Fluorescence study of the interaction of Suwannee River fulvic acid with metal ions and Al3+-metal ion competition

In this study emission and synchronous-scan fluorescence spectroscopy have been used to investigate the interaction of the class A (oxygen seeking 'hard acid') metal Al(3+), with Suwannee River fulvic acid (SRFA), as well as competition between Al(3+) and several other metal ions (Ca(2+), Mg(2+), Cu(2+), Pd(2+), La(3+), Tb(3+) and Fe(3+)) for binding sites on SRFA. Of the four metal ions possessing very similar (and relatively low) ionic indices (Ca(2+), Mg(2+), Cu(2+) and Pd(2+)) only the latter two paramagnetic ions significantly quenched SRFA fluorescence emission intensity. Of the four metal ions possessing very similar (and relatively low) covalent indices (Ca(2+), Mg(2+), La(3+) and Tb(3+)) only the last paramagnetic ion (Tb(3+)) significantly quenched SRFA fluorescence. None of these metals was able to significantly compete with SRFA-bound Al(3+).Fe(3+), which differs substantially from all of the other metals examined in this study in that it possesses a relatively high ionic index (but not as high as Al(3+)) and a relatively low covalent index (but not as low as Al(3+)), was able not only to quench SRFA fluorescence but also to compete (at least to some extent) with SRFA-bound Al(3+). Synchronous-scan fluorescence SRFA spectra taken in the absence and presence of Fe(3+) and/or Al(3+) support the view that these two metal ions can compete for sites on SRFA. The results of these fluorescence experiments further confirm the Al(3+), and metal ions that have electronic properties somewhat similar to Al(3+) (such as Fe(3+)) are somewhat unique in their ability to interact strongly with binding sites on fulvic acids.     

Fluorescence characterization of the interaction Suwannee river fulvic acid with the herbicide dichlorprop (2-(2,4-dichlorophenoxy)propionic acid) in the absence and presence of aluminum or erbium

This study uses fluorescence spectroscopy to better understand the role of environmental metal ions in the interaction of charged herbicides with biochemical degradation product Suwannee River fulvic acid (SRFA). The interactions between the widely-used herbicide dichlorprop (2-(2,4-dichlorophenoxy)propionic acid) (DCPPA) with Al³⁺ and the comparative metal Er³⁺ were probed at pH 4.0. Fluorescence experiments on binary solutions at pH 4.0 clearly indicated that Al³⁺ and Er³⁺ strongly interact with both SRFA and DCPPA alone in solution as demonstrated by fluorescence quenching with DCPPA and enhancement with SRFA by Al³⁺ and fluorescence quenching of both SRFA and DCPPA fluorescence by Er³⁺. Titrating Al³⁺ or Er³⁺ to SRFA-DCPPA quenched SRFA fluorescence as compared to the SRFA-metal ion binary complexes. Formation constants were determined using the Ryan-Weber model for the titration data. The DCPPA fluorescence results strongly support the formation of DCPPA-Al³⁺ and DCPPA-Er³⁺ complexes at pH values above the pK_a (3.0) of DCPPA. Excitation and emission data obtained on ternary solutions of SRFA-Al³⁺-DCPPA and SRFA-Er³⁺-DCPPA complexes at pH 4.0 suggest that at this pH where the predominant DCPPA species is negatively-charged, Al³⁺ and Er³⁺ metal ions may function to “bridge” negatively-charged fulvic acids to negatively-charged pesticides. Fluorescence data collected on UV-irradiated ternary complexes indicate that both metals can also bridge DCPPA interactions with SRFA under those conditions. The results of our studies suggest that creation of a herbicide-free boundary corridor is recommended near mines and runoff areas with metal ions in surface waters to control possible complexation among fulvic acids, DCPPA and metal ions that maintains these molecules in a bioavailable state to plants and animals.     

Fluorescence and FT-IR spectroscopic studies of Suwannee River fulvic acid complexation with aluminum, terbium and calcium.

