Circularly polarized luminescence of Sm (III) and Eu (III) complexes with chiral ligand (R/S)‐BINAPO

Luminescent lanthanide (III) ions have been exploited for circularly polarized luminescence (CPL) for decades. However, very few of these studies have involved chiral samarium (III) complexes. Complexes are prepared by mixing axial chiral ligands (R/S))‐2,2’‐bis(diphenylphosphoryl)‐1,1′‐binaphthyl (BINAPO) with europium and samarium Tris (trifluoromethane sulfonate) (Eu (OTf)₃ and Sm (OTf)₃). Luminescence‐based titration shows that the complex formed is Ln((R/S)‐BINAPO)₂(OTf)₃, where Ln = Eu or Sm. The CPL spectra are reported for Eu((R/S)‐BINAPO)₂(OTf)₃ and Sm((R/S)‐BINAPO)₂(OTf)₃. The sign of the dissymmetry factors, g_em, was dependent upon the chirality of the BINAPO ligand, and the magnitudes were relatively large. Of all of the complexes in this study, Sm((S)‐BINAPO)₂(OTf)₃ has the largest g_em = 0.272, which is one of the largest recorded for a chiral Sm³⁺ complex. A theoretical three‐dimensional structural model of the complex that is consistent with the experimental observations is developed and refined. This report also shows that (R/S)‐BINAPO are the only reported ligands where g_em (Sm³⁺) > g_em (Eu³⁺).     

A study of n-pentenylorthoesters having manno, gluco and galacto configurations in regioselective glycosylations

Kochetkov's extensive investigations of glycosyl orthoester and their analogs as glycosyl donors revealed that the alkyl derivatives were plagued by competition between the departing alcohol and the incoming acceptor. n-Pentenyl orthoesters (NPOEs) obviate competition by sequestering the departing pentenyl alcohol as a 2-halomethyl tetrahydrofuran. Exquisitely regioselective glycosidations of diol acceptors can be carried out with NPOEs triggered specifically with Yb(OTf)(3)/NIS. However with Sc(OTf)(3), double glycosidation is the major reaction. manno, gluco and galacto NPOEs have been investigated. The latter two, which require a different experimental procedure for the manno counterpart, also give an excellent regioselectivity with Yb(OTf)(3), but the yields are much lower than with manno.     

Glycosylation of sialyl acetates with a novel catalyst combination: bismuth triflate and BF3.OEt2 system

A combined system of bismuth triflate [Bi(OTf)(3)] and boron trifluoride etherate (BF(3).OEt(2)) in dichloromethane is an efficient promoter for the glycosylation of N-acetylneuraminic acid derivatives. The co-existence of two acid catalysts such as Bi(OTf)(3)-BF(3).OEt(2) or Bi(OTf)(3)-PPA is confirmed to be essential for obtaining high yields of glycosylation products with p-nitrobenzyl alcohol, which also turned to be superior to those reported previously.     

轻稀土离子对钙调蛋白激活的磷酸二酯酶活力作用的影响

研究了轻稀土离子（Ｌｎ３＋）对钙调蛋白（ＣａＭ）调控的磷酸二酯酶（ＰＤＥ）活力的影响。结果表明,在无Ｃａ２＋的ＣａＭ（Ａｐｏ—ＣａＭ）体系中,由ＣａＭ调节的ＰＤＥ的活力随Ｌｎ３＋浓度的变化曲线是双相效应,即在高浓度时,Ｌｎ３＋具有抑制ＣａＭ调节ＰＤＥ活力的能力;低浓度的Ｌｎ３＋可以提高ＣａＭ调节ＰＤＥ活力的能力。在Ｃａ２＋４－ＣａＭ－ＰＤＥ体系中,高浓度的Ｌｎ３＋的加入能抑制ＣｈＭ调节ＰＤＥ活力的能力,其抑制程度因其离子不同而异。ＣａＭ的两类拮抗剂ＪｕＡ（非竞争性抑制剂）和ＴＦＰ（竞争性抑制剂）都能抑制ＣａＭ－Ｌｎ３＋－ＰＤＥ系统的活性。最后对Ｌｎ３＋和ＣａＭ相互作用的分子机制进行了初步的讨论。     

