Kinetics of Metal Ion Exchange between Citric Acid and Serum Transferrin

Abstract

The exchange of Fe(3+), Tb(3+), In(3+), Ga(3+), and Al(3+) between the C-terminal metal-binding site of the serum iron transport protein transferrin and the low-molecular-mass serum chelating agent citrate has been studied at pH 7.4 and 25 degrees C. The removal of Ga(3+), In(3+), and Al(3+) follows simple saturation kinetics with respect to the citrate concentration. In contrast, removal of both Fe(3+) and Tb(3+) shows a combination of saturation and first-order kinetic behavior with respect to the citrate concentration. The saturation component is consistent with a mechanism for metal release in which access to the bound metal is controlled by a rate-limiting conformational change in the protein. The first-order kinetic pathway is very rapid for Tb(3+), and this is attributed to a direct attack of the citrate on the Tb(3+) ion within the closed protein conformation. It is suggested that this pathway is more readily available for Tb(3+) because of the larger coordination number for this cation and the presence of an aquated coordination site in the Tb(3+)-CO(3)-Tf ternary complex. There is relatively little variation in the k(max) values for the saturation pathway for Tb(3+), Ga(3+), Al(3+), and In(3+), but the k(max) value for Fe(3+) is significantly smaller. It is suggested that protein interactions across the interdomain cleft of transferrin largely control the release of the first group of metal ions, while the breaking of stronger metal-protein bonds slows the rate of iron release. The rates of metal binding to apotransferrin are clearly controlled in large part by the hydrolytic tendencies of the free metal ions. For the more amphoteric metal ions Al(3+) and Ga(3+), there is rapid protein binding, and the addition of citrate actually retards this reaction. In contrast, the nonamphoteric In(3+) ion binds very slowly in the absence of citrate, presumably due to the rapid formation of polymeric In-hydroxo complexes upon addition of the unchelated metal ion to the pH 7.4 protein solution. The addition of citrate to the reaction accelerates the binding of In(3+) to apoTf, presumably by forming soluble, mononuclear In-citrate complexes.

Spectroscopic analysis of the interaction between chromium (III) and apoovotransferrin

“…CD spectroscopy is commonly used for general structural characterization of soluble proteins; the technique is particularly suited for monitoring the conformational state of proteins in solution. Specifically, CD provides direct information on the secondary structure of proteins [21,23]. Our data show that Cr 3 + caused some significant CD spectral modifications in the intrinsic region, indicating a small decrease in alpha helix content, which is consistent with that induced by the addition of Al (V) [21].…”

“…Since this high ratio of K 1 /K 2 indicates that there does not appear to be significant cooperativity in the binding of Cr 3+ ion to the two ovotransferrin binding sites [21], the equilibration of r1 equiv of Cr 3+ ion with apoOTf should result in preferential binding to the more stable site. The stronger binding site can be identified by titrating with monoferric OTf [21,22].…”

“…The stronger binding site can be identified by titrating with monoferric OTf [21,22]. A sample of N-terminal monoferric OTf (Fe NOTf) was titrated with Cr 3+ (aq) ion.…”

The Crystal Structure of Iron-free Human Serum Transferrin Provides Insight into Inter-lobe Communication and Receptor Binding

“…The presence and location of citrate in the structure are notable due to the physiological relevance of citrate as both an anion and a chelator (53)(54)(55). Six of the seven citrates bound to the two NCS-related molecules are found within the open clefts of the N-and C-lobes (see supplemental Table S1).…”

A hypothesis for the basis of the pro-oxidant nature of calcium ions

“…proteins. Citrate is an important LMW ligand for many physiological metal ions and is also involved in the formation of ROS through its various metal complexes (Lobner et al 2003, Harris et al 2003, Chiueh 2001. Glutamate along with Ca 2+ is part of the excitotoxin response of inflammation.…”