In this study fluorescence emission and IR spectroscopy have been used to investigate the interaction of the class A (oxygen seeking "hard acid") metal Al(3+), with Suwannee River fulvic acid. Addition of Al(3+) ion results in a significant enhancement in fulvic acid fluorescence emission (at lambda(em)=424 nm) and significant red shift of the excitation wavelength (from lambda(ex)=324 nm to lambda(ex)=344 nm) at low pH values (pH approximately 4.0-5.0). At pH 4.0 (0.1 M ionic strength), where the predominant aluminum ion species is the "free" (aquo) ion, the fulvic acid fluorescence reaches 142% of the value in the absence of added metal ion. Analysis of the pH 4.0 and pH 5.0 fluorescence enhancement data with the nonlinear (single site) model of Ryan and Weber indicated binding constants in the range of 4.67.10(4)-2.87.10(6) M(-1) and concentrations of ligand sites in the range of 18.6.10(-6)-24.0.10(-6) M, both consistent with previous studies performed on both aquatic and soil fulvic acids. Companion fluorescence experiments performed on two other class A metal ions, Ca(2+) and Tb(3+), indicated no significant enhancement or quenching with Ca(2+) and only slight quenching with Tb(3+). Comparison of FT-IR spectra collected on fulvic acid alone and fulvic acid in the presence of the three class A metals (Al(3+), Ca(2+) and Tb(3+)) provides strong evidence for the involvement of carboxyl carbonyl functions in the binding of all three metal ions, which is not unexpected. The spectra also reveal, however, a very pronounced difference in the 4000-2000 cm(-1) IR spectral region between the Al(3+) spectrum and the Ca(2+) and Tb(3+) spectra. The -OH stretch spectral region in the Al(3+) spectrum has a major component shifted to higher energy (compared to fulvic acid alone or to fulvic acid in the presence of Ca(2+) or Tb(3+)). Even more striking is the emergence of a pronounced IR band at 2407 cm(-1), which is present only in the Al(3+) spectrum. The results of fluorescence and IR experiments with the model compounds salicylic acid and phthalic acid further confirm that both salicylic acid-like sites and phthalic acid-like sites are likely complexation sites for Al(3+) in fulvic acid and are major contributors to the observed spectroscopic changes associated with Al(3+) ion complexation. From a comparison of both the fluorescence and IR spectral results for all three class A metals, differing most strongly in the value of their ionic index, it seems clear that major sources of the deviation in spectral properties between Al(3+) and Ca(2+)/Tb(3+) is the unusually high value of its charge density and relatively low propensity for involvement in covalent bonding interactions (very high ionic index and relatively low covalent index in the Nieboer and Richardson classification of environmental metals), as well as affinity for certain functional groups.     

Thermodynamics and fluorescence studies of the interactions of cyclooctapeptides with Hg2+, Pb2+, and Cd2+

The purpose of this work is to characterize the interactions of cyclooctapeptides (CP) containing glutamyl and/or cysteinyl residues with common heavy-metal ions in order to facilitate the design of cyclopeptides as sensors for metal ions. Isothermal titration calorimetry studies show that cyclooctapeptides containing glutamyl and/or cysteinyl residues bind these Hg(2+) and Pb(2+) over Cd(2+) and other common metal ions. Differential binding isotherms, in their interactions with Hg(2+), support a two-binding site model, whereas pertinent interactions with Pb(2+) support a 2:1 stoichiometry, suggesting a CP/Pb(2+)/CP mode of complexation. The cyclooctapeptide containing both glutamyl and cysteinyl residues shows a significant binding affinity for Hg(2+) (K(a)=7.6x10(7)M(-1)), which is both enthalpically and entropically driven. The fluorescence of these cyclooctapeptides showed pronounced fluorescence quenching responses to Hg(2+) over Pd(2+) and Cd(2+). Stern-Volmer analyses of the dependence of fluorescence intensity on Hg(2+) and Pb(2+) are reported. The observed trends are useful for the design of Hg(2+) sensors based on fluorophore-tagged cyclooctapeptides.     

