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Transfer inovácií 28/2013 2013 BASIC PRINCIPLES OF ISOELECTRIC FOCUSING IN BIOMEDICAL ENGINEERING Dr.h.c. prof. Ing. Jozef Živčák, PhD. Department of Biomedical Engineering and Measurement DBEaM, TU Letná 9, 042 00 Kosice, Slovakia e-mail: [email protected] RNDr. Marianna Trebuňová, PhD. Associated Tissue Bank Rastislavova 43, Kosice, Slovakia e-mail: [email protected] Abstract Isoelectric focusing (IEF) is an electrophoretic technique for the separation of proteins based on their isoelectric point (pI). The pI is the pH at which a protein has no net charge and thus, does not migrate further in an electric field. IEF gels are used to determine the isoelectric point (pI) of a protein and to detect minor changes in the protein due to post-translational modifications such as phosphorylation and glycosylation [1]. Key words: Isoelectric focusing, isoelectric point, proteins electrical charge or the negative and positive charges are equal. Surfaces naturally charge to form a double layer. In the common case when the surface chargedetermining ions are H+/OH-, the net surface charge is affected by the pH of the liquid in which the solid is submerged. Again, the pI is the pH value of the solution at which the surfaces carries no net charge. The pI value can affect the solubility of a molecule at a given pH. Such molecules have minimum solubility in water or salt solutions at the pH which corresponds to their pI and often precipitate out of solution. Biological amphoteric molecules such as proteins contain both acidic and basic functional groups. Amino acids which make up proteins may be positive, negative, neutral or polar in nature, and together give a protein its overall charge. At a pH below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge. Proteins can thus be separated according to their isoelectric point (overall charge) on a polyacrylamide gel using a technique called isoelectric focusing, which uses a pH gradient to separate proteins. Isoelectric focusing is also the first step in 2-D gel polyacrylamide gel electrophoresis [2]. In t ro du ct io n In isoelectric focusing (IEF), proteins are applied to polyacrylamide gels (IEF gels) or immobilized pH gradient (IPG) strips containing a fixed pH gradient. An electrical field is applied and the protein sample containing a mixture of proteins migrates through the pH gradient. Individual proteins are immobilized in the pH gradient as they approach their specific pI. After staining the gel and documenting the results, proteins separated by pI can be separated by mass using 2D gel electrophoresis [1]. Isoelectric point The isoelectric point (pI), sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge. Amphoteric molecules called zwitterions contain both positive and negative charges depending on the functional groups present in the molecule. The net charge on the molecule is affected by pH of their surrounding environment and can become more positively or negatively charged due to the loss or gain of protons (H+). The pI is the pH value at which the molecule carries no 222 Theoretical aspects When a protein is at the position of its isoelectric point, it has no net charge and cannot be moved in a gel matrix by the electric field. It may, however, move from that position by diffusion. The pH gradient forces a protein to remain in its isoelectric point position, thus concentrating it; this concentrating effect is called "focusing". Increasing the applied voltage or reducing the sample load result in improved separation of bands. The applied voltage is limited by the heat generated, which must be dissipated. The use of thin gels and an efficient cooling plate controlled by a thermostatic circulator prevents the burning of the gel whilst allowing sharp focusing. The separation is estimated by determining the minimum pI difference (ΔpI), which is necessary to separate 2 neighbouring bands: Transfer inovácií 28/2013 2013 D = diffusion coefficient of the protein, • for positive charged macromolecules: = pH gradient, E = intensity of the electric field, in volts per centimetre, = variation of the solute mobility with the pH in the region close to the pI. Since D and for a given protein cannot be altered, the separation can be improved by using a narrower pH range and by increasing the intensity of the electric field. Resolution between protein bands on an IEF gel prepared with carrier ampholytes can be quite good. Improvements in resolution may be achieved by using immobilised pH gradients where the buffering species, which are analogous to carrier ampholytes, are copolymerised within the gel matrix. Proteins exhibiting pIs differing by as little as 0.02 pH units may be resolved using a gel prepared with carrier ampholytes while immobilised pH gradients can resolve proteins differing by approximately 0.001 pH units [3]. How to calculate isoelectric point of protein where pKp is the acid dissociation constant of positively charged amino acid. We can see, only pH of buffer is variable in equations. If we successively change this value, finally we will find isoelectric point of analyzed protein. The knowledge of isoelectric point is of great significance in biochemistry (mainly in elecrophoresis and isofocusing techniques), because it allows to match proper environment before the experiment starts [4]. Generally, macromolecules are positively charged and on the other hand, above proteins isoelectric point, their charge is negative.For example, during electrophoresis, direction of proteins migration, depends only from their charge. If buffer pH (and as a result gel pH) is higher than protein isoelectric point, the particles will migrate to the anode (negative electrode) and if the buffer pH is lower than isoelectric point they will go to the cathode. In situation when the gel pH and the protein isoelectric point are equal, proteins do not move at all (Picture 1) [4]. Isoelectric point (pI) is a pH in which net charge of protein is zero. In case of proteins isoelectric point mostly depends on seven charged amino acids: glutamate (δ-carboxyl group), aspartate (ß-carboxyl group), cysteine (thiol group), tyrosine (phenol group), histidine (imidazole side chains), lysine (ε-ammonium group) and arginine (guanidinium group). Additonally, one should take into account charge of protein terminal groups (NH2 i COOH). Each of them has its unique acid dissociation constant referred to as pK. Moreover, net charge of the protein is in tight relation with the solution (buffer) pH. Keeping in main this we can use Henderson-Hasselbach equation to calculate protein charge in certain pH: • for negative charged macromolecules: where pKn is the acid dissociation constant of negatively charged amino acid, Picture 1 Scheme of Isoelectric focusing Source: [5]. 223 Transfer inovácií 28/2013 CONCLUSION IEF is one of the principal techniques available for analyzing and characterizing proteins, peptides, glyco- or lipoproteins, cell membranes proteins, isoenzymes, etc. It can be very useful in studying factors that influence the net molecular charge of a molecule or a complex between macromolecules. It permits us to analyze ligand binding to proteins and to investigate the subunit structures in multimeric proteins [6]. 2013 [3] http://lib.njutcm.edu.cn/yaodian/ep/EP5.0/02_ methods_of_analysis/2.2.__physical_and_physicoc hemical_methods/2.2.54.%20Isoelectric%20focusin g.pdf [4] http://isoelectric.ovh.org/files/isoelectric-pointtheory.html. [5] http://en.wikipedia.org/wiki/File:Isoelectric_fo cusing_contribute2.jpg [6] Prats, M.: Minireview: Isoelectric Focusing in Experimental sectiojn, Biochemical education, 20 (2), 1992, p. 109 – 111 References [1] http://www.lifetechnologies.com/sk/en/home/li fe-science/protein-expression-and-analysis/proteingel-electrophoresis/protein-gels/specialized-proteinseparation/isoelectric-focusing.html [2] http://www.princeton.edu/~achaney/tmve/wiki 100k/docs/Isoelectric_point.html 224 Acknowledgement This study was supported by the grant VEGA 1/0515/13 of the Ministry of Education of the Slovak Republic and by the Agency of the Slovak Ministry of Education.