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PubMed2002

Carbohydrate-deficient transferrin isoforms measured by capillary zone electrophoresis for detection of alcohol abuse.

BACKGROUND: Measurements of carbohydrate-deficient transferrin (CDT) are used as markers of alcohol abuse. We developed a capillary zone electrophoresis (CZE) method aimed at improving accuracy of CDT testing. METHODS: We studied 111 alcohol abusers with Alcohol Use Disorders Identification Test scores >11 and 50 teetotalers. CZE was performed with a P/ACE 5500, fused-silica capillaries, and a CEofix CDT reagent set. After iron saturation, sera were loaded by low-pressure injection, separated at 28 kV, and monitored at 214 nm. We identified the transferrin isoforms by migration times, treatment with 100 U/L neuraminidase, and immunosubtraction with anti-human transferrin and anti-C-reactive protein antibodies. We compared CZE results with current biological markers of alcohol abuse, including the %CDT turbidimetric immunoassay. RESULTS: Migration times of the isoforms were identical in both populations. Asialotransferrin was missing in teetotalers but present in 92% of alcohol abusers. Disialotransferrin was higher in those who consumed excessive amounts of alcohol, whereas mean trisialotransferrin concentration was not affected by alcohol abuse. At cutoffs to maximize sensitivity and specificity, these values were 0.92 and 1 [mean ROC area (MRa), 0.96; 95% confidence interval (CI), 0.93-0.99] for asialotransferrin; 0.84 and 0.94 for the sum of asialo- + disialotransferrin (MRa, 0.94; 95% CI, 0.91-0.98); 0.79 and 0.94 for disialotransferrin (MRa, 0.89; 95% CI, 0.84-0.94); 0.62 and 0.53 for trisialotransferrin (MRa, 0.58; 95% CI, 0.49-0.68); 0.79 and 0.82 for a 3% %CDT; and 0.83 and 0.69 for a 2.6% cutoff (MRa, 0.87; 95% CI, 0.81-0.92). Current markers lack sensitivity (<0.65). Transferrins were not significantly correlated with serum enzymes and mean erythrocyte volume. CONCLUSIONS: CZE-isolated desialylated transferrin isoforms allowed differentiation between chronic alcohol abusers and teetotalers.

Adult↗
PubMed1992

Serotonin agonists increase transferrin levels via activation of 5-HT1C receptors in choroid plexus epithelium.

Choroid plexus epithelial cells are enriched in mRNA for proteins such as the iron carrier transferrin, which acts as a trophic factor in the brain. Choroid plexus epithelial cells also have a high density of 5-HT1C receptors linked to activation of the phosphoinositide (PI) hydrolysis second messenger system. The present studies show that the 5-HT1C/5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) potently increases PI hydrolysis and the levels of transferrin in primary cultures of rat choroid plexus epithelial cells. These effects are blocked by the 5-HT1C/5-HT2 receptor antagonists mesulergine and mianserin, but not by the 5-HT2 receptor-selective antagonist spiperone. Similarly, mesulergine and mianserin, but not spiperone, block the increases in transferrin levels and PI hydrolysis elicited by 5-carboxamidotryptamine (5-CT), a 5-HT1 receptor-selective agonist, and by serotonin. We conclude, therefore, that 5-HT1C receptor activation in the choroid plexus leads to an increase in the production of transferrin. By promoting transferrin synthesis in the choroid plexus, 5-HT may indirectly influence brain development and differentiation.

Amino Acid Isomerases↗
PubMed1992

Identification and quantification of transferrin receptors on trabecular cells.

The authors identified and quantified the receptor for transferrin on trabecular meshwork (TM) cells cultured from porcine eyes by using two receptor assays. Flow-cytometric analysis of TM cells that were incubated with a monoclonal antibody to the transferrin receptor revealed such receptors, which decreased in number as the cells passed from the pre- to the post-confluent phase. Quantitative characterization by radioligand binding of 125I-transferrin to trabecular cells followed by Scatchard analysis showed that pre-confluent cultures expressed 23,839 +/- 6746 high-affinity receptors/cell and post-confluent cultures expressed 5104 +/- 3639 receptors/cell. The expression of the receptor for transferrin reflects the index of mitotic activity and can be correlated with the proliferative state of the trabecular cells. Further characterization of the receptors for transferrin in the TM may open up the possibility of a pharmacologic approach that enhances the TM cell population, which is known to decrease with age and in glaucomatous states of the eye.

