Identification of important residues in metal-chelate recognition by monoclonal antibodies

The molecular characterization of antibodies directed against metal-chelate complexes will provide important insights into the design and development of radiotherapeutic and radioimaging reagents. In this study, two monoclonal antibodies directed against different metal-chelate complexes were expressed as recombinant Fab fragments. Covalent modification and site-directed mutagenesis were employed to ascertain those residues important in antigen recognition. Antibody 5B2 was raised to a Pb(II)-loaded isothiocyanatobenzyl-diethylenetriamine pentaacetic acid (DTPA)-protein conjugate. The native antibody bound to complexes of Pb(II)-p-aminobenzyl-DTPA with an affinity of 4.6 x 10(-9) M. A monovalent Fab fragment prepared from the native protein and a bivalent recombinant fragment exhibited comparable affinities for the same Pb(II)-chelate complex, approximately 6-fold lower than that of the intact antibody. Covalent modification and molecular modeling predicted that Lys(58) in the heavy chain contacted the Pb(II)-chelate ligand. Mutational analysis supported a role for Lys(58) in ion pair or hydrogen bond formation with the carboxylate groups on the chelate. Antibody E5 was directed toward an isothiocyanatobenzyl-ethylenediamine tetraacetic acid (EDTA)-protein conjugate loaded with ionic Cd(II). The native immunoglobulin recognized Cd(II)-p-aminobenzyl-EDTA with an affinity of 8.2 x 10(-12) M. A proteolytically derived fragment and a bivalent recombinant fragment bound to the same Cd(II)-chelate complex with affinities that were comparable to that of the native antibody. Homology modeling and mutagenesis identified three residues (Trp(52) and His(96) in the heavy chain and Arg(96) in the light chain) that were important for Cd(II)-chelate recognition. His(96) likely mediates a direct ligation to the Cd(II) ion and Trp(52) appears to be involved in hydrophobic stacking with the benzyl moiety of the chelator. Arg(96) appeared to mediate an electrostatic or hydrogen bond to the chelate portion of the complex. These studies demonstrate that antibody recognition of metal-chelate haptens occurs through a limited number of molecular contacts and that these molecular interactions involve both direct ligation between the antibody and the metal ion and interactions between the antibody and the chelator.     

Allosteric binding properties of a monoclonal antibody and its Fab fragment

Detailed equilibrium binding studies were conducted on a monoclonal antibody directed against Pb(II) complexed with a protein conjugate of diethylenetriaminepentaacetic acid (DTPA). Binding curves obtained with DTPA and a cyclohexyl derivative of DTPA in the presence and absence of metal ions were consistent with the anticipated one-site homogeneous binding model. Binding curves obtained with aminobenzyl-DTPA or its complexes with Ca(II), Sr(II), and Ba(II) were highly sigmoidal, characterized by Hill coefficients of 2.3-6.5. Binding curves obtained with the Pb(II) and In(III) complexes of aminobenzyl-DTPA were hyperbolic, but in each case the apparent affinity of the antibody for the chelator-metal complex was higher in the presence of excess chelator than it was in the presence of excess metal ion. In the presence of excess chelator, the equilibrium dissociation constant for the binding of aminobenzyl-DTPA-Pb(II) to the antibody was 9.5 x 10(-)(10) M. Binding curves obtained with the Hg(II) and Cd(II) complexes of aminobenzyl-DTPA were biphasic, indicative of negative cooperativity. Further binding studies demonstrated that aminobenzyl-DTPA-Hg(II) opposed the binding of additional chelator-metal complexes to the antibody more strongly than did aminobenzyl-DTPA-Cd(II). The Fab fragment differed from the intact antibody only in that the apparent affinity of the Fab was generally lower for a given chelator-metal complex. These data are interpreted in terms of a model in which (i) aminobenzyl-DTPA and its complexes bind both to the antigen binding site and to multiple charged sites on the surface of the compact immunoglobulin; and (ii) the bound, highly charged ligands interact in a complicated fashion through the apolar core of the folded antibody.     

