Novel immobilized Cu+2 ion grafted cellophane membranes for affinity separation of His-Tag Chitinase

M.S. Mohy Eldin; S. Abdel Rahman; G.F. El fawal

doi:10.1016/j.arabjc.2014.04.004

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Original article

10 (

2_suppl

); S3652-S3663

doi:

10.1016/j.arabjc.2014.04.004

Novel immobilized Cu⁺² ion grafted cellophane membranes for affinity separation of His-Tag Chitinase

M.S. Mohy Eldin^{^a,⁎}, S. Abdel Rahman^{^b}, G.F. El fawal^{^a}

a Department of polymers Material Research, Institute of Advanced Technology and New Materials, City for Scientific Research and Technology Applications, New Borg El-Arab City 21934, Alexandria, Egypt

b Department of Chemistry, Faculty of Science, Zagazig University, Zagazig, Egypt

⁎Corresponding author. Tel.: +20 1005318399. mohyeldinmohamed@yahoo.com (M.S. Mohy Eldin)

Received: 2013-6-16, Accepted: 2014-4-6, Published: 2014-05-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Two kinds of immobilized Cu⁺² ion grafted cellophane membrane were prepared using methacrylic acid (MAA) and methyl methacrylate (MMA). The affinity grafted membranes were tested in separation of His-Tag Chitinase enzyme from BSA-protein mixture. PMAA grafted membranes show a higher affinity than PMMA grafted ones towards the separation of Chitinase enzyme. Both types of affinity membranes show almost the same desorption capability for both proteins with small advantage for PMAA grafted affinity membranes. No leakage of Cu⁺² ions was detected during the protein elution process. The characters of the obtained Cu⁺² immobilized grafted membranes recommended them as novel immobilized metal affinity membranes (IMAMs) for His-Tag protein separation and presenting a solution for the metal ion leakage problem; the main reason for limiting the use of metal ion affinity chromatography technique.

Keywords

Chromatography

Graft copolymers

Proteins

Separation techniques

Membranes

Cellulose

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1 Introduction

Cellulose (CE) grafting is a process aimed to induce additional characters to the cellulose, without losing its natural ones, through attaching branches of synthetic polymer to the glucose backbone. This opens the way for using grafted cellulose matrices to have immobilized metal ion affinity chromatography (IMAC). Benefiting from the affinity of some specific functional groups like histidines, such cellulosic affinity matrices have been used as a protein purification method (Proath et al., 1975; Hemdan et al., 1989). Considering both stability and economy of the ligand in addition to high yields, IMAC is coming up as a good choice for protein purification (Arnold, 1991). Brandt et al. (1988) were the first who proposed affinity membrane chromatography as a strategy to overcome typical drawbacks of bead-based chromatography. During the last decade, high focus has been paid to the separation of biomolecules using affinity membranes due to the lower mass-transfer limitations observed in the membrane process than conventional column chromatography (Brandt et al., 1988; Roper and Lightfoot, 1995; Charcosset, 1998; Zou et al., 2001 ).

Different modes of affinity have been induced to membrane supports. The impact of different affinity modes on the relative adsorption properties and separation efficiencies of membranes has been deeply explored. Immobilized metal affinity membranes (IMAMs) Charcosset, 1998; Zou et al., 2001; Rodemann and Staude, 1994; Reif et al., 1994; Beeskow et al., 1995; Kubota et al., 1996; Arıca et al., 1998; Denizli et al., 1998; Camperi et al., 1998; Crawford et al., 1999; Yang et al., 1999; Hari et al., 2000; Senel et al., 2001; Tsai et al., 2002 are coming up as the most used affinity membrane techniques. Usually, they are used in the separation of surface-exposed amino acid proteins. Histidine, cysteine, tryptophan, and tyrosine, or polyhistidine-tagged biomolecules presented good examples. Different materials including cellulose, nylon, polysulfone, polyethylene, glass, synthetic copolymer, etc. have been studied for IMAMs application (Rodemann and Staude, 1994; Reif et al., 1994; Beeskow et al., 1995; Kubota et al., 1996; Arıca et al., 1998; Denizli et al., 1998; Camperi et al., 1998; Crawford et al., 1999; Yang et al., 1999; Hari et al., 2000; Senel et al., 2001; Tsai et al., 2002 ). Simple chemistry of binding hydroxyl groups with different chelating agents, gave the advantage of cellulose membranes over other materials as reported by Zou et al. in their review paper about previous cellulose-based IMAM studies; cellulose acetate and cellulose composite (Zou et al., 2001). The commercial availability of cellulose membranes such as cellulose acetate, cellulose nitrate, and regenerated cellulose is an additional benefit (Hari et al., 2000).

Cellulose acetate (Kubota et al., 1996) or nitrate need to transfer the functional groups back to hydroxyl groups before they can be used as IMAMs. Regenerated cellulose (RC) has no need for such regeneration step, in addition to its low nonspecific adsorption (Zou et al., 2001; Crawford et al., 1999) and good chemical stability which nominate it as good IMAMs. Inducing chelating agents into its structure is the most used technique in the literature to prepare IMAMs from regenerated cellulose (Roper and Lightfoot, 1995; Yang et al., 1999). One of the main drawbacks of using this technique is leakage of metal ions during the elution step (Kubota et al., 1996; Wu et al., 2003) which was mainly explained by insufficient chelating bonds with metal ions. Grafting technique has been persecuted as one of the solutions where unlimited numbers of functional groups with metal chelating character could be induced to have higher amounts and more stable immobilized metal ions (Roper and Lightfoot, 1995; Yang et al., 1999). In previous publication for the authors (Mohy Eldin et al., 2009), Cu⁺² immobilized cellophane PGMA grafted membranes were prepared and used in the separation of the beta Galactosidase from protein mixture solution. The IMAM shows promising results. This success encourages the authors to prepare immobilized metal ion affinity membranes through inducing carboxylic acid groups (Mohy Eldin et al., 2011) and hydroxyl-imide groups (unpublished data) via grafting of cellophane with PMAA and PMMA and then immobilized with Cu⁺² ions to be used finally in the separation of His-Tag Chitinase from its mixture with BSA protein. The affinity membranes show high affinity towards separation of Chitinase with no Cu⁺² ion leakage in the eluting solution.

Macroporous polymer beads from PMAA and PMMA have been used in various fields, for example as solid supports in solid-phase organic synthesis and heterogeneous catalysts and as adsorbents in column chromatography (Park et al., 2011).

In continuation of our research in this new direction for the preparation of novel IMAMs grafted polymer membranes, the conditions for affinity separation of His-Tag Chitinase from protein mixture using immobilized Cu⁺² ions grafted cellophane membranes were studied such as protein concentration, pH and adsorption time. In addition, the effect of grafting degree and amount of immobilized Cu⁺² ions were correlated to amount of adsorbed protein and membranes’ affinity.

