The pH-dependent conformational transition of β-lactoglobulin modulates the binding of protoporphyrin IX

We have investigated the interaction between PPIX and β-lactoglobulin (β-lg) as a function of the pH of the solution. β-lg is a small globular protein (MW ≈18 kDa) with a very well characterized structure that reveals several possible binding sites for ligands. The interaction with β-lg affects the photophysical properties of PPIX. The shift of PPIX emission maximum, excitation maximum and the increase of the fluorescence intensity is an indicator that binding between the porphyrin and β-lg occurs. The binding constant appears to be modulated by the pH of the solution. Spectroscopic measurements do not reveal any significant energy transfer between the Trp residues of β-lg and PPIX, however, fluorescence anisotropy decay measurements confirm the binding and the modulation introduced by the pH of the solution. Since β-lg has been shown to be stable within the range of pH adopted in our experiments (5.0–9.0), the results suggest that PPIX binds a site affected by the pH of the solution. Because of the crystallographic evidence an obvious site is near the aperture of the interior β-barrel however an alternative (or concurrent) binding site may still be present.     

Effects of photoproducts on the binding properties of protoporphyrin IX to proteins

Photosensitisers are the photoactive molecules used in photodynamic therapy (PDT) of cancer. Despite the importance of their interaction with polypeptides, only the binding to plasma proteins has been investigated in some detail. In our study we compared the binding of Protoporphyrin IX (a clinically useful photosensitiser) to an immunoglobulin G, with the binding to albumins. Binding to IgG is relevant because a possible method of increasing tumour specificity of photosensitisers is to bind them to tumour-specific antibodies. Binding constants to albumins and the immunoglobulin were comparable ( congruent with6 x 10(-6) M(-1)). The apparent number of PPIX molecules bound to each protein was also within a similar range (from 4 to 7). The absence of a shift in the emission spectrum of PPIX bound to IgG, however, indicates that either larger aggregates of PPIX bind to the immunoglobulin or that the binding site leaves PPIX exposed to the buffer. We observed that PPIX photoproducts compete with PPIX for the same binding sites. The number of PPIX molecules bound to each protein in the presence of photoproducts decreased by 50-80%. Due to the spectral overlap between PPIX and its photoproducts, the binding in the presence of photoproducts was investigated using Derivative Synchronous Fluorescence Spectroscopy (DSFS) to improve the spectral separation between chromophores in solution. We also concluded that fluorescence measurements underestimate the number of PPIX molecules binding each protein. In fact, non-linear Scatchard plots (in the case of albumin binding) by definition yield a minimum number of molecules attached to a protein. Moreover, the binding of large aggregates, formed by an unknown number of PPIX molecules, to IgG results in the underestimate of the number of molecules bound. The number of PPIX molecules bound to these proteins is also much larger than the number of sites estimated by protein fluorescence quenching.     

Spectroscopic studies of interaction of chlorobenzylidine with DNA

Electronic absorbance and fluorescence titrations are used to probe the interaction of chlorobenzylidine with DNA. The binding of chlorobenzylidine to DNA results in hypochromism, a small shift to a longer wavelength in the absorption spectra, and emission quenching in the fluorescence spectra. These spectral characteristics suggest that chlorobenzylidine binds to DNA by an intercalative mode. This conclusion is reinforced by fluorescence polarization measurements. Scatchard plots constructed from fluorescence titration data give a binding constant of 1.3 x 10(5) M(-1) and a binding site size of 10 base pairs. This indicates that chlorobenzylidine has a high affinity with DNA. The intercalative interaction is exothermic with a Van't Hoff enthalpy of -143 kJ/mol. This result is obtained from the temperature dependence of the binding constant. The interaction of chlorobenzylidine with DNA is affected by the pH value of the solution. The binding constant has its maximum at pH 3.0. Upon binding to DNA, the fluorescence from chlorobenzylidine is quenched efficiently by the DNA bases and the fluorescence intensity tends to be constant at high concentrations of DNA when the binding is saturated. The Stern-Volmer quenching constant obtained from the linear quenching plot is 1.6 x 10(4) M(-1) at 25 degrees C. The measurements of the fluorescence lifetime and the dependence of the quenching constant on the temperature indicate that the fluorescence quenching process is static. The fluorescence lifetime of chlorobenzylidine is 1.9 +/- 0.4 ns.     