Animals↗
PubMed1992

Effect of ethylenediamine di-o-hydroxyphenylacetic acid and transferrin on the growth of some bacterial strains in vitro.

The ability of some bacterial strains to obtain iron from ethylenediamine di-o-hydroxyphenylacetic acid (EDDA) or iron-free transferrin, and accordingly grow in their presence, was studied. Growth of Staphylococcus aureus and Yersinia pseudotuberculosis was inhibited by EDDA or by iron-free transferrin. Growth of Streptococcus faecalis, however, was inhibited by iron-free transferrin, but not by EDDA. The other bacterial strains, i.e.; Escherichia coli, Salmonella typhimurium and Shigella dysenteriae were able to grow both in the presence of EDDA or iron-free transferrin. All of the above bacterial strains grow in the presence of iron-saturated transferrin which was not able to bind the iron of the medium and accordingly left the iron of the medium available to them.

Bacteria↗
PubMed2005

High Risk Coronary Heart Disease, Lipid Fractions and Transferrin Saturation among Hypertensive Nigerians.

BACKGROUND AND OBJECTIVES: Hypertension and hypercholesterolemia are well established independent risk factors of coronary heart disease. Moreover, drug treatment of hypertension also affects lipid metabolism. Recently, body iron status has also been implicated as a risk factor of coronary heart disease. It is not however clear whether hypertension or hypercholesterolaemia directly or indirectly influences body iron status. This study was therefore decided to look at the association between transferrin saturation and hypercholesterolaemia in adult hypertensive Nigerians with high risk Coronary Heart Disease (CHD) lipid fraction. MATERIALS AND METHODS: The study group comprises eighty two adult Nigerians who are known hypertensives with high risk coronary heart disease lipid fraction: that is the ratio of high density lipoprotein cholesterol to total cholesterol (HDL-C/TC) <= 0.13 while the control subjects comprises eighty adult Nigerians who are also known hypertensives with normal HDL-C/TC ratio (>=0.30). RESULTS: The total iron binding capacity is not significantly different between the two groups. Also, serum iron and transferrin saturation were not significantly different in the two groups. Transferrin saturation does not show appreciable correlation with total cholesterol, LDL-cholesterol, HDL-cholesterol and triglyceride levels in any of the two groups. CONCLUSION: The association between transferrin saturation and hypercholesterolaemia in hypertensive is not strong enough to possibly suggest that one influences the other. However, the influence of antihypertensive drugs on lipid metabolism cannot be ruled out in this study because subjects and controls were not selected based on type or class of medication. KEYWORDS: transferrin saturation, high risk CHD lipid fraction, hypertensives.

Cholesterol, HDL↗
PubMed1991

Trafficking of the epidermal growth factor receptor and transferrin in three hepatocytic endosomal fractions.

Hepatocytes rapidly internalize epidermal growth factor (EGF) and transferrin by receptor-mediated endocytosis. Both EGF and its receptor are thought to be targeted for destruction in lysosomes, leading to down-regulation of the receptor, whereas transferrin, after unloading iron within the cell, is thought to recycle to the cell surface bound to its receptor. Previously, we isolated three endosomal fractions from livers of estradiol-treated rats and examined their roles in cellular trafficking of low density lipoproteins (LDL) and the LDL receptor, which cycles constitutively (Belcher, J. D., Hamilton, R. L., Brady, S. E., Hornick, C. A., Jäckle, S., Schneider, W. J., and Havel, R. J., Proc. Natl. Acad. Sci. U. S. A. (1987) 84, 6785-6789). In the current study we have taken advantage of the distinct trafficking of the EGF receptor and transferrin to evaluate further the functions of these endosome fractions. Intravenous injection of a saturating amount of EGF into estradiol-treated rats induced internalization of a single population of EGF receptors, which rapidly accumulated in the endosome fraction of intermediate density ("compartment of uncoupling of receptor and ligand" (CURL)) and subsequently in the low density endosome fraction (multivesicular bodies (MVBs)). The high density endosome fraction, whose membranes contain a high concentration of recycling receptors (designated receptor-recycling compartment (RRC)), failed to accumulate EGF receptors after injection of EGF. In livers of rats not given exogenous EGF, EGF receptors were found in small but comparable concentrations in RRC, CURL, and MVB membranes, consistent with other evidence that targeting of the EGF receptor to lysosomes is mediated by ligand-induced phosphorylation. Transferrin also accumulated first in CURL and later in MVBs, but it also accumulated rapidly in the RRC fraction, consistent with the proposed function of this fraction in receptor recycling. Since transferrin is not degraded during its endocytic cycle, these observations indicate that apotransferrin and its receptor recycle from late endosomes (MVBs) located at the apical pole of hepatocytes, as well as from early endosomes near the sinusoidal pole.

