Comments on the Article by Christensen U, Holm J and Hansen SI (2006) Bioscience Reports 26:291–299

Abstract

This is a comment on the article by Christensen et al. (2006) recently published in Bioscience Reports. The authors studied the interaction between folic acid and bovine folate-binding protein (FBP) using stopped-flow technique. They interpreted their binding data and concluded that the interaction consists of at least three steps at pH 7.4 and two steps at pH 5.0 and rejected a simple one-step binding model (A + B = AB). They did not take into consideration effects of FBP self-aggregation, even as this could be a source for parallel rate limiting reactions in their experiment. Christensen et al. (2006) conducted their study at a high concentration of FBP (0.5 lM). At such high FBP concentration (i.e. lM range) in neutral pH, selfaggregation of both unliganded [FBPM(FBP) n ] and liganded FBP [FBP*FolateM(FBP*Folate) n ] occurs to a high extent (Pedersen et al. 1980;Salter et al. 1981;Hansen et al. 1983); self-aggregation of liganded FBP occurs also to some extent in slightly acidic conditions (Pedersen et al. 1980). Because of the existing equilibriums of aggregates I suggest that there are rate limiting parallel reactions, where FBP forms inactive dimers and multimers that are affecting the results when using a rapid mixing technique such as stopped-flow for studying the interaction between folate and FBP. Under pseudo first-order conditions, the kinetic mechanism and parameters for a simple association between FBP and folate theoretically do not depend on the choice of which component is limiting. Using stopped-flow technique Slepenkov et al. (2006) found that the choice of which component is limiting changed the kinetics of the reaction. Further they suggest that there is a pre-existing equilibrium from the formation of inactive dimers (or multimers) that becomes important at high protein concentrations. When the concentrations of active monomer is lowered due to the interaction with the ligand during the stopped-flow experiment, the equilibrium shifts and the dimers slowly dissociate, yielding active monomers that then can bind ligand. This dissociation reaction produces a second, slow phase of the interaction intended to be studied. Therefore, it is likely that Christensen et al. (2006) would achieve different results if