Abstract
Molecularly imprinted PHEMAH cryogels were synthesized and used for purification of carbonic anhydrase from bovine erythrocyte. Cryogels were prepared with free radical cryopolymerization of 2-hydroxyethyl methacrylate and methacryloylamido histidine and characterized by swelling degree, macroporosity, FTIR, SEM, surface area and elemental analysis. Maximum carbonic anhydrase adsorption of molecularly imprinted PHEMAH cryogel was found to be 3.16 mg/g. Selectivity of the molecularly imprinted cryogel was investigated using albumin, hemoglobin, IgG, γ-globulin, and lysozyme as competitor proteins and selectivity ratios were found to be 15.26, 60.05, 21.88, 17.61, and 17.42, respectively. Carbonic anhydrase purity was demonstrated by SDS-PAGE and zymogram results.
Introduction
Carbonic anhydrase (CA; EC 4.2.1.1) is a zinc metallo-enzyme, which catalyzes a very simple but important physiological reaction: the hydrolysis of carbon dioxide to bicarbonate and proton.
CA is found in all living organisms of the phylogenetic tree (Bacteria, Archaea, Eucaria). The enzyme has independently evolved at least five times and there are five genetically different major enzyme family nowadays such as α-, β-, γ-, δ- and ϵ-CAs (Supuran Citation2010). α-CA is widespread in animals and is the only CA form expressed in vertebrates. Although, plants, algae and bacteria contain α-CA gene but β-CA is also a common CA type expressed in these organisms. Some invertebrates also carry β-CA gene. While γ-CA exists in Archaea and in some bacteria, marine diatoms have δ- and ϵ-CA (Gilmour Citation2010). CAs play critical role in respiration and CO2/bicarbonate transport, pH and CO2 homeostasis, electrolyte secretion in various organs and tissues, biosynthetic reactions (such as gluconeogenesis, lypogenesis, ureagenesis, and biosynthesis of pyrimidine nucleotide), bone resorption, calcification, tumourigenesis and other physiological and pathological processes in many organisms (Supuran and Scozzafava Citation2007, Banerjee et al. Citation2004). Various CA inhibitors have been used as antiglaucoma, anticonvulsant, antiepileptic, antiurolithic, and anticancer agent and some of these inhibitors were approved as a drug. In addition to its importance in metabolism and tumourigenesis, it was shown that CA is related to cell growth and apoptosis (Supuran et al. Citation2004). In order to carry out and precede these experiments, it is important to work with considerably pure and abundant amount of CA and because of this necessity, high-yield CA purification techniques have been developed. CA initially was purified with ethanol-chloroform pericipitation, and then classical protein purification steps (which follow ammonium sulfate precipitation, dialysis, and ion-exchange chromatography) were applied (Meldrum and Roughton Citation1933). After the exploration of specific CA inhibitors, purification of CA was carried out by using affinity chromatography techniques (Banerjee et al. Citation2004, Supuran et al. Citation2004, Meldrum and Roughton Citation1933, Ceyhun et al. Citation2011).
Molecularly imprinted polymers (MIP) are synthetic materials synthesized by cross linking of functional monomer or polymer in the presence of a template molecule (Bereli et al. Citation2008, Citation2013, Citation2011a, Tamahkar et al. Citation2011, Andaç et al. Citation2012a). When the template is removed, cavity forms which is complementary with template molecule with regard to size, shape, and orientation of its functional groups. The size and shape of the cavity allow the recognition of template molecule, while orientation of functional groups allows the binding of template molecule selectively (Janiak and Kofinas Citation2007). Molecular imprinting technology is extensively studied in fields of chemistry, biochemistry, and biotechnology nowadays. MIP are employed in three main research areas: i) separation and isolation; ii) antibody mimics (in biomimic analysis and sensors); and iii) catalysis and synthesis with enzyme mimics (Zhang et al. Citation2006).
Increasing demands for the biologically active and pure entities (i.e., low molecular weight compounds; biomolecules such as DNA and proteins; viruses; cell organelles; and even a whole cell) require the improvement of the polymeric materials (Dainiak et al. Citation2007). For this purpose, development of the new and original polymeric materials has gained great attention in the field of biotechnology (Lozinsky et al. Citation2002). Therefore, development of the macroporous polymeric materials draws great interest especially in the biomedical, biotechnological, and pharmacological sciences (Plieva et al. Citation2007). Cryogels are new polymeric gels which are synthesized by polymerizable monomers at frozen conditions. Cryogels are highly porous polymeric materials and can be synthesized with various morphology and porosity by using any gel preparing precursors (Özgür et al. Citation2011, Bereli et al. Citation2011b, Citation2012, Andaç et al. Citation2012b). Cryotropic gelation is a specific gel-preparation technique which takes place as a result of cryogenic application (freezing, incubation in frozen state, and defrosting) of polymerizable systems (Lozinsky et al. Citation2003). Basic applications of cryogels are defined with their porous structure and operational stability. Main areas of applications of cryogels include biocatalysis with immobilized enzymes and cells, bioseparation for purification of target molecules, chromatography of cell organelles, viruses, microbial and mammalian cells, and biomedical applications as three dimensional matrix for mammalian cell culture (Plieva et al. Citation2007).