Animals↗
PubMed1991

Interferon (IFN) alpha inhibits cell proliferation of UWOV2 cells without down-regulation of transferrin receptors.

It has been suggested that growth inhibition of cells by interferons may be mediated through interferon induced down-regulation of transferrin receptor expression. We describe a continuously growing cell line UWOV2 (pf) which expresses cell surface transferrin receptor but is able to grow in the absence of transferrin. This cell line is sensitive to the growth inhibitory effects of interferon alpha. Interferon alpha induced growth inhibition is not, however, accompanied by modulation of transferrin receptor expression suggesting that transferrin receptor modulation is not an essential component of the growth inhibitory effect of interferons.

Cell Division↗
PubMed1991

Use of indium-111-labeled transferrin to study plasma extravasation during endotoxin shock and the effects of the beta-2 agonist terbutaline.

Endotoxemia and sepsis are common causes of respiratory distress (ARDS), which is characterized by increased pulmonary vascular permeability to plasma proteins resulting in "noncardiac pulmonary edema." The aim of this series was to study the effects of the beta-2 receptor agonist, terbutaline, on plasma extravasation in multiple organs, in sheep exposed to endotoxin shock. A double isotope technique was used and the radioactivity was recorded in different organs (lungs, liver, spleen, kidneys, intestine) by a computerized gamma camera. Tc-99m-labeled erythrocytes were used as a marker for intravascular volume and In-111m-labeled transferrin for tracing extravascular plasma leakage. An organ-transferrin index (organ-TI) was calculated for each organ which corrects for changes in blood distribution. Fourteen sheep were anesthetized and ventilated. After stabilization (t = 0) all animals received E. coli endotoxin 10 micrograms/kg by IV infusion during 30 min. At t = 30, seven animals (group T) received IV infusion of terbutaline, 20 micrograms/kg/hr, during 4 h, while the other seven received normal saline and served as controls (group E). The endotoxin infusion caused an immediate and significant increase in the transferrin index in the lungs and in the liver in both groups. The transferrin index continued to rise in the control group towards the end of the experiment (t = 240), while in group T it reached a maximum 60 min after endotoxin. Four hours after endotoxin the transferrin index was significantly higher in the controls than in the terbutaline treated group, both in the lungs and in the liver (P less than 0.01). No significant changes were recorded in the kidneys or over the intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Selective modulation of the endocytic uptake of ricin and fluid phase markers without alteration in transferrin endocytosis.

Cytochalasin D was found to reduce the endocytosis of ricin and the fluid phase markers [14C]sucrose and Lucifer Yellow in Vero cells without reducing the uptake of transferrin. The number of coated pits at the plasma membrane was not affected by the treatment. Cytochalasin D also reduced the endocytosis of ricin in cells where uptake of transferrin from coated pits was blocked by low cytosolic pH. Colchicine had a similar effect as cytochalasin D. Both drugs inhibited the exocytosis of ricin from the cells, and they reduced the rate by which ricin intoxicated the cells. Cytochalasin D had essentially no effect on the ability of the cells to bind transferrin, whereas colchicine reduced the binding to some extent. Epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the endocytic uptake of ricin in A431 cells both under normal culture conditions and when the coated pit/coated vesicle pathway was blocked by acidification of the cytosol. In contrast, EGF and TPA had no stimulatory effect on the uptake of transferrin at normal cytoplasmic pH, and they did not abolish the ability of low cytoplasmic pH to inhibit endocytic uptake of transferrin. The results indicate that cytochalasin D and colchicine selectively inhibit endocytic uptake from non-clathrin-coated areas of the cell membrane whereas EGF and TPA stimulate it. The data support the view that there are different endocytic mechanisms, and they indicate that at least in some cell types the non-clathrin-coated endocytosis can be modulated.

