Synthesis of a novel affinity gel for the purification of carbonic anhydrases

Abstract

A new affinity gel was synthesized for the purification of carbonic anhydrase (CA, EC 4.2.1.1) isozymes from erythrocytes. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on ethylenediamine was covalently attached via CNBr activation, followed by reaction with the CA inhibitor 4-isothiocyanato-benzenesulfonamide. The derivatized gel incorporated thioureido-benzenesulfonamide moieties as CA ligand. The binding capacity of the new affinity gel was determined at different temperatures, pH values, ionic strengths and elution buffers. The maximum binding of various CAs was achieved at 25 °C with pH 8.5 and ionic strength around 0.4. The overall purifications for human (h) hCA I and hCA II were 672- and 580-fold, and with 62 and 43% yields, respectively. SDS–polyacrylamide gel electrophoresis showed single bands for each purified isozymes, corresponding to a molecular weight of approx. 29 kDa. This is an easily obtainable, efficient and robust affinity gel, useful for the purification of many other α-CAs.

• Affinity chromatography
• carbonic anhydrase
• purification

Introduction

Carbonic anhydrases (CA, EC 4.2.1.1) are a family of metalloenzymes that catalyzes the reversible hydration of carbon dioxide to form bicarbonate with generation of protons1. The widespread abundance of several CA isoforms in plants, animals and microorganisms suggests that this simple conversion of a membrane-permeable gas into a membrane-impermeable ionic product is vital to many important biological functions2. Sixteen CA isozymes have been described up to now in mammals, named CA I through CA XV, with CA II and CA IX being the most active catalysts of carbon dioxide hydration1^,2. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII and CA XIII), others are membrane bound (CA IV, CA IX, CA XII and CA XIV), two are mitochondrial (CA VA and CA VB) and one is secreted in saliva (CA VI)3. These isozymes are involved in the pH homeostasis, ion transport, water and electrolyte balance, bone resorption, calcification and tumorigenesis4^,5. CA isozymes are associated to many diseases such as glaucoma, obesity, cancer, epilepsy and osteoporosis6^,7. CA I is the most abundant isozymes with a relatively low enzyme activity. CA II is present in lower amounts, but it is referred to as the “high activity” isoenzyme because of its higher specific activity compared to CA I8. CA I is sensitive to sulfonamide inhibitors but CA II has even higher affinity for this class of inhibitors9–11. Over the years, CA has been purified from a number of sources. One of the cheapest and most readily available sources of CA is mammalian blood12^,13. There are several CA purification procedures known in the literature9^,12^,14^,15. However, the most commonly used purification method is the affinity chromatography, which gives high yields of the enzyme from a wide variety of sources16–19.

Affinity chromatography takes advantage of the purification of many proteins by using specific ligands or chemical groups with affinity for the protein to be purified. Aromatic and heteroaromatic sulfonamides are frequently used as ligands in these methods, as they are specific and strong inhibitors of many CAs19–21. A large number of affinity gels, using a variety of matrices, spacer arms and ligands, have been described in the literature19–25. The present report describes the successful purification of CA from erythrocytes using a novel affinity gel. The affinity gel was prepared on the Sepharose 4B matrix with a spacer arm incorporating ethylenediamine, to which 4-isothiocyanato-benzenesulfonamide was covalently attached, leading to the corresponding thioureido-benzenesulfonamide, as a new ligand for affinity gel purification of CAs.

Materials and methods

Materials

All chemicals were of analytical grade and obtained from Sigma-Aldrich (Taufkirchen, Germany) and Merck (Darmstadt, Germany). Human blood samples were obtained from the Balikesir University Faculty of Medicine.

Methods

Preparation of affinity gel

A total of 4 g CNBr was added to 20 mL Sepharose 4B and with 4 M NaOH the mixture’s pH was kept at 11. Mixture was transferred to a Buchner funnel and washed with 250 mL cold 0.1 M NaHCO₃ (pH: 10) buffer solution and was transferred to a beaker in the same buffer. Five millilitres of ethylene diamine was added to the suspension and the mixture was stirred with magnetic stirrer for 2 h. Then the suspension was kept at 4 °C for 16 h. At the end of this process, the gel was washed with water. Washing was repeated with 100 mL of 0.1 M NaHCO₃ (pH: 10) buffer. Ethylenediamine attached gel was taken up in 40 mL of the same buffer. 50 mg of 4-isothiocyanato-benzenesulfonamide were dissolved in 5 mL DMSO and added to the suspension. The mixture was stirred with magnetic stirrer for 3 h. The suspension is then transferred to the Buchner funnel and washed with 1 L distilled water and 200 mL of 0.05 M Tris-SO₄ (pH: 7.5) buffer and stored in the same buffer.

