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

Ovotransferrin is a main member of transferrin family and has a dual role in both the transport of iron and antibacterial function. Gallium-67 is widely used as an imaging agent for tumors. It has been reported that Ga3+ can bind to apoovotransferrin at two sites, one in the N-terminal lobe and another in the C-terminal lobe. However, several details of the interaction between Ga3+ and apoOTf remain unclear. Here, we report detailed investigations into the interactions of Ga3+ with apoovotransferrin at the molecular level. First, the characteristics of Ga3+ binding to apoovotransferrin were analyzed using UV difference spectra. The results show that Ga3+ prefers to bind to the N-terminal site rather than the C-terminal site under the experimental conditions. Effective stability constants of log KN = 18.88 ± 0.24 and log KC = 17.65 ± 0.12 were determined. Second, conformational changes in apoovotransferrin during Ga3+ binding were studied using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as a fluorescence probe. Apoovotransferrin undergoes a large conformational change when Ga3+ binds to the N-terminal site, and a smaller conformational change when the ion binds to the C-terminal site. UV difference spectra were also used to measure the rate at which EDTA removes Ga3+ from ovotransferrin carrying one Ga3+ at the N-terminal site. Ga3+ removal from the N-terminal binding site follows simple saturation kinetics.