Study on fisetin-aluminium(III) interaction in aqueous buffered solutions by spectroscopy and molecular modeling

Spectroscopic (UV/visible and IR) and theoretical studies were used to assess relevant interaction of fisetin, a tetrahydroxylated flavone molecule, and trivalent aluminium in a wide range of buffered aqueous solutions. The chelation sites, stoichiometry, stability and the dependence of the complexes structures on pH and aluminium/fisetin mole ratios were defined. Obtained results implicated successive formation of two complexes with aluminium(III)-fisetin stoichiometries of 1:1 and 2:1. Considering the fisetin molecular structure, results of vibrational analysis and theoretical calculations, it is possible to implicate 3-hydroxyl-4-carbonyl and 3′4′-dihydroxyl groups as those with the possible chelating power. The equilibrium geometries were optimized in vacuum at the B3LYP/6-31G(d) level of theory, which predict structural modifications between the ligand molecule in free state and in the complex structure. The theoretical model has been validated by both vibrational and electronic spectroscopies.     

Thermodynamic and spectroscopic studies of Cu(II) and Ni(II) complexes with a new proline-threonine dipeptide ligand

The interactions between a new proline-threonine dipeptide ligand with two metallic cations were investigated in aqueous solution. The metallic cations studied were the copper(II) and the nickel(II), which are involved in many biological processes. The combination of potentiometry, UV-visible spectrophotometry, EPR, and mass spectrometry was used to determine the formation constants of the complexes and their structure in solution. The complexation sites were identified using electronic absorption and EPR spectroscopies. Copper complexes were obtained as square planar or square pyramidal mononuclear species, whereas nickel complexes were obtained as dinuclear species with an octahedral geometry.     

Coordination of aluminium with purpurogallin and theaflavin digallate

Polyphenols are antioxidants, which are known to influence bioavailability of metals in the body. The theaflavins of black tea are important members of this family, which have been sparsely investigated. The complexation of aluminium with purpurogallin (2,3,4,6-tetrahydroxy-5H-benzocyclohepten-5-one) has been investigated using nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography-mass spectroscopy (LC-MS) and Fourier transform infrared (FT-IR) spectroscopy. 1H NMR was used to determine the coordination site of the aluminium ion and LC-MS to determine the stoichiometry and molecular weight of the major complex formed in solution. FT-IR spectral comparisons were used to corroborate the proposed chelating moiety. The complexation of aluminium with the high-molecular-weight, tea polyphenol theaflavin digallate was also investigated using 1H NMR and heteronuclear multiple quantum coherence experiments. Structures of the major aluminium purpurogallin and aluminium theaflavin digallate complexes are proposed.     

Complexes of cobalt (II) and manganese (II) with adenosine 5'-diphosphate and adenosine 5'-triphosphate. A circular dichroism study.

For studies of interactions between Co2+ and adenosine 5'-diphosphate or adenosine 5'-triphosphate (ADPH4+ and ATPH5+ in strongly acidic medium) visible circular dichroism (d-d transitions of Co2+) and ultraviolet circular dichroism (adenine transitions) have proven to be very sensitive to structural changes. Drastic variation of spectra as a function of pH and concentration enabled us to show the existence of various species, to state their stoichiometry and eventually, their self-association. With ATPH22-, C.D. results are in agreement with recent N.M.R. results. With ligands bearing three negative charges, complexes (1 metal:2 nucleotides)n are formed in which bases of the two nucleotides of the molecule are self-associated. With ADP3-, the visible C.D. spectrum of this complex is intense and hides the spectra of the complexes formed with other protonated species of ADP; this self-associated complex is detected up to a lower limit of 5 X 10(-4) M concentration. With ATPH3-, a complex exhibiting the same characteristics as the one with ADP3- is formed but in about twenty times less amount which explains why it was not detected by potentiometry. With 0.1 M ATP4-, dimeric (or polymeric) complexes, of 1:2 and 1:1 stoichiometry are observed. With 0.01 M ATP4-, 1:1 monomeric and 2:1 dimeric (or polymeric) complexes are detected. The interactions between Mn2+ ions and ADP or ATP have been studied by C.D. on the UV range. The same species as with Co2+ ions have been found but the 1:2 complex formation with ADP3- was shown to occur to a lesser extent and was not observed below a 10(-2) M ADP concentration.     