Purification of carbonic anhydrase from human erythrocytes

Human blood samples were obtained from the Balikesir University Faculty of Medicine, using bottles containing anticoagulant acid-citrate-dextrose (ACD). The blood samples were centrifuged at 5000 rpm for 20 min and the plasma and buffy coat were removed. After washing the packed red cells two times with NaCl (0.9%), the erythrocytes were hemolyzed with cold water. The ghosts and intact cells were removed by centrifugation at 15 000 rpm for 40 min at 4 °C. The hemolysate was applied to the Sepharose 4B-ethylene diamine-4-thioureido-benzenesulfonamide affinity column equilibrated with 25 mM Tris/0.1 M Na₂SO₄ (pH: 8.5) buffer. The affinity gel was washed with 25 mM Tris/22 mM Na₂SO₄ (pH 8.5) buffer, and CA isozymes were eluted under different elution conditions.

Protein determination

After elution step enzyme was determined spectrophotometrically at 280 nm and protein during the purification steps was determined spectrophotometrically at 595 nm according to the Bradford method, using bovine serum albumin as the standard26.

Enzyme assay

Carbonic anhydrase activity was measured by the Maren method27, which is based on the determination of the time required for solution pH to decrease from 10.0 to 7.4 due to hydration of CO₂.

SDS polyacrylamide gel electrophoresis

SDS polyacrylamide gel electrophoresis was performed after purification of the enzymes. It was carried out in 10 and 3% acrylamide for the running and the stacking gel, respectively, containing 0.1% SDS according to the Laemmli procedure. A 10 µL sample was applied to the electrophoresis medium. Gels were stained for 1.5 h in 0.1% Coommassie Brilliant Blue R-250 in 50% methanol and 10% acetic acid, then destained with several changes of the same solvent without the dye28.

Results and discussion

In this study, a new affinity gel was prepared for the purification of CA isozymes from a variety of sources. Sepharose 4B was selected as a matrix due to its long operational life, stability to mechanical stress and possession of favorable flow rates. These features are particularly important during routine purifications for large scale production of proteins. Several analogs of sulfonamides have been demonstrated to possess good binding affinities for CAs and they are also frequently used on affinity gels for the purification of these proteins29.

In the literature there are several types of methods that have been used with different matrices and different spacer arms that undergo activation methods. It is known that affinity gels prepared with these activation methods can undergo partial deterioration in the course of the reaction19^,25^,30. Affinity gel was prepared with Sepharose 4B, ethylenediamine and 4-isothiocyanato-benzenesulfonamide (Scheme 1). Ethylenediamine was covalently attached to the matrix after activation of sepharose with cyanogen bromide (Scheme 1). This leads initially to the formation of an iminocarbonate which is easily derivatized with primary amines in mild conditions, leading to the aminoethyl-arm derivatized sepharose. This intermediate was reacted with 4-isothiocyanato-benzenesulfonamide leading to the thioureido-benzenesulfonamide derivatized gel (Scheme 1).

Scheme 1. Preparation of the new affinity gel for purification of CAs.

The hemolysate was applied to the Sepharose 4B-ethylene diamine-4-thioureido-benzenesulfonamide affinity column equilibrated with 25 mM Tris/0.1 M Na₂SO₄ (pH: 8.5) buffer. The affinity gel was washed with 25 mM Tris/22 mM Na₂SO₄ (pH: 8.5) buffer, and CA isozymes were eluted under different elution conditions. The eluates were characterized by protein determination at 280 nm and assaying CO₂ hydratase activity for hCA I and hCA II. Specific activities for hCA I and hCA II were calculated by using hemolysate and purified enzyme solution. As a result, hCA I and hCA II were purified up to 672.72- and 580.68-fold with a recovery ratio of 62 and 43% compared to homogenate, respectively (Table 1). These values are better than some of the reported affinity gels and equivalent to others19–25. These values of the absorbance showed that some proteins, bound to the affinity material, have been removed from the column by the washing solutions. At the end of the last step, highly purified enzymes were obtained exhibiting a single band on SDS-PAGE. The human isozymes migrated as single bands in all cases, with apparently identical molecular masses (Figure 1). We used only one step for purification, Sepharose 4B-ethylene diamine-4-thioureido-benzenesulfonamide affinity chromatography by modification of elution conditions. hCA I and hCA II were purified using the affinity gel with different elution buffers (Figure 2). The most suitable elution buffers were 1 M NaCl/50 mM Na₂HPO₄ (pH: 6.3) for hCA I and 0.1 M CH₃COONa/0.5 M NaClO₄ (pH: 5.6) for hCA II (Figure 2). The binding capacities of the affinity gel for the hCA I and hCA II isozymes were determined at different temperatures (Figure 3) and ionic strengths (Figure 4). For this aim, buffers were prepared with different compounds at different concentrations for elution. For elution of hCA I and hCA II the buffers pH were 6.3 and 5.6, respectively. The binding capacities of the affinity gel for the hCA I and hCA II isozymes were determined at different temperatures in the range from 5 °C to 35 °C. For determination of optimum ionic strength, enzyme activity was determined using different concentrations of NaSO₄, in the range from 0.1 to 0.5. The enzyme was seen to exhibit the highest activity at 25 °C and maximum binding was achieved at ionic strength around 0.4. In order to determine the optimum pH for equilibration and washing buffers, 25 mM Tris/0.1 M Na₂SO₄ and 25 mM Tris/22 mM Na₂SO₄ buffers were used in the pH range of 7.0–9.5 (Figure 5). Maximum binding was determined for hCA I and hCA II at pH: 8.5 and 9.5, respectively. These results showed differences with other studies in the literature19–25^,30.

