Membrane chromatography effect of dye-ligand type on the purif by dffhrtcv3


                    CHROMATOGRAPHIC FIELDS

              M. Yakup Arıca, Meltem Yılmaz, Emine Yalçın, and Gülay Bayramoğlu
               Biochemical Processing and Biomaterial Research Laboratory, Faculty of Science,
                          Kırıkkale University, 71450-Yahşihan-Kırıkkale, Turkey

          In the affinity membrane chromatography, pseudo specific ligands, e.g., dyes, amino acids and metal
chelates have been used instead of biospecific ligands. The triazine dye ligands are usually smaller and simpler
molecules than biospecific ligands, with higher chemical and physical stability and cheaper.       There are many
reports that adsorption of cells and proteins on the polymer membrane surface are quantitatively changed, depending
on the type of ligands immobilised. The surface energy of solids cannot be measured directly. Numerical values for
surface energies can be obtained from contact angles measurements using different test liquids.
          We have studied the influence of the surface energy and its components of two different dye-ligand (i.e.,
Reactive Brown 10 and Reactive Green-5) immobilised poly(hydroxyethyl methacrylate) (pHEMA) membranes to
the adsorption of protein “lysozyme” from aqueous solutions. Experiments have been carried out to determine the
surface properties of the affinity membranes after ligands attachment by contact angles measurements. The
measurements of the contact angle for water, glycerol, formamide, diiodomethane (DIM), ethylene glycol and
dimethylsulphoxide (DMSO) on plain, both RB-10 and RG-5 immobilised and their lysozyme covered counterpart
pHEMA membranes were made. The highest contact angles were obtained with water, whereas DMSO gave the
lowest contact angles for all the tested membranes. The surface energy parameters of the investigated membranes
were calculated from the measured contact angle values using the mostly used three methods (i.e., the harmonic
mean by Wu, the geometric mean by Fowkes and acid-base by van Oss). The adsorption of lysozyme significantly
changed both the contact angles and component of surface free energy. It was found that the immobilisation of dye-
ligand increased the lysozyme adsorption capacity of the affinity membranes most significant, although there is not
very large difference of surface energy between plain and dye ligand immobilised pHEMA membranes. The results
of this investigation have important information on the design of a novel affinity adsorbent for the use in the
chromatographic field for separation of a target protein from biological fluids.


              Gülay Bayramoğlu, Meltem Yılmaz, Emine Yalçın and M. Yakup Arıca
  Biochemical Processing and Biomaterial Research Laboratory, Faculty of Science Kırıkkale University, 71450-
                                         Yahşihan-Kırıkkale, Turkey
          In recent years, the membrane chromatography has been introduced as an alternative to the traditional
column techniques. Affinity membranes operate in convective mode, which can significantly reduce diffusion
limitations commonly seen in column chromatography [1-5].
          In this study, poly(hydroxyethyl methacrylate), and a composite pHEMA/chitosan networks were
synthesized in the membrane form via UV initiated photo-polymerisation in the presence of an initiator
      azoisobutyronitrile. Reactive Yellow 2 (RY-2) wa covalently immobilized as a dye-ligand onto both
membranes. The polarity and surface energy of the investigated membranes were determined by contact angle
measurement. The incorporation of chitosan in the pHEMA networks produced more hydrophilic surface, as
indicated by contact angle analysis. The binding characteristic of lysozyme to pHEMA-RY-2 and
pHEMA/chitosan-RY-2 affinity membranes have been investigated from aqueous solution and their dye ligand
free forms were used as control systems. When chitosan was incorporated in the pHEMA network as a cationic
polymer led to higher adsorption capacity for the lysozyme. Selective adsorption behaviour was also observed in
the case of pHEMA/chitosan-RY-2 membrane for the lysozyme. The non-specific adsorptions of the lysozyme on
the pHEMA and pHEMA/chitosan membranes were about 1.9 and 7.2 mg/ml, respectively. These were
negligible for all others egg white proteins. The lysozyme adsorption data was analysed using the first order and
the second order models. The first-order equation in both affinity membrane systems is the most appropriate
equation to predict the adsorption capacities of the adsorbents. The adsorption isotherms well fitted the combined
Langmuir-Freundlich model. A theoretical analysis has been conducted to estimate the thermodynamic
contributions (changes in enthalpy, entropy and Gibbs free energy) for the adsorption of lysozyme to both dye-
ligand immobilised membranes. The adsorption capacities of both dye ligand immobilised membranes increased
with increasing the temperature while decreased with increasing the NaCl concentration.