Monoclonal antibodies that recognize minimal differences in the three-dimensional structures of metal-chelate complexes

Immunization of BALB/c mice with a cadmium-chelate-protein conjugate resulted in the isolation of two hybridoma cell lines (A4 and E5) that synthesized antibodies with different variable regions, but similar metal-chelate affinity. The ability of these two monoclonal antibodies to interact with 12 different metal-chelate complexes was studied using the KinExA 3000 immunoassay instrument. The two antibodies showed the highest affinity for cadmium and mercury complexes of ethylenediamine N,N,N',N'-tetraacetic acid (EDTA). The E5 antibody bound to EDTA complexes of cadmium and mercury with equilibrium dissociation constants (K(d)) of 1.62 x 10(-)(9) M and 3.64 x 10(-)(9) M, respectively. The corresponding values for the A4 antibody were 14.7 x 10(-)(9) M and 3.56 x 10(-)(9) M. Addition of a cyclohexyl ring to the EDTA backbone increased the affinity of E5 for the metal-chelate haptens, while decreasing the binding of A4 to the same haptens. Based on available crystal structures, molecular models were constructed for five different divalent metal-chelate complexes. The models were compared to determine structural features of the haptens that may influence antibody recognition. Difference distance matrixes were used to identify areas of the metal-chelate haptens that differed in three-dimensional space. Antibody affinity correlated well with the extent of total structural difference for these metal-EDTA complexes.     

Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone

Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to choose an approriate site for incorporating chelators such as DTPA. Otherwise, the bulky size of the chelator, depending on the site of incorporation, can affect yield, solubility, structure and pharmacological profile of the peptide.     

Antibody-based sensors for heavy metal ions

Competitive immunoassays for Cd(II), Co(II), Pb(II) and U(VI) were developed using identical reagents in two different assay formats, a competitive microwell format and an immunosensor format with the KinExA™ 3000. Four different monoclonal antibodies specific for complexes of EDTA–Cd(II), DTPA–Co(II), 2,9-dicarboxyl-1,10-phenanthroline–U(VI), or cyclohexyl–DTPA–Pb(II) were incubated with the appropriate soluble metal–chelate complex. In the microwell assay format, the immobilized version of the metal–chelate complex was present simultaneously in the assay mixture. In the KinExA format, the antibody was allowed to pre-equilibrate with the soluble metal-chelate complex, then the incubation mixture was rapidly passed through a microcolumn containing the immobilized metal-chelate complex. In all four assays, the KinExA format yielded an assay with 10–1000-fold greater sensitivity. The enhanced sensitivity of the KinExA format is most likely due to the differences in the affinity of the monoclonal antibodies for the soluble versus the immobilized metal–chelate complex. The KinExA 3000 instrument and the Cd(II)-specific antibody were used to construct a prototype assay that could correctly assess the concentration of cadmium spiked into a groundwater sample. Mean analytical recovery of added Cd(II) was 114.25±11.37%. The precision of the assay was satisfactory; coefficients of variation were 0.81–7.77% and 3.62–14.16% for within run and between run precision, respectively.     

Solid-phase synthesis of europium-labeled human INSL3 as a novel probe for the study of ligand-receptor interactions