Novel emissive bio-inspired non-proteinogenic coumarin-alanine amino acid: fluorescent probe for polyfunctional systems

Two new bio-inspired non-proteinogenic compounds L1 and L2, containing coumarin and/or acridine chromophores and bearing as spacer an alanine amino acid were successfully synthesized and fully characterized by elemental analysis, (1)H and (13)C NMR, infrared spectroscopy (KBr discs), melting point, ESI-TOF (electrospray ionization-time of flight-mass), UV-vis absorption and emission spectroscopy, fluorescence quantum yields and lifetime measurements. A relative fluorescence quantum yield of 0.02 was determined for both compounds. In L2 the presence of an intramolecular energy transfer from the coumarin to the acridine unit was observed. L1 and L2 are quite sensitive to the basicity of the environment. At alkaline values both compounds show a strong quenching in the fluorescence emission, attributed to the photoinduced electron transfer (PET). However, both deprotonated forms recover the emission with the addition of Zn(2+), Cd(2+) and Al(3+) metal ions. As multifunctional emissive probes, the titration of L1 and L2 with lanthanides (III), Eu(3+) and Tb(3+) was also explored as new visible bio-probes in the absence and in the presence of liposomes. In a liposomal environment a lower energy transfer was observed.     

Synthesis of a ratiometric fluorescent peptide sensor for the highly selective detection of Cd2+

A novel ratiometric fluorescent peptidyl chemosensor (Dansyl-Cys-Pro-Gly-Cys-Trp-NH(2), D-P5) for metal ions detection has been synthesized via Fmoc solid-phase peptide synthesis. The chemosensor exhibited a high selectivity for Cd(2+) over other metal ions including competitive transition and Group I and II metal ions in neutral pH. The fluorescence emission intensity of D-P5 was significantly enhanced in the presence of Cd(2+) by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The binding stoichiometry, detection limit, binding affinity, reversibility and pH sensitivity of the sensor for Cd(2+) were investigated.     

Effects of rare earth ions on the conformational stability of anticoagulation factor II from Agkistrodon acutus venom probed by fluorescent spectroscopy

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding of rare earth ions (RE(3+))-reconstituted ACF II in guanidine hydrochloride (GdnHCl) solution was studied by fluorescence. The GdnHCl-induced unfolding of RE(3+) (Nd(3+), Sm(3+), Eu(3+), Gd(3+))-reconstituted ACF II follows a three-state transition with a stable intermediate state. Substitutions of the RE(3+) ions for Ca(2+) in ACF II decrease the conformational stability of its native state but markedly increase the conformational stability of its intermediate state. The free energy change of RE(3+)-ACF II from the intermediate state to denatured state linearly increases with the increase of ionic potentials of bound metal ions (Ca(2+), Nd(3+), Sm(3+), Eu(3+), and Gd(3+)). The refolding of ACF II from the unfolded state to the intermediate state can be induced merely by adding 10 microM RE(3+) ions without changing the concentration of the denaturant. The kinetic results of the RE(3+)-induced refolding provide evidence indicating that the intermediate state of RE(3+)-ACF II consists of at least two refolding phases and that the refolding rate constant values of the faster phase decrease with the increase of the difference between the radii of Ca(2+) and RE(3+), but the refolding rate constant values of the slower phase are similar to each other. The results of this study indicate that the size of metal ion is the major factor responsible for the metal ion-induced conformational stabilization of the native ACF II, while the metal ionic potential plays a predominant role in stabilizing the conformation for the intermediate state.     

