Macromolecule–ligand binding studied by the Hummel and Dreyer method: current state of the methodology

Abstract

The use of the Hummel and Dreyer method to measure binding parameters of ligand–macromolecule associations is reviewed. The possibility to determine the number of binding sites and their association constants, even in the case of low affinity, and to control the free ligand concentration as an independent variable are the main advantages of the method. The conditions of the validity are rapid equilibrium kinetics, independence between ligand binding and macromolecule association, and identical retention rates between free and bound macromolecules. Initially developed on soft gels, the method has been applied to high-performance chromatography and capillary zone electrophoresis. Technical progress such as increase in resolution, detection sensitivity, and automation have improved its utilization. The binding parameters given by the Hummel and Dreyer method are in general similar to those obtained by other techniques, in comparable experimental conditions (equilibrium dialysis, ultrafiltration, frontal elution, vacancy peak method, vacancy affinity capillary electrophoresis, retention analysis, affinity chromatography and affinity capillary electrophoresis, physical methods). The choice between these methods is directed by material availability and practical constraints. Separation by new types of chromatographic columns or by capillary zone electrophoresis would enable the study of the simultaneous binding of different drugs on the same macromolecule and their competition.

Introduction

The study of macromolecule–ligand binding characteristics is a problem of practical interest for biology and medicine. The levels of free drugs in plasma (or their availability from a loosely bound state), on which depend the therapeutic effects, are determined by the affinities of the proteins (mainly albumin) for these drugs, and their knowledge is important for deciding their dosage. On the other hand, the saturation of biological receptors by specific small molecules (hormones, neurotransmitters) can be described by the same models.

A lot of very different methods have been developed to solve this problem. They have been already reviewed, in particular by Sebille et al. [1], Oravcova et al. [2], and Busch et al. [3]. A few of these methods involve previous separation of the constituents (filtration, precipitation, chromatography), which may disturb their mutual equilibrium. Those which do not require this step include physical methods, which make use of specific properties of the macromolecule–ligand systems (UV or IR spectrophotometry, fluorescence emission, circular dichroism, magnetic resonance), and analytical methods in which equilibrium is preserved. Among the latter, are equilibrium dialysis and different chromatographic methods, including that developed by Hummel and Dreyer [4], which is the subject of this review.

The principle of the Hummel and Dreyer method is as follows: a known quantity of a macromolecule (purified or not) is injected on a size exclusion chromatography column and eluted with a buffer containing a constant concentration of ligand. An amount of ligand, determined by the dissociation constant(s) of the equilibrium and the free ligand concentration, binds to the macromolecule and migrates with it, while a trough in the ligand concentration, corresponding to the quantity withdrawn from the solvent, migrates at its proper rate (Fig. 1). In these conditions, the macromolecule and the complex(es) remain in equilibrium with the ligand during the separation and no dissociation occurs, even in the case of weak associations.

Because of this interesting feature, this method has been used for numerous drug binding determinations in biochemistry and medicine. The most studied has been that of the anticoagulant warfarin on human (HSA) or bovine (BSA) serum albumin [1], [4], [5], [6], [7], [8], [9]. The affinities of several other drugs for these proteins have been determined by this method, in its original or modified form: furosemide [5], [10], ceftriaxone [10], [11], β₂-blocker ICI [10], phenobarbital and phenytoin [12], phenylbutazone [1], carvedilol [13], buspirone [14]. The binding of different drugs has also been investigated on glycosylated HSA [15] and on α₁ acid glycoprotein (AGP) [10], [13], [16].

Cation-binding capacities of particular proteins have been examined by this method: melanotropic-lipolytic peptide IIF for Ca²⁺, Mg²⁺, Na⁺, K⁺ [17], calcimedins [18], calcium-binding proteins from porcine liver [19] and arrestin [20] for Ca²⁺, BSA and bovine α lactalbumin for Sr²⁺ [21], collagen for Pb²⁺ [22].

The Hummel and Dreyer method has been also applied to the study of miscellaneous equilibria:

• enzymes and substrates or inhibitors [4], [23], [24], [25],

• tubulin and calmodulin [26] or antimitotic agents [27], [28],

• polysaccharides and flavour compounds [29],

• cyclodextrins and vitamin B-compounds [30], steroids [31] and various drugs [32],

• lipocalin proteins and biogenic amines [33] or nucleotides [34],

• cytochrome P450 and steroids [35].