Synthesis and characterisation of sulfated amphiphilic alpha-, beta- and gamma-cyclodextrins: application to the complexation of acyclovir

The synthesis of sulfated amphiphilic alpha-, beta- and gamma-cyclodextrins was achieved according to the standard protection-deprotection procedure. The formation of inclusion complexes between the amphiphilic alpha-, beta- and gamma-cyclodextrins and an antiviral molecule, acyclovir (ACV) was investigated by UV-visible spectroscopy (UV-Vis) and electrospray ionisation mass spectrometry (ESIMS). UV-Vis spectroscopy allowed determination of the stoichiometry and stability constants of complexes, whereas ESIMS, a soft ionisation technique, allowed the detection of the inclusion complexes. The results showed that the non-sulfated amphiphilic cyclodextrins exhibit a 1:2 stoichiometry with acyclovir, while sulfated amphiphilic cyclodextrins, except gamma-cyclodextrin, exhibit a 1:1 stoichiometry indicating the loss of one interaction site. Non-covalent interactions between acyclovir and non-sulfated amphiphilic cyclodextrins appear to take place both in the cavity of the cyclodextrin and inside the hydrophobic zone generated by alkanoyl chains. In contrast, in the case of sulfated amphiphilic cyclodextrins, the interactions appear to involve only the hydrophobic region of the alkanoyl chains.     

Immunoglobulin M possesses two binding sites for complement subcomponent C1q, and soluble 1:1 and 2:1 complexes are formed in solution at reduced ionic strength

Soluble complexes were formed between C1q, a subunit of the first component of human complement, and four different Waldenstr?m IgM proteins at reduced ionic strengths. The equilibria between these complexes and the free proteins were studied in the ultracentrifuge. Complex formation was found to be a very sensitive function of the salt concentration, and at physiological ionic strength complex formation was negligible. The complexes were cross-linked with a water-soluble carbodiimide and separated by sucrose gradient centrifugation. Both 22 S 1:1 and 26 S 2:1 C1q X IgM complexes were formed; stoichiometry was established by cross-linking 125I-C1q with 131I-IgM and determining the ratios of the specific activities of the gradient-purified materials. The association process was studied as a function of protein concentration and was analyzed by Scatchard and Hill plots to yield stoichiometry, association constant, and degree of cooperativity. The results indicated that IgM has two identical and independent binding sites for C1q. The intrinsic association constant was found to vary between 10(6) M-1 at 0.084 M ionic strength to 10(4) M-1 at physiological ionic strength; the slope of the log-log plot gave a value of -6.0. The cross-linked complexes were examined by electron microscopy, and the C1q appeared to be attached to the IgM through the C1q heads, implying that the biologically significant binding sites were involved in this interaction. For the 2:1 complexes, the two C1q appeared to attach to opposite surfaces of the IgM, suggesting the presence of a pseudo-2-fold axis lying in the plane of the IgM disk.     

The GTP-dependent restriction enzyme McrBC from Escherichia coli forms high-molecular mass complexes with DNA and produces a cleavage pattern with a characteristic 10-base pair repeat

The GTP-dependent restriction enzyme McrBC consists of two polypeptides: one (McrB) that is responsible for GTP binding and hydrolysis as well as DNA binding and another (McrC) that is responsible for DNA cleavage. It recognizes two methylated or hemimethylated RC sites (R(m)C) at a distance of approximately 30 to more than 2000 base pairs and cleaves the DNA close to one of the two R(m)C sites. This process is strictly coupled to GTP hydrolysis and involves the formation of high-molecular mass complexes. We show here using footprinting techniques, surface plasmon resonance, and scanning force microscopy experiments that in the absence of McrC, McrB binds to a single R(m)C site. If a second R(m)C site is present on the DNA, it is occupied independently by McrB. Whereas the DNA-binding domain of McrB forms 1:1 complexes with each R(m)C site and shows a clear footprint on both R(m)C sites, full-length McrB forms complexes with a stoichiometry of at least 4:1 at each R(m)C site, resulting in a slightly more extended footprint. In the presence of McrC, McrB forms high-molecular mass complexes of unknown stoichiometry, which are considerably larger than the complexes formed with McrB alone. In these complexes and when GTP is present, the DNA is cleaved next to one of the R(m)C sites at distances differing by one to five helical turns, suggesting that in the McrBC-DNA complex only a few topologically well-defined phosphodiester bonds of the DNA are accessible for the nucleolytic center of McrC.     