Palladium and platinum ions interfere with the measurement of erythrocyte vesiculation by inhibiting the acetylcholinesterase activity of the released spectrin-depleted microvesicles

Palladium (Pd(2+)) and platinum (Pt(2+)) ions were found to inhibit erythrocyte membrane-bound acetylcholinesterase (AChE) with Ki values of 6.0 and 6.5 microg/ml, respectively. Lineweaver-Burke plots revealed that the inhibition of erythrocyte AChE by both metal ions was competitive in nature. Binding studies using alkaline phosphatase as a reporting enzyme confirmed that both metal ions indeed did bind to the enzyme molecules. In the process of red cell vesiculation, membrane-bound AChE is shed along with vesicles. The measurement of AChE activities in the medium containing vesiculated RBC could potentially be served as an index of vesiculation. Inhibition of AChE activities by both metal ions can thus constitute a potential source of error in vesiculation measurement. To illustrate these effects, a simulated vesiculation system, using green tea polyphenol in the presence (25 microg/ml) or absence of Pd(2+) ion was simultaneously examined by the electronmicrography and the AChE method. We observed vesiculation under the experimental condition in Pd(2+)-free controls that was associated with a time-dependent increase in AChE activity were barely detected in the Pd(2+)-spiked specimen because of the masking effect exerted by the metal ions themselves.     

Acidity of exogenous metal ion in the activation of calcineurin

The pH dependent activation of calcineurin by exogenous metal ion was studied over the pH range from 6.5 to 9.0 in increments of 0.5 pH units. Calcineurin activated by Co2+, Ni2+, or Mg2+ was characterized and compared to the pH dependency of the Mn(2+)-activated enzyme (Martin, B.L., and Graves, D.J. (1986) J. Biol. Chem. 261, 14545-14550). The pH dependency of the kinetic parameters varied with metal ion and subsequent analysis yielded estimates for the pKa values for the enzyme-metal ion and the enzyme-metal ion-substrate complexes with each of the exogenous metal ions characterized. The evaluated pK(a)s for enzyme-metal ion (EM) complexes showed an inverse relationship with the pK(a)s of the M(2+)-H2O complex. In contrast, variation of the pK(a)s for the enzyme-metal ion-substrate (EMS) complexes showed no trend. These data support the hypothesis that exogenous metal ion functions to facilitate a proton transfer before the turnover of substrate with the acidity of the exogenous metal ion as a primary determinant of its participation.     

A study of GDP binding to purified thermogenin protein from brown adipose tissue.

1. The binding of GDP to purified thermogenin protein was studied by using fluorescence spectroscopy and equilibrium dialysis. 2. GDP binding to thermogenin diminished fluorescence emission in a concentration-dependent manner that exhibited saturation. 3. Kd values for binding of nucleoside di- and tri-phosphates were lower than those for nucleoside monophosphates. 4. The GDP-induced fluorescence quenching was decreased by increasing pH, but the apparent Kd was unaltered by pH changes. 5. Equilibrium dialysis showed a Kd change from 3 to 6 microM when the pH was increased from 6.6 to 8.5. 6. The apparent pK of the fluorescence changes induced by pH (8.3) was identical with the apparent pK of the GDP-binding response. 7. The data are consistent with the existence of protonated and non-protonated forms of thermogenin protein that both bind GDP.     

Kinetics of conformational changes induced by the binding of various metal ions to bovine alpha-lactalbumin.

The kinetic effects of the binding of various metal ions (Ca(2+), Cd(2+), Co(2+), Mg(2+), Mn(2+), Sr(2+) and Zn(2+)) to apo bovine alpha-lactalbumin has been monitored by means of stopped-flow fluorescence spectroscopy. Our results show that the measured rate constant for the binding of metal ions to the Ca(2+)-site increases with increasing binding constant. This is, however, not the case for metal ions binding to the Zn(2+)-site. The binding experiments performed at different temperatures allowed us to calculate the activation energy for the transition from the metal-free to the metal-loaded state of the protein. These values do not depend on the nature of the metal ion but are correlated with the type of binding site. As a result, we were able to demonstrate that Mg(2+), a metal ion which was thought to bind to the Ca(2+)-site, shows the same binding characteristics as Co(2+) and Zn(2+) and therefore most likely interacts with the residues belonging to the Zn(2+)-binding site.     