Finally, the process of adsorbed protein elution was studied and affecting factors such as eluent concentration, pH and elution time were explored.

2 Experimental

2.1

2.1 Materials

Cellophane sheets, kindly supplied by Misr Rayon Co. Kafr Eldawar (Egypt), were used after extraction with hot distilled water to remove the surface additives (20% glycerol and Na₂SO₃), Potassium Persulphate (KPS) (purity 99%, M.wt. 270.31), sodium bisulphite (SBS) (purity 98%, M.wt. 190.11), hydroxylamine hydrochloride (purity 99%), methacrylic acid (MAA) (purity 98%) and methyl methacrylate (MMA) (purity 98%) were obtained from Sigma–Aldrich (Germany). Copper sulfate (purity 98%, M.Wt. 249.68) was obtained from El-Naser Pharmaceutical Co. for Chemicals, Egypt. His-Tag Chitinase enzyme was kindly supplied by Dr. El-Sayed Hafez (Nucleic Acid Research Department, GEBRI, Alexandria, Egypt). BSA protein (fraction V., minimum 96% electrophoresis, nitrogen content 16.2%) was supplied from Sigma–Aldrich Chemical Ltd. (Germany). Albumin kit and total protein kit were supplied from Diamond Diagnostics Co. for Modern Laboratory Chemicals (Egypt).

2.2

2.2 Methods

2.2.1

2.2.1 Cellophane membrane grafting

All the polymerization reactions were carried out in air atmosphere (El-Awady et al., 1999; Lepoutre and Hui, 1973; Kulkarni and Mehta, 1968 ). A piece of cellophane (5 cm × 5 cm) was placed in a test tube with fitted stopper in a thermo stated water bath. Known concentration of KPS in ethanol–water (1:1) aqueous solution was added and left for 30 min before adding definite amount of single monomer (MMA or MAA) and left to copolymerize for a definite time at a definite temperature. After completion of the polymerization time, the test tube was left at R.T over night to complete the grafting process. The grafted membranes were then removed and washed thoroughly with appropriate solvent, hot distilled water for PMAA and hot ethanol for PMMA, to remove homopolymers. After extraction, the grafted membranes were dried at 60 °C in air drier and weighted to evaluate the grafting percentage according to the following equation (El-Awady et al., 2002):

(1)

GP % = \frac{Wt. of grafted membrane - Wt. of original membrane}{Wt. of original membrane} \times 100

2.2.2

2.2.2 Grafted membrane functionalization

Polymethyl methacrylate grafted membranes have gained ester groups which do not have the capability to immobilize Cu⁺² ions, so further functionalization step is need. Amination is a process that occurs through the reaction of terminal ester groups with hydroxyl amine. As a result; terminal hydroxyl groups and secondary-tertiary amines are introduced. The grafting and functionalization (amination) processes are illustrated in Scheme 1.

Schematic diagram of amination process of cellophane-g-PMMA membranes.

2.2.3

2.2.3 FT-IR analysis

FT-IR spectra of origin and grafted cellophane membranes were recorded on a Fourier Transform Infrared spectrophotometer (Shimadzu FTIR-8400S, Japan).

2.2.4

2.2.4 Drying

The moist samples were placed in a suitable oven and dried to constant weight at 60 °C overnight.

2.2.5

2.2.5 Copper ion immobilization

To immobilize copper ions, the grafted membrane (5 cm × 5 cm) was incubated with 20 mL of CuSO₄ (150 ppm) solution and shaken at 50 rpm for 30 min. at 30 °C. After incubation, the membrane was sequentially washed with DI water in order to remove the unbounded or weakly linked copper ions. The membrane turns to blue colour as indicator of copper ions immobilization.

2.2.6

2.2.6 Copper ion recovery

Elution process was successfully performed by using 20 mL of 0.1 N HCl, for 30 min., in 30 °C at 150 rpm. The amount of copper immobilized and eluted was determined by using an atomic absorption spectrophotometer (A Analysis T300, Perkin Elmer, USA).

2.2.7

2.2.7 Water uptake (W%)

To determine the water uptake percent, the membrane was previously immersed in distilled water at room temperature for 24 h. The membrane was then removed and dried by wiping between two filter papers and then weighted (Vita et al., 1984).

(2)

W % = {[W 1 - W 2] / W 2} \times 100

where W1 is the Wt. of wet membrane and W2 the wt. of dry membrane.

2.2.8

2.2.8 Mechanical analysis

Mechanical analysis is a technique in which the tensile strength and elongation of the sample are monitored under force. Mechanical analysis was carried out using a Shimadzu tensile test machine, Model AG-IS (Japan).

2.2.9

2.2.9 Dimension stability

A ruler was used to measure the dimension of the membrane before and after soaking in distilled water at room temperature for 24 h. The dimension stability was calculated as follows (El-Awady et al., 2002);

(3)

Dimension stability = ([D 2 - D 1] / D 1) \times 100

where: D1 is the dimension of membrane before soaking and D2 is the dimension of membrane after soaking.

2.2.10

2.2.10 Surface roughness

The changes on the surface roughness were monitored by using Surface Roughness Tester model SJ-201P. (Japan).

2.3

2.3 Protein separation

In batch adsorption experiment, a piece of dry affinity membrane, 5 cm × 5 cm, was incubated in 20 mL phosphate buffer solution (pH 7) of Chitinase–BSA (1:1) under gentle shaking at room temperature for 1 h. Different concentrations of protein mixtures were tested. After adsorption, the membrane was washed with phosphate buffer (loading buffer) and then air-dried. In this study, the method used to determine the amount of adsorbed protein in batch mode depends on calculated difference between the amounts of protein in the original adsorbed solution and those which remain after adsorption plus those eluted in washing buffer solution.

In order to investigate the adsorption of both proteins onto our membranes, the adsorption of both individual proteins has been investigated and then we study the adsorption of the proteins mixture to check the membranes’ affinity towards His-Tag protein. Different factors affecting the adsorption process have been studied. These factors are summarized as follows: Protein concentration (w/v, %); 0.025–0.25%, adsorption pH; 4–8, adsorption time; 15–120 min.

In the elution process, the immobilized membrane with adsorbed proteins was immersed in 20 mL imidazole solution using different concentrations (0.2–0.6 M) of selected pH (6–8) at fixed temperature (30 °C) with gentile shaking for different time (15–90 min).