An efficient solid-phase synthesis protocol has been developed which, together with regioselective sequential formation of the three disulfide bonds, enabled the preparation of specifically monolanthanide (europium)-labeled human insulin-like peptide 3 (INSL3) for the study of its interaction with its G-protein-coupled receptor, RXFP2, via time-resolved fluorometry. A commercially available chelator, diethylene triamine pentaacetic acid (DTPA), was coupled to the N-terminus of the INSL3 A-chain on the solid phase, and then a coordination complex between europium ion and DTPA was formed using EuCl 3 to protect the chelator from production of an unidentified adduct during subsequent combination of the A- and B-chains. The labeled peptide was purified in high yield using high-performance liquid chromatography with nearly neutral pH buffers to prevent the liberation of Eu (3+) from the chelator. Using time-resolved fluorometry, saturation binding assays were undertaken to determine the binding affinity (p K d) of labeled INSL3 for RXFP2 in HEK-293T cells stably expressing RXFP2. The dissociation constant of DTPA-labeled INSL3 (9.05 +/- 0.03, n = 3) that was obtained from saturation binding experiments was comparable to that of (125)I-labeled INSL3 (9.59 +/- 0.09, n = 3). The receptor binding affinity (p K i) of human INSL3 was determined to be 9.27 +/- 0.06, n = 3, using Eu-DTPA-INSL3 as a labeled ligand, which again is similar to that obtained when (125)I-INSL3 was used as labeled ligand (9.34 +/- 0.02, n = 4). This novel lanthanide-coordinated, DTPA-labeled INSL3 has excellent sensitivity, stability, and high specific activity, properties that will be particularly beneficial in high-throughput screening of INSL3 analogues in structure-activity studies.     

Novel monoclonal antibodies with specificity for chelated uranium(VI): isolation and binding properties

A derivative of 1,10-phenanthroline that binds to UO(2)(2+) with nanomolar affinity was found to be a very effective immunogen for the generation of antibodies directed toward chelated complexes of hexavalent uranium. This study describes the synthesis of 5-isothiocyanato-1,10-phenanthroline-2,9-dicarboxylic acid and its use in the generation and functional characterization of a group of monoclonal antibodies that recognize the most soluble and toxic form of uranium, the hexavalent uranyl ion (UO(2)(2+)). Three different monoclonal antibodies (8A11, 10A3, and 12F6) that recognize the 1:1 complex between UO(2)(2+) and 2,9-dicarboxy-1,10-phenanthroline (DCP) were produced by the injection of BALB/c mice with DCP-UO(2)(2+) covalently coupled to a carrier protein. Equilibrium dissociation constants for the binding of DCP-UO(2)(2+) to antibodies 8A11, 10A3, and 12F6 were 5.5, 2.4, and 0.9 nM, respectively. All three antibodies bound the metal-free DCP with roughly 1000-fold lower affinity. The second-order rate constants for the bimolecular association of each antibody with soluble DCP-UO(2)(2+) were in the range of 1 to 2 x 10(7) M(-1) s(-1). Binding studies conducted with structurally related chelators and 21 metal ions demonstrated that each of these three antibodies was highly specific for the soluble DCP-UO(2)(2+) complex. Detailed equilibrium binding studies conducted with three other derivatives of DCP, either complexed with UO(2)(2+) or metal-free, suggested that the antigen binding sites on the three antibodies have significant functional and structural similarities. Biomolecules that bind specifically to uranium will be at the heart of any new biotechnology developed to monitor and control uranium contamination. The three antibodies described herein possess sufficient affinity and specificity to support the development of immunoassays for hexavalent uranium in environmental and clinical samples.     

Apoptosis induced by chelation of intracellular zinc is associated with depletion of cellular reduced glutathione level in rat hepatocytes

Zn(2+) has multiple implications in cellular metabolism, including free radicals metabolism and cell death by apoptosis. In the present study, we examined the role of Zn(2+) in the regulation of apoptosis in cultured rat hepatocytes. The chelation of Zn(2+) by a membrane permeable metal ion chelator, N, N, N', N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), induced apoptosis. Addition of ZnSO(4) prevented TPEN-induced apoptosis. Unlike the effect of TPEN, a membrane impermeable metal ion chelator, diethylenetriamine pentaacetic acid (DTPA), did not induce apoptosis, indicating that chelation of intracellular Zn(2+) was required to trigger apoptosis. Caspase-3-like proteolytic activity, a general biochemical mediator of apoptosis in a variety of cells and tissues, was also activated with the treatment of TPEN but not DTPA. TPEN treatment, but not DTPA, also resulted in the depletion of intracellular reduced glutathione (GSH) but addition of Zn(2+) recovered the GSH level. N-acetyl-L-cysteine (NAC), a thiol antioxidant, prevented TPEN-induced apoptosis. These results taken together suggest that intracellular Zn(2+) interfere with the apoptosis process, possibly through the regulation of cellular redox potential involving GSH.     