In this work, CA-imprinted poly(2-hydroxyethyl methacrylate-co-methacryloylamido histidine) [PHEMAH] cryogels were prepared for purification of CA from bovine erythrocyte. Imprinted cryogels were characterized with swelling studies, macroporosity, flow resistance, Fourier transform infrared (FTIR), scanning electron microscope (SEM), elemental analysis, and surface area measurements. Effects of pH, buffer type, CA concentration, ionic strength, temperature, chromatographic flow rate on the adsorption of CA were also investigated. Reusability and stability of the CA-imprinted cryogels were also studied. Selectivity of the CA-imprinted PHEMAH cryogels to CA was searched using albumin (bovine serum), hemoglobin (bovine), lysozyme (egg white), IgG (bovine serum), and γ-globulin (bovine serum) as competing proteins. CA-imprinted PHEMAH cryogels were used for the purification of CA from bovine erythrocyte, and purity of the CA was demonstrated with SDS-PAGE and zymogram.
Materials and methods
Materials
CA II (from bovine erythrocyte), 2-hydroxyethyl methacrylate (HEMA), N,N’-methylene bis(acrylamide) (MBAAm), ammonium persulfate (APS), N,N,N’,N’-tetramethylene diamine (TEMED), p-nitrophenyl acetate (p-NPA), bromothymol blue, potassium thiocyanate, boric acid, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), albumin (from bovine serum), hemoglobin (from bovine), IgG (from bovine serum), γ-globulin (from bovine serum), sodium dodecyl sulfate (SDS), Tris-Base, acrylamide, glycerol, bromophenol blue, glycine, ethanol, histidine methyl ester, hydroquinone, triethylamine, methacryloyl chloride and electrophoresis standard proteins (myosin [rabbit blood, 205 kDa], β-galactosidase [E. coli, 116 kDa], phosphorylase B [rabbit blood, 97 kDa], albumin [bovine serum, 66 kDa], albumin [egg white, 45 kDa], and CA [bovine erythrocyte, 29 kDa]) were purchased from Sigma (Steinheim, Germany). All other chemicals were of reagent grade and used without any purification step.
Methods
Synthesis of N-methacryloyl-(L)-histidine monomer
In order to prepare CA-imprinted cryogel, N-methacryloyl-(L)-histidine (MAH) was used as a functional monomer (Garipcan and Denizli Citation2002). For synthesis of MAH, 5.0 g of histidine methyl ester and 0.2 g of hydroquinone were dissolved in 100 mL of CH2Cl2 and the solution was cooled at 0°C. After addition of 12.75 g of triethylamine, 5.0 mL of methacryloyl chloride was added to the solution under nitrogen atmosphere and resultant solution was mixed for 2 h at room temperature. Then, unreacted methacryloyl chloride was extracted with 50.0 mL of NaOH (10%, w/v). Organic phase was evaporated and MAH was crystallized with ethanol and ethyl acetate.
Synthesis of CA-imprinted PHEMAH cryogels
In order to form a pre-complex between CA and MAH, MAH monomer (0.1, 0.2, and 0.3 mmol) and CA (5.0, 10.0, 15.0, and 20.0 mg) were dissolved in 2.0 mL of phosphate buffer solution (pH 6.0, 0.1 M) and mixed for 4 h at 50 rpm and + 4°C. Then, 1.0 mL of this complex was mixed with 1.3 mL of HEMA and volume of the solution was made up to 5.0 mL with distilled water. This solution was mixed with 10.0 mL of MBAAm solution (0.283 g) and then 20.0 mg of APS was added to the solution. After adding 25.0 μL of TEMED, 3.5 mL of the solution was immediately poured to a syringe (0.8 cm diameter and 5.0 mL volume). Then, solution was incubated at − 12°C for 24 h. Synthesized cryogels were kept at room temperature to melt the frozen water. After that the cryogels were washed with distilled water (200 mL) in order to remove the unreacted monomers. The plain PHEMA and non-imprinted PHEMAH cryogels were also prepared by using the same method without addition of MAH/CA and CA, respectively.
Characterization of cryogels
Determination of swelling degree. PHEMA, non-imprinted PHEMAH, and CA-imprinted PHEMAH cryogels were dried at 60°C for 24 h in order to determine the swelling degree. Dried cryogels were weighed, and then were put in a water bath (25°C) and adsorbed water amount was recorded at defined time intervals. Swelling degree of the cryogels was calculated using the following equation:
Determination of macroporosity. In order to determine the macroporosity cryogels were swelled up to equilibrium and weighed (mswollen). Then swollen cryogels were squeezed, weighed (msqueezed), and macroporosity was calculated as follows:
Determination of flow resistance. For this, 200 mL of water was siphonaged from a breaker to the cryogel column by using 1.0 m silicon pipe and volume of passed through water with hydrostatic pressure was measured in one min.