Cobalt- and nickel-binding property of cullin-2.

Treatment with divalent metal ions such as cobalt (Co(2+)) or nickel (Ni(2+)) result in the stabilization of hypoxia-inducible factor-1alpha (HIF1alpha). Recently, HIF1alpha was shown to be ubiquitinated by an E3-ligase complex and be subsequently targeted for proteasomal degradation. In this study, we demonstrated that Co(2+) and Ni(2+) specifically bind to cullin-2. Mutant analysis revealed that cullin-2 possesses at least three sites for the binding. Furthermore, fluorescence spectroscopy revealed that only Co(2+) and Ni(2+) have the binding activity to cullin-2, but other metal ions, including Cu(2+), Ca(2+), Mg(2+), Mn(2+), and Zn(2+), did not. Finally, we found that Co(2+) and Ni(2+) do not bind to any components of the E3-ligase other than cullin-2, suggesting that cullin-2 is a key target of Co(2+) and Ni(2+). Interestingly, Co(2+) did not affect the complex formation of the ligase, suggesting that the metal binding to cullin-2 affects the function, but not the assembly of the E3-ligase.     

The interaction of native DNA with Zn(II) and Cu(II) complexes of 5-triethyl ammonium methyl salicylidene orto-phenylendiimine

The interaction of native calf thymus DNA with the Zn(II) and Cu(II) complexes of 5-triethyl ammonium methyl salicylidene orto-phenylendiimine (ZnL(2+) and CuL(2+)), in 1 mM Tris-HCl aqueous solutions at neutral pH, has been monitored as a function of the metal complex-DNA molar ratio by UV absorption spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. The results support for an intercalative interaction of both ZnL(2+) and CuL(2+) with DNA, showing CuL(2+) an affinity of approximately 10 times higher than ZnL(2+). In particular, the values of the binding constant, determined by UV spectrophotometric titration, equal to 7.3x10(4) and 1.3x10(6)M(-1), for ZnL(2+) and CuL(2+), respectively, indicate the occurrence of a marked interaction with a binding size of about 0.7 in base pairs. The temperature dependence of the absorbance at 258 nm suggests that both complexes strongly increase the DNA melting temperature (Tm) already at metal complex-DNA molar ratios equal to 0.1. As evidenced by the quenching of the fluorescence of ethidium bromide-DNA solutions in the presence of increasing amounts of metal complex, ZnL(2+) and CuL(2+) are able to displace the ethidium cation intercalated into DNA. A tight ZnL(2+)-DNA and CuL(2+)-DNA binding has been also proven by the appearance, in both metal complex-DNA solutions, of a broad induced CD band in the range 350-450 nm. In the case of the CuL(2+)-DNA system, the shape of the CD spectrum, at high CuL(2+) content, is similar to that observed for psi-DNA solutions. Such result allowed us to hypothesize that CuL(2+) induces the formation of supramolecular aggregates of DNA in aqueous solutions.     

Distinct effects of Zn2+, Cu2+, Fe3+, and Al3+ on amyloid-beta stability, oligomerization, and aggregation: amyloid-beta destabilization promotes annular protofibril formation