Animals↗
PubMed1990

An investigation of placental transferrin processing: influence of N-ethylmaleimide.

The uptake and processing of 125I-59Fe labelled diferric transferrin was studied with the single bolus technique using the isolated guinea-pig placenta. Following preperfusion of the placenta with the SH-alkylating agent N-ethylmaleimide preferential iron uptake was inhibited and a total blockade of transplacental iron transfer was obtained. It can be shown by means of subcellular fractionation studies that neither transferrin binding nor endocytosis of the transferrin receptor complex were affected by N-ethylmaleimide. Additional experiments performed with microvillous membrane vesicles isolated from control placentas or from placentas preperfused with N-ethylmaleimide demonstrated that N-ethylmaleimide does not affect the affinity of the transferrin receptor for its ligand. The number of receptors per mg membrane protein remains unchanged as well. Ka = 1.4 x 10(8) M-1, n = 3.6 x 10(12). The results show that the blockade is located at the level of endosomal iron release. Since it is known that N-ethylmaleimide inhibits the endosomal proton pump, our results strongly suggest that the endocytotic pathway is a necessary route in transferrin mediated transplacental iron transfer.

Animals↗
PubMed1990

Regulation of ferritin and transferrin receptor mRNAs.

Iron regulates the synthesis of two proteins critical for iron metabolism, ferritin and the transferrin receptor, through novel mRNA/protein interactions. The mRNA regulatory sequence (iron-responsive element (IRE)) occurs in the 5'-untranslated region of all ferritin mRNAs and is repeated as five variations in the 3'-untranslated region of transferrin receptor mRNA. When iron is in excess, ferritin synthesis and iron storage increase. At the same time, transferrin receptor synthesis and iron uptake decrease. Location of the common IRE regulatory sequence in different noncoding regions of the two mRNAs may explain how iron can have opposite metabolic effects; when the IRE is in the 5'-untranslated region of ferritin mRNA, translation is enhanced by excess iron whereas the presence of the IREs in the 3'-untranslated region of the transferrin receptor mRNA leads to iron-dependent degradation. How and where iron actually acts is not yet known. A soluble 90-kDa regulatory protein which has been recently purified to homogeneity from liver and red cells specifically blocks translation of ferritin mRNA and binds IRE sequences but does not appear to be an iron-binding protein. The protein is the first specific eukaryotic mRNA regulator identified and confirms predictions 20 years old. Concerted regulation by iron of ferritin and transferrin receptor mRNAs may also define a more general strategy for using common mRNA sequences to coordinate the synthesis of metabolically related proteins.

Animals↗
PubMed1990

5-HT-induced transferrin production by choroid plexus epithelial cells in culture: role of 5-HT1c receptor.

Previous studies in our laboratory have demonstrated that serotonin (5-HT) elevates transferrin production by choroid plexus epithelial cells in primary culture in a time- and concentration-dependent fashion. The present study shows that 5-HT stimulates phosphoinositide hydrolysis in these cells and further demonstrates that the phosphoinositide hydrolysis response is mediated by the 5-HT1c receptor. To determine if the effect on transferrin is also mediated by the 5-HT1c receptor, the effects of 5-HT receptor agonists and antagonists were examined in choroid plexus epithelial cells in primary culture. The stereoisomers of lysergic acid diethylamide (LSD) and the piperazine derivative 6-chloro-2-[1-piperazinyl]-piperazine (MK-212) were evaluated as potential agonists. MK-212 and (+)LSD mimicked 5-HT, increasing transferrin levels to the same extent. The levorotary isomer, (-)LSD, had no effect. This agonist profile agrees with that previously found for 5-HT1c receptor-mediated phosphoinositide hydrolysis. Three antagonists with varying potencies to block 5-HT1c receptor-mediated phosphoinositide hydrolysis were examined: ritanserin, mianserin and spiperone. The results of these studies were less clear-cut. Neither mianserin nor ritanserin significantly reduced the effects of 5-HT on transferrin, even though they markedly reduced 5-HT-induced phosphoinositide hydrolysis. Consistent with its low potency at the 5-HT1c receptor, spiperone, a 5-HT2 and 5-HT1a antagonist, was a less effective antagonist of the phosphoinositide hydrolysis response than were ritanserin and mianserin. Spiperone also failed to block the effect of 5-HT on transferrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Human transferrin. Expression and iron modulation of chimeric genes in transgenic mice.

