A magnificent enzyme superfamily: carbonic anhydrases, their purification and characterization

Abstract

In this paper, we reviewed the purification and characterization methods of the α-carbonic anhydrase (CA, EC 4.2.1.1) class. Six genetic families (α-, β-, γ-, δ-, ζ- and η-CAs) all know to date, all encoding such enzymes in organisms widely distributed over the phylogenetic tree. Starting from the manuscripts published in the 1930s on the isolation and purification of α-CAs from blood and other tissues, and ending with the recent discovery of the last genetic family in protozoa, the η-CAs, considered for long time an α-CA, we present historically the numerous and different procedures which were employed for obtaining these catalysts in pure form. α-CAs possess important application in medicine (as many human α-CA isoforms are drug targets) as well as biotechnological processes, in which the enzymes are ultimately used for CO₂ capture in order to mitigate the global warming effects due to greenhouse gases. Recently, it was discovered an involvement of CAs in cancerogenesis as well as infection caused by pathogenic agents such as bacteria, fungi and protozoa. Inhibition studies of CAs identified in the genome of the aforementioned organisms might lead to the discovery of innovative drugs with a novel mechanism of action.

• Affinity chromatography
• carbonic anhydrase
• characterization
• inhibitor

Introduction

Carbonic anhydrase (CA, EC 4.2.1.1.) is a zinc-containing metalloenzyme that is widespread in nature and catalyzes the reversible hydration of CO₂ to and H⁺. CA plays an important role in many physiologic processes, such as, for example, respiration, by facilitating the transport of CO₂ from metabolizing tissues to lungs. In plants, CAs are involved in the photosynthetic fixation of CO₂. Mammalian erythrocytes, where CA was originally discovered, contain two distinct isozymes (CAI and CAII) differing in their catalytic activities. Nowadays, 16 isoforms are known in vertebrates, all belongings to the α class. They also differ in their subcellular localization: CAI, CAII, CAIII, CAVII, CA VIII, CA X, CA XI and CA XIII are found in cytoplasm; CAIV, IX, XII, XIV and XV are membrane-bound forms; two mitochondrial forms, CAVA and VB; and CAVI is a secretory form. All these isoforms have been described in detail1^,2.

The CAs have been the targets of drug development for the treatments of glaucoma, obesity, osteoporosis, epilepsy, high altitude sickness, as well as cancer1–3. This is one of the reasons that promted us to evaluate the purification and characterization methods of the α-CA family, since such a review article has not yet been published.

Initially, the α-CA was purified from bovine erythrocytes4. Human erythrocyte revealed three CA isoforms on basis of the electrophoretic analysis, which were designated as the A, B and C isoforms5–10. It should be mentioned that this nomenclature is no longer used for decades. The A and B isoforms, in fact, showed an indistinguishable amino acid composition, while the C isoform was characterized by a unique amino acid composition as well as a higher catalytic activity for carbon dioxide hydration when compared with the A and B isoforms11. The A isozyme, identical to the B isoforms, and the C enzyme were later designated as CAI and CAII, respectively, and this is still their acronyms. During 1970s, the amino acid sequences and X-ray crystal structures were reported for both human CAI and CAII12–16. During the same decade, a sulfonamide-resistant CA activity was discovered in male rat liver homogenates17^,18 and in chicken muscle tissue19. It later became clear that sulfonamide-resistant CA enzyme was the basic muscle protein purified from rabbit skeletal muscle20. This enzyme was subsequently named as CAIII21 and had a very low catalytic activity compared with CA I and, especially, CA II1–3.

The identification of these three isoforms was just the beginning of the α-CA family history1–3. In 1979, a secreted CA enzyme (CAVI) was isolated from the saliva of sheep22 and the homologous human enzyme was characterized in 198723. During the 1980s, a membrane-associated enzyme CAIV was purified for the first time from bovine24 and human tissues25^,26. In the 1990s, the cytosolic CAVII27, the transmembrane isozymes CAIX28^,29, CAXII30, CAXIV31^,32, the mitochondrial enzymes, CA VA33^,34 and VB35 were discovered. Afterwards, the cytosolic CAXIII has been purified and characterized36, whereas the three acatalytic isoforms CA VIII, X and XI were shown to be cytosolic proteins and named with the acronym CARP (carbonic anhydrase-related polypeptide)1–3. In the following, we will present a historical overview on the purification and characterization of α-CA class1–3.

1920–1930

The first indication of CA activity was observed in the late 1920s, when experiments performed with hemolyzed blood demonstrated that the rate of carbon dioxide release from the blood was higher than expected and it was expected that blood could contain a catalyst for this reaction37^,38.