In this study PMAA grafted cellophane membranes with Cu⁺² ions, 119 μg/g and GP% (144%) and PMMA grafted cellophane membranes with Cu⁺² ions, 47.2 μg/g and GP% (58%) have been used according to the best results obtained in previously published work (Mohy Eldin et al., 2011).

2.3.1

2.3.1 Determination of bovine serum albumin amount

Using Albumin kit, 10 μL of protein solution, adsorbed and/or eluted, was mixed with 2 mL of reagent (2), [succinate buffer 75 mmol/L PH 4.2, BCG 0.12 mmol/L, tensioactive 2 g/L (W/V)], (A_sample) and 10μL of reagent (1), [protein standard 5 g/dl], with 2 mL of reagent (2); (A_stander). The solution was mixed well and allowed to stand for 10 min. at room temperature. The absorbance (A) of the samples and the standard was measured against the reagent blank at 630 nm. The amount of the BSA was calculated according to the following formula (Perry et al., 1979);

(4)

Bovine Serum Albumin amount (mg) = [A_{sample} / A_{stander}] \times 5

2.3.2

2.3.2 Determination of Chitinase amount

Using total protein kit, 20 μL of protein solution, adsorbed and/or eluted, was mixed with 1 mL reagent (2), (A_sample), or 20 μL of reagent (1) with 1 mL reagent (2); (A_stander). The samples were mixed well and incubated for 10 min. at 37 °C. The absorbance (A) of the samples and the standard was measured against the reagent blank at 540 nm. The amount of measured protein was calculated according to the following equation.

(5)

Total protein amount (mg) = [A_{sample} / A_{stander}] \times 7

Where reagent 1: protein standard; 7 g/dl, Reagent 2: NaOH; 0.75 M, K-Na. tartarate; 21 mmol/L, Pot. Iodide; 6 mmol/L, cupric sulphate; 6 mmol/L.

Chitinase amount (mg) was calculated by the difference between total protein amount calculated by Eq. (5) and Albumin amount calculated by Eq. (4).

3 Results and discussion

3.1

3.1 Membrane preparation and characterization

In this study, PMAA grafted cellophane membranes (GP%; 144) with immobilized Cu⁺² ions (119 μg/g) and PMMA grafted cellophane membranes (GP%; 58) with immobilized Cu⁺² ions (47.2 μg/g) have been used according to the best results obtained in previously published work (Mohy Eldin et al., 2011). Verification of the grafting and further more functionalization processes were obtained from FT-IR data. Physico-chemical characters of the prepared grafted affinity membranes including thickness, water uptake, mechanical properties, and finally surface roughness were evaluated. Morphological changes of membranes’ surface upon modification were monitored through SEM examinations.

Figs. 1 and 2 illustrate the FT-IR spectra of un-grafted, grafted membranes and immobilized Cu⁺² ions grafted membranes. From figures, the IR-spectrum shows that absorption bands between 3250 and 3500 cm⁻¹ arising from hydroxyl groups were located on the un-grafted membranes. The IR spectra of poly methacrylic acid grafted cellophane membrane showed a characteristic band at 1731 cm⁻¹ which is peculiar to the carbonyl group of acid stretching vibrations; Fig. 1B. The absence of this band in the spectra of un-grafted cellulose (Fig. 1A) proves the formation of PMAA grafted cellulose. Loading of Cu⁺² ions onto the PMAA grafted membranes greatly masked this band. This confirms the interaction between Cu⁺² ions and carboxylic groups (Fig. 1C).

FT-IR spectra of un-grafted cellophane membrane (A), PMAA grafted membrane, GP = 63% (B), Cu+2-immobilized PMAA-grafted membrane (C).

FT-IR spectra of PMMA grafted membrane treated with hydroxyl amine, GP = 42% (A) and Cu+2-immobilized aminated PMMA-grafted membrane (B).

The IR spectra of PMMA grafted cellophane showed a characteristic band at 1728–1731 cm⁻¹, arising from C⚌O stretching of ester group of PMMA. This confirms the grafting of PMMA onto the membranes. Other band between 1100 and 1200 cm⁻¹ arising from amine groups resulted from the amination process of the PMMA grafted membranes (Fig. 2A). It can be seen that loading Cu⁺² ions masked completely the character bands mentioned above of grafted membranes where bands’ intensity was reduced and a shift in the wavelength of those bands was monitored. This gives an indication for succeeding of Cu⁺² immobilization process (Fig. 2B).

The physical change observed upon grafting of cellophane membranes with individual PMAA (GP; 140%) and PMMA (GP; 50%) was the thickness increment. This increment was proportional to the grafting percentage. Indeed, the increment of PMMA grafted membranes’ thickness is much higher than that of PMAA grafted ones. This could be referred to the hydrophobic nature of PMMA grafted polymer branches and the presence of ester groups in their structure. The thickness of un-grafted, PMMA, and PMAA grafted membranes was found to be 13, 56, and 44 (μm), respectively.

Both tensile strength and elongation characters were monitored for un-grafted and grafted membranes (Table 1). It is obvious that the grafting process in general improves the tensile strength of the grafted membranes. The tensile strength of PMMA grafted membranes was found to be higher than that of PMAA grafted ones. On the other hand, the grafting process has a negative effect on the elongation character of the grafted membranes in general. PMAA grafted membranes have been much affected. This behaviour may be referred to the degree of hydrogen bond formation which is expected to be formed between the carboxylic groups of the PMAA grafted branches. This degree is higher than that formed between the hydroxyl and imide groups of the aminated PMMA grafted branches. Moreover, the double grafting percentage of PMAA and the cross-linking effect of Cu⁺² ions have to be taken into account. Overall, the dimension stability of the grafted membranes is good enough compared to the un-grafted cellophane membranes. Improvement of the mechanical properties and the dimension stability of the prepared IMAMs are essential requirements for membranes working in the field of protein separation.

Table 1 Mechanical properties of Cu⁺² immobilized cellophane grafted membranes.

Membranes type	GP (%)	Dimension satiability	Tensile strength (N)	Elongation (mm)
Cellophane	Zero	9% ± 0.7	15.28 ± 1.2	3.26 ± 0.24
PMMA	56 ± 5	10.7% ± 0.9	26.19 ± 1.9	2.03 ± 0.18
PMAA	120 ± 9.6	10.4% ± 0.8	22.57 ± 1.8	0.59 ± 0.04

Water absorption in cellulose films is attributed mostly to the hydroxyl groups located on polysaccharides chains. Nearly one-third of the cellulose in cellophane membranes is amorphous in nature and this is responsible for water absorption, while crystalline portion being impenetrable.

