Identification of platination sites on human serum transferrin using (13)C and (15)N NMR spectroscopy.
Reactions between various apo and metal-bound forms of human serum transferrin (80 kDa) and the recombinant N-lobe (40 kDa) with [Pt(en)Cl(2)] or cis-[PtCl(2)(NH(3))(2)] have been investigated in solution via observation of [(1)H,(15)N] NMR resonances of the Pt complexes, [(1)H,(13)C] resonances of the eCH(3) groups of the protein methionine residues, and by chromatographic analysis of single-site methionine mutants. For the whole protein, the preferred Pt binding site appears to be Met256. Additional binding occurs at the other surface-exposed methionine (Met499), which is platinated at a slower rate than Met256. In contrast, binding of similar Pt compounds to the N-lobe of the protein occurs at Met313, rather than Met256. Met313 is buried in the interlobe contact region of intact transferrin. After loss of one chloride ligand from Pt and binding to methionine sulfur of the N-lobe, chelate-ring closure appears to occur with binding to a deprotonated backbone amide nitrogen, and the loss of the other chloride ligand. Such chelate-ring closure was not observed during reactions of the whole protein, even after several days.
Cox
M C
MC
Department of Chemistry, University of Edinburgh West Mains Road, Edinburgh EH9 3JJ, UK.
Barnham
K J
KJ
Frenkiel
T A
TA
Hoeschele
J D
JD
Mason
A B
AB
He
Q Y
QY
Woodworth
R C
RC
Sadler
P J
PJ
D000970:Antineoplastic Agents / Q000378:metabolism; D001665:Binding Sites; D002247:Carbon Isotopes; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D009944:Organoplatinum Compounds / Q000378:metabolism; D010984:Platinum / Q000378:metabolism*; D014168:Transferrin / Q000235:genetics / Q000378:metabolism*
D000970:Antineoplastic Agents; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D009944:Organoplatinum Compounds; D014168:Transferrin; D010984:Platinum; D008715:Methionine