Abnormally high concentrations of Zn(2+), Cu(2+), and Fe(3+) are present along with amyloid-β (Aβ) in the senile plaques in Alzheimer disease, where Al(3+) is also detected. Aβ aggregation is the key pathogenic event in Alzheimer disease, where Aβ oligomers are the major culprits. The fundamental mechanism of these metal ions on Aβ remains elusive. Here, we employ 4,4'-Bis(1-anilinonaphthalene 8-sulfonate) and tyrosine fluorescence, CD, stopped flow fluorescence, guanidine hydrochloride denaturation, and photo-induced cross-linking to elucidate the effect of Zn(2+), Cu(2+), Fe(3+), and Al(3+) on Aβ at the early stage of the aggregation. Furthermore, thioflavin T assay, dot blotting, and transmission electron microscopy are utilized to examine Aβ aggregation. Our results show that Al(3+) and Zn(2+), but not Cu(2+) and Fe(3+), induce larger hydrophobic exposures of Aβ conformation, resulting in its significant destabilization at the early stage. The metal ion binding induces Aβ conformational changes with micromolar binding affinities and millisecond binding kinetics. Cu(2+) and Zn(2+) induce similar assembly of transiently appearing Aβ oligomers at the early state. During the aggregation, we found that Zn(2+) exclusively promotes the annular protofibril formation without undergoing a nucleation process, whereas Cu(2+) and Fe(3+) inhibit fibril formation by prolonging the nucleation phases. Al(3+) also inhibits fibril formation; however, the annular oligomers co-exist in the aggregation pathway. In conclusion, Zn(2+), Cu(2+), Fe(3+), and Al(3+) adopt distinct folding and aggregation mechanisms to affect Aβ, where Aβ destabilization promotes annular protofibril formation. Our study facilitates the understanding of annular Aβ oligomer formation upon metal ion binding.     

The removal by crab shell of mixed heavy metal ions in aqueous solution

In order to examine the inhibition effect of other heavy metal ions on the removal by crab shell of heavy metal ions in aqueous solutions, three ions (Pb(2+), Cd(2+), Cr(3+)) were used in single, binary and ternary systems. In single heavy metal ion systems, the removals of Cr(3+) and Pb(2+) were much higher than that of Cd(2+). In binary heavy metal ions systems, Cd(2+) did not affect Pb(2+) removal while Cr(3+) had a severe inhibition effect on the removal of Pb(2+). Cd(2+) removal was slightly affected by the presence of Pb(2+); however, it was severely affected by the presence of Cr(3+). The inhibitory effect of Cd(2+) on Cr(3+) was relatively lower than that of Pb(2+).     

Fluorescence quenching of beta-carboline alkaloids in micellar media. A study to select the adequate surfactant to use in analytical techniques.

The study of fluorescence quenching of the fluorophores allows the localization of the alkaloids (harmane and harmine) in the micelles (SDS, CTAB, Brij-35) to be established. In aqueous micellar solutions (SDS and Brij-35) at pH 13.0, emission corresponding to the neutral or zwitterionic forms can be observed. In the presence of CTAB (pH = 13.0) it was possible to observe the emission of anionic form. These species are not present in buffered aqueous solutions at these pH values. Bromide ion was added to the different surfactant solutions and the quenching effect was studied according to the Stern-Volmer equation. In the presence of SDS the quenching effect is considerably reduced compared to the aqueous solutions without surfactants, while for Brij-35 micelles were similar to those observed in homogeneous aqueous solution. For CTAB micelles a notable fluorescence quenching was observed for the different pH values studied. The fluorescence quenching studies show that the neutral species are associated inside the micelles, instead of the ionic species (cationic, zwitterionic or anionic) remaining on the surface of the micelles. The anionic surface of SDS micelles prevents the quenching effect by anionic quenchers for both neutral and charged species.     

Effect of metal ions on the interaction between bovine serum albumin and berberine chloride extracted from a traditional Chinese Herb coptis chinensis franch

The effect of four metal ions Cu(2+), Ni(2+), Zn(2+) and Co(2+) on the interaction between bovine serum albumin (BSA) and berberine chloride (BC) extracted from a traditional Chinese Herb coptis chinensis franch, was investigated mainly by means of UV and fluorescence spectroscopy in this paper. The four metal ions make the quenching efficacy of BC to BSA higher than that in the absence of these metal ions because of the possible transition of BSA molecular conformation caused by metal ions. It was found that the quenching mechanism is a combination of static quenching with nonradiative energy transfer. In the presence of metal ions, the apparent association constant K(A) and the number of binding sites of BC on BSA are both decreased in a range of 8-19% and 25-28%, respectively, which indicates that the metal ions decrease the binding efficacy of BC on BSA and increase the concentration of free BC simultaneously. The scheme of interaction between BC and BSA in the presence of metal ions is a strong quenching but a weak binding.     

