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 Ga(3+) 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 Ga(3+) and apoOTf remain unclear. Here, we report detailed investigations into the interactions of Ga(3+) with apoovotransferrin at the molecular level. First, the characteristics of Ga(3+) binding to apoovotransferrin were analyzed using UV difference spectra. The results show that Ga(3+) prefers to bind to the N-terminal site rather than the C-terminal site under the experimental conditions. Effective stability constants of logK(N)=18.88+/-0.24 and logK(C)=17.65+/-0.12 were determined. Second, conformational changes in apoovotransferrin during Ga(3+) binding were studied using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as a fluorescence probe. Apoovotransferrin undergoes a large conformational change when Ga(3+) 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 Ga(3+) from ovotransferrin carrying one Ga(3+) at the N-terminal site. Ga(3+) removal from the N-terminal binding site follows simple saturation kinetics.
Li
Yingqi
Y
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006, PR China.
Liu
Bin
B
Ge
Zhongjie
Z
Yang
Binsheng
B
D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003207:Conalbumin / Q000737:chemistry* / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005708:Gallium / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D009282:Naphthalenesulfonates / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D001059:Apoproteins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D003207:Conalbumin; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D004492:Edetic Acid; D005708:Gallium