Raman spectra and normal coordinate analyses of low-frequency vibrations of oxo-bridged manganese complexes

The active sites of certain metalloenzymes involved in oxygen metabolism, such as manganese catalase and the oxygen-evolving complex of photosystem II, contain micro -oxo-bridged Mn clusters with ligands that include H(2)O and micro (1,3)-carboxylato bridges provided by protein side chains. In order to understand better the vibrational spectra of such clusters, the low-frequency resonance Raman spectra of a series of structurally characterized Mn-oxo model complexes were examined. The series includes complexes of the type [Mn(2)(O)(OAc)(2)(bpy)(2)(L)(2)] and [Mn(2)(O)(2)(OAc)(bpy)(2)(L)(2)], where bpy=2,2'-bipyridine, OAc=acetate and L=H(2)O or Cl(-). Complexes containing the isotopomers OAc- d(3) and D(2)O, as well as those containing both isotopomers, were also examined. Normal coordinate analyses (NCA) were performed on the various complexes using theGF matrix method. Selected vibrational modes in the 200-600 cm(-1) region were assigned based on the spectra and NCA, which identify vibrational modes arising from the metal-ligand bonds. These results will be useful in interpreting the vibrational spectra obtained from metalloproteins containing Mn-oxo complexes in their active sites.     

Effect of inhibitors on the ubiquinone binding capacity of the primary energy conversion site in the Rhodobacter capsulatus cytochrome bc(1) complex.

A key issue concerning the primary conversion (Q(O)) site function in the cytochrome bc(1) complex is the stoichiometry of ubiquinone/ubihydroquinone occupancy. Previous evidence suggests that the Q(O) site is able to accommodate two ubiquinone molecules, the double occupancy model [Ding, H., Robertson, D. E., Daldal, F., and Dutton, P. L. (1992) Biochemistry 31, 3144-3158]. In the recently reported crystal structures of the cytochrome bc(1) complex, no electron density was identified in the Q(O) site that could be ascribed to ubiquinone. To provide further insight into this issue, we have manipulated the cytochrome bc(1) complex Q(O) site occupancy in photosynthetic membranes from Rhodobacter capsulatus by using inhibitor titrations and ubiquinone extraction to modulate the amount of ubiquinone bound in the site. The nature of the Q(O) site occupants was probed via the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectra to modulation of Q(O) site occupancy. Diphenylamine (DPA) and methoxyacrylate (MOA)-stilbene are known Q(O) site inhibitors of the cytochrome bc(1) complex. Addition of stoichiometric concentrations of MOA-stilbene or excess DPA to cytochrome bc(1) complexes with natural levels of ubiquinone elicits the same change in the [2Fe-2S] cluster EPR spectra; the g(x)() resonance broadens and shifts from 1. 800 to 1.783. This is exactly the same signal as that obtained when there is only one ubiquinone present in the Q(O) site. Furthermore, addition of MOA-stilbene or DPA to the cytochrome bc(1) complex depleted of ubiquinone does not alter the [2Fe-2S] cluster EPR spectral line shapes, which remain indicative of one ubiquinone or zero ubiquinones in the Q(O) site, with broad g(x)() resonances at 1. 783 or 1.765, respectively. The results are quite consistent with the Q(O) site double occupancy model, in which MOA-stilbene and DPA inhibit by displacing one, but not both, of the Q(O) site ubiquinones.     

Induced circular dichroism of the interaction between pinacyanol and algal alginates

The interaction between pinacyanol chloride and sodium alginate or guluronate-rich alginate is found to effect profound changes in the visible absorbance and circular dichroism spectra. Two different types of aggregates are observed depending on the relative dye/alginate concentrations. With a dye/alginate ratio at 1:1, a complex is deduced based on an analysis of Job’s method and conductometric titrations. Another complex forms at 1:10 dye/alginate ratio and only in the presence of alginate or guluronate-rich alginate. The two aggregates are in dynamic equilibrium according to the presence of isosbestic points in the visible spectra. The effects of pH and divalent cations on the spectra are studied. The 1:10 complex is damaged by addition of hydrochloric acid and divalent cations; however, at low concentration of these agents the spectra indicate conversion of the complex into the 1:1 aggregate. Models for the two complexes are proposed taking into account the preference of guluronate binding sites for chelating ions.     

