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Biomedical subjects

D C Harris

Publications and source records attributed to D C Harris.

At least 91 records · Page 5Linked to original sources
PubMed1985

Reduction potential of iron in transferrin.

The reduction potential of Fe3+ in transferrin was measured spectrophotometrically by equilibration with methyl viologen in the presence of sodium dithionite. For an ionic strength near 0.1 M at 25 degrees C and pH 7.3 under 0.048 atm. CO2, half of the iron is reduced at a potential near -0.40 V (vs. standard hydrogen electrode). At least one disulfide bond of the protein is partially reduced at a potential of -0.44 V, as evidenced by reaction with [14C]iodoacetate.

Disulfides↗
PubMed1985

Microsequence analysis of peptides and proteins. V. Design and performance of a novel gas-liquid-solid phase instrument.

We describe the construction and performance of a novel, automated, Edman chemistry-based microsequencer. The reagent and solvent delivery system, the reaction cartridge for coupling and cleavage, and the conversion flask are all constructed from chemically inert perfluoroelastomers. The delivery valves are of a new design incorporating the use of electromagnetically actuated solenoids and zero-dead-volume construction, and may be connected in a modular fashion resulting in multiple inputs with a single output line which can be flushed with inert gas. The bottle closures are of a new design based on an all-Teflon compression fitting. The reaction cartridge and conversion flask are thermostated by solid-state heaters in an aluminum block. The overall size of the instrument is 25 X 34 X 14 in. The chemistry utilizes 2% aqueous triethylamine as the coupling base which is delivered to the reaction cartridge via a stream of nitrogen. The "gas-phase" delivery of the coupling base and the cleavage acid (trifluoroacetic acid) is modeled after the method described by R. M. Hewick et al. (J. Biol. Chem. 256, 7990-7997,1981). The instrument has performed well over a period of 3 years in terms of low background peaks, sensitivity in the picomole range, and reliability of operation. The use of economical components, ease of construction and operation, and sensitive analytical capability make this instrument a useful tool for microsequence analysis of peptides and proteins.

Amino Acid Sequence↗
PubMed1983

Fate of oxygen during ferritin iron incorporation.

The deposition of Fe(II) into ferritin using 18O2 as the oxidant was investigated. Only 3-4% of the oxygen atoms in the FeOOH core of ferritin were derived from the oxidant. This was true whether large (1200-1900 Fe/molecule) or small (220-240 Fe/molecule) amounts of iron were added to apoferritin or when iron was added to ferritin already containing 1000 Fe/molecule. Experiments using H218O in the solvent showed that nearly all of the oxygen atoms in the core were derived from solvent. The stoichiometry of the reaction was close to 2 Fe(II)/O2, instead of the expected value of 4 Fe(II)/O2. Reactions of 18O2 with Fe(II) in the absence of apoferritin had a similar 18O distribution and stoichiometry. Our results are most consistent with the crystal growth model of ferritin iron deposition.

Animals↗
PubMed1983

Multiple myeloma with renal failure. A case for intensive treatment.

A series of nine consecutive patients with multiple myeloma and renal failure is presented. All patients were treated with urinary alkalinisation with sodium bicarbonate and/or acetazolamide, diuresis with saline, mannitol and/or furosemide, pulse melphalan and prednisone and, where indicated, allopurinol and aluminium hydroxide. A substantial and sustained improvement in renal function has been achieved in all nine patients. Of five patients with a urea more than 25 mmol/L at presentation, the median survival to date is 64 weeks. Of these patients only one has died --not from renal failure but pneumonia, eighteen months after presentation. The others are alive and well. The results confirm the effectiveness of these measures in both improving renal function and prolonging survival, and suggest a more optimistic prognosis for patients with multiple myeloma and renal failure.

Adult↗
PubMed1982

Model system oxidations supporting the crystal growth model of ferritin iron uptake.

Fe2+ is oxidized and taken up by ferritin or ápoferritin in the presence of dioxygen. Iodate causes Fe2+ oxidation and uptake by ferritin, but not by apoferritin. Synthetic iron polymer facilitates Fe2+ oxidation by either dioxygen or iodate. Nitrilotriacetic acid or iminodiacetic acid facilitate oxidation of Fe2+ by oxygen but not by iodate. These results support the crystal growth model of ferritin iron uptake, with iron polymer serving as a model for the ferritin core and aminocarboxylic acids mimicking the metal-binding sites of apoferritin.

