Incorporation of 5-hydroxytryptophan into transferrin and its receptor allows assignment of the pH induced changes in intrinsic fluorescence when iron is released.
Human serum transferrin (hTF) is a bilobal glycoprotein that transports iron to cells. At neutral pH, diferric hTF binds with nM affinity to the transferrin receptor (TFR) on the cell surface. The complex is taken into the cell where, at the acidic pH of the endosome ( approximately pH 5.6), iron is released. Since iron coordination strongly quenches the intrinsic tryptophan fluorescence of hTF, the increase in the fluorescent signal reports the rate constant(s) of iron release. At pH 5.6, the TFR considerably enhances iron release from the C-lobe (with little effect on iron release from the N-lobe). The recombinant soluble TFR is a dimer with 11 tryptophan residues per monomer. In the hTF/TFR complex these residues could contribute to and compromise the readout ascribed to iron release from hTF. We report that compared to Fe(C) hTF alone, the increase in the fluorescent signal from the preformed complex of Fe(C) hTF and the TFR at pH 5.6 is significantly quenched (75%). To dissect the contributions of hTF and the TFR to the change in fluorescence, 5-hydroxytryptophan was incorporated into each using our mammalian expression system. Selective excitation of the samples at 280 or 315 nm shows that the TFR contributes little or nothing to the increase in fluorescence when ferric iron is released from Fe(C) hTF. Quantum yield determinations of TFR, Fe(C) hTF and the Fe(C) hTF/TFR complex strongly support our interpretation of the kinetic data.
James
Nicholas G
NG
Department of Biochemistry, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405-0068, USA.
Byrne
Shaina L
SL
Mason
Anne B
AB
D006916:5-Hydroxytryptophan / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D011789:Quantum Theory; D011990:Receptors, Transferrin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism*
D011990:Receptors, Transferrin; D014168:Transferrin; C028283:diferric transferrin; D006916:5-Hydroxytryptophan; D007501:Iron