NMR studies of carbonic anhydrase-fluorinated benzenesulfonamide complexes

Fluorine NMR has been used to examine complexes formed by 2-fluoro-, 3-fluoro-, and 2,5-difluorobenzenesulfonamide and human carbonic anhydrases I and II. The results show that all three inhibitors form complexes with both isozymes that have 2:1 inhibitor/enzyme stoichiometry. The fluorine spectra observed for all inhibitor-isozyme combinations are consistent either with rapid rotation of the aromatic ring of the inhibitor in the complexes or with preferential binding of only one of the two possible conformations of the inhibitors that are isomeric by virtue of rotation about the C1-C4 bond of the fluoro aromatic ring. Because ring rotation is slow in the case of the pentafluorobenzenesulfonamide-CA I complex, selective binding of rotamers is the explanation of these observations presently favored. A computer graphics study shows that formation of 2:1 complexes of CA I is feasible without appreciable distortion of the protein tertiary structure found in the crystalline state.     

The first intermediates in the bromination of bicyclo[3.3.1]nonylidenebicyclo[3.3.1]nonane, combination of experiments and theoretical results

Bicyclo[3.3.1]nonylidenebicyclo[3.3.1]nonane (1) and adamantylideneadamantane (Ad=Ad) are two caged olefins with closely related structures at the double bond. Both compounds react instantaneously with Br2 in chlorinated hydrocarbon solvents to give mixtures of olefin-Br2 aggregates identified as the 1:1 pi-complex and bromonium tribromide, bromonium pentabromide ion pairs. The stoichiometry, formation constants and the electronic spectra of all the species present at equilibrium (pi-complex and bromonium ions), obtained by addition of bromine to alkene 1, have been determined in 1,2-dichloroethane at 25 degrees C and compared with the values that characterize the corresponding aggregates arising from Ad=Ad. The absence of the two bridging CH2 groups in 1 significantly affects all the formation constants. Moreover, at variance with Ad=Ad, olefin 1 reacts with bromine to give, depending on reagent concentration, a substitution product. DFT (B3LYP) and ONIOM computations of 1:1 Br2-olefin complexes for 1 and Ad=Ad confirm that the association energy is larger for the complex 1-Br2. The higher stability of this species seems to be correlated to the greater IP of 1 with respect to Ad=Ad which is able to compensate the reduced polarizability. The experimental value of the formation constant found for the complex 1-Br2, 643 vs 289 M(-1) further supports the primary role exerted by dispersion interactions in alkene-Br2 pi-complexes.     

Nuclear magnetic resonance studies of the hammerhead ribozyme domain. Secondary structure formation and magnesium ion dependence

Proton nuclear magnetic resonance (n.m.r.) experiments were used to probe base-pair formation in several hammerhead RNA enzyme (ribozyme) domains. The hammerhead domains consist of a 34 nucleotide ribozyme bound to a complementary 13 nucleotide non-cleavable DNA substrate. Three hammerhead domains were studied that differ in the sequence and stability of one of the helices involved in recognition of the substrate by the ribozyme. The n.m.r. data show a 1:1 stoichiometry for the ribozyme-substrate complexes. The imino proton resonances in the hammerhead complexes were assigned by two-dimensional nuclear Overhauser effect experiments. These data confirm the presence of two of the three helical regions in the hammerhead domain, predicted from phylogenetic data; and are also consistent with the formation of the third helix. Since a divalent cation is required for efficient catalytic activity of the hammerhead domain, the magnesium ion dependence of the n.m.r. spectra was studied for two of the hammerhead complexes. One of the complexes showed very large spectral changes upon addition of magnesium ions. However, the complex that has the most C.G base-pairs in one of the recognition helices shows essentially no spectral (and therefore presumably structural) changes upon addition of magnesium. These data are consistent with a model where the magnesium binding site already exists in the magnesium-free complex, suggesting that the magnesium ion serves primarily a catalytic, and not a structural, role under the conditions used here.     

Role of magnesium ion in the interaction between chromomycin A3 and DNA: binding of chromomycin A3-Mg2+ complexes with DNA.