FTIR measurements. In order to get the FTIR spectrum of synthesized cryogels, samples were dried and powdered. Powdered cryogels were mixed homogenously with KBr and pressed in a pellet form. Pellets were mounted in the FTIR spectrophotometer (Varian, FTS 7000, USA) and spectra of samples were taken.
Surface morphology analysis. Surface morphology of synthesized cryogels was analyzed using SEM (FEI, Quanta 250 FEG, USA). The swollen cryogels were mounted in the SEM device and SEM photographs were taken in Environmental SEM (ESEM) mode.
Elemental analysis. MAH incorporation onto polymeric structure was determined by elemental analysis. For this purpose, total nitrogen amount of synthesized cryogels was determined by using elemental analysis device (Leco, CHNS-932, USA).
Determination of surface area. Surface area of the cryogels was determined by the Brunauer-Emmett-Teller (BET) analysis. For this, powdered samples of cryogels were mounted in the BET devices (Micromeritics, Gemini V, USA) and surface area of the cryogels was determined.
Protein determination
All protein determinations were carried out according to method of Bradford (Citation1976). In this method, 50.0 μL of protein solution was mixed with 2.5 mL of Commasie Brilliant Blue G-250 solution and absorbance of the solution was read against the blank using a spectrophotometer (Shimadzu, UV1601, Japan). As a blank, distilled water was used instead of the protein sample.
CA activity studies
CA enzyme activities were carried out by using synthetic substrate p-nitro phenylacetate (p-NPA) according to method by Pocker and Ng (Citation1973). Absorbance of the yellow-colored product which was formed by esterase activity of the CA was read spectrophotometrically at 400 nm. Since the bovine blood and erythrocytes contain some other enzymes which possess esterase activity, in all CA purification studies CA activity studies were carried out using its natural substrate CO2 by method of Rickli (Rickli et al. Citation1964). This method involves the measurement of pH changes which occur when CO2 is converted to HCO3−. The change of pH was monitored with pH indicator bromothymol blue when the blue color of solution was converted to yellow. Duration for color change was measured with a chronometer, and enzymatic and specific activities were calculated using following equations:
where t0 is the color change time for enzyme-free test and t is the color change time for enzymatic test.
Optimization of CA adsorption conditions
CA adsorption studies were carried out in a continuous system. At a typical adsorption study, firstly cryogels were equilibrated with appropriate buffer, and then CA solution was passed through the cryogel column for 2 h. Adsorbed CA amount was calculated with initial and final CA concentration of the solution.
The effect of different buffers on the CA adsorption onto CA-imprinted PHEMAH cryogels was investigated and for this purpose; HEPES (pH 7.0, 7.5, and 8.0), MES (pH 5.5, 6.0, and 6.5), MOPS (pH 6.5, 7.0, 7.5, and 8.0), Tris-HCl (pH 7.0, 7.5, 8.0, 8.5, and 9.0), acetate (pH 4.0, 4.5, 5.0, and 5.5), phosphate (pH 6.0, 6.5, 7.0, and 7.5), and borate (pH 8.0, 8.5, 9.0, 9.5, and 10.0) buffers were used.
In order to investigate the CA adsorption capacity of imprinted cryogels, CA concentration was varied between 0.05 and 2.00 mg/mL. Experiments were also repeated for non-imprinted cryogels for demonstration of non-specific CA adsorption.
Adsorption behavior of imprinted cryogels was studied by using CA adsorption data. For this purposes, two main adsorption isotherms (Langmuir and Freundlich) were used.
Langmuir isotherm can be expressed as follows:
where q is the Langmuir single layer adsorption capacity (mg/g), Ce is the equilibrium CA concentration (mg/mL), b is the Langmuir adsorption equilibrium constant.
Freundlich isotherm is defined as follows:
where KF is the Freundlich adsorption constant (mg/g), Ce is the equilibrium CA concentration (mg/mL), n is the Freundlich constant which express the heterogeneity of the system (Derazshamshir et al. Citation2010).
Another important parameter that affects adsorption is the ionic strength of the medium. In order to investigate the effect of ionic strength, medium's ionic strength was changed between 0 and 1.0 M by using NaCl.
The effect of medium temperature on the CA adsorption was investigated in the temperature range of 4.0–60°C.
Chromatographic flow rate was changed between 0.1 and 4.0 mL/min, to examine the effect of chromatographic flow rate on the CA adsorption.
Fluorescence spectrophotometry analysis
In an attempt to investigate the effect of adsorption process on the conformation of CA enzyme, fluorescence spectrophotometry (Shimadzu, RF53010, Japan) analysis was carried out using pure and desorbed CA in order to compare the shifts (if any) on the excitation and emission wave lengths.