1930–1940

A few years later, it was discovered that the catalyst was an enzyme and it was named CA. Subsequently, when it was partially isolated and purified for the first time39^,40, the catalyst was shown to contain one zinc ion per molecule and appeared to have a molecular weight of approximately 30 kDa41. It was determined that bovine erythrocytes contained up to 2 g of enzyme per liter, making CA one of the most abundant proteins in blood42.

1960s

Lindskog purified CA for the first time in 1960, from bovine erythrocytes4 using the ion-exchange chromatographic method on diethylaminoethyl (DEAE) cellulose and the zone electrophoresis on columns containing a cellulose matrix. The purified enzyme obtained with these two chromatographic techniques showed a high degree of homogeneity.

In 1961, Nyman purified CA from human erythrocytes5. The catalyst was purified applying the same chromatographic techniques used by Lindskog. Two CAs isoforms were detected in the human erythrocytes by a procedure involving ethanol – chloroform extraction. These isoforms were called hCA I and hCA II and they were purified with a high degree of homogeneity by ion-exchange chromatography or zone electrophoresis on cellulose columns5–10.

In 1968, Furth purified horse CAs using chromatographic columns, such as DEAE Sephadex and Sulfoethyl-Sephadex. B and C isoforms were purified to homogeneity by DEAE Sephadex chromatographic column, whereas Sulfoethyl-Sephadex was used for obtaining the isoform A43.

In the same years, CAs were purified by Falkbring et al.44 by the affinity chromatography technique. Falkbring et al.44 and Whitney45 used affinity gels formed by coupling p-aminobenzenesulfonamide or p-(aminomethyl)benzenesulfonamide to Sepharose polysaccharides by means of cyanogen bromide activation. This method gives a stable and a high capacity affinity gel suitable for the purification of CAs from a variety of sources. The azo-sulfonamide coupled gels have several advantages. They can be reused several times with no loss of efficiency, have a higher binding capacity and the red-colored product enabled visual estimation of coupling efficiency. The separation of the secondary isozymes from the major forms of CAs requires the use of an ion-exchange column. Secondary isozymes purification has been discussed by Funakoshi and Deutsch46. The higher yields of the azo gels is due to the length of the coupled inhibitor. In fact the azobenzene spacer between the gel and the sulfamoyl moiety binding to the catalytic Zn(II) seems to be beneficial for an efficient complexation of the protein to the affinity gel.

1970s

DEAE-cellulose (Whatman, microgranular pre-swollen columns) was used by Carter and Parsons for purifying CAs in a homogeneous form47.

In 1971, after ammonium sulfate precipitation, the bovine CA was purified by size exclusion chromatography using a Sephadex G-75 column. However, the observed specific activity of the purified enzyme was low. Subsequently, it was demonstrated that an initial precipitation of non-CA components by the addition of cold chloroform and ethanol, followed by ammonium sulfate fractionation48 was a powerful strategy for obtaining a CA with a specific activity not altered by the salt precipitation protocol.

In 1977, Khalifah et al.49 explored the use of carboxymethyl (CM) Bio-Gel columns, which have an excellent stability to variation of pH and ionic strength that are frequently encountered in affinity chromatography purification procedures. The CM Bio-Gel had a binding capacity of 15–20 mg of CA/mL of bed volume similar to the values reported by Osborne and Tashian50. The only difference is that the gel of Osborne and Tashian had a bed volume sensitive to pH and ionic strength variations. In 1979, Fernley and colleagues purified CA VI from the ovine parotid gland with Sephadex G-7522; while Shinar and Navon51 purified bovine CA using Lindskog’s method.

1980s

Wistrand and Knuuttila25 used ion-exchange chromatography and affinity chromatography methods for purifying bovine lens CA. Salmo gairdneri CA was purified by Hall and Schraer using three purification steps: chloroform ethanol extraction, size exclusion chromatography (Sephadex G-75) and anion exchange chromatography (DEAE Bio-Gel)52. As described in the literature, for a large-scale preparation of bovine and porcine CAs was used the rapid ion-exchange chromatography technique on DEAE-cellulose column to obtain the catalysts in its pure form. Rapid ion-exchange chromatography on DEAE-cellulose should be generally applicable in protein purification, and especially advantageous in purification of unstable proteins where time-consuming separations often give rise to low yields of active material53.

In 1985, Yang et al. used the p-aminomethylbenzene sulfanomide-coupled affinity column (Sepharose 6B) to purify CA54. This is still one of the most frequently used affinity columns for the purification of α-CAs.