The data of water uptake percentage of un-grafted and grafted cellophane membranes are presented in Table 2. PMMA grafted membranes’ water uptake percentage was found, as expected, less than that of un-grafted ones. This is may be due to partial blockage of the internal membranes’ amorphous part by the hydrophobic ester and methyl groups of PMMA graft side chains. These results are agree with previous results obtained by the authors (Mohy Eldin et al., 2011) where the grafting of hydrophobic monomers such as MMA resulted in reduction of water up take character. PMAA grafted membranes show a higher water uptake compared to un-grafted cellophane ones. This is due to an additional induced number of (OH) groups on the cellulose backbone results from grafted carboxylic groups of PMAA graft chains. This trend is in agreement with previously published data by Kulkarni and Mehta (1968) in which they grafted poly acrylic acid onto a cellophane. The obtained grafting percentage values were very close to those obtained in the current study but with doubled value of water uptake. This could be attributed to the presence of methyl groups in the structure of MAA which is known by its hydrophobic character.

Table 2 Water uptake percentage for cellophane, cellophane grafted membranes and Cu⁺² immobilized cellophane grafted membranes.

Membranes type	Cellophane	PMAA-g-cellophane	PMMA-g-cellophane
GP%	0	144 ± 12	50.6 ± 3.8
Grafted membranes	50.2 ± 4.2	62.1 ± 5.3	29 ± 2.6
Cu⁺²-immobilized cellophane grafted membranes	48.2 ± 4.3	69.7 ± 5.6	59.1 ± 4.8

In general, immobilized grafted membranes with Cu⁺² ions show a higher water uptake percentage for both types comparing with un-grafted cellophane membranes. This is may be due to the chelating nature of immobilized cupper ions with water molecules. Water uptake is a reflection of the membrane hydrophilicity which is known as the main factor that inhibits non-specific protein adsorption resulted from interactions between hydrophobic moieties in both polymer matrix and protein molecules.

One of the main advantages of the cellophane membranes is their low non-specific protein adsorption character. This character is directly affected by the surface roughness. In general, it was found that the grafting process increases the surface roughness of grafted matrices where the nature of grafted branches has a direct effect. Although the grafting percentage of PMMA (GP%; 56%) is almost half of PMAA (GP%; 120%), the surface roughness was found to be 4.2, 6.6, and 6.1 um for un-grafted, PMMA and PMAA grafted membranes, respectively. These results may be responsible in part about the non-specific adsorption of both proteins on grafted membranes.

Fig. 3 shows SEM micrograph of un-grafted and grafted membranes. The figure revealed that no phase separation has been noticed which is considered as a good indication for grafting homogeneity which consequently affects the porous structure of the grafted membranes to be less porous. This results an increase in the thickness of the grafted membranes.

SEM Micrograph of un-grafted cellophane membrane (A), PMAA-grafted membranes; GP% = 37 (B), PMMA-grafted membranes; GP% = 39 (C).

3.2

3.2 Protein separation

Chitinolytic enzymes have wide ranging applications such as preparation of pharmaceutically important chitooligosaccharides and N-acetyl d-glucosamine, preparation of single-cell protein, isolation of protoplasts from fungi and yeast, control of pathogenic fungi, treatment of chitinous waste, and control of malaria transmission (Dahiya et al., 2006).

Chitinase separation from equal mixture with BSA using Cu⁺² ion immobilized PMAA and PMMA grafted membranes, as an application, was investigated. The results are discussed in the following.

To exclude the effect of protein isoelectric point on the separation process, BSA has been selected based on its PI (4.9), which is very close to that of Chitinase; PI (5.0). First, control membranes were used to test the nonspecific interaction between the equal mixture of proteins and the un-grafted cellophane membranes to prove later on that the adsorption is due to the affinity of copper ligand. The nonspecific interaction of protein equal mixture and the cellophane membranes in different stages has been investigated. It was found that no nonspecific interactions between both proteins and cellophane membranes were observed. This finding is in agreement with the results obtained by other authors (Wu et al., 2003). They explained the obtained results according to “the anionic nature of both proteins in the pH used in the study and the anionic nature of bearing carboxylic groups on the grafted membranes”. On the other hand, PMAA grafted membrane adsorbed about 3% and 6% of BSA and Chitinase, respectively, from 0.25% protein mixture solution. A higher adsorption percentage was observed with PMMA grafted membrane where 6% BSA and 12% Chitinase were adsorbed. This could be referred to the hydrophilicity and the surface roughness increments of the grafted membranes relative to un-grafted ones.

3.2.1

3.2.1 Protein concentration

To examine the affinity of the immobilized Cu⁺² ion grafted membranes towards separation of His-Tag Chitinase–BSA protein mixture (1:1) with different concentrations ranging from 0. 025% to 0.25% (w/v), the adsorption of individual Chitinase and BSA proteins in the same concentrations range was tested separately (Fig. 4).

Effect of BSA and Chitinase concentrations on adsorption capacity of Cu+2-immobilized grafted membranes.

At lowest BSA concentration tested, 0.025%, no adsorption at all was observed on both types of grafted membranes (Fig. 4). At concentrations beyond 0.1%, the adsorption of BSA onto immobilized Cu⁺² ions PMAA grafted membranes was found to be higher than its counterpart onto immobilized Cu⁺² ion PMMA grafted membranes. The same trend for His-Tag Chitinase adsorption was found (Fig. 4). At all studied concentrations, the adsorption of Chitinase onto immobilized Cu⁺² ions PMAA grafted membranes was found to be higher than its counterpart onto immobilized Cu⁺² ion PMMA grafted membranes. At the lowest concentration tested, 0.025%, the adsorption was observed on both types of grafted membranes (Fig. 4). In general, the most reasonable explanations for the obtained results are the high concentration of immobilized Cu⁺² ions onto the PMAA in addition to the fact that BSA has less numbers of adjacent exposed histidine (Gaberc-Porekar and Menart, 2001) compared to Chitinase protein which contains His-Tag (6 histidine). This leads consequently to less adsorption affinity since it is well known that the histidine is the center for making bond with Cu⁺² ions. This explained the higher affinity towards Chitinase.

To examine the affinity of the immobilized Cu⁺² ions PMAA grafted membranes towards His-Tag Chitinase protein, different concentrations of Chitinase–BSA (1:1) ranging from 0.025% to 0.25% (w/v) were tested. Fig. 5A shows the affinity of immobilized Cu⁺² ions PMAA grafted membranes towards Chitinase protein. The results reveal that increase in the Chitinase and BSA concentration leads to an increase in the percentage of adsorption onto the membrane. Highest adsorption observed for Chitinase is 435 mg/g and for BSA is 150 mg/g. The presence of BSA has a little effect on the adsorption affinity of membranes towards Chitinase; Fig. 4. The adsorption percentage of Chitinase decreases with increase of protein concentration then leveling off at high concentrations. These results prove the affinity of membranes towards Chitinase.

