Steric restrictions on the binding of large metal ions to serum transferrin.
Apotransferrin in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid at 25 degrees C and pH 7.4 was titrated with acidic solutions of Lu3+, Tb3+, and Eu3+. Metal binding at the two specific metal-binding sites of transferrin was followed from changes in the difference UV spectra at 245 nm. The binding of Tb3+ was also followed from changes in the fluorescence emission spectrum at 549 nm. Apotransferrin was titrated with solutions containing varying ratios of the metal ion and the competitive chelating agent nitrilotriacetic acid, and metal-transferrin binding constants were calculated by nonlinear least-squares fits of the absorbance as a function of titrant added. The sequential carbonate-independent equilibrium constants for the binding of two metal ions are log KM1 = 11.08 and log KM2 = 7.93 for Lu3+, log KM1 = 11.20 and log KM2 = 7.61 for Tb3+, and log KM1 = 9.66 and log KM2 = 7.27 for Eu3+. Titrations of both C-terminal and N-terminal monoferric transferrins indicate that all of these metal ions bind more strongly to the C-terminal binding site. The trend in log K values as a function of the lanthanide ionic radius has been evaluated both by plots of log K versus the metal ion charge/radius ratio and by linear free-energy relationships in which binding constants for complexes of the larger lanthanides are plotted versus the binding constants for complexes with the smallest lanthanide, Lu3+. Both methods indicate that there is a sharp drop in the binding constants for the C-terminal binding site for metals larger than Tb3+. This decrease is attributed to a steric hindrance to the binding of the larger cations. The steric effect is not as strong for metal binding at the N-terminal site. As a result, the selectivity for binding to the C-terminal site, which is quite high for the smaller lanthanides, drops sharply on going from Tb3+ to Nd3+.
Harris
W R
WR
Department of Chemistry, University of Missouri-St. Louis 63121, USA. wharris@umsl.edu
Yang
B
B
Abdollahi
S
S
Hamada
Y
Y
D000495:Allosteric Site; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism*; D005063:Europium / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008187:Lutetium / Q000378:metabolism; D008674:Metals, Rare Earth / Q000378:metabolism; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013725:Terbium / Q000378:metabolism; D013816:Thermodynamics; D014168:Transferrin / Q000737:chemistry* / Q000378:metabolism*
D001059:Apoproteins; D008674:Metals, Rare Earth; D014168:Transferrin; C028284:apotransferrin; D013725:Terbium; D005063:Europium; D008187:Lutetium; D009571:Nitrilotriacetic Acid