稀土离子对CaM及Ca2+-Mg2+-ATPase活力及CD研究

研究了稀土离子（Ｌｎ３＋）对钙调蛋白（ＣａＭ）调控的Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的活力影响。结果表明,在ＣａＭ和Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的体系中,一些Ｌｎ３＋（Ｌａ３＋、Ｇｄ３＋）对由ＣａＭ调节的Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的活力影响呈现双相效应,即Ｌｎ３＋在低浓度时,能提高激活Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的水解活力;在高浓度时,则抑制ＣａＭ调节Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ活力的能力;少数Ｌｎ３＋（Ｓｍ３＋）仅表现出抑制效应。在无ＣａＭ的Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ体系中,高浓度的Ｌｎ３＋抑制Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的基础活力。结合圆二色（ＣＤ）谱信息对Ｌｎ３＋和ＣａＭ相互作用的分子机制进行了初步的探讨。     

苯丙氨酸稀土配合物与磷脂脂质体相互作用的研究

用ＤＳＣ、ＦＴ－ＩＲ和Ｒａｍａｎ光谱法研究了苯丙氨酸（Ｐｈｅ）及其稀土配合物Ｌｎ（Ｐｈｅ）Ｃｌ３．６Ｈ２Ｏ（Ｌｎ＝Ｓｍ．ｌａ．Ｇａ．Ｙｂ）对ＤＰＰＣ热致性的影响,并从分子水平上探讨了它们与ＤＰＰＣ相互作用的机理。结果表明：Ｐｈｅ对ＤＰＰＣ从凝胶态到液晶态的相变温度Ｔｍ影响不大,但磷脂酰链的构象有序度降低;Ｌｎ（Ｐｈｅ）Ｃｌ３．６Ｈ２Ｏ像Ｌｎ３＋一样,使ＤＰＰＣ的Ｔｍ显著升高,但前者的升高程度小于后者,同时磷脂链的构象有序度升高。上述ＤＰＰＣ脂质体二元体系的铸膜ＦＴＩＲ结果与ＤＰＰＣ脂质体基本上相同,配合物Ｌｎ（Ｐｈｅ）Ｃｌ３．６Ｈ２Ｏ的配合键是离子型的．     

Dehydration of fructose to 5-hydroxymethylfurfural by rare earth metal trifluoromethanesulfonates in organic solvents

The catalytic dehydration of fructose to 5-hydroxymethylfurfural (HMF) was investigated by using various rare earth metal trifluoromethanesulfonates, that is, Yb(OTf)₃, Sc(OTf)₃, Ho(OTf)₃, Sm(OTf)₃, Nd(OTf)₃ as catalysts in DMSO. It is found that the catalytic activity increases with decreasing ionic radius of rare earth metal cations. Among the examined catalysts, Sc(OTf)₃ exhibits the highest catalytic activity. Fructose conversion of 100% and a HMF yield of 83.3% are obtained at 120 °C after 2 h by using Sc(OTf)₃ as the catalyst. Moreover, the catalytic dehydration of fructose was also carried out in different solvents, for example, DMA, 1,4-dioxane, and a mixture of PEG-400 and water. The results show that among the solvents DMSO is the most efficient in promoting the dehydration of fructose to HMF, and no rehydration byproducts such as levulinic acid and formic acid are detected.     

Expression of laminins 1 and 10 in carcinoma cells and comparison of their roles in cell adhesion

The expression pattern of laminin (Ln) alpha1 chain has been a controversial topic due to discrepancies between mRNA and protein studies. Recently it was reported that the monoclonal antibody 4C7, previously thought to recognize Ln alpha1 chain, actually detects Ln alpha5 chain. This finding makes it necessary to reestimate the role of Ln alpha1 chain and to compare the expression and functions of Ln alpha1 and alpha5 chains. We studied the expression of Ln alpha1 and alpha5 chains and production of Ln-1 and Ln-10 in cultured human carcinoma cells. Ln alpha1 chain mRNA was detected in JAR choriocarcinoma cells and in all four renal cell carcinoma cell lines studied. In contrast, pancreatic, colon, and lung alveolar carcinoma cell lines did not express or produce Ln alpha1 chain, suggesting that Ln-1 (alpha1 beta1 gamma1) is produced only by certain carcinoma cells. Ln alpha5 chain mRNA was expressed in all carcinoma cells, but was not incorporated into extracellular matrix in vitro, as shown with JAR cells. Immunoprecipitation of metabolically labeled cells showed that cells expressing Ln alpha1 mRNA also produced 400-kDa Ln alpha1 chain, whereas all cells produced 380-kDa Ln alpha5 chain. Adhesion to Ln-1 was inhibited by a functionally blocking antibody against alpha6-integrin subunit, whereas adhesion to Ln-10 was inhibited by an antibody against alpha6-integrin in JAR cells and by an antibody against alpha3-integrin in PANC-1 cells. The results suggest that Ln-10 is a ubiquitously expressed Ln isoform in carcinoma cells, and the mechanism of adhesion to Ln-10 is cell-type specific.     