Effect of Chitinase:BSA (1:1) concentration on proteins’ adsorption affinity of Cu+2-immobilized PMAA grafted membranes.

Fig. 5B shows the affinity of immobilized Cu⁺² ion PMMA grafted membranes towards Chitinase protein. The results reveal that increase in the Chitinase and BSA concentration leads to an increase in the percentage of protein adsorption onto the membrane. Highest adsorption observed for Chitinase is 290 mg/g and for BSA is 130 mg/g. No significant effect of BSA presence on the adsorption affinity of membranes towards Chitinase was observed; Fig. 4. The same trend was obtained with PMAA grafted membranes, but with less adsorption capacity, where the adsorption percentage of Chitinase decreases with increase of protein concentration. These results may be due to the variation of immobilized amount of Cu⁺² ions.

Effect of Chitinase:BSA (1:1) concentration on proteins’ adsorption affinity of Cu+2-immobilized PMMA grafted membranes.

3.2.2

3.2.2 Adsorption pH

The role of the medium pH is fairly complex in the adsorption and elution processes of proteins, because it influences a number of properties including electron-donor acceptor properties of the solutes and metal stability. The pH range 6–8 favours retention of histidine and cysteine residues, at a more alkaline range, coordinations with amino functional groups which result in a decreasing selectivity (Wong et al., 1991).

Fig. 6A and B have showed the adsorption results as a function of pH variation from 4 to 8. In general, BSA and Chitinase adsorption increases with increasing pH. Fig. 6A shows that PMAA grafted membranes adsorbed 437 and 152 mg/g of Chitinase and BSA, respectively. On the other hand, PMMA grafted membranes adsorbed 283 and 128 mg/g of Chitinase and BSA, respectively (Fig. 6B). The figures also reveal that the percentage of adsorbed Chitinase is double that of BSA, although the concentration of BSA and Chitinase is equal. This behaviour could be referred to the number of the incorporated amino acids located on the surface of both proteins since Chitinase has at least double number of histidine than BSA. The reason of getting the higher percentage of proteins adsorption near pH 7 is that all the histidine, cysteine and tryptophan residues become neutral at this pH and play an important role as a ligand can coordinate to the immobilized Cu⁺² ions (Kubota, 1996).

Effect of adsorption pH on proteins’ adsorption affinity of Cu+2-immobilized PMAA grafted membranes.

Effect of adsorption pH on proteins’ adsorption affinity of Cu+2-immobilized PMMA grafted membranes.

In conclusion, pH has showed an effect on both the protonation and consequently the conformation structure of proteins. This leads to a change in the available number of histidine residues for coordination (Sharma and Agarwal, 2001; Gaberc-Porekar and Menart, 2001).

3.2.3

3.2.3 Adsorption time

Fig. 7A and B show the effect of variation adsorption time on the adsorption capacity of immobilized Cu⁺² ion PMMA and PMAA grafted membranes. From the figures we can see that the highest adsorption capacity for immobilized Cu⁺² ions PMAA grafted membranes was obtained after 1 h and after 45 min for immobilized Cu⁺² ion PMMA grafted membranes. This time was found enough for proteins to adsorb onto the membranes as a result of histidine chelating with immobilized metal ions. Further increase of adsorption time has no effect on the capacity of the membranes which is considered as an advantage in shortening the time needed for separation process.

Effect of adsorption time on proteins’ adsorption affinity of Cu+2-immobilized PMAA grafted membranes.

Effect of adsorption time on proteins’ adsorption affinity of Cu+2-immobilized PMMA grafted membranes.

The benefit in Chitinase adsorption over BSA is due to the presence of a higher histidine number in Chitinase protein structure than BSA which induced higher affinity towards Chitinase protein (Sharma and Agarwal, 2001; Gaberc-Porekar and Menart, 2001).

3.3

3.3 Protein elution

Elution of adsorbed proteins occurs when the imidazole nitrogen is protonated by decreasing pH, generating a positively charged ammonium ion which is repelled by the positively charged metal ion. A good method to achieve good resolution is the incorporation of the eluent compounds known to present a higher affinity for the adsorption sites than proteins. Addition of histidine or imidazole has been used in gradient mode elution as an effective way for selective protein elution especially polyhistidine-tagged protein (Wong et al., 1991). In order to investigate the elution of both proteins from our membranes, different factors affecting the elution step have been studied as follows.

3.3.1

3.3.1 Imidazole concentration

To study the effect of variation of imidazole concentration on the Chitinase and BSA elution step, different concentrations of imidazole eluent have been used; Fig. 8A and B. From the figures it can be observed that the percentage of eluted proteins increases with increasing imidazole concentration. Fig. 8A shows that the maximum recovery percentage of proteins, 313.5 mg/g (95%) of Chitinase and 135 mg/g (90%) of BSA, had been obtained using 0.4 M imidazole with immobilized Cu⁺² ion PMMA grafted membranes. On the other hand, Fig. 8B shows that 264.4 mg/g (88%) of Chitinase and 100 mg/g (80%) of BSA were recovered from immobilized Cu⁺² ion PMMA grafted membranes using 0.3 M imidazole. This difference in imidazole concentration used with both membranes could be attributed to the difference of immobilized Cu⁺² amounts which are in favour of immobilized Cu⁺² ions PMAA grafted membranes since it has a higher grafting percentage, 144%, compared with 58% for immobilized Cu⁺² ion PMMA grafted membranes. Over these concentrations, the protein recovery percentage decreases. As it can be seen, recovery percentage of Chitinase is higher than BSA. This is may be attributable to the difference in the histidine residue content.

Effect of imidazole concentration on proteins’ desorption affinity of Cu+2-immobilized PMAA grafted membranes.

Effect of imidazole concentration on proteins’ desorption affinity of Cu+2-immobilized PMMA grafted membranes.

3.3.2

3.3.2 Elution pH

Fig. 9A and B reveal the pH dependence of elution process for both adsorbed Chitinase and BSA on both types of membranes. Imidazole solution with different pH values was used for PMAA grafted membranes (0.4 M) and for PMMA grafted membranes (0.3 M) as eluent. Fig. 9A shows that from PMAA grafted membranes; 404.5 mg/g (93%) of adsorbed Chitinase and 135 mg/g (90%) of adsorbed BSA were eluted. On the other hand, Fig. 9B shows that from PMMA grafted membranes 252 mg/g (90%) of adsorbed Chitinase and 107.5 mg/g (86%) of adsorbed BSA were eluted. From Fig. 9 it can be seen that pH 7 was effective to obtain the higher elution percentage for both adsorbed proteins. At this pH (7.0), histidine, cystein and tryptophan residues of adsorbed proteins, Chitinase and BSA, become neutral which were further displaced by imidazole and finally eluted as a result (Kubota, 1996). Our findings are in agreement with published results by other authors (Sharma and Agarwal, 2001; Gaberc-Porekar and Menart, 2001). They referred the obtained results to “the protonation of proteins which lead to conformation changes of their structures and hence the availability and number of coordinated histidine residues”.