An optimized antibody-chelator conjugate for imaging of carcinoembryonic antigen with indium-111

A monoclonal antibody to carcinoembryonic antigen showing minimal cross-reactivity with blood cells and normal tissues was derivatized with benzylisothiocyanate derivatives of EDTA and DTPA. Seven chelators per immunoglobulin could be incorporated without loss of immunoreactivity. The resulting conjugates, labeled with indium-111, showed low liver uptake in animals. A cold kit, comprising the DTPA conjugate at a molarity of antibody bound chelator exceeding 1 x 10⁻⁴M, gave radiochemical yields of indium labeled antibody of ⩾95% and was stable for 1 yr.     

Characterization of antibody-chelator conjugates: determination of chelator content by terbium fluorescence titration

Fluorescence titrations were performed by adding varying mole ratios of terbium(III) to antibody conjugates formed by benzyl isothiocyanate derivatives of three different polyaminopolycarboxylate chelators (NTA, EDTA, and DTPA) and the results compared to values for average chelator content obtained by cobalt-57 binding assays. For two different murine monoclonal antibodies, the average chelator content obtained by terbium fluorescence titration correlated closely with that measured by the cobalt-57 binding assay. It is concluded that lanthanide fluorescence titrations provide a useful alternative to radiometal binding assays for the determination of chelator content in protein-chelator conjugates.     

Binding of polyaminocarboxylate chelators to the active-site copper inhibits the GSNO-reductase activity but not the superoxide dismutase activity of Cu,Zn-superoxide dismutase

In addition to its superoxide dismutase (SOD) activity, Cu,Zn-superoxide dismutase (CuZnSOD) catalyzes the reductive decomposition of S-nitroso-L-glutathione (GSNO) in the presence of thiols such as L-glutathione (GSH). The GSNO-reductase activity but not the superoxide dismutase (SOD) activity of CuZnSOD is inhibited by the commonly used polyaminocarboxylate metal ion chelators, EDTA and DTPA. The basis for this selective inhibition is systematically investigated here. Incubation with EDTA or DTPA caused a time-dependent decrease in the 680 nm d-d absorption of Cu(II)ZnSOD but no loss in SOD activity or in the level of metal loading of the enzyme as determined by ICP-MS. The chelators also protected the SOD activity against inhibition by the arginine-specific reagent, phenylglyoxal. Measurements of both the time course of SNO absorption decay at 333 nm and oxymyoglobin scavenging of the NO that is released confirmed that the chelators inhibit CuZnSOD catalysis of GSNO reductive decomposition by GSH. The decreased GSNO-reductase activity is correlated with decreased rates of Cu(II)ZnSOD reduction by GSH in the presence of the chelators as monitored spectrophotometrically at 680 nm. The aggregate data suggest binding of the chelators to CuZnSOD, which was detected by isothermal titration calorimetry (ITC). Dissociation constants of 0.08 +/- 0.02 and 8.3 +/- 0.2 microM were calculated from the ITC thermograms for the binding of a single EDTA and DTPA, respectively, to the CuZnSOD homodimer. No association was detected under the same conditions with the metal-free enzyme (EESOD). Thus, EDTA and DTPA must bind to the solvent-exposed active-site copper of one subunit without removing the metal. This induces a conformational change at the second active site that inhibits the GSNO-reductase but not the SOD activity of the enzyme.     