Lanthanide ions induce hydrolysis of hemoglobin-bound 2,3-diphosphoglycerate (2,3-DPG), conformational changes of globin and bidirectional changes of 2,3-DPG-hemoglobin's oxygen affinity

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln(3+) binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln(3+) binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln(3+) binding sites for Hb can be classified into three categories: i.e. positive cooperative sites (N(I)), non-cooperative strong sites (N(S)) and non-cooperative weak sites (N(W)) with binding constants in decreasing order: K(I)>K(S)>K(W). The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln(3+) concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln(3+) binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln(3+) concentration. At the range of [Ln(3+)]/[Hb]<2, the marked increase of oxygen affinity (P(50) decrease) with the Ln(3+) concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln(3+) concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln(3+) binding. This was indicated by the changes in secondary structure characterized by the decrease of alpha-helix content.     

Epitope mapping of function‐blocking monoclonal antibody CM6 suggests a “weak” integrin binding site on the laminin‐332 LG2 domain

Laminin‐332 (Ln‐332) is an extracellular matrix molecule that regulates cell adhesion, spreading, and migration by interaction with cell surface receptors such as α3β1 and α6β4. Previously, we developed a function‐blocking monoclonal antibody against rat Ln‐332, CM6, which blocks hemidesmosome assembly induced by Ln‐332‐α6β4 interactions. However, the location of its epitope on Ln‐332 has remained unclear. In this study, we show that the CM6 epitope is located on the laminin G‐like (LG)2 module of the Ln‐332 α3 chain. To specify the residues involved in this epitope, we produced a series of GST‐fused α3 LG2 mutant proteins in which rat‐specific acids were replaced with human acids by a site‐directed mutagenesis strategy. CM6 reactivity against these proteins showed that CM6 binds to the ¹⁰⁸⁹NERSVR¹⁰⁹⁴ sequence of rat Ln‐332 LG2 module. In a structural model, this sequence maps to an LG2 loop sequence that is exposed to solvent according to predictions, consistent with its accessibility to antibody. CM6 inhibits integrin‐dependent cell adhesion on Ln‐332 and inhibits cell spreading on both Ln‐332 and recombinant LG2 (rLG2; but not rLG3), suggesting the presence of an α3β1 binding site on LG2. However, we were unable to show that rLG2 supports adhesion in standard assays, suggesting that LG2 may contain a “weak” integrin binding site, only detectable in spreading assays that do not require washes. These results, together with our previous findings, indicate that binding sites for α3β1 and α6β4 are closely spaced in the Ln‐332 LG domains where they regulate alternative cell functions, namely adhesion/migration or hemidesmosome anchoring. J. Cell. Physiol. 223:541–548, 2010. © 2010 Wiley‐Liss, Inc.     

Indium triflate catalyzed peracetylation of carbohydrates

Peracetylation is a very common protection strategy that is widely implemented in carbohydrate synthesis. Here, a method for the peracetylation of carbohydrates using catalytic In(OTf)(3) in neat acetic anhydride is reported. In(OTf)(3) has low toxicity and is mild and water tolerant, and the reactions are high yielding and efficient. Details regarding the scope and mechanism of the reaction are briefly discussed.     