Reusability and stability of the cryogel
Reusability of the CA-imprinted cryogels was investigated for 10 adsorption/desorption cycle. For this, after each adsorption step CA was desorbed from the cryogel and cryogel was equilibrated with appropriate buffer and then cryogel was used for the next adsorption step. In order to determine the operational stability of the cryogel, after each desorption step activity of the CA was measured.
Selectivity of the CA imprinted cryogel
Selectivity of the CA-imprinted PHEMAH cryogel toward the CA protein was investigated using competitive proteins (e.g., albumin, hemoglobin, lysozyme, IgG, and γ-globulin). Molecular recognition selectivity of the molecular imprinted polymers was evaluated by dispersion coefficient (Kd) and selectivity coefficient (k) (Kuchen and Schram Citation1988, Dai et al. Citation1999). Kd was calculated for CA and other competitive proteins using following equation:
where Ci and Cf are the initial and final protein concentrations, respectively. V is the volume of the solution and W is the mass of the cryogel. k was calculated as follows:
where Kd1 and Kd2 are the dispersion coefficients of the template and competitive protein. In order to show the effect of imprinting over the protein selectivity, relative selectivity coefficient (k’) was used and defined as:
where kimprinted and knon-imprinted are the selectivity coefficients of the imprinted and non-imprinted cryogels, respectively.
Purification of CA from bovine erythrocyte
Fresh bovine blood was provided from the abattoir of the Aydın municipality. To prevent coagulation, bovine blood was collected in EDTA (0.1 M) containing glass bottle. Blood samples (50 mL) were centrifuged at 1500 rpm for 15 min in order to separate the plasma and white blood cells. Precipitated erythrocytes were washed twice with 0.9% NaCl solution and were hemolyzed with cold distilled water. Erythrocyte membranes and whole cells were precipitated by centrifugation at + 4°C, at 20,000 rpm for 30 min (Şentürk et al. Citation2009). The pH of obtained hemolysate was adjusted using solid MES. Then, hemolysate was directly used for the CA adsorption experiments. Purity of the purified CA using CA-imprinted PHEMAH cryogels was demonstrated with SDS-PAGE according to Laemmli (Citation1970) and protein bands were displayed using Commasie Brilliant Blue G-250 dye. In order to show the CA bands selectively, zymogram dyeing was carried out. For this, electrophoresis was performed under the SDS-free conditions. At the end of the electrophoresis period, gels were incubated in 0.1% bromothymol solution (in 0.1 M borate buffer, pH 9.0) for 10 min. Then gels were dried with filter paper and were put in saturated CO2 solution. Yellow band with the blue background demonstrated that the band belongs to CA and the enzyme was in active form (Manchenko Citation2003).
Results and discussion
Characterization studies
CA adsorption capacities of all synthesized cryogels are summarized in , which demonstrates that adsorption capacities of plain PHEMA and non-imprinted PHEMAH cryogels were lower than that of imprinted ones. Maximum CA adsorption capacity was found to be with CA10-encoded cryogel (2.7 mg/g cryogel), which contains 0.3 mmol MAH monomer and 10 mg CA in 15.0 mL of polymerization solution. With this result, CA10-encoded cryogel was used for the further adsorption and purification studies. Polymerization yield, swelling degree, macroporosity, and flow rate of synthesized cryogels are demonstrated in . As seen in table, for most combinations polymerization yields were above 95%. Swelling degrees of PHEMA and non-imprinted PHEMAH cryogels were found to be higher than that of CA-imprinted PHEMAH cryogel. Maximum swelling degree of imprinted cryogels was found to be 6.53 g H2O/g cryogel by using CA2-encoded cryogel. Macroporosity of imprinted cryogels were increased with increasing MAH concentration. It was also observed that cryogels which had high swelling degree had also high macroporosity. Maximum flow rate was found to be with CA6-encoded cryogel and flow rate generally decreased with MAH concentration. As expected, cryogels which had high macroporosity exhibited high flow rate.
Table I. CA adsorption capacities and characteristics of synthesized cryogels.
Subtraction of two FTIR spectra (also known as difference spectrum) is a method used for cleaning of overlapping bands and/or simplification of experimental spectrum. Difference spectrum is a sensitive spectroscopic method for all type of band alteration and is an ideal tool for exposition of the spectrums (Gradadolnik Citation2003). Since the FTIR spectra of PHEMA and PHEMAH cryogels were very similar, revelation of the incorporation of the MAH monomer into the polymeric structure was too difficult using these FTIR spectra. For this reason, difference spectrum was used with substraction of PHEMA spectrum from PHEMAH spectrum. In this way, only MAH spectrum was visualized. shows the difference spectrum of the PHEMAH–PHEMA cryogel. As seen in figure, characteristic stretching bands of –NH and CH3 and C = O stretching bands of carbonyl group were clearly seen around 3400 and 1720 cm− 1, respectively. Stretching vibrations of amide I bond around 1659 cm− 1 and amide II bond around 1540 cm− 1 arise from MAH monomer. –CH2 and CH3 stretching vibrations were observed at 1450 and 1390 cm− 1, respectively while, C–O stretching bands of carbonyl group can be seen at 1270 cm− 1. Stretching band of C–C–C was found around 1100 cm− 1. All these FTIR bands belong to the MAH monomer and these results showed that MAH monomer was incorporated into polymeric structure.
