enzyme shows a rather broad peak in the 208 to 212 rnp region ([m’] value, - ! 74), whereas the corresponding peak with the C enzyme is at 204 rnp ([m’] value, +817), which is perhaps more typical for carbonic anhydrases.

DISCUSSION

The two forms of horse red cell carbonic anhydrase studied here seem to show t.he same unusual type of polymorphism found with the primate enzymes. If one generalizes from these two species, the characteristic features could be defined firstly by the presence of two chemically distinct proteins in unequal amounts, and secondly by the very different specific activities. Consist- ently, the more abundant form is the less active.

The nomenclature of the various forms of carbonic anhydrase isolated in different laboratories was first standardized on the basis of behavior on ion exchange chromatography. The first letter of the alphabet was given to the most acidic form, which would presumably emerge last on DEAE-Sephadex chromatog-

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Issue of September 25, 1968 A. J. Furth

raphy (14). At that time only the bovine and human enzymes had been purified, thereby giving the two bovine components A and B and the three human components A, B, and C. Since then the picture has changed. The human A component has been little studied, and most of the emphasis has been on the B and C components which can be clearly distinguished from each other by both activity and composition. The A component seems to be a minor constituent of low activity, which closely re- sembles enzyme 13 in its composition, activity, and structure (4). Similarly, the bovine .I component, which resembles bovine B in bot,h activity and composition, has been largely neglected.

At this stage it would seem more logical to classify the various forms of carbonic nnhydrase in terms of activity rather than ion exchange properties. X11 the carbonic anhydrases purified up to no\\- fall very readily into one of two classes. The high specific act,ivit>* forms include the enzymes human C, bovine I<, and, more recently, rhesus monkey C (2), whereas the low specific act,ivitJ. forms include human B and rhesus monkey B enzymes. On this basis there is no difficulty in classifying the two horse carbonic anhydrases. The low specific activity form has been designated enzyme B and the high specific activity form, enzyme C. The B enzyme is then also the more acidic and so conforms with the original method of nomenclature, but more important, it also resembles the primate B enzymes in being more abundant and less active than its C counterpart.

The significance of the two minor horse C components isolated by sulfoethyl-Sephades chromatography is not clear. On electrophoresis of the crude C enzyme immediately after its iso- lation by DE.iE-Sel)hades chromatography, they appeared merely as minor constituents, yet occasionally on sulfocthyl- Sephadex runs they accounted for as much as 6OG/,, of the total protein. In these casts, in which it was possible to isolate enough for amino acid analysis, there was no significant diffcrencc from that of the exllected composition of the C enzyme. Yet the specific activities were only in the order of 15,000 to 25,000 units per mg, compared with 40,000 units per mg for the major C component. The highest specific activity was associated with the fraction which was nlost retarded on the colun~n, a11d which could bc associated with the elcctrophoretic Band I so prominent in the starting nlaterinl. Therefore it, seems probable that this is t,hc major, if not the only, C component present in the red cell, and that the minor components (electrophoretic bands 2 and 3) are artifacts produced from it during the course of purification. Duff and (‘olcman (2) have suggested that partial denat uration in the monkey enzyme may produce stable conformers with the same chemical conlposition but different elrctrophoretic bchuvior, depending on the dcgrce of exposure of charged groups. The C enzyme, at Icast, in primates, is less stable than the 13 enzyme (28) and a partial denaturntion of horse C protein could account for both the lower sl)ecific activity and the lower net positive charge of these two acidic C components.

If the characteristic I)olymorphism of carbonic anhydrase is found in two such distantly related species as man and horse, it may be fairly widrs;l)read. It is unwise to draw conclusions about nrultiple enzyme forms before they are purified and chem- ically analyzed, but the evidence available at present suggests that polymorl;hism occurs in at least three different orders of mammals, the primates, ungulates, and rodents. Among t’he rodents, guinea pig enzyme has been separated, with the use of DEAE-Sephadcx chromatography (7)) into two components with different specific activities. Rabbit enzyme has been

separated into chemically distinct high and low specific activity forms in this laboratory.’ Preliminary data suggest that the polymorphism of the rabbit enzyme has the characteristic features enumerated for human and horse enzymes.

