Robert C. Woodworth

Identification of platination sites on human serum transferrin using (13)C and (15)N NMR spectroscopy.

Abstract Reactions between various apo and metal-bound forms of human serum transferrin (80 kDa) and the recombinant N-lobe (40 kDa) with [Pt(en)Cl2] or cis-[PtCl2(NH3)2] have been investigated in solution via observation of [1H,15N] NMR resonances of the Pt complexes, [1H,13C] resonances of the eCH3 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.

Competitive Binding of Bismuth to Transferrin and Albumin in Aqueous Solution and in Blood Plasma

Several bismuth compounds are currently used as antiulcer drugs, but their mechanism of action is not well established. Proteins are thought to be target sites. In this work we establish that the competitive binding of Bi3+ to the blood serum proteins albumin and transferrin, as isolated proteins and in blood plasma, can be monitored via observation of 1H and13C NMR resonances of isotopically labeled [ε-13C]Met transferrin. We show that Met132 in the I132M recombinant N-lobe transferrin mutant is a sensitive indicator of N-lobe metal binding. Bi3+binds to the specific Fe3+ sites of transferrin and the observed shifts of Met resonances suggest that Bi3+ induces similar conformational changes in the N-lobe of transferrin in aqueous solution and plasma. Bi3+ binding to albumin is nonspecific and Cys34 is not a major binding site, which is surprising because Bi3+ has a high affinity for thiolate sulfur. This illustrates that the potential target sites for metals (in this case Bi3+) in proteins depend not only on their presence but also on their accessibility. Bi3+ binds to transferrin in preference to albumin both in aqueous solution and in blood plasma.

An improved vertical polyacrylamide gel electrophoresis apparatus: Application to typing and subtyping of haptoglobins

Abstract A vertical gel electrophoresis apparatus allowing the casting of two parallel polyacrylamide gel slabs or of single slabs of various thicknesses is described. Techniques for both typing and subtyping of human haptoglobins with the apparatus are presented.

Efficient production and isolation of recombinant amino-terminal half-molecule of human serum transferrin from baby hamster kidney cells

Expression of the amino-terminal lobe of human serum transferrin secreted into the culture medium by transformed baby hamster kidney (BHK) cells has been increased from the levels reported originally of 10-15 micrograms/ml to 55-120 micrograms/ml. Use of the serum substitute, Ultraser G, has facilitated isolation of the recombinant protein, resulting in approximately 80% recovery of expressed hTF/2N from the culture medium. In the three experiments described, 300-750 mg of recombinant protein was collected over a period of 25 days from five expanded surface roller bottles each containing 200 ml of medium (seven to nine collections). The use of alginate beads to encapsulate the transformed BHK cells provided no advantage over normal culturing over 25 days. A lag in production resulting in 30% less recombinant protein over this time period was observed. The production and isolation procedures described are easily handled by one person. The system is amenable to incorporation of isotopically substituted amino acids useful in NMR studies.

N-lobe versus C-lobe complexation of bismuth by human transferrin

Interactions of recombinant N-lobe of human serum transferrin (hTF/2N) with Bi3+, a metal ion widely used in medicine, have been investigated by both UV and NMR spectroscopy. The bicarbonate-independent stability constant for Bi3+ binding (K*) to hTF/2N was determined to be log K* 18.9+/-0.2 in 5 mM bicarbonate/10 mM Hepes buffer at 310 K, pH7.4. The presence of Fe3+ in the C-lobe of intact hTF perturbed Bi3+ binding to the N-lobe, whereas binding of Bi3+ to the C-lobe was unaffected by the presence of Fe3+ in the N-lobe. Reactions of Bi3+ (as bismuth nitrilotriacetate or ranitidine bismuth citrate) with hTF/2N in solutions containing 10 mM bicarbonate induced specific changes to high-field 1H-NMR peaks. The 1H co-ordination shifts induced by Bi3+ were similar to those induced by Fe3+ and Ga3+, suggesting that Bi3+ binding causes similar structural changes to those induced by hTF/2N. 13C-NMR data showed that carbonate binds to hTF/2N concomitantly with Bi3+.