Figure 1. Difference spectrum of PHEMAH–PHEMA cryogels.
SEM photographs of the PHEMA, non-imprinted PHEMAH, and CA-imprinted PHEMAH are demonstrated in . As shown here, synthesized cryogels were quite porous, and pore diameter of the cryogels was measured to be around 10–100 μm. Pores were interconnected, separated with thin pore walls, and it was observed that cryogels were intensively macroporous. Cryogel morphology was changed with incorporation of MAH monomer to the polymeric structure, but surface morphologies of the imprinted and non-imprinted cryogels were found to be similar.
Figure 2. SEM photograph of PHEMA (a and b), non-imprinted PHEMAH (c and d), and CA-imprinted PHEMAH cryogels (e and f).
Incorporated MAH amount onto polymeric structure was calculated to be 7.38 μmol/g cryogel by using nitrogen stoichiometry by elemental analysis results.
According to BET analysis results, specific surface areas of PHEMA, non-imprinted PHEMAH, and CA-imprinted PHEMAH cryogels were found to be 23.6, 27.9, and 29.3 m2/g, respectively. It was clearly concluded that specific surface area of the cryogels was increased with incorporation of MAH monomer and surface area of the imprinted cryogel was greater than non-imprinted one, which may be due to the creation of cavities with removal of the template molecule as a result of imprinting process (Aslıyüce et al. Citation2010).
Optimization of CA adsorption conditions
The effect of the different buffers on the CA adsorption onto CA-imprinted cryogels was investigated and experimental results are demonstrated in . As seen from the figure, maximum CA adsorption was achieved by using pH 6.0 MES buffer. CA adsorption capacities were decreased at below and above pH 6.0. MES, MOPS, and HEPES are zwitter-ionic buffers and they carry two or more oppoite charges. These buffers may interact with CA binding region with ion–ion interaction and may decrease the CA adsorption capacity. But in the case of MES buffer, this interaction was found minimum and for this reason the maximum CA adsorption occurred with this buffer (Akgöl et al. Citation2007).
Figure 3. Effects of different buffer types on the CA adsorption onto CA-imprinted PHEMAH cryogel. Temperature: 25°C, CA concentration: 0.5 mg/mL, chromatographic flow rate: 0.5 mL/min.
In order to investigate the effect of CA concentration on the adsorption, the experiments were carried out using 0.05–2.00 mg/mL CA concentrations. demonstrates that CA adsorption capacity of the cryogel increased with increasing CA concentration, but it reached a saturation level at 0.5 mg/mL CA concentration (3.48 mg/g cryogel). It may be speculated that at this concentration, all the CA binding regions in the cryogel were filled and the cryogel cannot bind CA molecules any more. Nonspecific CA adsorption onto PHEMA and non-imprinted PHEMAH cryogel was found to be quite low. Adsorption behavior of CA onto imprinted PHEMAH cryogel was investigated using results of CA concentration study and adsorption constants of Langmuir and Freundlich adsorption isotherm are summarized in . It was concluded that CA adsorption onto CA-imprinted cryogel fits the Langmuir adsorption isotherm. As expected, CA was bound to the binding regions of the imprinted cryogel as a monolayer and similar energies.
Figure 4. Effects of CA concentration on the CA adsorption onto CA-imprinted PHEMAH cryogel. Temperature: 25°C, pH: 6.0 MES buffer, chromatographic flow rate: 0.5 mL/min.
Table II. Kinetic constants of Langmuir and Freundlich isotherms.
Another important parameter which affects the adsorption is ionic strength. For this, CA solutions were prepared with different ionic strength (0–1.0 M NaCl) and were used for adsorption experiments. Experimental findings are given in . As seen in figure, CA adsorption capacity decreased rapidly with increasing ionic strength from 0 to 1.0 M. Adsorption process is affected highly by ionic strength and adsorption capacity is generally decreased with increasing ionic strength. In this work, CA adsorption capacity of the CA imprinted cryogel decreased from 3.25 to 0.25 mg/g cryogel with increasing the ionic strength for 0 to 1.0 M by using NaCl.
Figure 5. Effects of ionic strength on the CA adsorption onto CA-imprinted PHEMAH cryogel. Temperature: 25°C, CA concentration: 0.5 mg/mL, pH: 6.0 MES buffer, chromatographic flow rate: 0.5 mL/min.