The bovine enzyme does not, fit into the general pattern, unless the low specific activity form mentioned by Nyman (13) is present in much greater amounts than he implies. The original purification procedure separated only two very reactive com- ponents, A and 1~. These have almost) identical composition and so do not correspond in any way to the polymorphism discussed here. The purification method gave good recoveries, but this was in terms of aci,ivity. It has already been pointed out, in describing the isolation of the horse B enzyme, how easy it is to overlook a low specific activity component, especially when the main contaminant, is hemoglobin. The enzyme yields cannot then be followed in terms of protein and further, the henloplobin tends to obscure any enzyme assay based on calorimetry. With the horse enzyme, the low specific activity form has the furt,her unfortunate property of an isoelectric point much closer to that of hemoglobin than t,hat of the high specific activity form, so that it was much more difhcult to isolat,e by ion exchange chromatog- raphy. If the bovine enzyme also combined a similar low iso- electric point with a low specific activit’y, the situation would be very similar, so that this could still prove to be the major bovine protein. The polymorphism would then follow the gclleral pattern.

In discussing the genetic origin of the two forms of carbonic anhydrase, Kyman and Lindskog (4) have suggested that a gene duplication ocscurred, similar to that leading to the present forms of hemoglobin. (‘ertninly the two proteins stem to be under separate genetic control. Electrophoretic studies, such as the work of Tashian (5) and ‘l’ashian, Douglas and Yu (8) on primate enzymes, show that the II component can vary fairly frequently, and quite independently from the C enzyme, and :i sirnilnr electrophoretic study wit,h horse carbonic anhydrase would be very profitable, since the very basic C enzyme can be so readily distinguished. Furthrr, a comparison of the amino acid con- positions of B and C forms as in Table III gives very convincing evidence for separate genetic control, particularly as the coding for asparagine and glutamine is distinct from that for the parrnt acids. The high amide content of enzyme C then means that the number of nucleotide substitutions in the two genes must be rather greater than is at first obvious when the amino acid con- positions of t,hc two proteins are compared.

Duff and Coleman (2) have pointed out t’hat’ wit,11 t,he human and rhesus monkey enzymes, there is more similarity between corresponding high (or low) specific a&v&y forms of different species than there is between two components from the same species. Hence, one composition might be called characteristic of low specific activity and another of high specific activity. In Table IV the compositions of seven purified carbonic anhydrases have been compared on this basis. h high scrinr content may be characteristic of the low specific activity forms, but otherwise it is difficult to see any marked similarity between the proteins of either group. This might be expected if polymorphism arose sufficiently early for any composition characteristic of one com- ponent, to be obscured by later independent mutations.

Aside from the genetic quest’ion, the most interesting feature of the two groups of carbonic anhydrase is their very different rates

1 D. Drescher, unpublished results.

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4840 Horse Erythmcyte Carbonic Anhydrases Vol. 243, n-o. 18

of reaction. The seven purified proteins of Table IV were readily classified by their sllecific act,ivities, and &xc this property must be some reflection of events at the active site, one might expect other kinetic parameters to be characteristic of these two groups. The kinetic data available in the literature at present do not permit many comparisons between B and C forms, because of varying conditions of pH and ot,her factors used by different workers, but certain generalizations are possible. Firstly, wherever K,,, values have been measured under comparable conditions for the two forms, they have been found to be signifi- cantly lower for the B enzymes. This is true for the reaction of human carbonic anhydrases with CO?, bicarbonate, or p-nitro- phenyl esters (11, 29), and for the reaction of horse carbonic anhydrases with p-nitrophcnyl ester, as shown here. A4t the same time the IT,,,;,, values are always much higher for the C enzymes in the reactions with CO2 and p-nitrophenyl acetate (although the hun~n 1’1 enz)-me is mole effective than C as a

catalyst, for the hydrolysis of o-nitrophenyl acetate (9, 11)). The differences in I;,,;,, may be less important in v&o, if the C enzymes, with their lower substrate affinity, are operating under conditions more removed from saturation t,han the 13 enzymes. Differcrices in inhibitor binding const,ants have been foulid for human 15 and C rnzyrucs (ll), and further studies &)rIg these lines would obviously be of interest.