Microstructures,interactions and dynamics properties studies of N-methyldiethanolamine + guanidinium triflate ionic liquid + water tertiary system at the standard temperature

Molecular dynamics simulations with an all-atom force field have been carried out in order to understand the phase equilibrium behaviour of ternary aqueous mixtures containing guanidinium triflate ionic liquid [gua][OTf] and water mixed with N-methyldiethanolamine (MDEA) in different function composition at the standard temperature of 298.15 K. A very good numerical agreement has been obtained for the prediction of the mixture densities. The analysis of structural and dynamic properties showed that the molecular level of ternary mixtures is slightly affected by the presence of MDEA and [gua][OTf] molar fractions. For MDEA–water interactions in [gua][OTf] media, we found that MDEA prefers to be surrounded by water molecules rather than by MDEA molecules even at a high MDEA molar fraction. While for [gua][OTf]–water interaction in MDEA media, as [gua][OTf] molar fraction increases, water molecules replace counterions in the coordination shell of both ions, thus weakening their interaction. On the other hand, for MDEA–[gua][OTf] interactions in water media, we have found that as the molar fraction of [gua][OTf] increases, a sulfonate group from anion appears to have a stronger association by making hydrogen bonding with MDEA molecules. The chemical process using ionic liquids (ILs) as solvents is commonly limited by their high viscosity. Based on their physical properties such as viscosities, these ternary solvents can be applied in natural gas industry, such as removing carbon dioxide using aqueous MDEA and IL at high pressure.     

A Biologically Active Sequence of the Laminin ��2 Large Globular 1 Domain Promotes Cell Adhesion through Syndecan-1 by Inducing Phosphorylation and Membrane Localization of Protein Kinase C��

Laminin-2 promotes basement membrane assembly and peripheral myelinogenesis; however, a receptor-binding motif within laminin-2 and the downstream signaling pathways for motif-mediated cell adhesion have not been fully established. The human laminin-2 α2 chain cDNAs cloned from human keratinocytes and fibroblasts correspond to the laminin α2 chain variant sequence from the human brain. Individually expressed recombinant large globular (LG) 1 protein promotes cell adhesion and has heparin binding activities. Studies with synthetic peptides delineate the DLTIDDSYWYRI motif (Ln2-P3) within the LG1 as a major site for both heparin and cell binding. Cell adhesion to LG1 and Ln2-P3 is inhibited by treatment of heparitinase I and chondroitinase ABC. Syndecan-1 from PC12 cells binds to LG1 and Ln2-P3 and colocalizes with both molecules. Suppression of syndecan-1 with RNA interference inhibits cell adhesion to LG1 and Ln2-P3. The binding of syndecan-1 with LG1 and Ln2-P3 induces the recruitment of protein kinase Cδ (PKCδ) into the membrane and stimulates its tyrosine phosphorylation. A decrease in PKCδ activity significantly reduces cell adhesion to LG1 and Ln2-P3. Taken together, these results indicate that the Ln2-P3 motif and LG1 domain, containing the motif, within the human laminin-2 α2 chain are major ligands for syndecan-1, which mediates cell adhesion through the PKCδ signaling pathway.     

Binding and iron delivering of monoferric ovotransferrins to chick-embryo red blood cells (CERBC)

1. Both monoferric forms of OTf, each of about 80 kDa, bound to CERBC enough tightly, but at a lesser extent with respect to Fe2OTf with a Bmax in the order: 59Fe2OTf greater than OTf59FeC much greater than 59FeNOTf. 2. Fe2OTf competed, in equimolar ratio, with 59FeNOTf or OTf59FeC, lowering the Bmax value; a 10-fold molar excess of Fe2OTf almost abolished the binding of both labelled monoferric forms. Apo-OTf did not compete with the monoferric forms for binding to CERBC. Iron-saturated N- or C-terminal OTf half-molecules, each of about 40 kDa, were unable to displace the monoferric form. By contrast, the mixture of both half-molecules gave results very similar to Fe2OTf. 59FeNOTf and OTf59FeC were displaced by a molar excess of both unlabelled monoferric forms. 3. Uptake experiments showed that both monoferric forms of OTf were less effective in delivering iron to CERBC with respect to the diferric form, but, nevertheless, there was still an appreciable iron uptake which paralleled the binding behaviour, being the C-form slightly more efficient than the N-form.     