                         M. Yakup Arıca, Emine Yalçın and Gülay Bayramoğlu
               Biochemical Processing and Biomaterial Research Laboratory, Faculty of Science,
                            Kırıkkale University, 71450-Yahşihan-Kırıkkale, Turkey

          Immobilized-metal affinity chromatography (IMAC) is a separation technique that uses covalently
bound chelating compounds on solid chromatographic supports to immobilize metal ions, which serve as affinity
ligands for various proteins, making use of coordinative binding of some amino acid residues (such as, histidine,
cysteine and trytophan) exposed on the surface. The selectivity of the separation can be tailored through the
choice of metal ions, solvent conditions, or modification of the target protein (e.g., the adition of histidine-rich
residues). The nature of protein binding has important implications for the design of efficient separations as well
as for the design of new materials for IMAC support.
          In this work, a copolymer membrane was synthesized from 2-hydroxyethyl methacrylate and 2-
methacrylolyamido-histidine (pHEMA-MAAH) via UV initiated photopolymerization in the presence of an
initiator         a
                 ’- zoisobutyronitrile. L-histidine was covalently incorporated to monomer (i.e.,
methacryloylchloride) as a metal chelating ligand. After preparation of membrane, different metal ions were
immobilized onto L-hisdidine-ligand to use in the immobilized metal affinity chromatography (IMAC). The
binding characteristics of human immunoglobulin G (IgG) to IMAC adsorbents and selectivity of immobilized
metals ion (i.e. Fe(III)) and Cu (II) to the IgG have been investigated from aqueous solution using L-histidine
attached copolymer membrane as a control system. The experimental data was analyzed using two adsorption
kinetic models the pseudo-first order and the pseudo-second order to determine the best-fit equation for the
adsorption of IgG onto L-histidine incorporated and/or different metals ion immobilized affinity membranes. The
first-order equation in the affinity membrane systems is the most appropriate equation to predict the adsorption
capacity for all the tested adsorbents. The IgG adsorption capacity of the pHEMA-MAAH, pHEMA-MAAH-Fe
(III) and pHEMA-MAAH-Cu(II) affinity membranes were 13.1, and 23.3 and 29.9 mg ml-1, respectively.


             M. Yakup Arıca, Bülent Kaya, Ayşegül Ulkü Şenel and Gülay Bayramoğlu
               Biochemical Processing and Biomaterial Research Laboratory, Faculty of Science,
                          Kırıkkale University, 71450-Yahşihan-Kırıkkale, Turkey
          Lipase (triacylglycerol ester hydrolyses, EC is an important enzyme in biological systems,
where it catalyses the hydrolysis of triacylglycerol to glycerol and fatty acids. The enzyme is distributed among
higher animals, plants and microorganisms in which its plays a key role in the lipids metabolism. Lipase has been
widely used in the enzymatic organic synthesis and clinical analysis.
          The availability of large number support materials and methods of enzyme immobilisation leave
virtually no bioactive species without a feasible route of immobilisation. It is, thus, important that the choice of
support material and immobilisation method over the free bioactive agent should be well justified
          In this work, the epoxy group containing poly(GMA-HEMA) microspheres were prepared by
suspension polymerisation. The epoxy groups of the poly(GMA-HEMA) microspheres were used for the covalent
attachment of Candida rugosa lipase and 1,6 diaminohexane (i.e., spacer-arm). Candida rugosa lipase was also
covalently immobilised onto the spacer-arm-attached poly(GMA-HEMA) microspheres using glutaric dialdehyde
as a coupling agent. The maximum lipase immobilization capacity of the poly(GMA-HEMA-) and poly(GMA-
HEMA)-spacer-arm attached microspheres was 16.1 and 28.3 mg g-1, respectively. The attachment of the spacer-
arm resulted an increase in the apparent activity of the immobilised lipase with respect to the enzyme
immobilised via the epoxy groups of the microspheres. The activity yield of the lipase immobilised on the spacer-
arm attached microspheres was up to 45%, and this was 14% for the enzyme immobilized through epoxy groups.
Therefore, the rest of the immobilization study was carried out using only spacer-arm attached microspheres. The
optimum temperature for lipase immobilised on the spacer-arm attached microspheres was 5 C higher than that
of the free enzyme and was also significantly broader. The immobilised lipase has resistance to temperature
inactivation as compared to that of the free form.