Apoferritins↗
PubMed1980

Binding of xylenol orange to transferrin. Demonstration of metal-anion linkage.

The iron transport protein, transferrin, binds two metal ions and, concomitantly, two carboxylate anions. The metal ion indicators, xylenol orange and semi-xylenol orange are carboxylate anions which exhibit a characteristic visible spectrum when attached to a metal. We prepared the ternary complexes VO2+-transferrin-xylenol orange and VO2+-transferrin-semi-xylenol orange. The EPR spectra show that the vanadyl ion is attached to the protein and the visible spectra show that the xylenol orange or semi-xylenol orange is attached to the metal. The implication is that in other metal-transferrin-anion complexes, the anion is directly attached to the metal.

Chemical Phenomena↗
PubMed1979

Insensitivity of the ferritin iron core to heat treatment.

To test whether the reactivity of ferritin iron is affected by the heat treatment used in ferritin isolation, we prepared ferritin from the same horse spleen with or without heating. Both samples exhibited similar reactivity upon reduction or chelation of iron.

Animals↗
PubMed1978

Iron exchange between ferritin and transferrin in vitro.

The transfer of iron between horse spleen [55Fe]ferritin and human apotransferrin or [59Fe]transferrin in homogeneous solution was investigated. Transfer between the two proteins in the presence of citrate, ATP, or ascorbate occurs in both direction, but the net flow is always from ferritin to transferrin. Ferritin which is ca. 1/3 to 1/2 saturated with iron appears to be most reactive. Chemically prepared apoferritin does not accept iron from diferric transferrin. Citrate-mediated transfer of iron from ferritin to apotransferrin is first order with respect to ferritin, zero order with respect to transferrin, and has a complex dependence upon citrate concentration. Direct transfer of iron from native or reconstituted ferritin to apotransferrin in the absence of any identifiable mediating agent was observed to occur at about half the rate attained in the presence of 1 mM citrate. No transfer of iron between the two proteins occurs across a dialysis membrane in the absence of a mediating agent. No binding of transferrin and ferritin to each other was demonstrable. One possible explanation for these observations is that iron from the core of ferritin is in equilibrium with iron near the outer surface of the protein, where the metal would be available to transferrin.

Animals↗
PubMed1978

Oxalate and spin-labeled oxalate as probes of the anion binding site of human transferrin. Metal to anion distance.

The spin-labeled anion N-[4-(2,2,6,6-tetramethylpiperidin-1-oxyl)] oxamate has been synthesized and characterized. In the presence of this compound, a specific iron-transferrin-anion complex is formed, as evidenced by the development of a characteristic red color. No EPR signal was observed for the nitroxyl radical in the protein complex, presumably due to broadening of the signal by the paramagnetic metal ion. Failure to observe a signal implies that the metal to nitroxyl distance is less than or equal to 6 A. This suggests that the anion is directly attached to the metal ion in the protein. The pH dependence of iron dissociation from iron-transferrin-oxalate is also reported. This complex is more stable at low pH than iron-transferrin-carbonate.

Binding Sites↗
PubMed1977

Functional equivalence of iron bound to human transferrin at low pH or high pH.

Human transferrin was labeled with 59Fe at one of its two metal-binding sites (designated A) at pH 6.0. 55Fe was then added to site B at pH 7.5. Both isotopes of iron were taken up in equal proportions by human reticulocytes. These experiments do not support the hypothesis that each binding site of transferrin has a different physiologic function.

Binding Sites↗
PubMed1977

Different metal-binding properties of the two sites of human transferrin.

Transferrin, the serum serum iron-transport protein which can bind two metal ions at physiologic pH, binds just one Fe3+, VO2+, or Cr3+ ion at pH 6.0. Fe3+ and VO2+ appear to be bound at the same site, designated A, based on electron paramagnetic resonance (EPR) spectra of VO2+-transferrin and (Fe3+)1(VO2+)1-transferrin. The EPR spectra of (Cr3+)1(VO2+)1-transferrin and of (Cr3+), (FE3+)1-transferrin indicate that that Cr3+ is bound to site B at pH 6.0. Transferrin was labeled at site A with 59Fe at pH 6.0 and at site B with 55Fe at pH 7.5. When the pH of the resulting preparation was lowered to 6.3 and the dissociated iron was separated by gel filtration, about ten times as much 55Fe as 59Fe was lost. The same EPR and isotopic-labeling experiments showed that Fe3+ added to transferrin at pH 7.5 binds to site A with about 90% selectivity.

Binding Sites↗