Dual actions of lanthanides on ACTH-inhibited leak K(+) channels

Bovine adrenal zona fasciculata cells express background K(+) channels (I(AC) channels) whose activity is potently inhibited by ACTH. In whole cell patch clamp recordings, it was discovered that the trivalent lanthanides (Ln(3+)s) lanthanum and ytterbium interact with two binding sites to modulate K(+) flow through these channels. Despite large differences in ionic radii, these Ln(3+)s inhibited I(AC) channels half-maximally with IC(50) values near 50 microM. In addition, these Ln(3+)s blocked and reversed ACTH-mediated inhibition of I(AC) K(+) channels at similar concentrations. The Ln(3+)s did not alter inhibition of I(AC) by angiotensin II or cAMP. Ln(3+)-induced uncoupling of ACTH receptor activation from I(AC) inhibition was prevented by raising the external Ca(2+) concentration from 2 to 10 mM. The divalent cation Ni(2+) (500 microM) also blocked ACTH-dependent inhibition of I(AC) through a Ca(2+)-sensitive mechanism. The results are consistent with a model in which Ln(3+)s produce opposing actions on I(AC) K(+) currents through two separate binding sites. In addition to directly inhibiting I(AC), Ln(3+)s (and Ni(2+)) bind with high affinity to a Ca(2+)-selective site associated with the ACTH receptor. By displacing Ca(2+) from this site, Ln(3+)s prevent ACTH from binding and accelerate its dissociation. These results identify Ln(3+)s as a relatively potent group of noncompetitive ACTH receptor antagonists. Allosteric actions of trivalent and divalent metal cations on hormone binding, mediated through Ca(2+)-specific sites, may be common to a variety of peptide hormone receptors.     

Quantitative determination of the conformation of ATP in aqueous solution using the lanthanide cations as nuclear-magnetic-resonance probes.

Chemical shift perturbations of the eight 1H resonances and of the three 31P resonances in the nuclear magnetic resonance spectra of ATP in 2H2O, pH 6.0, have been induced by specifically bound lanthanide cations Ln3+ (Ln = Pr, Nd, Eu, Yb). After separation of contact (through bond) perturbations the resultant through-space shifts, which are found to have axial symmetry, are used in an analysis of the conformation of the Ln3+ -ATP complex. A computer program was used to search for the conformations of the molecule which fit the nuclear magnetic resonance data. The "best" solutions obtained represent a small closely interrelated family of conformations. Effects of the cation Gd3+ on the longitudinal relaxation rates of five of the protons of ATP were also measured and used to confirm the conformational family. One of these conformations corresponds closely to one of the crystal structure forms, with an anti arrangement of the base-ribose unit and and a right-hand helical phosphate chain folded towards the adenine part of the molecule. The lanthanide ion binds predominantly to the beta and gamma phosphates and does not interact with the purine ring, these two centres being separated by at least one water molecule.     

Influence of lactose-citrate co-metabolism on the differences of growth and energetics in Leuconostoc lactis, Leuconostoc mesenteroides ssp. mesenteroides and Leuconostoc mesenteroides ssp. cremoris

The biodiversity of growth and energetics in Leuconostoc sp. has been studied in MRS lactose medium with and without citrate. On lactose alone, Ln. lactis has a growth rate double that of Ln. cremoris and Ln. mesenteroides. The pH is a more critical parameter for Ln. mesenteroides than for Ln. lactis or Ln. cremoris; without pH control Ln. mesenteroides is unable to acidify the medium under pH 4.5, while with pH control and as a consequence of a high Y(ATP) its growth is greater than Ln. lactis and Ln. cremoris. In general, lactose-citrate co-metabolism increases the growth rate, the biomass synthesis, the lactose utilisation ratio, and the production of lactate and acetate from lactose catabolism. The combined effect of the pH and the co-metabolism lactose-citrate on the two components of the proton motive force (deltap = deltapsi - ZdeltapH) has been studied using resting-cell experiments. At neutral pH deltap is nearly entirely due to the deltapsi, whereas at acidic pH the deltapH is the major component. On lactose alone, strains have a different aptitude to regulate their intracellular pH value, for Ln. mesenteroides it drastically decreases at acidic pH values (pH, = 5.2 for pH 4), while for Ln. lactis and Ln. cremoris it remains above pH 6. Lactose-citrate co-metabolism allows a better control of pH homeostasis in Ln. mesenteroides, consequently the pHi becomes homogeneous between the three strains studied, for pH 4 it is in an interval of 0.3 pH unit (from pHi = 6.4 to pHi = 6.7). In this metabolic state, and as a consequence of the variation in deltapH, and to some extent in the deltapsi, the difference of deltap between the three strains is restricted to an interval of 20 mV.     