Acid pairs increase the N-terminal Ca2+ affinity of CaM by increasing the rate of Ca2+ association

A series of N-terminal calmodulin (CaM) mutants was generated to probe the relationship between the N-terminal Ca(2+) affinity and the number of paired, negatively charged Ca(2+) chelating residues in the N-terminal Ca(2+)-binding sites of CaM. When the number of acid pairs [negatively charged residues at positions +x and -x (X-axis), +y and -y (Y-axis), and +z and -z (Z-axis)] was increased from zero to one and then to two, a progressive increase was seen in the N-terminal Ca(2+) affinities. The maximal ranges of the increases observed in the N-terminal Ca(2+) affinity were approximately 8-8.5-fold for site I, approximately 4.5-5-fold for site II, and approximately 11-fold for both sites, in comparison to the mutants containing no acid pairs. The maximal values of N-terminal Ca(2+) affinity were bestowed by the presence of five acidic chelating residues in site I or II, individually. Addition of the sixth acidic chelating residue (third acid pair) to both N-terminal Ca(2+)-binding sites reduced the N-terminal Ca(2+) affinity. The increases in Ca(2+) affinity observed were caused by an increase in the Ca(2+) association rates for the Y- and Z-axis acid pairs, while the X-axis acid pair caused a reduction in the Ca(2+) dissociation rates.     

Mobilisation of iron and manganese by plant-borne and synthetic metal chelators

Phytosiderophores released by roots of iron-deficient grasses mobilise Fe, Zn, Mn and Cu in calcareous soils. Mobilisation of Fe, Zn and Cu can be explained as the chelation of these metal cations by phytosiderophores. Mobilisation of Mn could not be so explained because phytosiderophores have a much smaller affinity for Mn than for Fe, Cu and Zn. Model experiments have been made with freshly precipitated Fe(OH)₃ and different soils to study the mobilisation of iron and manganese by plant-borne chelating phytosiderophores, the synthetic metal chelators DTPA and the microbial metal chelator sulphonated ferrioxamine B (FOB). Compared with the synthetic chelator DTPA, the plant-borne chelating phytosiderophores mobilised Fe very efficiently, but no change was observed in the Mn mobilisation by phytosiderophores.Different phytosiderophores, as well as the microbial metal chelator FOB, were used to compare the mobilisation of iron and manganese in a calcareous soil.     

De novo synthesis of a new diethylenetriaminepentaacetic acid (DTPA) bifunctional chelating agent

Diethylene triamine pentaacetic acid (DTPA) has been in extensive use as a metal chelator in the development of radiopharmaceuticals and contrast agents. The former application uses DTPA mostly as a bifunctional chelating agent (BCA) conjugated to tumor-targeting vehicles (TTVs) such as monoclonal antibodies (MAbs) and receptor-directed peptides. A new bifunctional DTPA derivative was synthesized by a fully organic scheme. This compound, N(4),N(alpha),N(alpha),N(epsilon),N(epsilon)-[pentakis(carboxymethyl)]-N(4)-(carboxymethyl)-2,6-diamino-4-azahexanoic hydrazide (20) was prepared by a convergent synthesis strategy using N(alpha)-benzyloxycarbonyl-2,3-diaminopropionic acid as the starting compound. This commercially available material was used to build a functionalized triamine which served as the molecular core template for assembling the target molecule. To evaluate the conjugation and radiolabeling capabilities of this new molecule, it was covalently attached to the anti-TAG-72 MAb, Delta CH2HuCC49, and the conjugate was radiolabeled in near-quantitative yields with yttrium-90 ((90)Y) and lutetium-177 ((177)Lu). Biodistribution of the (177)Lu-labeled DTPA-Delta CH2HuCC49 in tumor-bearing nude mice demonstrated preservation of the immunoreactivity of the MAb as indicated by high tumor uptake. In addition to the introduction of a new bifunctional DTPA, this work reports on a novel synthetic approach for preparation of this useful metal chelator and introduces a new conjugation protocol.     

Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of inactivation by chelators and steady-state kinetic properties

Dihydropyrimidine amidohydrolase (EC 3.5.2.2) catalyzes the reversible hydrolysis of 5,6-dihydropyrimidines to the corresponding beta-ureido acids. Previous work has shown that incubation of this Zn2+ metalloenzyme with 2,6-dipicolinic acid, 8-hydroxyquinoline-5-sulfonic acid, or o-phenanthroline results in inactivation by Zn2+ removal by a reaction pathway involving formation of a ternary enzyme-Zn2+-chelator complex which subsequently dissociates to yield apoenzyme and the Zn2+-chelate (K. P. Brooks, E. A. Jones, B. D. Kim, and E. G. Sander, (1983) Arch. Biochem. Biophys. 226, 469-483). In the present work, the pH dependence of chelator inactivation is studied. The equilibrium constant for formation of the ternary complex is strongly pH dependent and increases with decreasing pH for all three chelators. There is a positive correlation between the value of the equilibrium constant observed for each chelator and the value of its stability constant for formation of Zn2+-chelate. The affinity of the chelators for the enzyme increases in the order 8-hydroxyquinoline-5-sulfonic acid greater than o-phenanthroline greater than 2,6-dipicolinic acid. The first-order rate constant for breakdown of the ternary complex to yield apoenzyme and Zn2+-chelate is invariant with pH for a given chelator but is different for each chelator, increasing in the reverse order. The pH dependence of the inactivation shows that two ionizable groups on the enzyme are involved in the inactivation. On the other hand, the steady-state kinetic behavior of the enzyme is well-described by ionization of a single group with a pK of 6.0 in the free enzyme. The basic form of the group is required for catalysis; protonation of the group decreases both Vmax and the apparent affinity for substrate. Conversely, binding of substrate decreases the pK of this group to about 5. L-Dihydroorotic acid is shown to be a competitive inhibitor of dihydropyrimidine amidohydrolase. Binding of L-dihydroorotic acid increases the pK of the ionizable group to 6.5. The agreement between the pK in the enzyme-L-dihydroorotic acid complex and the higher pK observed in the pH dependence of inactivation by chelators suggests that the same group is involved in the binding of acid, and chelators. The different effects of substrate and L-dihydroorotic acid on the pK suggest that the binding modes of these two ligands may be different and suggest a structural basis for the mutally exclusive substrate specificities of dihydropyrimidine amidohydrolase and dihydroorotase.     

Monoclonal antibodies against human abnormal (des-gamma-carboxy)prothrombin specific for the calcium-free conformer of prothrombin

A monoclonal antibody JO1 X 1 was prepared against human abnormal prothrombin using the hybridoma technique. The clone secreting this antibody was selected on the basis of the ability of this antibody to bind to abnormal prothrombin, but not to prothrombin, in the presence of calcium ions. The antibodies were purified by affinity chromatography in EDTA on columns of prothrombin-Sepharose. Bound antibodies were eluted with 15 mM CaCl2. The kinetics of dissociation of antibody from the antibody-prothrombin complex with the addition of calcium ions fit a first-order kinetic model. Increasing CaCl2 concentration increased the rate of antibody-prothrombin dissociation. Ca(II) and Mn(II) inhibited antibody-prothrombin interaction; half-maximal binding was observed at 0.9 and 4 mM, respectively. Mg(II) had little effect on antibody-antigen interaction. The JO1 X 1 antibody bound fragment 1, fragment (1-39), abnormal prothrombin, and prothrombin equivalently in the presence of EDTA, but did not bind to des(1-44)prothrombin in the presence of EDTA or prothrombin in the presence of CaCl2. These results indicate that the monoclonal antibody JO1 X 1 is conformation specific for the calcium-free conformer of prothrombin and directed against an antigenic determinant near the NH2 terminus of prothrombin expressed in the 1-39 region of the protein. This analysis provides confirmation of the presence of a metal-free conformer of prothrombin.     

