Quantitive Analysis Of Biospec

Per Lundahl, Andreas Lundqvist, Eva Greijer

• Publisher: Routledge
• Year: 2021
• Print ISBN: 9789057023781
• eBook ISBN: 9781317836537
• Edition: 1
• Topic: Sciences physiques
• Subtopic: Chimie

First published in 2004, this book collects several up-to-date methods for quantitative analysis of biospecific interactions, a field that has a long history that perhaps can be said to have begun with the classical paper of G. Scatchard in 1949 (The attractions of proteins for small molecules and ions, but which has advanced impressively during the last few years. A precise spatial arrangement of just a few hydrogen bonds can confer a remarkably specific reversible association between two molecules. A web of weak interactions governs biospecific recognition in general. The binding equilibria in living cells tune and coordinate a multitude of functions. The thermodynamic properties of such interactions are often studied by binding experiments in simplified and essentially ideal systems. However, similar types of studies may elucidate the biologically relevant dynamic steady-state conditions in living cells and organisms, allowing for the very wide range of interactant concentrations and the interplay between the many reactions and interactions. The development in biosciences will continue with in-depth studies of macromolecules and membranes. More detailed knowledge will allow analyses of delicate balances between substances and events in the complex systems involved in life processes. Methods to study biospecific affinities are thus highly important tools for understanding mechanisms and effects of molecular binding events in vivo and in vitro, e.g., in biochemical, biomedical and pharmaceutical research, and for biotechnological research and production.

1. ELECTROPHORETIC ANALYSIS OF REVERSIBLE INTERACTIONS

NIELS H.H. HEEGAARD

Department of Autoimmunology, Statens Serum Institut, Copenhagen S, Denmark

INTRODUCTION

To understand how biological systems function, non-covalent interactions that occur in these systems must be described both qualitatively and in terms of equilibrium binding constants. For this purpose numerous approaches exist including precipitation techniques, analytical ultracentrifugation and filtration assays (Hulme, 1990), spectroscopic measurements (Chriswell and Schilt, 1975), active site titration (in the case of enzymes), and equilibrium dialysis methods (Klotz, 1989). Many of these approaches require special characteristics of the molecules under study, consume quite large amounts of material and/or involve molecular modifications by immobilization or labelling procedures.

Electrophoresis may under the right circumstances be considered an advantageous alternative because reversible interactions that take place during electrophoresis and influence the separation patterns (affinity electrophoresis) (Bøg-Hansen, 1973) can make it possible to identify and quantitate binding phenomena.

Affinity electrophoresis is most often carried out using non-denaturing electrophoresis techniques in gels or in free solution in capillaries (Chapter 2). The study of most binding interactions or other functional characterization (e.g., of enzymatic activity) will not be possible in the presence of strongly charged detergents (e.g., sodium dodecyl sulfate) or other denaturing chemicals.

In affinity electrophoresis, molecular recognition is an additional factor that influences the separation process and thereby enhances the selectivity. The ensuing changes in molecular mobility have been used in numerous applications for the identification and quantitative characterization of the interactions of biomolecules (Takeo, 1987; Heegaard, 1994b).

In the present chapter, the basic principles governing the uses of affinity electrophoresis and the assumptions, requirements, and derivation of equations for binding constant determination will be introduced together with chosen examples of applications.

BACKGROUND AND THEORY

History

Electrophoresis has a long history as a separation method in biology (Tiselius, 1937; Tiselius et al., 1963). Its potential as a means of mixing interacting molecules and measuring specific interaction patterns was realized more than 40 years ago with antigens and antibodies (Macheboeuf et al., 1953; Nakamura and Ueta, 1958; Bussard, 1959; Ressler, 1960a), enzymes and substrates/inhibitors (Nakamura and Wakeyama, 1961), and lectins and glycoproteins (Nakamura and Suzuno, 1965). Chosen highlights in the development of electrophoretic techniques from the perspective of this chapter are shown in Table 1.

Table 1 Important milestones in the development of electrophoresis for binding studies

Theory of adsorption in chromatography	Wilson, 1940; Weiss, 1943; DeVault, 1943
Electrophoresis in gel supports	Smithies, 1955; Raymond and Weintraub, 1959; Hjertén, 1961
Biospecific interaction in electrophoresis	Nakamura and Ueta, 1958; Grabar and Williams, 1953; Macheboeuf et al., 1953
Theory of electrophoretic mobility shift analysis	Nakamura and Wakeyama, 1961
Quantitative estimates by affinity gel electrophoresis	Takeo and Nakamura, 1972; Bøg-Hansen and Takeo, 1980
Modern capillary electrophoresis	Mikkers et al., 1979; Jorgenson and Lukacs, 1981; 1983
Quantitative studies of binding in capillary electrophoresis	Heegaard and Robey, 1992; Kraak et al., 1992; Chu and Whitesides, 1992; Chu et al., 1992; Honda et al., 1992; Carpenter et al., 1992

With respect to antigens and antibodies, the analysis of their interactions has grown into a subspecialty with numerous immunoelectrophoretic techniques that have been of considerable value for detection and quantitation of specific molecules in complex fluids (Axelsen et al., 1973; Axelsen, 1983). Another class of specific reagents, lectins, has found widespread use for the identification and detailed structural characterization of glycoconjugates in various gel electro-phoretic techniques (Bpg-Hansen, 1973; Bøg-Hansen et al., 1975).

