Chemistry
Isoelectric Point
The isoelectric point is the pH at which a molecule carries no net electrical charge. It is the point at which the molecule is electrically neutral, with equal numbers of positive and negative charges. At the isoelectric point, a molecule will not migrate in an electric field.
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6 Key excerpts on "Isoelectric Point"
eBook - PDF- Sankhavaram R. Panini(Author)
- 2021(Publication Date)
- Thieme(Publisher)
Exami-nation of the titration curve allows for the identifcation of the amino acid based on the number of p K a points and their val-ues. The titra-tion curves of glycine, aspartic acid, and arginine are depicted in ▶ Fig. 4.3. Isoelectric Point Amino acids are zwitterions because they possess both positive and negative charges at neutral pH. The sum of the charges varies with pH such that an amino acid that has one α -carboxyl group and one α -amino group will be positively charged at low pH values (< 2.8). As the pH of the environment is elevated, the net charge of the amino acid becomes zero. At even higher pH, the charge becomes negative. The pH at which the charge is zero is called the Isoelectric Point (p I ). The p I of an amino acid is obtained by calculating the average of its two p K a values that are closest in value to each other. Importantly, the p I defines the pH at which molecules in solution will not migrate in an electric field. This behavior is observed in amino acids with uncharged side-groups. Foundations Protein charge A protein ’ s overall charge at a particular pH is directly related to its amino acid composition. Proteins are composed of neutral, hydrophilic, and charged (both positive and negative) amino acids. The net electrical charge of a protein is determined by a combination of charges on its component amino acids at a given pH. The Isoelectric Point (p I ) of a protein is defined as the pH at which the protein has no net charge. A protein carries a net positive charge at pH values below its p I and a net negative charge at values above its p I . The property of protein charge can be used to separate the proteins in a complex mixture using an electrical field. For example, native gel electrophoresis is employed to resolve the major serum proteins such as the albumin and the α 1 -, α 2 -, β -, and γ -globulins.
eBook - PDF- Satinder Ahuja(Author)
- 2000(Publication Date)
- Academic Press(Publisher)
Amines (-NH2) and other basic functions of proteins, such as guanidines, are uncharged at alkaline pH, but are cationic below about pH 10 (e.g., -NH3). The overall compositions and structures of proteins and the properties of the separation media affect the pH at which individual ionizable side chains actually dissociate. As a result, each individual ionizable group in a protein has a nearly unique dissociation point. The key to understanding lEF is the recogni-tion that the net charges carried by proteins are pH dependent. The net charge on a protein is the algebraic sum of all its positive and negative charges. There is a specific pH for every protein at which the net charge it carries is zero. This isoelectric pH value, termed the Isoelectric Pointy or pi, is a characteristic physicochemical property of every protein. The definition of pi for molecules as complex as proteins is more or less an operational one and is taken to be that pH at which a protein has zero electrophoretic mobility in an isoelectric focusing run. Nevertheless, it has been shown that the pis of some acidic proteins (up to about pH 7) can be calculated from their amino acid compositions.^^ If the number of acidic groups in a protein exceeds the number of basic groups, the pi of that protein will be at a low pH value and the protein is classified as being acidic. On the other hand, if basic groups outnumber acidic groups, the pi will be high, with the protein classified as basic. Proteins show considerable variation in Isoelectric Points, but pi values usually fall in the range of pH 3 to pH 12 with a great many having pis between pH 4 and ISOELECTRIC FOCUSING 265 pH 7/^ Some peptides are too small to have a balance of positive and negative charge groups. These peptides do not focus into sharp bands. Isoelectric focusing v^as developed to separate proteins on the basis of differences in their pi values.
- David Garfin, Satinder Ahuja(Authors)
- 2005(Publication Date)
- Academic Press(Publisher)
3 THEORY AND SIMULATION OF ISOELECTRIC FOCUSING ToL. SOUNART a, P.A. SAFIER b, AND J.C. BAYGENTS b ~ National Laboratories,Albuquerque, NM 87185-141 I bThe University of Arizona, Tucson,AZ 85721 I. PRINCIPLES OF ISOELECTRIC FOCUSING A. Steady Focusing and the Isoelectric Point B. FocusingTransients in a Steady pH Gradient II. NUMERICAL SIMULATION OF IEF A. Balance Laws B. Initial and Boundary Conditions C. Numerical Implementation III. ILLUSTRATIVE SIMULATIONS OF IEF IV. SUMMARY REFERENCES I. PRINCIPLES OF ISOELECTRIC FOCUSING Isoelectric focusing (IEF) is an electrophoretic separation scheme tail- ored to amphoteric compounds. IEF is used primarily to resolve mixtures of proteins and/or peptides. Similar to any other charged solute, an amphoteric compound translates under the action of an externally applied electric field~a process known alternatively as electromigration or eleco trophoresis. Owing to the chemical composition of amphoteric substances, their electrophoretic mobility is a function of pH: at low pH, the mobility is positive; at high pH, the mobility is negative. The generic relationship between electrophoretic mobility and pH is sketched for an amphoteric compound in Figure 1, where ~/E denotes the electrophoretic mobility, and the curve is drawn for a solution of (approximately) constant ionic strength. The Isoelectric Point (pI) is the pH at which the electrophoretic mobility of the compound is nil. At a given ionic strength, each ampho- teric species evinces a different pI and, in the IEF scheme, these differences in pI serve as the basis to resolve separands. 9 2005Elsevier Inc.All rightsreserved. Handbookof IsoelectricFocusing and Proteomics D. Garfinand S. Ahuja,editors. 41 42 T.L. SOUNART et al. PE pl pH FIGURE I Generic behavior of electrophoretic mobility versus pH for an ampho- teric species at constant ionic strength.