The effect of medium temperature on the CA adsorption capacity of imprinted cryogel was investigated in the temperature range of 4.0–60.0°C, and is demonstrated in . From the figure, it can be concluded that adsorption of CA onto CA-imprinted cryogel was not temperature-dependant. CA adsorption at 4.0°C was 1.84 mg/g, while adsorption at 60°C was 1.88 mg/g and the maximum CA adsorption was found at 25°C.
Figure 6. Effects of temperature on the CA adsorption onto CA-imprinted PHEMAH cryogel. CA concentration: 0.5 mg/mL, pH: 6.0 MES buffer, chromatographic flow rate: 0.5 mL/min.
shows the effect of chromatographic flow rate on the CA adsorption onto imprinted cryogel. As seen here, CA adsorption capacity of the CA-imprinted cryogel decreased with increasing chromatographic flow rate. CA adsorption capacity decreased from 2.401 to 0.185 mg/g cryogel when the flow rate increased from 0.1 to 4.0 mL/min. This decrease at high flow rates was may be due to decrease in interaction time between CA molecules and CA-binding regions of the cryogel (Aslıyüce et al. Citation2010).
Figure 7. Effects chromatographic flow rate on the CA adsorption onto CA-imprinted PHEMAH cryogel. Temperature: 25°C, CA concentration: 0.5 mg/mL, pH: 6.0 MES buffer.
Fluorescence spectrophotometry analysis
Florescence spectrum of pure and desorbed CA are shown in . With this result, it was demonstrated that, CA had two different excitation (291 and 581 nm) and two different emission (341 and 673 nm) wave lengths. The pure and the desorbed CAs gave the excitations and the emissions at the same wave lengths. In the light of this information it can be concluded that, conformations of the purified or adsorbed CA did not change with adsorption onto CA-imprinted cryogels and the protein did not denatured throughout the adsorption studies.
Figure 8. Florescence spectrum of pure and desorbed CAs.
Reusability and stability of the cryogel
Synthesized CA-imprinted PHEMAH cryogels were used for CA adsorption studies for 10 cycles. After each cycle, CA was desorbed from the cryogel using 1.0 M of NaCl solution (at pH 4.0, acetate buffer) and cryogel was washed with distilled water and was equilibrated for next cycle. Operational stability of the cryogel was also investigated by measuring the enzymatic activity of the desorbed CA. Reusability and the operational stability profile of the CA-imprinted cryogels are given in . As seen here, CA adsorption capacity did not significantly change within 10 adsorption/desorption cycles, and it was found that adsorption capacity of the CA-imprinted cryogel decreased only about 10.2% after the 10 cycles. Also, operational stability of the cryogel was found to be very high and 80.71% of CA initial activity was protected at the end of 10 cycles.
Figure 9. Reusability and operational stability of the CA imprinted PHEMAH cryogel. Temperature: 25°C, CA concentration: 0.5 mg/mL, pH: 6.0 MES buffer, chromatographic flow rate: 0.5 mL/min.
Selectivity of the CA-imprinted cryogel
Selectivity of the CA-imprinted PHEMAH cryogel was investigated against albumin, hemoglobin, IgG, γ-globulin, and lysozyme. Albumin, hemoglobin, IgG, and γ-globulin were found in bovine blood and were used for the examination of possible interference. Lysozyme is a small (14000 Da) enzyme and was used for the investigation of the effect of molecular size on the adsorption. Experimental findings are summarized in . It was observed that, CA-imprinted cryogels were more selective to CA than other competitor proteins by comparing their k’ values. Selectivity of the CA-imprinted cryogel toward the CA was higher than that of albumin, hemoglobin, IgG, and γ-globulin and selectivity to the CA was found to be 15.6, 60.05, 21.88, and 17.61 times greater, respectively. It was also found that synthesized cryogels selectively bound the CA 17.4 times greater than lysozyme. MIP can recognize the template molecules due to the specific binding interactions, binding regions, shape and size accordance between target molecule, and binding region of the polymer. Moreover, power of the interaction between template and binding regions determine the selectivity of the molecularly imprinted polymers (Dai et al. Citation2011).
Table III. Selectivity of the CA-imprinted cryogels.
Purification of CA from bovine erythrocyte
CA was purified from bovine blood under the optimized conditions. After the typical CA adsorption experiments, CA was desorbed and purity of the enzyme was demonstrated with SDS-PAGE (). A purification table was also prepared by using the enzymatic analysis experiment in purification studies (). As seen in , CA was successfully purified from bovine erythrocytes with a single-step application. As demonstrated in , CA was purified with yield of 79.41% and the CA purification fold was calculated to be 84. In order to visualize the purified CA by its enzymatic activity, zymogram dying was carried out and photographs of zymogram of the pure and the purified CAs are demonstrated in . As seen in figure, pure and purified CAs can be seen as yellow bands above the blue background and it was concluded that purified CA protected its enzymatic activity after the purification steps.