The field of electrophoretic interaction analysis has been rejuvenated by the advent of capillary electrophoresis (CE) (Mikkers et al., 1979; Jorgenson and Lukacs, 1981) and a wide number of both qualitative and quantitative binding studies have now appeared using this technique (cf. reviews by Monnig and Kennedy, 1994; Chu et al., 1995; Heegaard, 1997).

Theory

The experimental set-up depends on the interaction kinetics of the system (Matousek and Horejsí, 1982) but the fundamental requirement is that the electrophoretic mobility of complexed molecules is different from the mobility of free molecules. Binding assays can in some cases be performed by quantitation of ligated and/or free molecules after electrophoretic separation of pre-equilibrated samples. The requirement in this case is that the preformed complexes are stable enough to maintain complex formation during electrophoresis—at least until a clear separation between free and bound molecules is possible.

The pre-equilibration approach has been much used e.g., to characterize strong protein–DNA complex formation in native gel electrophoresis (Fried, 1989) (gel shift or mobility shift assays) and relies on quantitation of DNA separated from DNA complexes at different DNA–protein ratios. CE has been used in a similar way (Heegaard and Robey, 1992), and with pre-equilibrated samples analyzed in the presence of saturating concentrations of ligand in the electrophoresis buffer the approach may also be used to study lower affinity binding (fast off rates) e.g., when estimating the stoichiometry of binding (Chu et al., 1994). As a rule, the kinetics of binding and the detection sensitivity determine if a pre-equilibration experimental set-up will be feasible. The combination of separation (by electrophoresis) and quantitation (by the detection method) of free and/or bound forms of pre-incubated analyte make this use of electrophoresis, including the data handling, equivalent with traditional approaches such as equilibrium dialysis and filtration assays.

Another electrophoretic approach is analogous to affinity chromatography because the analyte (the receptor molecule) is electrophoresed through a gel or buffer where the ligand is present. This is what is generally understood as affinity electrophoresis even though the term has been used about pre-equilibration experiments where molecules are also interacting during electrophoresis. The term was originally introduced for qualitative studies in agarose gels (Bíø-Hansen, 1973; Bøg-Hansen et al., 1975). Obviously, this method depends on the differential electrophoretic mobility of free and complexed molecules as well, but migration shifts are measured in this approach instead of changes in peak areas.

The first affinity electrophoresis experiments were performed in polyacrylamide gels where complexed molecules had no mobility either because the ligand was immobilized or because of the high molecular weight of complexes. When it was realized (Nakamura and Wakeyama, 1961) that the situation was analogous, equations from the theory of adsorption chromatography (Wilson, 1940; Weiss, 1943; DeVault, 1943) were transformed into equations applicable to the electrophoretic interaction analysis. The assumptions were: establishment of instantaneous equilibrium, no diffusion, a homogeneously distributed ligand with a single binding site, a binding stoichiometry of 1 : 1,...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• Preface
• Contributors
• 1 Electrophoretic Analysis of Reversible Interactions
• 2 Determination of Affinity Constants by Capillary Electrophoresis
• 3 Measurement of Binding Constants by Frontal Affinity Chromatography
• 4 Determination of Affinity Constants by Partition Equilibrium Methods
• 5 Determination of Transmembrane Protein Affinities for Solutes by Frontal Chromatography
• 6 Modeling of Chiral Liquid Chromatography using an Immobilized Protein as the Selector
• 7 Determination of Receptor-Ligand Affinity Constants
• 8 Determination of Equilibrium and Kinetic Constants for the Interaction of GTPases with Nucleotides, Regulators and Effectors
• 9 Biospecific Interaction Analysis using Integrated Optics Techniques
• 10 Receptor Binding Studies on Lipid Vesicles using the BIAcore
• 11 The Analysis of Surface Plasmon Resonance-based Biosensor Data using Numerical Integration: The Epidermal Growth Factor Receptor-Ligand Interaction as an Example
• 12 Affinity Chromatography for the Determination of Interactions between Drugs and Proteins: HPLC and CE Methods Employing ‘Secondary and Dynamic’ Equilibria
• Index