Effect of desorption pH on proteins’ desorption affinity of Cu+2-immobilized PMAA grafted membranes.

Effect of desorption pH on proteins’ desorption affinity of Cu+2-immobilized PMMA grafted membranes.

3.3.3

3.3.3 Elution time

The effect of variation elution time on protein recovery percentage is shown in Fig. 10. Fig. 10A show that maximum protein recovery percentages have been obtained after 30 min of elution time (pH 7 at 30 °C) using 0.4 M imidazole with immobilized Cu⁺² ion PMAA grafted membranes where 400 mg/g (92%) of Chitinase and 135 mg/g (90%) of BSA were eluted. On the other hand, Fig. 10B show that 0.3 M imidazole was used with immobilized Cu⁺² ion PMMA grafted membranes where 252 mg/g (90%) of Chitinase and 112.5 mg/g (90%) of BSA were eluted. Prolongation of elution time over 30 min leads to decrease in protein recovery percentage. Since 30 min is enough for imidazole to displace the adsorbed protein from the membrane surface which leads consequently to reduce the protein crowdedness onto the membrane surface, prolongation of time may lead to protein adsorption again on the membrane surface and so recovery percentage decreases.

Effect of desorption time on proteins’ desorption affinity of Cu+2-immobilized PMAA grafted membranes.

Effect of desorption time on proteins’ desorption affinity of Cu+2-immobilized PMMA grafted membranes.

4 Conclusion

Methacrylic acid (MAA) and methyl methacrylate (MMA) were used to graft cellophane membranes. To have affinity for His-Tag proteins, the grafted membranes were further immobilized with copper ions before they were tested in separation of His-Tag Chitinase enzyme from protein mixture with BSA. Immobilized Cu⁺² ion PMAA grafted affinity membranes show high affinity towards the separation of Chitinase enzyme which adsorbed 80% of Chitinase and only 28% of BSA from 0.25% protein mixture solution. At lower concentrations, 0.025%-0.050%, the membranes show complete selectivity towards Chitinase. No leakage of Cu⁺² ions was detected in the elution solution while 95% of Chitinase and 90% of BSA were eluted. On the other hand, immobilized Cu⁺² ion PMMA grafted affinity membranes show lower affinity towards the separation of Chitinase enzyme which adsorbed 56% of Chitinase and 25% of BSA from 0.5% protein mixture solution (1:1). At lower concentrations, 0.05%, the membranes show complete selectivity towards Chitinase where 30% was adsorbed compared with only 5% of BSA. No leakage of Cu⁺² ions was detected in the eluting solution while 88% of adsorbed Chitinase and 80% of adsorbed BSA were eluted.

In conclusion, the prepared immobilized Cu⁺² ion grafted membranes present as novel materials in the field of membrane affinity chromatography recommended for applications in separation and purification of His-Tag proteins. Moreover, a new solution for the metal ion leakage problem; the main reason for limiting the use of metal ion affinity chromatography technique, has been presented.

The selectivity of the immobilized metal ion membranes can be controlled and changed by immobilizing the membranes with specific metal ions. This will open the possibility to use the same membranes in separation of other His-Tag proteins. On the other hand, immobilizing the membranes with a mixture of metal ions may contribute in improving the affinity of the prepared membranes towards either natural or His-Tag proteins. Further investigations in this direction are now in processing in our laboratories.

