Aggregation of a folate-binding protein from cow's milk

Abstract

Sedimentation equilibrium experiments of folate-binding protein from cow's milk performed in the analytical ultracentrifuge at pH 5 9 showed a single molecular species with a molecular weight of 30,000 9 In contrast, at pH 7.4 sedimentation equilibrium indicated molecular weights ranging from about 30,000 to at least 300,000. OSedimentation velocity experiments at pH 5.0 showed a single symmetrical peak with s20,w = 2.8 S in agreement with a molecular weight of about 30,000. At pH 6.0 was seen a faster moving boundary (s20,w~ 6S) in addition to the slow one. At higher pH two peak maxima were seen: a slow one with sedimentation coefficients of about 5S and a fast one increasing from 9S to 1 IS with increasing pH. Addition of folate further increased the sedimentation rates. By assuming that G.LBERT'S rapid monomer-n-mer equilibrium theory was valid also for a lower polymer in equilibrium with a higher polymer our data can be interpreted as an equilibrium between a tetramer and a polymer consisting of more than 16 monomers. In the case of folate addition the results may similarly be interpreted as an equilibrium between an octamer and a polymer composed of more than 32 monomers.

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

“…Hence, this conformational change increases the overall affinity by 4-5 orders of magnitude! Apart from the third step probably due to the well-known phenomenon of ligandinduced polymerization (Pedersen et al 1980;Hansen et al 1983) the reaction schemes at pH 5 (monomeric FBP) and pH 7.4 are qualitatively similar consistent with the notion that folate only binds to monomeric FBP. At pH 7.4 and at the present FBP concentration polymeric (apo) FBP prevails, and as argued dissociation to active monomers could theoretically be a rate limiting step decreasing the apparent affinity for folate.…”

“…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.…”

High-Affinity Folate Binding in Leukocyte Lysate from Normal Subjects: Effect of Concentration of Binding Protein, Temperature and pH

“…positive cooperativity and a ligand affini ty that increases with a decreasing concen tration of the binding protein. These two phenomena are also associated with the high-affinity folate-binding systems in milk and serum [5,6,8,10]; they may sug gest the involvement of a polymerizing system in which the oligomer prevailing at low protein concentrations has a greater affinity for the ligand than does the po lymer [11], So far this hypothesis has gained some support from ultracentrifu gation and gel filtration studies demon strating a remarkably great aggregation tendency of purfied folate binder from cow's whey [12,13]. The folate binder in leukocyte lysate did moreover resemble the milk and serum binders in the follow ing respects, the effects of incubation tem perature and pH on binding, affinity con stants in the range of 109-1010 1/mol, a molecular weight of 25,000-30,000 and a weak affinity to anion exchange resins at near-neutral pH [5,6,9,10],…”

Stopped-Flow Kinetic Studies of the Interaction of Bovine Folate Binding Protein (FBP) and Folate

“…The FBP-folate complex shows less surface hydrophobicity than free FBP [10,11], thus conformational changes are expected to occur during the reaction. At neutral pH some polymerisation of FBP may occur, in particular in the presence of ligand [12,13]. At pH 5 FBP is monomeric, and polymeric forms have been observed only at concentrations >10 lM in the presence of ligand [12,14].…”

“…At neutral pH some polymerisation of FBP may occur, in particular in the presence of ligand [12,13]. At pH 5 FBP is monomeric, and polymeric forms have been observed only at concentrations >10 lM in the presence of ligand [12,14].…”