Figure 10. SDS-PAGE photograph of CA purification steps. Line 1: protein standards [myosin (rabbit blood, 205 kDa), β-galactosidase (E. coli, 116 kDa), phosphorylase B (rabbit blood, 97 kDa), albumin (bovine blood, 66 kDa), albumin (egg white, 45 kDa) and carbonic anhydrase (bovine erythrocyte, 29 kDa); line 2: Pure CA; line 3: crude hemolysate; line 4: purified CA desorbed from CA-imprinted PHEMAH cryogel.
Figure 11. Zymogram photographs of pure CA (1) and purified CA (2) desorbed from CA-imprinted PHEMAH cryogel.
Table IV. Purification of CA from bovine erythrocyte by using CA-imprinted PHEMAH cryogel.
Conclusions
In this work, CA-imprinted PHEMAH cryogels were synthesized for purification of CA from bovine blood. Synthesized cryogels had sponge-like structure and easily lost their water inside pores when the cryogels compressed. Dried cryogels swelled fast when submerged in water and reach their original size in a few minutes. Characterization of the cryogels was carried out with FTIR, SEM, elemental analysis, and surface area measurements. It was found that the cryogels were considerably porous and the pore diameter was found to be about 10–100 μm by using SEM. Specific surface area of PHEMA, non-imprinted PHEMAH, and CA-imprinted PHEMAH cryogels were found to be 23.6, 27.9, and 29.3 m2/g, respectively. The effects of pH and buffer type, CA concentration, ionic strength, temperature, and chromatographic flow rate on the CA adsorption were optimized. For the desorption of CA from cryogel, 1.0 M NaCl solution (in acetate buffer, pH 4.0) was used. Florescence spectrums of CA were taken to show if any structural changes or denaturation of CA occurred in the adsorption studies. Cryogels were used for 10 adsorption/desorption cycles and CA adsorption capacity of the cryogels decreased only about 10% at the end of 10 cycles. Selectivity of the CA-imprinted cryogels was investigated against the competitor proteins albumin, hemoglobin, IgG, γ-globulin, and lysozyme, its selectivity toward the CA was found to be 15.26, 60.05, 21.88, 17.61, and 17.42 times, respectively. Synthesized CA-imprinted PHEMAH cryogels were used for the purification of CA from bovine blood and CA was purified with 79.41% recovery. The purity was showed with SDS-PAGE and the purification fold is estimated as 83.98. This study is a rare example in relation to use of the molecular imprinting technology for purification of enzymes and this study is the first report about purification of CA by using molecular imprinted cryogels.
Declaration of interest
The authors report no declarations of interest. The authors alone are responsible for the content and wirting of the paper.
References
- Akgöl S, Özkara S, Uzun L, Yılmaz F, Denizli A. 2007. Pseudospecific magnetic affinity beads for immunoglobulin-G depletion from human serum. J Appl Polym Sci. 106:2405–2412.
- Andaç M, Baydemir G, Yavuz H, Denizli A. 2012a. Molecularly imprinted composite cryogel for albumin depletion from human serum. J Mol Recog. 25:555–563.
- Andaç M, Galaev IY, Denizli A. 2012b. Dye attached poly(hydroxyethyl methacrylate) cryogel for albumin depletion from human serum. J Sep Sci. 35:1173–1182.
- Aslıyüce S, Bereli N, Uzun L, Onur MA, Say R, Denizli A. 2010. Ion-imprinted supermacroporous cryogel, for in vitro removal of iron out of human plasma with beta thalassemia. Sep Purif Technol. 73:243–249.
- Banerjee AL, Swanson M, Mallik S, Srivastava DK. 2004. Purification of recombinant human carbonic anhydrase-II by metal affinity chromatography without incorporating histidine tags. Protein Expres Purif. 37:450–454.
- Bereli N, Andaç M, Baydemir G, Say R, Galaev IY, Denizli A. 2008. Protein recognition via ion-coordinated molecularly imprinted supermacroporous cryogels. J Chromatogr A. 1190:18–26.
- Bereli N, Saylan Y, Uzun L, Say R, Denizli A. 2011a. L-Histidine imprinted supermacroporous cryogels for protein recognition. Sep Purif Technol. 82:28–35.
- Bereli N, Şener G, Yavuz H, Denizli A. 2011b. Oriented immobilized anti-LDL antibody carrying poly(hydroxyethyl methacrylate) cryogel for cholesterol removal from human plasma. Mater Sci Eng C. 31:1078–1083.
- Bereli N, Ertürk G, Denizli A. 2012. Histidine containing macroporous affinity cryogels for Immunoglobulin G purification. Sep Sci Technol. 47:1813–1820.
- Bereli N, Ertürk G, Tümer MA, Say R, Denizli A. 2013. Oriented immobilized anti-hIgG via Fc fragment-imprinted PHEMA cryogel for IgG purification. Biomed Chromatogr. 27:599–607.