Chromomycin A3 is an antitumor antibiotic which blocks macromolecular synthesis via reversible interaction with DNA template only in the presence of divalent metal ions such as Mg2+. The role of Mg2+ in this antibiotic-DNA interaction is not well understood. We approached the problem in two steps via studies on the interaction of (i) chromomycin A3 and Mg2+ and (ii) chromomycin A3-Mg2+ complex(es) and DNA. Spectroscopic techniques such as absorption, fluorescence, and CD were employed for this purpose. The results could be summed up in two parts. Absorption, fluorescence, and CD spectra of the antibiotic change upon addition of Mg2+ due to complex formation between them. Analysis of the quantitative dependence of change in absorbance of chromomycin A3 (at 440 nm) upon input concentration of Mg2+ indicates formation of two types of complexes with different stoichiometries and formation constants. Trends in change of fluorescence and CD spectroscopic features of the antibiotic in the presence of Mg2+ at different concentrations further corroborate this result. The two complexes are referred to as complex I (with 1:1 stoichiometry in terms of chromomycin A3:Mg2+) and complex II (with 2:1 stoichiometry in terms of chromomycin A3:Mg2+), respectively, in future discussions. The interactions of these complexes with calf thymus DNA were examined to check whether they bind differently to the same DNA. Evaluation of binding parameters, intrinsic binding constants, and binding stoichiometry, by means of spectrophotometric and fluorescence titrations, shows that they are different. Distinctive spectroscopic features of complexes I and II, when they are bound to DNA, also support that they bind differently to the above DNA. Measurement of thermodynamic parameters characterizing their interactions with calf thymus DNA shows that complex I-DNA interaction is exothermic, in contrast to complex II-DNA interaction, which is endothermic. This feature implies a difference in the molecular nature of the interactions between the complexes and calf thymus DNA. These observations are novel and significant to understand the antitumor property of the antibiotic. They are also discussed to provide explanations for the earlier reports that in some cases appeared to be contradictory.     

A Highlights from MBoC Selection: The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution

The nuclear pore complex (NPC) regulates transport between the nucleus and cytoplasm. Soluble cargo-protein complexes navigate through the pore by binding to phenylalanine-glycine (FG)-repeat proteins attached to the channel walls. The Nup62 complex contains the FG-repeat proteins Nup62, Nup54, and Nup58 and is located in the center of the NPC. The three proteins bind each other via conserved coiled-coil segments. To determine the stoichiometry of the Nup62 complex, we undertook an in vitro study using gel filtration and analytical ultracentrifugation. Our results reveal a 1:1:1 stoichiometry of the Nup62 complex, where Nup54 is central with direct binding to Nup62 and Nup58. At high protein concentration, the complex forms larger assemblies while maintaining the Nup62:Nup54:Nup58 ratio. For the homologous Nsp1 complex from Saccharomyces cerevisiae, we determine the same stoichiometry, indicating evolutionary conservation. Furthermore, we observe that eliminating one binding partner can result in the formation of complexes with noncanonical stoichiometry, presumably because unpaired coiled-coil elements tend to find a promiscuous binding partner. We suggest that these noncanonical stoichiometries observed in vitro are unlikely to be physiologically relevant.     

Interaction of mithramycin with chromatin

Mithramycin (MTR) is an anti-cancer antibiotic that blocks the macromolecular biosynthesis via reversible interaction with DNA template in the presence of bivalent metal ion such as Mg2+. In absence of DNA, mithramycin forms two types of complexes with Mg2+, complex I (with 1:1 stoichiometry in terms of MTR: Mg2+) and complex II (with 1:2 stoichiometry in terms of MTR: Mg2+). In an eukaryotic system, the drug would interact with chromatin, a protein-DNA complex. We have employed the spectroscopic techniques such as absorption and fluorescence to study the interaction of MTR: Mg2+ complexes with rat liver chromatin. In this report, we have shown that the two types of ligands have different binding potentials with the same chromatin. This supports our proposition that complexes I and II, are different molecular species. We have also shown that the histone protein(s) reduce the binding potential and the number of available sites for both ligands.     