ADSORPTION OF FULVIC ACID ON ALGAL SURFACES AND ITS EFFECT ON CARBON UPTAKE

Adsorption of Suwannee River fulvic acid (SRFA) to algal surfaces of three green algae was studied at environmentally relevant pH values (4 –7) and SRFA concentrations (5–100 mg·L ^{− 1}). The influence of adsorbed SRFA on carbon uptake of Scenedesmus subspicatus Chodat was also examined. Although no adsorption was observed at neutral pH values (pH 6 and 7), at pH 4 up to 31 mg SRFA·m ^{− 2} and at pH 5 up to 4 mg SRFA·m ^{− 2} was adsorbed to the algal surfaces. Electrophoretic mobility measurements of S. subspicatus demonstrated an increase in the negative surface charge of the alga in the presence of SRFA at pH 4. The adsorbed SRFA also influenced ¹⁴C uptake in S. subspicatus; in this case, enhanced carbon uptake could be related to the amount of adsorbed SRFA. The binding of humic substances by algal surfaces was interpreted as the result of hydrogen bonding and hydrophobic interactions.     

The intrinsic tryptophan fluorescence of beta 1-bungarotoxin and the Ca2+-binding domains of the toxin as probed with Tb3+ luminescence.

beta 1-Bungarotoxin has only one tryptophan residue, namely Trp-19 in the phospholipase A2 subunit. The environment of Trp-19 was studied by intrinsic fluorescence and solute quenching. The native protein showed an emission peak at 330 nm. About 90% of the fluorescent tryptophan was accessible to quenching by either acrylamide or KI but not to CsCl. A red-shift in the emission peak occurred between 2.0 M- and 4.0 M-guanidinium chloride, and the helix-coil transition of the polypeptide backbone occurred between 4.0 M- and 6.0 M-guanidinium chloride. These results suggested that Trp-19 was in a less polar medium but near a positive charge. The local conformation around Trp-19 could be disturbed by binding of Tb3+ or Ca2+ or Sr2+ to the toxin molecule. Tb3+ a tervalent lanthanide ion, effectively substituted for Ca2+ in stimulating the phospholipase A2 activity of beta 1-bungarotoxin. Upon the binding of Tb3+ to the toxin, the Tb3+ fluorescence in the 450-650 nm region was enhanced. This resulted from the energy transfer from Trp-19 to Tb3+. The distance between the energy-transfer pair was estimated to be 0.376-0.473 nm at pH 7.6 and 0.486-0.609 nm at pH 6.3. Assuming that there were two Tb3+-binding sites on the toxin molecule, at pH 7.6 the association constants of the high-affinity and the low-affinity sites were determined to be 3.82 x 10(3) M-1 and 2.85 x 10(2) M-1 respectively. At between pH 6.0 and 7.0 Tb3+ bound to the high-affinity site decreased greatly but did not disappear entirely. Both Ca2+ and Sr2+ competed with Tb3+ at the high-affinity sites, but Sr2+ could not substitute for Ca2+ in stimulating the phospholipase A2 activity.     

Comparative studies on the iron chelators O-TRENSOX and TRENCAMS: selectivity of the complexation towards other biologically relevant metal ions and Al(3+)

Complexation constants have been determined by potentiometric titration and spectrophotometric measurements for several biologically relevant divalent metals (Ca(2+), Cu(2+), Zn(2+)) as well as Al(3+) with the sulfonated tris(8-hydroxyquinolinate) tripodal ligand O-TRENSOX. The values demonstrate great selectivity of O-TRENSOX for Fe(3+) according to the sequence Fe(3+) >Cu(2+)>Zn(2+)>Ca(2+). This selectivity is compared to that shown by tris(hydroxamate) and tris(catecholate) ligands. (1)H NMR spectroscopy of the diamagnetic complexes have been carried out in (2)H(2)O solutions.