eBook - PDFHandbook of Nanophysics
Nanoparticles and Quantum Dots
- Klaus D. Sattler(Author)
- 2016(Publication Date)
- CRC Press(Publisher)
18 -10 Rongjun Pan Guangxi University of Technology Kongyong Liew South-Central University for Nationalities and University Malaysia Pahang 18 -2 Handbook of Nanophysics: Nanoparticles and Quantum Dots of zero charge. The Isoelectric Point is defined as the pH value at which the electrokinetic potential equals zero. The points of zero charge are defined as the pH values at which one of the catego-ries of surface charge equals zero. Three operational categories of surface charge could be identified: (1) structural, denoted by σ st ; (2) adsorbed proton, σ H ; and (3) adsorbed ion, denoted by Δ q . Thus, two points of zero charge can be defined: (1) point of zero net proton charge (PZNPC, σ H = 0) and (2) point of zero net charge (PZNC, σ st + σ H = 0) (Li et al., 2004). For pure materi-als or those metal oxides without specific adsorption, the iso-electric point is equal to its point of zero charge (Kosmulski and Saneluta, 2004). 18.3 Origin of Nanoparticles’ Surface Charge It has been confirmed experimentally that nanoparticles’ sur-face is positively or negatively charged. However, the origin of the surface charge is uncertain when the particles are dispersed in aqueous or in nonaqueous medium, even if in the same matrix. 18.3.1 Origin of Surface Charge in Aqueous Medium Generally, when the nanoparticles are dispersed in aqueous medium, the origin of surface charge is considered to occur through the following routes. 18.3.1.1 Dissociation of Functional Group(s) of the Nanoparticles Usually, some particles, such as those in polymer colloids, are charged because of the dissociation of functional group(s). For example, when nano-sized SiO 2 are dispersed in water with dif-ferent pH value, equilibration with surface Si–OH will take place as follows: Si Si Si OH 2 + OH – O – + OH H 2 O H + Therefore, the surface charge condition varies with the pH of the medium.
eBook - PDFGel Electrophoresis and Isoelectric Focusing of Proteins
Selected Techniques
- Robert C. Allen, Calvin A. Saravis, H.R. Maurer(Authors)
- 2019(Publication Date)
- De Gruyter(Publisher)
While at its Isoelectric Point, it will achieve a steady state of zero migration. The development of practical laboratory methods f o r isoelectric focusing has added a new dimension to electrophoretic techniques in polyacrylamide and charge-free agarose support media for the separation of proteins and other amphoteric substances. Isoelectric focusing, which has been recently demonstrated to be a special case of the moving boundary theory (12) is thus, basically a method for separation of substances according to their Isoelectric Points in a Isoelectric Focusing stable pH gradient whose range may be preselected. For all practical purposes, this may be considered an equilibrium process where zone width is at a minimum at the completion of separation in contrast to zone and moving boundary electrophoresis where the zone width increases with run time due to diffusion. This method offers the advantage of being able to separate amphoteric substances based on their Isoelectric Points, which is a more valuable characteristic than their mobiliy at a certain pH. Further, the technique may be employed for both analytical and preparative purposes on a variety of supporting media, such as sucrose density gradients, acrylamide, granular gel beds and charge-free agarose gels. The procedure has been refined to the point where one can reproduceably separate substances differing by less than 0.005 pH unit when narrow pH range ampholytes at high voltage gradients are used (13) and to 0.001 pH unit on the newer immobilized pH gradients (14). 3.3. Background Kolin (15) described the separation of proteins by electrical transport in a pH gradient generated in a sucrose density gradient contained in a Tiselius - like apparatus. The material to be separated was placed at the interface of an acidic and basic buffer which were allowed to diffuse against one another, while at the same time being subjected to an electromotive force.
eBook - PDFModern methods in protein chemistry
Review articles following the joint meeting of the Nordic Biochemical Societies Damp/Kiel, FR of Germany, September 27–29, 1982
- Harald Tschesche(Author)
- 2019(Publication Date)
- De Gruyter(Publisher)
HIGH RESOLUTION ANALYTICAL AND PREPARATIVE ISOELECTRIC FOCUSING OF PROTEINS: PRINCIPLES AND STRATEGY Bertold J. Radola Institut für Lebensmitteltechnologie und Analytische Chemie Technische Universität München, D-8050 Freising-Weihenstephan Introduction Over the past few years some notable advances in isoelectric focusing have provided the basis for separations with improved resolution, speed and high capacity. By analogy to recent developments in the field of high performance liquid chromato-graphy it is suggested to describe the practice delineated by the indicated criteria as high resolution isoelectric focusing. A substantial reduction in the thickness of gel layers allows for use of high field strengths and shorter separation distan-ces reduce the focusing time from hours, in traditional iso-electric focusing, to minutes. In preparative separations under appropriately chosen conditions both high sample load and high resolution can be obtained. Isoelectric focusing is a method for the separation of amphoteric substances in a stable continuous pH gradient according to differences in iso-electric points (1). The Isoelectric Point (pi) is defined as the pH at which the electrophoretic mobility passes through zero. The Isoelectric Point is a much more valuable charac-teristic of a protein than its mobility at a certain pH. Iso-electric focusing is a steady-state method which offers two distinct advantages over the more usual kinetic methods of electrophoresis.(i) After the steady-state has been attained, the process is time-independent so that zone definition does not deteriorate with further passage of time. In practice some minor deterioration becomes apparent after extended focusing Modern Methods in Protein Chemistry - Review Articles © 1983 by Walter de Gruyter & Co. - Berlin • New York 22 periods, particularly in gel-stabilized systems, due to the operation of certain non-ideal effects (2).
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