Antimicrobial study on trivalent lighter rare-earth complexes of 2-pyrazinecarboxylate with hydrazinium cation

The hydrazinium lanthanide metal complexes of 2-pyrazinecarboxylic acid (HpyzCOO) of the formulae (N₂H₅)₂[Ln(pyzCOO)₅]·2H₂O, where Ln=La or Ce and (N₂H₅)₃[Ln(pyzCOO)₄(H₂O)]·2NO₃, where Ln=Pr, Nd, Sm or Dy have been synthesized by the addition of an aqueous solution of the corresponding metal nitrate hydrates to an aqueous mixture of the respective carboxylic acids and hydrazine hydrate. The in vitro antibacterial screening of the free acid and its metal complexes has been carried out against Escherichia coli, Salmonella typhi and Vibrio cholerae. Antifungal activities of all the synthesized compounds were screened for in vitro growth inhibitory activity against Aspergillus fumigatus and Aspergillus niger by using the disc diffusion method. The antimicrobial activities of the prepared metal complexes show more promising activity than the corresponding free acid, its hydrazinium salts, and the standard control antibiotics, Co-trimoxazole and Carbendazim.     

Heterobinuclear Zn‐Ln (Ln = La,Nd, Eu,Gd, Tb,Er and Yb) complexes based on asymmetric Schiff‐base ligand: synthesis,characterization and photophysical properties

With a novel asymmetric Schiff‐base zinc complex ZnL (H₂L = N‐(3‐methoxysalicylidene)‐N′‐(5‐bromo‐3‐methoxysalicylidene)phenylene‐1,2‐diamine), obtained from phenylene‐1,2‐diamine, 3‐methoxysalicylaldehyde and 5‐bromo‐3‐methoxysalicylaldehyde, as the precursor, a series of heterobinuclear Zn‐Ln complexes [ZnLnL(NO₃)₃(CH₃CN)] (Ln = La, 1; Ln = Nd, 2; Ln = Eu, 3; Ln = Gd, 4; Ln = Tb, 5; Ln = Er, 6; Ln = Yb, 7) were synthesized by the further reaction with Ln(NO₃)₃·6H₂O, and characterized by Fourier transform‐infrared, fast atom bombardment mass spectroscopy and elemental analysis. Photophysical studies of these complexes show that the strong and characteristic near‐infrared luminescence of Nd³⁺, Yb³⁺and Er³⁺ with emissive lifetimes in the microsecond range has been sensitized from the excited state of the asymmetric Schiff‐base ligand due to effective intramolecular energy transfer; the other complexes do not show characteristic emission due to the energy gap between the chromophore and lanthanide ions. Copyright © 2012 John Wiley & Sons, Ltd.     

Reversible association of half-molecules of ovotransferrin in solution. Basis of co-operative binding to reticulocytes.

In the present paper, gel-filtration studies of diferric-ovotransferrin (Fe2OTf), the individual half-molecules of ovotransferrin (OTf) and equimolar mixtures of half-molecules have been interpreted according to the Gilbert theory as developed by Ackers & Thompson [(1965) Proc. Natl. Acad. Sci. U.S.A. 53, 342-349]. The data indicate that the half-molecules associate reversibly in solution and allow determination of a dissociation constant, Kd' = 8.0 (+/- 2.7) microM. Equilibrium binding studies have been performed using NH4Cl to block removal of iron from equimolar differentially iodine-labelled half-molecules (125I and 131I), in order to evaluate the binding of each to chick-embryo red blood cells under identical conditions. The amount of associated half-molecules over a range of concentrations has been calculated using the constant derived from the gel-filtration experiments described above. A computerized non-linear least-squares regression analysis of the data leads to determination of Kd* (the apparent dissociation constant for the interaction between OTf or half-molecules and the transferrin (Tf) receptors of chick-embryo red blood cells) and Bmax (binding at infinite free-ligand concentration) for the half-molecules similar to those found for Fe2OTf. Recent reports confirm that the two iron-binding domains of both OTf and human lactotransferrin associate non-covalently in solution. Our work shows that the isolated half-molecules of OTf are able to reassociate in solution and that this reassociation has functional significance by allowing the complex to be recognized by the Tf receptor.