References

Arıca M.Y., Testereci H.N., Denizli A., . Dye-ligand and metal chelate poly(2-hydroxyethylmethacrylate) membranes for affinity separation of proteins. J. Chromatogr. A. 1998;799:83.
[Google Scholar]
Arnold F.h., . Metal affinity separation: a new dimension in protein processing. Bio/Technology. 1991;9:151.
[Google Scholar]
Beeskow T.C., Kusharyoto W., Anspach F.B., Kroner K.H., Deckwer W.-D., . Surface modification of microporous polyamide membranes with hydroxyethyl cellulose and their application as affinity membranes. J. Chromatogr. A. 1995;715:49.
[Google Scholar]
Brandt S., Goffe R.A., Kessler S.B., O’Connor J.L., Zale S.E., . Membrane-based affinity technology for commercial scale purification. Bio/Technology. 1988;6:779.
[Google Scholar]
Camperi S.A., Grasselli M., Navarro del Cañizo A.A., Smolko E.E., Cascone O., . Chromatographic characterization of immobilized metal ion hollow-fiber affinity membranes obtained by direct grafting. J. Liq. Chromatogr. Relat. Technol.. 1998;21:1283.
[Google Scholar]
Charcosset C., . Purification of proteins by membrane chromatography. J. Chem. Technol. Biotechnol.. 1998;71:95.
[Google Scholar]
Crawford J., Ramakrishnan S., Periera P., Gardner S., Coleman M., Beitle R., . Immobilized metal affinity membrane separation: characteristics of two materials of differing preparation chemistries. Sep. Sci. Technol.. 1999;34:2793.
[Google Scholar]
Dahiya N., Tewari R., Hoondal G.S., . Biotechnological aspects of chitinolytic enzymes: a review. Appl. Microbiol. Biotechnol.. 2006;71:773-782.
[Google Scholar]
Denizli A., Senel S., Arica M.Y., . Cibacron Blue F3GA and Cu(II) derived poly(2-hydroxyethylmethacrylate) membranes for lysozyme adsorption. Colloids Surf. B. 1998;11:113.
[Google Scholar]
El-Awady N.I., El-Awady M.M., Mohy Eldin M.S., . Cerric ion-initiated grafting of acrylonitrile onto cellophane films. Egypt. J. Text. Polym. Sci. Technol.. 1999;3:25-41.
[Google Scholar]
El-Awady M.M., El-Awady N.I., Mohy Eldin M.S., . Chemical induced graft copolymerization of acrylic acid onto cellophane films. Int. J. Polym. Mater.. 2002;51:209-223.
[Google Scholar]
Gaberc-Porekar V., Menart V., . Perspectives of immobilized-metal affinity chromatography. J. Biochem. Biophys. Methods. 2001;49:335.
[Google Scholar]
Gaberc-Porekar V., Menart V.J., . Perspectives of immobilized-metal affinity chromatography. Biochem. Biophys. Methods Publication. 2001;49:335-360.
[Google Scholar]
Hari P.R., Paul W., Sharma C.P., . Adsorption of human IgG on Cu²⁺-immobilized cellulose affinity membrane: preliminary study. J. Biomed. Mater. Res.. 2000;50:110.
[Google Scholar]
Hemdan E.S., Zhao Y., Sulkowski E., Porath J., . Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. Proc. Natl. Acad. Sci. USA. 1989;86:1811.
[Google Scholar]
Kubota Naoji, Nakagawa Yasuhiro, Eguchi Yukari, . Recovery of serum proteins using cellulosic affinity membrane modified by immobilization of Cu²⁺ ion. J. Appl. Poly. Sci.. 1996;62:1153-1160.
[Google Scholar]
Kubota N., Nakagawa Y., Eguchi Y., . Recovery of serum proteins using cellulosic affinity membrane modified by immobilization of Cu²⁺ ion. J. Appl. Poly. Sci.. 1996;62:1153.
[Google Scholar]
Kulkarni A.Y., Mehta P.c., . Ceric ion induced redox polymerization of acrylonitrile grafted cellulose fibers. J. Appl. Poly. Sci.. 1968;12:1321-1342.
[Google Scholar]
Lepoutre P., Hui S.H., . Grafting acrylonitrile onto wood pulp: influence of process variables. J. Appl. Poly. Sci.. 1973;19:1257-1268.
[Google Scholar]
Mohy Eldin M.S., Soliman E.A., Hassan E.A., Abu-Saied M.A., . Immobilized metal ions cellophane – PGMA grafted membranes for affinity separation of β-galactosidase enzyme: preparation and characterization. J. Appl. Poly. Sci.. 2009;111:2647-2656.
[Google Scholar]
Mohy Eldin M.S., Abdel Rahman S., El-fawal G.F., . Preparation and characterization of grafted cellophane membranes for affinity separation of His-Tag Chitinase. Adv. Polym. Technol.. 2001;30:191-202.
[Google Scholar]
Park Juyoung, Kim Yo-Han, Yoon Hyo-Jin, Jun Bong-Hyun, Lee Yoon-Sik, . Preparation of pore size controllable macroporous polymer beads. J. Ind. Eng. Chem.. 2011;17:794-798.
[Google Scholar]
Perry B.W., Doumas B.T., . Effect of heparin on albumin determination by use of bromcresol green and bromcresol purple. Clin. Chem.. 1979;25:1520-1522.
[Google Scholar]
Proath J., Carlsson J., Olsson I., Belfrage G., . Metal chelate affinity chromatography, a new approach to protein fractionation. Nature. 1975;258:598.
[Google Scholar]
Reif O.W., Nier V., Bahr U., Freitag R., . Immobilized metal affinity membrane adsorbers as stationary phases for metal interaction protein separation. J. Chromatogr. A. 1994;664:13.
[Google Scholar]
Rodemann K., Staude E., . Synthesis and characterization of affinity membranes made from polysulfone. J. Membr. Sci.. 1994;88:271.
[Google Scholar]
Roper D.K., Lightfoot E.N., . Separation of biomolecules using adsorptive membranes. Chromatogr. A. 1995;702(1995):3.
[Google Scholar]
Senel S., Arica M.Y., Denizli A., . Zinc ion-promoted adsorption of lysozyme to Cibacron Blue F3GA-attached microporous polyamide hollow-fiber membranes. Colloids Surf. A. 2001;182:161.
[Google Scholar]
Sharma S., Agarwal G.P., . Interactions of proteins with immobilized metal ions: a comparative analysis using various isotherm models. Anal. Biochem.. 2001;288:126-140.
[Google Scholar]
Tsai Y.H., Wang M.-Y., Suen S.-Y., . Purification of hepatocyte growth factor using polyvinyldiene fluoride-based immobilized metal affinity membranes: equilibrium adsorption study. J. Chromatogr. B. 2002;766:133.
[Google Scholar]
Vita, S.B., 1984. Ion-exchange and water sorbent materials produced by graft copolymerization to cellulose (Ph.D thesis). North Caroline State University, USA.
Wong J.W., Albright R.L., Wang N.-H., . Immobilized metal ion affinity chromatography (IMAC) chemistry and bioseparation applications. Sep. Purif. Methods. 1991;20:49-106.
[Google Scholar]
Wu C., Suen S., Chen S., Tzeng J., . Analysis of protein adsorption on regenerated cellulose-based immobilized copper ion affinity membranes. J. Chromatogr. A. 2003;996:53-70.
[Google Scholar]
Yang L., Jia L., Zou H., Zhang Y., . Immobilized iminodiacetic acid (IDA)-type Cu²⁺-chelating membrane affinity chromatography for purification of bovine liver catalase. Biomed. Chromatogr.. 1999;13:229.
[Google Scholar]
Zou H., Luo Q., Zhou D., . Affinity membrane chromatography for the analysis and purification of proteins. J. Biochem. Biophys. Methods. 2001;49:199.
[Google Scholar]

Introduction

Experimental

Materials
Methods
Protein separation

Results and discussion

Membrane preparation and characterization
Protein separation
Protein elution

Conclusion

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[1] Arıca M.Y., Testereci H.N., Denizli A., . Dye-ligand and metal chelate poly(2-hydroxyethylmethacrylate) membranes for affinity separation of proteins. J. Chromatogr. A. 1998;799:83.
[Google Scholar]

[2] Arnold F.h., . Metal affinity separation: a new dimension in protein processing. Bio/Technology. 1991;9:151.
[Google Scholar]

[3] Beeskow T.C., Kusharyoto W., Anspach F.B., Kroner K.H., Deckwer W.-D., . Surface modification of microporous polyamide membranes with hydroxyethyl cellulose and their application as affinity membranes. J. Chromatogr. A. 1995;715:49.
[Google Scholar]

[4] Brandt S., Goffe R.A., Kessler S.B., O’Connor J.L., Zale S.E., . Membrane-based affinity technology for commercial scale purification. Bio/Technology. 1988;6:779.
[Google Scholar]

[5] Camperi S.A., Grasselli M., Navarro del Cañizo A.A., Smolko E.E., Cascone O., . Chromatographic characterization of immobilized metal ion hollow-fiber affinity membranes obtained by direct grafting. J. Liq. Chromatogr. Relat. Technol.. 1998;21:1283.
[Google Scholar]

[6] Charcosset C., . Purification of proteins by membrane chromatography. J. Chem. Technol. Biotechnol.. 1998;71:95.
[Google Scholar]