1990s

α-CA isozymes were purified by a combination of affinity and hydrophobic interaction chromatography. Immobilization of sulfonamides on an epoxy-activated solid support provided a stationary phase for affinity chromatography. First, the enzyme is directly isolated from the homogenate of human erythrocytes or rat stomach by an affinity chromatography. Then, the CA obtained by the affinity chromatography was salt precipitated with ammonium sulfate and purified to homogeneity by a hydrophobic interaction chromatography. The enzyme was recovered with a high yield55.

In 1996, Schmidt et al. used His₃ metal binding site for removing from the substrate pocket of an enzyme and expose to solvent on a protein surface, it showed clear selectivity for Zn(II) compared with Cu(II) and Ni(II) via immobilized metal affinity chromatography (IMAC), allowing highly selective purification of RBP/H₃(A), retinol binding protein resulting form and of His6-tagged RBP as well56.

Küfrevioğlu and colleagues also purified CAs with a Sepharose-4B-l-tyrosine-sulfanilamide affinity column. CAI and CAII were separated from each other efficiently57.

In 1998, Bergenhem et al. characterized by h CARP VIII (human CARP) and reported on the expression, purification and determination of apparent molecular weight, CD-spectra and stability toward denaturation, respectively. The recombinant h CARP VIII was cloned into a glutathione-S-transferase fusion vector by this group58. Also, in the same year, Vince and Reithmeier purified glutathione S-transferase (GST)-fusion protein (GST-Ct) or GST was immobilized on glutathione-Sepharose 4B59.

2000s

In 2000, Vince et al. indicated a specific interaction between the carboxy terminal (Ct) of anion exchanger 1 (AE1) and CAII using cosolubilization, coimmunoprecipitation, immunofluorescence, peptide competition and a sensitive microtiter plate assay utilizing binding of a GST fusion protein of the Ct (GST-Ct) to immobilized CAII60.

The singly spin-labeled variants, hCAII206 and hCAII67, and doubly spin-labeled hCAII67–206 and hCAII59–174 were purified by affinity chromatography on a gel matrix incorporating sulfonamide groups that bind efficiently to the active site of hCAII49^,61. In this procedure only the hCAII molecules that possess activity bound to the column, whereas aggregated and misfolded proteins washed through the column62.

Krungkrai et al. applied a three steps purification procedure of the malarial CA (pfCA) by using combinations of cation-exchange Mono S column (fractions eluting at pH 6.5) and anion-exchange Mono Q column (fractions eluting at 80 mM KCl), as well as gel filtration on Superose 12 column or on a FPLC system63.

Different from other studies, cytosolic and integral CA isozymes were purified with a Sepharose-4B-L-Tyrosine-Sulfanilamide affinity column and characterized extensively64. Demir et al. stated that leukocyte CA was not purified previously. In their study, leukocyte CA was purified and characterized according to its localization within the cell65.

In 2004, Ozensoy et al.66 synthesized a new affinity gel for purıfıcation of CAs. They used EUPERGIT C-250 L as matrix and p-aminobenzensulfonamide (sulfanilamide) as ligand. EUPERGIT C-250 L was selected as a matrix due to its long time stability, high resistance to mechanical stress and low viscosity. Several analogs of sulfanilamide have been demonstrated to possess good binding affinities for CAs67. According to this procedure, p-aminoethylbenzenesulfonamide was chosen as a ligand, since it is a specific and strong inhibitor of CA. p-Aminoethylbenzenesulfonamide was bound to the oxirane groups on EUPERGIT C-250 L by means of a covalent amide bond by Ozensoy et al. The oxirane groups on EUPERGIT C-250L also served as spacer-arms on the affinity gel. Using this affinity gel, they purified hCAI and hCAII from erythrocytes with a high yield66.

For the first time, Demir et al. purified CAs from outer peripheral, cytosolic, inner peripheral and integral of bovine stomach by an affinity column containing Sepharose-4B-L-tyrosine-sulfanilamide68.