Transferrin (TF) is a plasma protein that transports and is regulated by iron. The aim of this study was to characterize human TF gene sequences that respond in vivo to cellular signals affecting expression in various tissues and to iron administration. Chimeric genes were constructed containing 152, 622, and 1152 base pairs (bp) of the human TF5'-flanking region with the coding region of a reporter gene, CAT (chloramphenicol acetyltransferase), and introduced into the germ line of mice. Transgenes containing TF 5'-flanking sequences to -152 bp were expressed poorly in all tissues examined. In contrast, transgenes containing TF sequences to -622 or -1152 bp were expressed at high levels in brain and liver, greater than or equal to 1000-fold higher than tissues such as heart and testes. Liver and brain are major sites of endogenous TF mRNA synthesis, but liver mRNA levels are 10-fold higher than brain. A significant diminution of CAT enzymatic activity in liver accompanied iron administration in both TF(0.67) and TF(1.2)CAT transgenic mice, mimicking the decrease of transferrin in humans following iron overload. Levels of endogenous plasma transferrin also decreased in iron-treated transgenic mice. Transgenic mouse lines carrying human TF chimeric genes will be useful models for analyzing the regulation of human transferrin by iron and for determining the molecular basis of transferrin regulation throughout mammalian development into the aging process.

Animals↗
PubMed1989

Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma.

The sugar chains of transferrin samples, purified from sera of patients with hepatocellular carcinoma and of healthy individuals, were released quantitatively as radioactive oligosaccharides by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Comparative study of their structure by serial lectin column chromatography, by Bio-Gel P-4 column chromatography, and by sequential exoglycosidase digestion revealed that prominently altered glycosylation is commonly found in the hepatoma transferrins, although they all contain two complex-type asparagine-linked sugar chains in one molecule like in the case of normal transferrins. The alteration is quite various, including the increase of highly branched sugar chains, of those with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc beta 1----groups in their outer chain moieties and of those with a fucosylated trimannosyl core. Many but not all of the hepatoma transferrin samples contained a small amount of a bisected biantennary sugar chain, which was not detected in the normal transferrin samples.

Biomarkers, Tumor↗
PubMed1989

Comparison of the electron spin echo envelope modulation (ESEEM) for human lactoferrin and transferrin complexes of copper(II) and vanadyl ion.

Copper(II) and vanadyl ions were bound to human milk lactoferrin or serum transferrin with carbonate or oxalate as the synergistic anion. Electron spin echo envelope modulation (ESEEM) due to nitrogen of a coordinated histidine imidazole was observed for both the copper and vanadyl complexes. For both metals, the modulation frequencies in the Fourier transforms of the data were similar for the two proteins and were weakly dependent on anion. When data in D2O/glycerol-d3 were compared with data in H2O/glycerol, the deep deuterium modulation indicated multiple exchangeable protons in the vicinity of the metals with at most one proton within about 2.9 A of the metal. The distribution of exchangeable protons around the metals as probed by ESEEM was the same, within experimental uncertainty, for the copper or vanadyl complexes with either carbonate or oxalate as the anion. When 13C-labeled oxalate was used as the synergistic anion, 13C-ESEEM was observed for both the copper and vanadyl complexes of lactoferrin and transferrin. The deeper 13C modulation for copper and vanadyl transferrin [13C]oxalate than for vanadyl transferrin [13C]carbonate suggests that both ends of the oxalate are bound to the metal in the transferrin and lactoferrin complexes.

Copper↗
PubMed1989

The clinical measurement of serum transferrin receptor.