[7] Crawford J., Ramakrishnan S., Periera P., Gardner S., Coleman M., Beitle R., . Immobilized metal affinity membrane separation: characteristics of two materials of differing preparation chemistries. Sep. Sci. Technol.. 1999;34:2793.
[Google Scholar]

[8] Dahiya N., Tewari R., Hoondal G.S., . Biotechnological aspects of chitinolytic enzymes: a review. Appl. Microbiol. Biotechnol.. 2006;71:773-782.
[Google Scholar]

[9] Denizli A., Senel S., Arica M.Y., . Cibacron Blue F3GA and Cu(II) derived poly(2-hydroxyethylmethacrylate) membranes for lysozyme adsorption. Colloids Surf. B. 1998;11:113.
[Google Scholar]

[10] El-Awady N.I., El-Awady M.M., Mohy Eldin M.S., . Cerric ion-initiated grafting of acrylonitrile onto cellophane films. Egypt. J. Text. Polym. Sci. Technol.. 1999;3:25-41.
[Google Scholar]

[11] El-Awady M.M., El-Awady N.I., Mohy Eldin M.S., . Chemical induced graft copolymerization of acrylic acid onto cellophane films. Int. J. Polym. Mater.. 2002;51:209-223.
[Google Scholar]

[12] Gaberc-Porekar V., Menart V., . Perspectives of immobilized-metal affinity chromatography. J. Biochem. Biophys. Methods. 2001;49:335.
[Google Scholar]

[13] Gaberc-Porekar V., Menart V.J., . Perspectives of immobilized-metal affinity chromatography. Biochem. Biophys. Methods Publication. 2001;49:335-360.
[Google Scholar]

[14] Hari P.R., Paul W., Sharma C.P., . Adsorption of human IgG on Cu²⁺-immobilized cellulose affinity membrane: preliminary study. J. Biomed. Mater. Res.. 2000;50:110.
[Google Scholar]

[15] Hemdan E.S., Zhao Y., Sulkowski E., Porath J., . Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. Proc. Natl. Acad. Sci. USA. 1989;86:1811.
[Google Scholar]

[16] Kubota Naoji, Nakagawa Yasuhiro, Eguchi Yukari, . Recovery of serum proteins using cellulosic affinity membrane modified by immobilization of Cu²⁺ ion. J. Appl. Poly. Sci.. 1996;62:1153-1160.
[Google Scholar]

[17] Kubota N., Nakagawa Y., Eguchi Y., . Recovery of serum proteins using cellulosic affinity membrane modified by immobilization of Cu²⁺ ion. J. Appl. Poly. Sci.. 1996;62:1153.
[Google Scholar]

[18] Kulkarni A.Y., Mehta P.c., . Ceric ion induced redox polymerization of acrylonitrile grafted cellulose fibers. J. Appl. Poly. Sci.. 1968;12:1321-1342.
[Google Scholar]

[19] Lepoutre P., Hui S.H., . Grafting acrylonitrile onto wood pulp: influence of process variables. J. Appl. Poly. Sci.. 1973;19:1257-1268.
[Google Scholar]

[20] Mohy Eldin M.S., Soliman E.A., Hassan E.A., Abu-Saied M.A., . Immobilized metal ions cellophane – PGMA grafted membranes for affinity separation of β-galactosidase enzyme: preparation and characterization. J. Appl. Poly. Sci.. 2009;111:2647-2656.
[Google Scholar]

[21] Mohy Eldin M.S., Abdel Rahman S., El-fawal G.F., . Preparation and characterization of grafted cellophane membranes for affinity separation of His-Tag Chitinase. Adv. Polym. Technol.. 2001;30:191-202.
[Google Scholar]

[22] Park Juyoung, Kim Yo-Han, Yoon Hyo-Jin, Jun Bong-Hyun, Lee Yoon-Sik, . Preparation of pore size controllable macroporous polymer beads. J. Ind. Eng. Chem.. 2011;17:794-798.
[Google Scholar]

[23] Perry B.W., Doumas B.T., . Effect of heparin on albumin determination by use of bromcresol green and bromcresol purple. Clin. Chem.. 1979;25:1520-1522.
[Google Scholar]

[24] Proath J., Carlsson J., Olsson I., Belfrage G., . Metal chelate affinity chromatography, a new approach to protein fractionation. Nature. 1975;258:598.
[Google Scholar]

[25] Reif O.W., Nier V., Bahr U., Freitag R., . Immobilized metal affinity membrane adsorbers as stationary phases for metal interaction protein separation. J. Chromatogr. A. 1994;664:13.
[Google Scholar]

[26] Rodemann K., Staude E., . Synthesis and characterization of affinity membranes made from polysulfone. J. Membr. Sci.. 1994;88:271.
[Google Scholar]

[27] Roper D.K., Lightfoot E.N., . Separation of biomolecules using adsorptive membranes. Chromatogr. A. 1995;702(1995):3.
[Google Scholar]

[28] Senel S., Arica M.Y., Denizli A., . Zinc ion-promoted adsorption of lysozyme to Cibacron Blue F3GA-attached microporous polyamide hollow-fiber membranes. Colloids Surf. A. 2001;182:161.
[Google Scholar]

[29] Sharma S., Agarwal G.P., . Interactions of proteins with immobilized metal ions: a comparative analysis using various isotherm models. Anal. Biochem.. 2001;288:126-140.
[Google Scholar]

[30] Tsai Y.H., Wang M.-Y., Suen S.-Y., . Purification of hepatocyte growth factor using polyvinyldiene fluoride-based immobilized metal affinity membranes: equilibrium adsorption study. J. Chromatogr. B. 2002;766:133.
[Google Scholar]

[31] Vita, S.B., 1984. Ion-exchange and water sorbent materials produced by graft copolymerization to cellulose (Ph.D thesis). North Caroline State University, USA.

[32] Wong J.W., Albright R.L., Wang N.-H., . Immobilized metal ion affinity chromatography (IMAC) chemistry and bioseparation applications. Sep. Purif. Methods. 1991;20:49-106.
[Google Scholar]

[33] Wu C., Suen S., Chen S., Tzeng J., . Analysis of protein adsorption on regenerated cellulose-based immobilized copper ion affinity membranes. J. Chromatogr. A. 2003;996:53-70.
[Google Scholar]

[34] Yang L., Jia L., Zou H., Zhang Y., . Immobilized iminodiacetic acid (IDA)-type Cu²⁺-chelating membrane affinity chromatography for purification of bovine liver catalase. Biomed. Chromatogr.. 1999;13:229.
[Google Scholar]

[35] Zou H., Luo Q., Zhou D., . Affinity membrane chromatography for the analysis and purification of proteins. J. Biochem. Biophys. Methods. 2001;49:199.
[Google Scholar]