Chelator-facilitated removal of iron from transferrin: relevance to combined chelation therapy

Current iron chelation therapy consists primarily of DFO (desferrioxamine), which has to be administered via intravenous infusion, together with deferiprone and deferasirox, which are orally-active chelators. These chelators, although effective at decreasing the iron load, are associated with a number of side effects. Grady suggested that the combined administration of a smaller bidentate chelator and a larger hexadentate chelator, such as DFO, would result in greater iron removal than either chelator alone [Grady, Bardoukas and Giardina (1998) Blood 92, 16b]. This in turn could lead to a decrease in the chelator dose required. To test this hypothesis, the rate of iron transfer from a range of bidentate HPO (hydroxypyridin-4-one) chelators to DFO was monitored. Spectroscopic methods were utilized to monitor the decrease in the concentration of the Fe-HPO complex. Having established that the shuttling of iron from the bidentate chelator to DFO does occur under clinically relevant concentrations of chelator, studies were undertaken to evaluate whether this mechanism of transfer would apply to iron removal from transferrin. Again, the simultaneous presence of both a bidentate chelator and DFO was found to enhance the rate of iron chelation from transferrin at clinically relevant chelator levels. Deferiprone was found to be particularly effective at 'shuttling' iron from transferrin to DFO, probably as a result of its small size and relative low affinity for iron compared with other analogous HPO chelators.     

Investigations of N-linked macrocycles for 111in and 90Y labeling of proteins

To simplify the synthesis of macrocyclic chelators, commercially available macrocyclic amines were condensed with halogenated acetic acid to prepare the five chelators 12N4 (DOTA), 14N4 (TETA), 15N4, 9N3 and 12N3. Only 12N4 and 9N3 showed efficient labeling of the free chelator with ¹¹¹In and ⁹⁰Y. Serum stability studies at 37 °C with In-labeled DTPA, 12N4 and 9N3 showed no loss of label over 2 days whereas, with ⁹⁰Y, only 12N4 showed stabilities comparable to DTPA. The 12N4 chelator was derivatized by attaching biotin on one N-acetate group to simulate the attachment to protein. The serum stability for both ¹¹¹In and ⁹⁰Y was identical to that of biotin derivatized DTPA and lower than that of the free chelators. Biodistribution studies in normal mice of a model protein (avidin) labeled with ⁹⁰Y via biotinylated 12N4 and biotinylated DTPA showed identical distribution at 1 day except in bone where the %ID/g for the macrocyclic-conjugated protein (3.4 ± 0.5, N = 8) was significantly (P < 0.001) lower than that of the DTPA-conjugated protein (9.4 ± 0.9, N = 7). In conclusion, macrocycles may be readily synthesized from the macrocyclic amines and several show useful stabilities with In and Y. When N-linked to a protein, the Y biodistribution was found to be superior to that of the corresponding DTPA-coupled protein.     

Molecular characterization of calmodulin trapping by calcium/calmodulin-dependent protein kinase II

Autophosphorylation of alpha-Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) at Thr(286) results in calmodulin (CaM) trapping, a >10,000-fold decrease in the dissociation rate of CaM from the enzyme. Here we present the first site-directed mutagenesis study on the dissociation of the high affinity complex between CaM and full-length CaM kinase II. We measured dissociation kinetics of CaM and CaM kinase II proteins by using a fluorescently modified CaM that is sensitive to binding to target proteins. In low [Ca(2+)], the phosphorylated mutant kinase F293A and the CaM mutant E120A/M124A exhibited deficient trapping compared with wild-type. In high [Ca(2+)], the CaM mutations E120A, M124A, and E120A/M124A and the CaM kinase II mutations F293A, F293E, N294A, N294P, and R297E increased dissociation rate constants by factors ranging from 2.3 to 116. We have also identified residues in CaM and CaM kinase II that interact in the trapped state by mutant cycle-based analysis, which suggests that interactions between Phe(293) in the kinase and Glu(120) and Met(124) in CaM specifically stabilize the trapped CaM-CaM kinase II complex. Our studies further show that Phe(293) and Asn(294) in CaM kinase II play dual roles, because they likely destabilize the low affinity state of CaM complexed to unphosphorylated kinase but stabilize the trapped state of CaM bound to phosphorylated kinase.