The oliticnl rotatory dispersion patterns of the horse carboriic airhydrascs show a general similarity to those observed for the human (al), rhesus monkey (a), and bovine (27) enzymes. The position of the trough near 222 nib: in t,he two horse enzymes (Fig. 5) is very sin&r to what is observed for the hunian rrizynies (Reference 21, Fip. l), although the trough is even shallower for t,he cnzynics of the horse than it is for those of man. The peaks in the 200 to 210 111~: range show a close correspondence between the 1% c~lz~-nm of horse and man, with a small nrgati\-e \-Ale at

the IIC:A in each C:IS~, and also between the C enzymes of the t,wo slrecics, for which the peaks are positive. The height of the positive lIeal< for the horse C enzyme, [1x’] = 800 dcg cm2 per decimolc, however, is considerably lower t,h:m that for the human C rnzyme, [Tr!‘] = 2T50.

The pattern of (‘otton effects at longer wave lengths (Fig. 5), in the region of the aromatic absorption bands, shows some features characteristic of almost all carbonic anhydrascs, and others that vary with the individual enzymes. -1 peak at 292 to 293 nip and :I trough near 288 mc: appear to be lircscrit in all carbonic :anhytlrases. The decrement in [m'], l’rom peak to

trough, is close to TO tlcg cm2 per decimole for the horse C enzyme, but is considerably smaller, near 45, for the 1~ enzyme. Both values are lower than those observed for the human enzymes 1% and C (21, 2i, 30).

The ~cneral olnical rotatory dispersion lxrt,tern, between 295 and 250 III~, is very similar for the horse and human C enzymes. The horse 15 enzyme, however, in the region below 280 mp, differs markedly from the human II enzyme; the curve for the former descends steeply as the wave leiigt,h decreases, wit,h no sign of the peak near 260 nip that is characteristic of the human B en zyme in this repion. Indeed this ljortion of the curve, for horse carboriic anhydrase II, is very similar to that reported by Rosen- berg (2T) for bovine enzyme 13, even though the latmr enzynie, unlike horse enzyme I<, is :I “high activity” enzyme.

It is clear that the optical rotatory disliersion patterns of the various carbonic anhydrases, in the region of the aromatic ab- sorption bands, show considerable individuality. Until more

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knowledge of the demiled structure of the native enzymes is available, it would be premature to attempt interpretation of these individual differences, or to pick out any features which could be c.rlled characteristic of &her high or low specific ac- tivity proteins.

One of the most interesting: aspects of carbonic anhgdrase chemistry is the difference in activity of two rather similar pro- t,eins from the same species. It is to be hoped that more data comparing the properties of B and C enzymes will reveal some of the underlying structural differences responsible for this.

Note ;Zdded in Proof (July 196S)-The recent work of Byvoet and Gotti (31) suggests that erythrocyte carbonic anhgdrase from the dog may also show polymorphism. In one esperiment, using DEAE-cellulose, these workers succeeded in isolating a minor carbonic anhydrase component which appeared to have a higher specific activity than the major more basic carbonic anhydrase protein. This minor component would then to some extent be comparable to other carbonic anhydrase C enzymes, since in spite of its more acidic character it is both more reactive and less abundant than the major component. Furt,her studies on carbonic anhydrases from the dog would clearly be necessary to settle this question.

~lcknowledgment-I would like to thank Dr. John T. Edsall for much hell) and encouragement during the course of this work.

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Issue of September 2.5, 1968 A. J. Fwth 4841

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Anna J. FurthPurification and Properties of Horse Erythrocyte Carbonic Anhydrases

1968, 243:4832-4841.J. Biol. Chem. 

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