                                 Gülay Bayramoğlu and M. Yakup Arıca
  Biochemical Processing and Biomaterials Research Laboratory, Faculty of Science, Kırıkkale University, TR-
                                    71450 Yahşihan-Kırıkkale, Turkey

           Copolymer membranes are widely used in the fields of medicine and biotechnology, for clinical
applications, for affinity separation of a target protein from biological fluids, for the construction of enzyme
reactors and enzyme electrode or in scientific investigations.
           In this work, epoxy group-containing poly(hydroxyethylmethacrylate-co-glycidyl methacrylate), poly
(HEMA-co-GMA), membranes were prepared by UV initiated photopolymerization in the presence of an
initiator. The epoxy group content of the membrane was changed by varying the comonomer ratio (i.e., glycidyl
methacrylate) in the polymerisation mixture. The membrane was then grafted with an ionic polyethylenimine
polymer (PEI). The amount of grafted polyethylenimine increased in accordance with the epoxy group content.
The PEI grafted membrane surface was then used for glucose oxidase (GOD) immobilization via ionic
interactions. The immobilization of enzyme onto the poly(HEMA-co-GMA-4)-PEI membranes from aqueous
solutions containing different amounts of GOD at different pH was investigated in a batch system. The amount of
GOD immobilized on the modified membrane surface increased with increasing concentration of GOD in the
immobilization medium. The maximum GOD immobilization capacities of the poly(HEMA-co-GMA-4)-PEI
membrane were 84           g/cm2. The activities of GOD-immobilized onto poly(HEMA-co-GMA-1-5)-PEI
membranes were in the range 7.6-14.5 U/cm2. The pH and temperature profile was slightly broader for
immobilized preparations than that of the free enzyme. The storage conditions for the immobilized enzyme
preparations were studied and the immobilized enzymes had a long-storage stability (only 7% and 24% activity
decrease in 3 months under wet and dry storage conditions, respectively. After inactivation of enzyme, the poly
(HEMA-co-GMA)-PEI membrane can be easily regenerated and reloaded with the enzyme for repeated use. In
addition, the poly(HEMA-co-GMA-1-5) and PEI grafted membranes surfaces were characterized by contact
angle measurements. An increase in the glycidyl methacrylate ratio in the co-polymer structure led to a decrease
on the surface wettability of the membranes whereas incorporation of PEI on the membrane surface resulted an
increase on the surface wettability of the membranes.

1. M. Yakup Arıca, Meltem Yılmaz, Emine Yalçın, and Gülay Bayramoğlu, Evaluation of lysozyme adsorptive
   behaviour of poly(hydroxyethylmethacrylate) affinity membranes related to the surface energy and its
   components to be used in chromatographic fields. (SOZLÜ SUNUM)
2. Gülay Bayramoğlu, Meltem Yılmaz, Emine Yalçın and M. Yakup Arıca, Purification of Lysozyme from Whole
   Egg White Using Reactive Yellow-2 Immobilised Poly(hydroxyethylmethacrylate)                and Poly
   (hydroxyethylmethacrylate)/Chitosan Membranes: Evaluation of Effective Parameters (POSTER)
3. M. Yakup Arıca, Emine Yalçın and Gülay Bayramoğlu, Separation of human IgG by histidine ligand attached
   and metal ions-immobilized poly(hydroxyethylmethacrylate-co-methacrylolyamido-histidine) membranes.
4. M. Yakup Arıca, Ayşegül Ulkü Şenel and Gülay Bayramoğlu, Preparation of spacer-arm attached poly
   (glycidylmethacrylate-co-hydroxyethylmethacrylate) microspheres and utilized for biomacromolecule
   immobilization (POSTER)
5. Gülay Bayramoğlu and M. Yakup Arıca, Polyethyleneimine grafted poly(hydroxyethylmethacrylate-co-glycidyl
   methacrylate) ion-exchange membranes for reversible immobilization of proteins(POSTER)

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