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Human serum transferrin: a tale of two lobes. Urea gel and steady state fluorescence analysis of recombinant transferrins as a function of pH, time, and the soluble portion of the transferrin receptor

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Abstract

Iron release from human serum transferrin (hTF) has been studied extensively; however, the molecular details of the mechanism(s) remain incomplete. This is in part due to the complexity of this process, which is influenced by lobe–lobe interactions, the transferrin receptor (TFR), the salt effect, the presence of a chelator, and acidification within the endosome, resulting in iron release. The present work brings together many of the concepts and assertions derived from previous studies in a methodical, uniform, and visual manner. Examination of earlier work reveals some uncertainty due to sample and technical limitations. We have used a combination of steady-state fluorescence and urea gels to evaluate the effect of conformation, pH, time, and the soluble portion of the TFR (sTFR) on iron release from each lobe of hTF. The use of authentic recombinant monoferric and locked species removes any possibility of cross-contamination by acquisition of iron. Elimination of detergent by use of the sTFR provides a further technical advantage. We find that iron release from the N-lobe is very sensitive to the conformation of the C-lobe, but is insensitive to the presence of the sTFR or to changes in pH (between 5.6 and 6.4). Specifically, when the cleft of the C-lobe is locked, the urea gels indicate that only about half of the iron is completely removed from the cleft of the N-lobe. Iron release from the C-lobe is most affected by the presence of the sTFR and changes in pH, but is unaffected by the conformation of the N-lobe. A model for iron release from diferric hTF is provided to delineate our findings.

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Abbreviations

BHK:

Baby hamster kidney cells

DMEM-F12:

Dulbecco’s modified Eagle’s medium containing Ham F-12 nutrient mixture

FeC hTF:

Nonglycosylated recombinant monoferric C-lobe human serum transferrin (mutations Y95F and Y188F preclude binding in the N-lobe) that contains an N-terminal His6 tag

Fe2 hTF:

Nonglycosylated recombinant diferric human serum transferrin that contains an N-terminal His6 tag

FeN hTF:

Nonglycosylated recombinant monoferric N-lobe human serum transferrin (mutations Y426F and Y517F preclude binding in the C-lobe) that contains an N-terminal His6 tag

hTF:

Human serum transferrin

LockC hTF:

Nonglycosylated recombinant diferric hTF that contains an N-terminal His6 tag and where mutation R632A locks iron in the C-lobe

LockN hTF:

Nonglycosylated recombinant diferric hTF that contains an N-terminal His6 tag and where mutation K206E locks iron in the N-lobe

MES:

2-Morpholinoethanesulfonic acid

sTFR:

Soluble portion of the transferrin receptor (residues 121–760) expressed as a recombinant entity that contains an N-terminal His6 tag

TBE:

Tris(hydroxymethyl)aminomethane–borate–EDTA

TFR:

Human serum transferrin receptor

Tris:

Tris(hydroxymethyl)aminomethane

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Acknowledgments

This work was supported by USPHS Grant R01 (DK 21739) to A.B.M.. Support for S.L.B. came from Hemostasis and Thrombosis Training Grant (5T32HL007594), issued to K. G. Mann at the University of Vermont by the National Heart, Lung, and Blood Institute.

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Authors and Affiliations

  1. Department of Biochemistry, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT, 05405-0068, USA

    Shaina L. Byrne & Anne B. Mason

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  1. Shaina L. Byrne
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  2. Anne B. Mason
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Correspondence to Anne B. Mason.

Electronic supplementary material

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775_2009_491_MOESM1_ESM.pdf

Maximum fluorescence emission from Fe2 hTF, FeN hTF, FeC hTF, LockC hTF and LockN hTF at pH 7.4 (100 mM HEPES) and pH 6.4–5.6 (100 mM MES, 300 mM KCl, 4 mM EDTA). All samples were incubated at least 15 minutes to try to assure complete iron removal. Samples were excited at 280 nm and emission was monitored between 300–400 nm using a 320 nm cut-on filter. (PDF 281 kb)

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Byrne, S.L., Mason, A.B. Human serum transferrin: a tale of two lobes. Urea gel and steady state fluorescence analysis of recombinant transferrins as a function of pH, time, and the soluble portion of the transferrin receptor. J Biol Inorg Chem 14, 771–781 (2009). https://doi.org/10.1007/s00775-009-0491-y

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  • DOI: https://doi.org/10.1007/s00775-009-0491-y

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