Monoclonal antibody reagents were used to develop a sensitive enzyme-linked immunoassay for clinical measurement of circulating transferrin receptor. By using transferrin-bound receptor for the preparation of the immunologic reagents, we developed an assay that gives an identical dose-response curve with either free or transferrin-bound receptor. The mean concentration of circulating receptor in 82 normal male and female volunteers was 5.63 +/- 1.42 mg/L. The level was reduced significantly in patients with primary aplastic anemia and post-transplant aplasia (2.58 +/- 1.07 mg/L and 2.32 +/- 0.48 mg/L, respectively) and was sharply elevated in patients with hemolytic anemia and iron deficiency anemia (33.1 +/- 17 and 18.0 +/- 11.4 mg/L, respectively). Our assay values are approximately 20-fold higher than results published previously in a study that used an immunoradiometric assay. The disparity apparently relates to a difference in sensitivity of the latter assay for free and transferrin-bound receptor. Measurements of serum transferrin receptor provide a useful clinical index of either total or iron-deficiency erythropoiesis.

Adult↗
PubMed1989

Regulation of transferrin receptor in myeloid and monocytic differentiation of HL-60 leukemia cells.

Modulation of surface transferrin receptor activity has been associated with leukemia cell differentiation and proliferation. To examine the mechanisms involved in regulating this event, receptor protein and mRNA levels were measured in HL-60 promyelocytic leukemia cells induced to differentiate along the myelocytic and monocytic pathways. Transferrin receptor down-regulation which occurs during granulocytic differentiation by dimethyl sulfoxide, retinoic acid, or aclacinomycin A appears to be kinetically compatible with reduced biosynthesis resulting from reductions in the level of steady-state mRNA. In contrast, genetic modulation does not appear to mediate the initial receptor down-regulation seen during 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation. However, a reduction in levels of receptor message appears to contribute to the maintenance of diminished transferrin receptor activity in these 12-O-tetradecanoylphorbol-13-acetate-treated cells. A common feature of myelocytic and monocytic differentiating cells is the complete inhibition of cellular proliferation observed within 10 to 16 h following a four-fold reduction in surface transferrin receptor. We conclude that the early decline in transferrin receptor levels precludes its regulation as a consequence of the decrease in proliferation, but rather implicates its role in the programmed cessation of growth which is requisite for the terminal differentiation of these cells.

Aclarubicin↗
PubMed1986

The exchange of Fe3+ between pyrophosphate and transferrin. Probing the nature of an intermediate complex with stopped flow kinetics, rapid multimixing, and electron paramagnetic resonance spectroscopy.

A detailed study of the exchange of Fe3+ between pyrophosphate and human serum transferrin was undertaken to test the hypothesis of a generalized reaction route for exchange of Fe3+ between transferrin and chelators. The initial rate of Fe3+ transfer from pyrophosphate to apotransferrin-CO2-3 is highly sensitive to the pyrophosphate to iron ratio with a maximal rate being observed at a ratio of 3:1, consistent with the presence of slowly reactive polymeric species at ratios less than 3:1 as revealed by EPR and kinetic measurements. At a ratio of 4:1 the reaction is distinctly biphasic. The rapid first phase results in the formation of an intermediate postulated as a mixedligand complex of the type PPi-Fe3+-transferrin-CO2-3. The intermediate has a distinct EPR spectrum and an absorption spectrum similar to that of Fe3+-transferrin-CO2-3, but with a spectral maximum at 450 nm rather than 465 nm. The second phase principally arises from the slow reaction of polymeric iron-pyrophosphate with the apoprotein and has contributions from the breakdown of the intermediate formed in the first phase. The rate of formation of the intermediate shows a hyperbolic dependence on NaHCO3 and apotransferrin concentrations, the latter suggesting a rate-limiting labilization of Fe3+(PPi)3, perhaps to form species of the type Fe3+(PPi)2, prior to attack by apotransferrin-CO2-3. Multimixing stopped flow spectrophotometry was employed to test the chemical reactivity of the Fe3+ to reduction at various times during the first phase. Surprisingly, a diminution of reactivity of 1000-fold was noted after only 2% of the first phase was completed, indicating a fast initial reaction which is not observed by normal rapid flow spectrophotometry. This initial reaction may involve the binding of iron-pyrophosphate to allosteric sites on the protein. The kinetics of iron removal from Fe3+-transferrin-CO2-3 by PPi are consistent with a rate-limiting conformational change in the protein as proposed earlier.

Apoproteins↗