Complexation of ytterbium to human transferrin and its uptake by K562 cells.
There is an increasing interest in the use of lanthanides in medicine. However, the mechanism of their accumulation in cells is not well understood. Lanthanide cations are similar to ferric ions with regard to transferrin binding, suggesting transferrin-receptor mediated transport is possible; however, this has not yet been confirmed. In order to clarify this mechanism, we investigated the binding of Yb3+ to apotransferrin by UV-Vis spectroscopy and stopped-flow spectrophotometry, and found that Yb3+ binds to apotransferrin at the specific iron sites in the presence of bicarbonate. The apparent binding constants of these sites showed that the affinity of Yb3+ is lower than that of Fe3+and binding of Yb3+ in the N-lobe is kinetically favored while the C-lobe is thermodynamically favored. The first Yb3+ bound to the C-lobe quantitatively with a Yb/apotransferrin molar ratio of < 1, whereas the binding to the other site is weaker and approaches completeness by a higher molar ratio only. As demonstrated by 1H NMR spectra, Yb3+ binding disturbed the conformation of apotransferrin in a manner similar to Fe3+. Flow cytometric studies on the uptake of fluorescein isothiocyanate labeled Yb3+-bound transferrin species by K562 cells showed that they bind to the cell receptors. Laser scanning confocal microscopic studies with fluorescein isothiocyanate labeled Yb3+-bound transferrin and propidium iodide labeled DNA and RNA in cells indicated that the Yb3+ entered the cells. The Yb3+-transferrin complex inhibited the uptake of the fluorescein labeled ferric-saturated transferrin (Fe2-transferrin) complex into K562 cells. The results demonstrate that the complex of Yb3+-transferrin complex was recognized by the transferrin receptor and that the transferrin-receptor-mediated mechanism is a possible pathway for Yb3+ accumulation in cells.
Du
Xiu-Lian
XL
Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing, China.
Zhang
Tian-Lan
TL
Yuan
Lan
L
Zhao
Yong-Yuan
YY
Li
Rong-Chang
RC
Wang
Kui
K
Yan
Siu Cheong
SC
Zhang
Li
L
Sun
Hongzhe
H
Qian
Zhong-Ming
ZM
D001667:Binding, Competitive; D002412:Cations; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007501:Iron; D020014:K562 Cells; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D018613:Microscopy, Confocal; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013816:Thermodynamics; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism*; D014466:Ultraviolet Rays; D015018:Ytterbium / Q000737:chemistry* / Q000378:metabolism
D002412:Cations; D014168:Transferrin; D007501:Iron; D015018:Ytterbium