- Bradford MM. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72:248–251.
- Ceyhun SB, Şentürk M, Yerlikaya E, Erdoğan O, Küfrevioğlu Öİ, Ekinci D. 2011. Purification and characterization of carbonic anhydrase from the teleost fish Dicentrarchus labrax (European seabass) liver and toxicological effects of metal on enzyme activity. Environ Toxicol Phar. 32:69–74.
- Dai S, Burleigh MC, Shin Y, Morrow CC, Barnes CE, Xue Z. 1999. Imprint coating: a novel synthesis of selective functionalized ordered mesoporous sorbents. Angew Chem Int Edit. 38:1235–1239.
- Dainiak MB, Galaev IY, Kumar A, Plieva FM, Mattiasson B. 2007. Chromatography of living cells using supermacroporous hydrogels, cryogels. Adv Biochem Eng Biot. 106:101–127.
- Dai CM, Geissen SU, Zhang YL, Zhang YJ, Zhou XF. 2011. Selective removal of diclofenac from contamined water using molecularly imprinted polymer microspheres. Environ Pollut. 159:1660–1666.
- Derazshamshir A, Baydemir G, Andaç M, Say R, Galaev IY, Denizli A. 2010. Molecularly imprinted PHEMA-based cryogel for depletion of hemoglobin from human blood. Macromol Chem Phys. 211: 657–668.
- Garipcan B, Denizli A. 2002. A novel affinity support material for the separation of Immunoglobulin G from human plasma. Macromol Biosci. 2:135–144.
- Gilmour KM. 2010. Perspectives on carbonic anhydrase. Comp Biochem Phys. 157:193–197.
- Gradadolnik J. 2003. Infrared difference spectroscopy: Part I. Interpretation of the difference spectrum. Vib Spectrosc. 31: 279–288.
- Janiak DS, Kofinas P. 2007. Molecular imprinting of peptides and proteins in aqueous media. Anal Bioanal Chem. 389:399–404.
- Kuchen W, Schram J. 1988. Metal-ion selective exchange resins by matrix imprint with methacrylates. Angew Chem Int Edit. 27: 1695–1697.
- Laemmli UK. 1970. Cleavage of structure protein during the assembly of the head of bacteriophage T4. Nature. 277:680–685.
- Lozinsky VI, Plieva FM, Galaev IY, Mattiasson B. 2002. The potential of polymeric cryogels in bioseparation. Bioseparation. 10:163–188.
- Lozinsky VI, Galaev IY, Plieva FM, Savina IN, Jungvid H, Mattiasson B. 2003. Polymeric cryogels as promising materials of biotechnological interest. Trends Biotechnol. 21:445–451.
- Manchenko GP. 2003. Handbook of Detection of Enzymes on Electrophoretic Gels. Boca Raton: CRC Press, pp. 482–483.
- Meldrum NU, Roughton FJW. 1933. Carbonic anhydrase. Its preparation and properties. J Physiol. 80:113–142.
- Özgür E, Bereli N, Türkmen D, Ünal S, Denizli A. 2011. PHEMA cryogel for in-vitro removal of anti-dsDNA antibodies from SLE plasma. Mater Sci Eng C. 31:915–920.
- Plieva FM, Galaev IY, Mattiasson B. 2007. Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulate-containing fluids and cell culture applications. J Sep Sci. 30:1657–1671.
- Pocker Y, Ng JSY. 1973. Properties and carbon dioxide hydration kinetics. Biochemistry. 12:5127–5134.
- Rickli EE, Ghazanfar SAS, Gibbons BH, Edsall JT. 1964. Carbonic anhydrases from human erythrocytes. J Biol Chem. 239:1065–1078.
- Supuran CT, Scozzafava A, Conway J. 2004. Carbonic Anhydrase. Its Inhibitors and Activators. Boca Raton: CRC Press.
- Supuran CT, Scozzafava A. 2007. Carbonic anhydrase as targets for medicinal chemistry. Bioorgan Med Chem. 15:4336–4350.
- Supuran CT. 2010. Carbonic anhydrase inhibitors. Bioorg Med Chem Lett. 20:3467–3474.
- Şentürk M, Gülçin İ, Daştan A, Küfrevioğlu Öİ, Supuran CT. 2009. Carbonic anhydrase inhibitors. Inhibition of human erythrocyte isozyme I and II with a series of antioxidant phenols.Bioorgan Med Chem. 17:3207–3211.
- Tamahkar E, Bereli N, Say R, Denizli A. 2011. Molecularly imprinted supermacroporous cryogels for cytochrome c recognition. J Sep Sci. 34:3433–3440.
- Zhang H, Ye L, Mosbach K. 2006. Non-covalent molecular imprinting with emphasis on its application in separation and drug development. J Mol Recognit. 19:248–259.