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Fe(III) uptake by the iron-delivery and iron-scavenging protein, hen ovotransferrin has been investigated in vitro between pH 6.5 and 9. In the absence of any ferric chelate, apo-ovotransferrin loses two protons with K1a = 50 +/- 1 nM and K2a = 4.0 +/- 0.1 nM. These acid-base equilibria are independent of the interaction of the protein with bicarbonate. The interaction with bicarbonate occurs with two different affinity constants, KC = 9.95 +/- 0.15 mM and KN = 110 +/- 10 mM. FeNAc3 exchanges its Fe(III) with the C-site of the protein in interaction with bicarbonate, direct rate constants k1 = 650 +/- 25 M-1 s-1, reverse rate constant k-1 = (6.0 +/- 0.1) x 10(3) M-1 s-1 and equilibrium constant K1 = 0.11 +/- 0.01. This iron-protein intermediate loses then a single proton, K3a = 3.50 +/- 0.35 nM, and undergoes a first change in conformation followed by a two or three proton loss, first order rate constant k2 = 0.30 +/- 0.01 s-1. This induces a new modification in conformation followed by the loss of one or two protons, first order rate constant k3 = (1.50 +/- 0.05) x 10(-2) s-1. These modifications in the monoferric protein conformation are essential for iron uptake by the N-site of the protein. In the last step, the monoferric and diferric proteins attain their final state of equilibrium in about 15,000 s. The overall mechanism of iron uptake by ovotransferrin is similar but not identical to those of serum transferrin and lactoferrin. The rates involved are, however, closer to lactoferrin than serum transferrin, whereas the affinities for Fe(III) are lower than those of serum transferrin and lactoferrin. Does this imply that the metabolic function transferrins is more related to kinetics than to thermodynamics?

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Iron release from ovotransferrin in acidic media (3 , pH , 6) occurs in at least six kinetic steps. The first is a very fast (# 5 ms) decarbonation of the iron-loaded protein. Iron release from both sites of the protein is controlled by what appear to be slow proton transfers. The N-site loses its iron first in two steps, the first occurring in the tenth of a second range with second order rate constant k 1 = (2.30^0.10) Â 10 4 m 21´s21 , first order rate constant k 21 = (1.40^0.10) s 21 and equilibrium constant K 1a = (60^6) mm. The second step occurs in the second range with a second order rate constant k 2 = (5.2^0.15) Â 10 3 m 21´s21 , first order rate constant k 22 = (0.2^0.02) s 21 and equilibrium constant K 2a = (39^5) mm. Iron is afterward lost from the C-site of the protein by two different pathways, one in the presence of a strong Fe(III) ligand such as citrate and the other in the presence of weak ligands such as formate or acetate. The first step, common to both paths, is a slow proton uptake which occurs in the tens of second range with a second order rate constant k 3 = (1.22^0.03) Â 10 3 m 21´s21 and equilibrium constant K 3a = (1.0^0.1) mm. In the presence of citrate, this step is followed by formation of an intermediate complex with monoferric ovotransferrin; stability constant K LC = (0.435^0.015) mm. This last step is rate-controlled by slow proton gain which occurs in the hundred second range with a second order rate constant k 4 = (1.05^0.05) Â 10 4 m 21´s21 , first order rate constant k 24 = (1.0^0.1) Â 10 22 s 21 and equilibrium constant K 4a = (0.95^0.15) mm. In the presence of a weak iron(III) ligand such as acetate or formate, formation of an intermediate complex is not detected and iron release is controlled by two final slow proton uptakes. The first occurs in the hundred to thousand second range, second order rate constant k 5 = (6.90^0.30) Â 10 6 m 21´s21 . The last step occurs in the thousand second range. Iron release by ovotransferrin is similar but not identical to that of serum-transferrin. It is slower and occurs at lower pH values. However, as seen for serum-transferrin, it seems to involve the protonation of the amino acid sidechains involved in iron co-ordination and perhaps those implicated in interdomain H-bonds. The observed proton transfers are, then, probably controlled by the change in conformation of the binding lobes from closed when iron-loaded to open in the apo-form.Keywords: transferrin; ovotransferrin; iron metabolism; stopped-flow.Transferrins constitute the most important iron regulation system in vertebrates and some invertebrates, such as worms and insects [1]. Soluble transferrins, such as serum-transferrin, lactoferrin and ovotransferrin, are glycoproteins consisting of a single chain of about 700 amino acids and 80 kDa. These proteins have very similar 3D structures and are all bilobal. Each lobe contains an iron complexation cleft, in which the metal is co-ordinated to the side-chains of four amino acid ligands and a synergistic carbonate or bicarbonate...

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Background: The acetylcholine activated inward rectifier potassium current (IKACh) was shown to be constitutively active in chronic atrial fibrillation. Its blockade has been proposed to be a possible antifibrillatory pharmacotherapy. We therefore tested the hypothesis that blocking IKACh with the bee venom peptide tertiapin, or the small molecule chloroquine, terminates chronic AF in the sheep heart. Methods and Results: We tested our hypothesis using biochemistry, electrophysiology and molecular modeling. In patch clamp, the IC50 of IKACh block by tertiapin, and chloroquine was 60 nM, and 710 nM respectively, while dofetilde, a currently used class III antiarrhythmic, did not block the current. On the other hand, dofetilide and chloroquine blocked the rapid delayed rectifier potassium current (IKr) with an IC50 of 50nM, and 2.3 microM respectively, while tertiapine had no effects. Furthermore, molecular modeling indicated that 1 chloroquine molecule blocks the intracellular ion permeation vestibule of the tetrameric Kir3.1, a molecular correlate of IKACh, by interacting with amino acids important for the channel’s rectification. Dofetilide did not interact with Kir3.1. In vitro fluorescence measurements of chloroquine titration into the purified Kir3.1 intracellular domain protein confirmed the computational results and indicated that chloroquine directly binds Kir3.1 with a stoichiometry of 1 chloroquine molecule per Kir3.1 tetramer. Finally, optical mapping of the chronically fibrillating sheep atria showed that while tertiapine, chloroquine, and dofetilide slowed down chronic AF and prolonged the action potential, only tertiapine and chloroquine (the IKACh blockers) restored normal sinus rhythm. Conclusions: IKACh could be a more powerful anti atrial fibrillation target compared to IKr. Pharmacological blockade of IKACh with a peptide, or a small molecule similar to chloroquine could terminate chronic AF and restore normal sinus rhythm.