Determination of binding constants by affinity chromatography

This review summarizes developments in the use of affinity chromatography to characterize biospecific interactions in terms of reaction stoichiometry and equilibrium constant. In that regard, the biospecificity incorporated into the design of the experiment ensures applicability of the method regardless of the sizes of the reacting solutes. By the adoption of different experimental strategies (column chromatography, simple partition equilibrium, solid-phase immunoassay and biosensor technology protocols) quantitatiative affinity chromatography can be used to characterize interactions governed by an extremely broad range of binding affinities. In addition, the link between ligand-binding studies and quantitative affinity chromatography is illustrated by means of partition equilibrium studies of glycolytic enzyme interactions with muscle myofibrils. an exercise which emphasizes that the same theoretical expressions apply to naturally occurring examples of affinity chromatography in the cellular environment. (C) 2004 Elsevier B.V. All rights reserved.

Interpretation of the temperature dependence of rate constants in biosensor studies

A comparison is made between Arrhenius and transition-state analyses of the temperature dependence of rate constants reported in four published biosensor studies. Although the Eyring transition-state theory seemingly affords a more definitive solution to the problem of characterizing the activation energetics, the analysis is equivocal because of inherent assumptions about reaction mechanism and the magnitude of the transmission coefficient. In view of those uncertainties it is suggested that a preferable course of action entails reversion to the empirical Arrhenius analysis with regard to the energy of activation and a preexponential factor. The former is essentially equivalent to the enthalpy of activation, whereas the magnitude of the latter indicates directly the extent of disparity between the frequency of product formation and the universal frequency factor (temperature multiplied by the ratio of the Boltzmann and Planck constants) and hence the likelihood of a more complicated kinetic mechanism than that encompassed by the Eyring transition-state theory. (C) 2004 Elsevier Inc. All rights reserved.

Electronic energy-transfer rate constants for geometrical isomers of a bichromophoric macrocyclic complex

The rate of electronic energy transfer (EET) between a naphthalene donor and an anthracene acceptor in [ZnL3]-(ClO4)(2) and [ZnL4](ClO4)(2) was determined by time-resolved fluorescence measurements, where L 3 and L 4 are the geometrical isomers of 6-[(anthracen-9-ylmethyl)amino]-trans-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-13-amine (L-2), substituted with either a naphthalen-1-ylmethyl or naphthalen-2-ylmethyl donor, respectively. The energy transfer rate constant, k(EET), was determined to be (0.92 +/- 0.02) x 10(9) s(-1) for the naphthalen-1-ylmethyl-substituted isomer, while that for the naphthalen-2-ylmethyl-substituted isomer is somewhat faster, with k(EET) = (1.31 +/- 0.01) x 10(9) s(-1). The solid-state structure of [(ZnLCl)-Cl-3]ClO4 has been determined, and using molecular modeling calculations, the likely distributions of solution conformations in CH3CN have been evaluated for both complexes. The calculated conformational distributions in the common trans-III N-based isomeric form gave Forster EET rate constants that account for the differences observed and are in excellent agreement with the experimental values. It is shown that the full range of conformers must be considered to accurately reproduce the observed EET kinetics.

Interpretation of the temperature dependence of equilibrium and rate constants

The objective of this review is to draw attention to potential pitfalls in attempts to glean mechanistic information from the magnitudes of standard enthalpies and entropies derived from the temperature dependence of equilibrium and rate constants for protein interactions. Problems arise because the minimalist model that suffices to describe the energy differences between initial and final states usually comprises a set of linked equilibria, each of which is characterized by its own energetics. For example, because the overall standard enthalpy is a composite of those individual values, a positive magnitude for AHO can still arise despite all reactions within the subset being characterized by negative enthalpy changes: designation of the reaction as being entropy driven is thus equivocal. An experimenter must always bear in mind the fact that any mechanistic interpretation of the magnitudes of thermodynamic parameters refers to the reaction model rather than the experimental system For the same reason there is little point in subjecting the temperature dependence of rate constants for protein interactions to transition-state analysis. If comparisons with reported values of standard enthalpy and entropy of activation are needed, they are readily calculated from the empirical Arrhenius parameters. Copyright (c) 2006 John Wiley & Sons, Ltd.