In 2007, Supuran et al. have been cloned and purified hCAIII by the GST-fusion protein method. GST-hCA III fusion protein was produced into E. coli strain BL21. The GST-hCA III fusion protein has been purified in two steps by affinity chromatography. One of the steps is Glutathione Sepharose 4B column for obtaining supernatant69. Moreover, Piermarini et al. compared the ability of the Ct domain of three SLC4 transporters, SLC4-A1 (AE1), SLC4-A4 (NBCe1) and SLC4-A8 (NDCBE), to bind immobilized CA II, using enzyme-linked immunosorbent assay detection. This group discovered that when expressed as GST fusion proteins, all three bind to CAII (Kd 300–600 nM) better than does pure GST70.

da Costa Ores and colleagues purified CA with ethanol and chloroform extraction and ammonium sulfate precipitation. The purification of CA by ethanol/chloroform extraction resulted in an enzyme-specific activity value of 2623 U mL⁻¹, a recovery of 98% and a purification factor of 104-fold. The fraction precipitated by 60% ammonium sulfate saturation showed an enzyme activity of 2162 U mL⁻¹, a recovery of 66% and purification factor of 1.4-fold using. The purified enzyme extracts obtained by both procedures showed the potential for use in carbon dioxide capturing processes. Therefore, these purification techniques represent feasible alternatives for the industrial capture of CO₂71.

Oviya et al. using a combination of chromatographic techniques (Sephadex G-75 and DEAE cellulose columns; Genei, India) obtained a 4.64-fold purification of CA72.

Alternatively, Uygun et al. synthesized molecularly imprinted PHEMAH cryogels to purify CAs. The synthesized CA-imprinted PHEMAH cryogels were used for the purification of CA from bovine blood and the enzyme was purified with 79.4% recovery. The purity was assessed by SDS-PAGE and the purification fold was 83.98. This study represents a rare example of the molecular imprinting technology to purify an enzyme and it was applied to the purification of CA73.

More recently, histidine-based tags were engineered onto the surface of a protein rather than restricting an engineered tag to the N- or C-terminal of the amino acid sequence of the molecule. Hoffman et al. presented the Arg160His mutation of Haemophilus influenza β-CA (HICA), which mimics the endogenous metal affinity of E. coli enzyme (ECCA, also belongs to the β-class). This is the first step toward developing a general method to engineer the protein surface to make it capable to bind a metal ion, such as nickel, and make possible it purification by a metal affinity chromatography. Compared with the published three chromatography techniques (cation-exchange, hydrophobic exchange and gel exclusion), metal affinity purification was a substantial improvement in time and resources74.

Different from past studies66, Ozensoy et al. prepared a new affinity gel for the purification of α-CAs with some modifications. The new affinity gel reported in this study was prepared using EUPERGIT C-250L as a chromatographic bed material. Etylenediamine spacer-arms were attached to EUPERGIT C-250L to prevent steric hindrance between the matrix and ligand. This strategy improves the effective binding of the CA to a specific ligand of the aromatic sulfonamide type75. An overview of the affinity gels/ligands used for CA purification is shown in Table 1.

Table 1. Isolation of carbonic anhydrase (E.C. 4.2.1.1) isozymes by using affinity gels.

Affinity ligands	Solid supports or immobilized affinity ligands	References
Sulfanilamide	Sephadex G-150	Falkbring et al.44
(p-Aminomethylbenzene-sulfonamide)	Sepharose 4B	Whitney45
2-Amino-1,3,4-thiadiazole-5-sulfonamide	Sepharose with NH2(CH2)10COOH	Champagnol76
p-[(2,4-Diaminophenyl)azo]benzene-sulfonamide(Prontosil)	CM-Sephadex	Osborne and Tashian50

Conclusions

CAs were much investigated in the past decades1–3^,77–82, especially for their involvement in a multitude of physiologic and pathologic processes in all organisms, from prokaryotes to eukaryotes. Apart from the classical applications of the CA inhibitors (CAIs) as diuretics, antiglaucoma and antiepileptic agents83–91 of pharmaceuticals that suppress the activity of carbonic anhydrase, ultimately many such agents were shown to be effective as antiobesity and anticancer agents91–93. Moreover, the inhibition of CAs from pathogenic organisms (such as bacteria, fungi and protozoa) started to be considered as an interesting approach for designing anti-infective agents with a new mechanism of action1–3^,94. These studies allowed the identification of new CAs in the genome of pathogens, such as bacteria, fungi and protozoa. In the last years, in fact, Capasso and Supuran95 groups, cloned, purified and characterized several CAs from different sources to carry out biochemical, structural and phylogenetic studies. All such investigations require CAs with a reasonable grade of purity and large amount of enzyme. Here, the purification procedures for the α-CAs are extensively reviewed, as historically these are the first class of such enzymes discovered. An overview on the purifications methods of the α-CAs, starting from their discovery to date, might be of great interest to all researchers involved in the fascinating field.

Declaration of interest

The authors declare no conflict of interest. Research from Supuran group was supported by several 6th and 7th framework EU programmes (Euroxy, Metoxia, DeZnIT and Dynano).