Figure 1. SDS-PAGE pattern. Molecular mass standards: β-galactosidase (116 kDa), bovine serum albumin (66.2 kDa), egg albumin (45.0 kDa), lactate dehydrogenase (35 kDa), REase Bsp981 (Escherichia coli) (25 kDa).

Figure 2. Effect of different buffers for elution of hCA I and hCA II. 1. 50 mM Na₂HPO₄/1 M NaCl – 50 mM Na₂HPO₄/0.2 M KSCN; 2. 0.1 M KI/0.1 M Tris-SO₄ – 50 mM Na₂HPO₄/0.2 M KSCN; 3. 0.1 M KI/0.1 M Tris-SO₄ – 0.1 M NaCH₃COO/0.5 M NaCIO₄; 4. 50 mM Na₂HPO₄/1 M NaCl – 0.4 M NaN3/0.1 M Tris-SO₄; 5. KI/Tris-SO₄ – 0.4 M NaN₃/0.1 M Tris-SO₄; 6. 50 mM Na₂HPO₄/1 M NaCl – 0.1 M NaCH₃COO/0.5 M NaCIO₄.

Figure 3. Effect of temperature.

Figure 4. Effect of ionic strength.

Figure 5. Effect of pH.

Table 1. Purification scheme of hCAI and hCAII from human erythrocytes by Sepharose 4B-ethylene diamine-4-isothiocyanato-benzenesulfonamide affinity chromatography. Experimental analysis were performed at +4 °C. CA enzyme activity was determined colorimetrically using CO₂-hydration method of Maren.

Purification step	Volume (mL)	Activity (U/mL)	Total activity (U)	Proteinamount (mg/mL)	Total protein (mg)	Specificactivity (U/mg)	Overall yield (%)	Overall purification (fold)
Purification scheme of hCAI
Hemolysate	85	2.51	213.35	56	4760	0.44	100	–
Affinity chromatography	6	15.33	91.98	0.01	0.06	255.5	43.11	580.68
Purification scheme of hCAII
Hemolysate	85	2.51	213.35	56	4760	0.44	100	–
Affinity chromatography	15	8.88	133.2	0.030	0.45	296	62.43	672.72

hCA: Human carbonic anhydrase.

hCA II has generally a higher affinity for sulfonamides than hCA I31. 4-isothiocyanato-benzenesulfonamide exhibited quite effective inhibition to hCA II, but rather different from hCA I. K_i value for hCA I and hCA II were 5 µM and 0.185 µM for 4-isothiocyanato-benzenesulfonamide, respectively32. K_i values for p-amino benzenesulfonamide were found to be 28 µM for hCA I33 and 10.1 µM for hCA II34. 4-isothiocyanato-benzenesulfonamide was more effective inhibitor than p-amino benzenesulfonamide for hCA I and hCA II. So, we used this compound for a ligand to synthesize the affinity gel. The purification fold and yield of Sepharose 4B-L Tyrosine-p-aminobenzene sulfonamide gel for hCA I was better than hCAII30. However, Sepharose 4B-ethylene diamine-4-thioureido-benzenesulfonamide gel purification fold and yield for hCA II was better than for hCAI. In addition, the binding capacities of the affinity gel for the hCA I and hCAII were better than other studies19–25^,30. Thus, Sepharose 4B-ethylene diamine-4-thioureido-benzenesulfonamide affinity gel is shown to be favourable for the purification of hCA I and hCA II in active form. These results mean that the procedure used in the purification is good enough to be used in further studies and also has an advantage of taking very short experimental period.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

This work has been supported by Balikesir University Research Project (2012/16).