The nucleotide sequence of rabbit liver transferrin cDNA

The cDNA sequence of rabbit liver transferrin has been determined. The largest cDNA was 2279 base pairs (bp) in size and encoded 694 amino acids consisting of a putative 19 amino acid signal peptide and 675 amino acids of plasma transferrin. The deduced amino acid sequence of rabbit liver transferrin shares 78.5% identity with human liver transferrin and 69.1% and 44.8% identity with porcine and Xenopus transferrins, respectively. At the amino acid level, vertebrate transferrins share 26.4% identity and 56.5% similarity. The most conserved regions correspond to the iron ligands and the anion binding region. Optimal alignment of transferrin sequences required the insertion of a number of gaps in the region corresponding to the N-lobe. In addition, the N-lobes of transferrins share less amino acid sequence similarity than the C-lobes.

Spectral and metal-binding properties of three single-point tryptophan mutants of the human transferrin N-lobe.

Human serum transferrin N-lobe (hTF/2N) contains three conserved tryptophan residues, Trp(8), Trp(128) and Trp(264), located in three different environments. The present report addresses the different contributions of the three tryptophan residues to the UV-visible, fluorescence and NMR spectra of hTF/2N and the effect of the mutations at each tryptophan residue on the iron-binding properties of the protein. Trp(8) resides in a hydrophobic box containing a cluster of three phenylalanine side chains and is H bonded through the indole N to an adjacent water cluster lying between two beta-sheets containing Trp(8) and Lys(296) respectively. The fluorescence of Trp(8) may be quenched by the benzene rings. The apparent increase in the rate of iron release from the Trp(8)-->Tyr mutant could be due to the interference of the mutation with the H-bond linkage resulting in an effect on the second shell network. The partial quenching in the fluorescence of Trp(128) results from the nearby His(119) residue. Difference-fluorescence spectra reveal that any protein containing Trp(128) shows a blue shift upon binding metal ion, and the NMR signal of Trp(128) broadens out and disappears upon the binding of paramagnetic metals to the protein. These data imply that Trp(128) is a major fluorescent and NMR reporter group for metal binding, and possibly for cleft closure in hTF/2N. Trp(264) is located on the surface of the protein and does not connect to any functional residues. This explains the facts that Trp(264) is the major contributor to both the absorbance and fluorescence spectra, has a strong NMR signal and the mutation at Trp(264) has little effect on the iron-binding and release behaviours of the protein.

[1H,13C] NMR determination of the order of lobe loading of human transferrin with iron: comparison with other metal ions

Human serum transferrin (hTF) is a single‐chain bilobal glycoprotein (80 kDa) which transports Fe3+ and a variety of other metal ions in blood. Only diferric transferrin, not the apo‐protein, binds strongly to transferrin receptors and is taken up by cells via receptor‐mediated endocytosis. We show here that 2D [1H,13C] NMR studies of recombinant ϵ‐[13C]Met‐hTF allow the order of lobe loading with various metal ions, including Fe3+, to be determined. In particular, the resonance for Met‐464, a residue in the hydrophobic patch of helix 5, is very sensitive to iron binding in the C‐lobe. The selectivity of lobe loading with Fe3+ is compared to loading with Fe2+ (which binds as Fe3+), Al3+, Ga3+ and Bi3+. Similar changes in shifts of the Met residues are observed for these metal ions, suggesting that they induce similar conformational changes in the protein.

A pmr study of the effects of pH and anion and metal ion binding of the histidyl residues of ovotransferrin

High resolution proton magnetic resonance studies of ovotransferrin show clear resolution of four groups of C(2)-H histidyl resonances to low field of the major aromatic envelope. Titrations of the protein in the absence and presence of synergistic anions, oxalic acid, malonic acid, and 2,6-dipicolinic acid, and anions plus metal ions reveal that six histidines are involved in the binding sites. These histidines, three in each binding site, are near to one another. In each binding site one histidine is involved in binding to anions and two are involved in binding to metal ions.

The low pKa value of iron-binding ligand Tyr188 and its implication in iron release and anion binding of human transferrin.

2D NMR–pH titrations were used to determine pK a values for four conserved tyrosine residues, Tyr45, Tyr85, Tyr96 and Tyr188, in human transferrin. The low pK a of Tyr188 is due to the fact that the iron‐binding ligand interacts with Lys206 in open‐form and with Lys296 in the closed‐form of the protein. Our current results also confirm the anion binding of sulfate and arsenate to transferrin and further suggest that Tyr188 is the actual binding site for the anions in solution. These data indicate that Tyr188 is a critical residue not only for iron binding but also for chelator binding and iron release in transferrin.