Fluorescence spectroscopic and (19)f NMR studies of human thymidylate synthase with its cognate RNA

In order to investigate the interaction between hTS protein and its cognate mRNA, a 29nt fragment of TS mRNA was synthesized. This region has been suggested as a putative stem-loop involved in translational autoregulation. The melting temperature of the 29ntRNA was 65 degrees C, suggesting that this region does indeed form a stem-loop. Fluorescence spectroscopy was used to monitor the RNA: hTS protein interaction [dissociation constant (K(d)) 3.9 +/- 0.8 nM; stoichiometry of binding 1dimeric hTS: 1RNA]. When hTS was titrated against FdUMP, this gave the expected stoichiometry of 1dimeric hTS: 1.7 FdUMP but in the presence of the 29ntRNA, the stoichiometry of binding changed to 1dimeric hTS: 1RNA: 1FdUMP. Experiments using methotrexate (MTX) gave a stoichiometry of 1dimeric hTS: 1MTX and in the presence of 29ntRNA, the stoichiometry was unchanged. (19)F-NMR spectra of human TS: FdUMP complexes were found to be strikingly similar to analogous NMR spectra of complexes formed by L.casei TS and mouse TS. In the presence of FdUMP, spectra exhibited two additional resonances (-1.50 ppm and -34.4 ppm). The resonance at -1.50 ppm represents non-covalently bound FdUMP, the peak at -34.4 ppm represents covalently bound FdUMP. The addition of methotrexate to the binary TS-FdUMP complex caused a displacement of the internal equilibrium, with only the covalently-bound form seen, and with a slightly disturbed (19)F chemical shift (-36.5 ppm). Similar results were found when MTX was replaced by folinic or folic acid. The addition of 29ntRNA caused no changes to the (19)F spectra of either the binary or ternary complexes.     

Adsorption of peripheral enzymes to membrane anchor proteins

The character of the isotherms of specific adsorption of peripheral enzymes to dimeric anchor proteins embedded in the membrane has been analysed. The situations are discussed when adsorption corresponds to the stoichiometry of one or two molecules of peripheral enzyme per dimeric binding site. The corresponding expressions describing the competitive interrelationships between peripheral enzymes adsorbed to the same binding sites have been derived. The experimental data on the adsorption of glycolytic enzymes to erythrocyte membranes are used for the illustration of the theoretical predictions. The physiological role of enzyme self-association which leads to the formation of enzyme oligomers of unlimited length is discussed. It is assumed that under in vivo conditions the association sites of such enzymes are saturated through interactions with anchor proteins of subcellular structures and with the enzymes of the corresponding metabolic pathways. Therefore the linearly associating enzymes play the key role in the formation of multienzyme complexes attached to subcellular structures. The significance of 6-phosphofructokinase adsorption to erythrocyte membranes in the formation of the complex of glycolytic enzymes is discussed.     

The complexation of metal cations by D-galacturonic acid: a spectroscopic study

Solid complexes of D-galacturonic acid (GalA) with cobalt(II), copper(II), nickel(II) and oxovanadium(IV) (1-4) were prepared and characterised. The metal-to-ligand molar ratio was 1:2 for complexes 1-3 and 1:1 for complex 4. The alpha- and beta-anomers of GalA were detected in all the complexes in solid state and in solutions. An addition of small amounts of the paramagnetic complexes to the D2O solution of pure ligand led to NMR line broadening of some 1H and 13C nuclei. This broadening was sensitive to the anomeric state of GalA in the case of complexes 1 and 4. NMR and vibrational spectroscopic data indicate the formation of carboxylate complexes of all the cations, while noncarboxylic oxygens are also involved into the metal bonding in some cases. VCD spectra of complexes 1-4 in D2O and Me2SO-d6 solutions confirm that GalA carboxylic group may participate in the formation of optically active species around the metal cation. Possible ways of GalA coordination by metal cations of this study were proposed and discussed.     

Complexes of mycobactin fromMycobacterium smegmatis with scandium,yttrium and lanthanum

Summary The interaction of cations of group IIIb elements (Sc, Y, La) with mycobactin S in ethanol leads to the formation of 1:1 complexes which closely resemble the known aluminium compound with respect to ultraviolet absorption and fluorescence emission spectra. Determination of molar stoichiometry by spectrophotometry shows that this method can be conveniently applied to the estimation of purity in mycobactin samples. Hydrolytic dissociation measurements based on aqueous extraction of the labelled complexes in heterogeneous phase indicate a pronounced gradation in cationbinding stability, which increases from La (rapid and complete dissociation) to Sc ( 24% dissociation under similar conditions). The observed properties of the complexes are rationalized by semi-empirical model calculations, which suggest that ionic radius effects resulting from interaction of the IIIb cations with mycobactin S would not favour octahedral coordination of these elements as in the stable Fe(III) complex.