Interaction of zinc protoporphyrin with intact oxyhemoglobin

In erythropoietic protoporphyria and lead poisoning, free protoporphyrin (PPIX) and zinc protoporphyrin (ZPP), respectively, accumulate in erythrocytes. That PPIX and ZPP bind to human hemoglobin A (Hb4) is established, but the site of binding is still a matter of controversy. We investigated the interaction of ZPP with intact, tetrameric oxy Hb4, using batch microcalorimetry, front-face fluorometry, absorption difference spectroscopy, oxygen equilibrium studies, and isoelectric focusing (IEF). In the presence of oxy Hb4 (pH 7.35, 0.05 M phosphate), the fluorescence emission maximum (excitation at 420 nm) of ZPP immediately shifts from 587 nm (ZPP alone) to 594 nm, as expected when binding to protein. The fluorescence intensity increases with time and is correlated with the ZPP:Hb4 mole ratio. A slow, time-dependent reaction is also observed with microcalorimetry: the rate of heat of reaction exhibits both a fast and a slow component. The heats of reaction range from -2.1 to -14.8 mcal depending upon the ZPP:Hb4 ratio of 4:1 (0.4 mM:0.1 mM) to 38:1 (3.8 mM:0.1 mM), respectively, and are typical of weak, noncovalent protein-ligand interactions. The optical difference spectra are a function of the ZPP:Hb4 molar ratio and also exhibit a slow increase in intensity over time. No time-dependent optical difference spectra are observed with ZPP or with Hb4 alone. The oxygen affinity of Hb4 in the presence of ZPP decreases with increasing mole ratio. During IEF, all ZPP separates from Hb4, consistent with a weak, noncovalent interaction at a non-heme pocket site. We conclude that ZPP binds to intact, tetrameric hemoglobin at non-heme pocket sites in a nonspecific, weak, noncovalent interaction.     

Binding characteristics of a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine, to bovine serum albumin as measured by fluorescence

The interaction between a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine and bovine serum albumin was investigated by fluorescence measurements. The apparent binding constants were obtained at various pHs assuming the equivalence and independence of the interaction sites on the protein from the fluorescence titration curves. The maximum binding was attained at pH 8.0, and the apparent binding constant was (5.28 +/- 0.13).10(5) M-1 with one binding site per albumin molecule. Thermodynamic parameters were also determined from the van't Hoff plot of the apparent binding constants at pH 7.5. The free energy change, enthalpy change and entropy change were -7.70 +/- 0.09 kcal.mol-1, -4.59 kcal.mol-1 and 10.2 e.u., respectively.     

Interaction of alpha-chymotrypsin with the fluorescent probe 1-anilinonaphthalene-8-sulfonate in solution.

The binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonate (Ans) to alpha-chymotrypsin (alpha-CHT) at pH 3.6 is accompanied by a dramatic enhancement of Ans fluorescence and a shift of the emission maximum to shorter wavelengths. Our study reveals that one Ans molecule binds to alpha-CHT at a site different from either the active site of alpha-CHT or the 2-p-toluidinylnapthalene-6-sulfonate binding site. the binding constant of Ans is about the same (10(4) M-1) at pH 3.6 and 6.4. Nanosecond fluorescence depolarization data indicate that Ans is rigidly bound to alpha-CHT. The fluorescence enhancement due to binding of Ans to alpha-CHT at low pH could be due to binding either to a hydrophobic site or to a site where local dipoles do not relax during the excited-state lifetime of Ans. As the pH is increased, fluorescence intensity of the Ans-alpha-CHT complex decreases appreciably; and the emission maximum shifts to longer wavelengths. The fluorescence decay curves exhibit a corresponding sensitivity to pH. The pH effect on the fluorescence of Ans-alpha-CHT can be interpreted in terms of a pH-dependent equilibrium between alpha-CHT conformers differing in the degree of mobility of polar residues and water molecules at the Ans binding site or structural changes in the Ans binding site.     

1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain

The molten globule (MG) state of proteins is widely detected through binding with 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent dye. This strategy is based upon the assumption that when in molten globule state, the exposed hydrophobic clusters of protein are readily bound by the nonpolar anilino-naphthalene moiety of ANS molecules which then produce brilliant fluorescence. In this work, we explored the acid-induced unfolding pathway of chymopapain, a cysteine proteases from Carica papaya, by monitoring the conformational changes over a pH range 1.0–7.4 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The spectroscopic measurements showed that although maximum ANS fluorescence intensity was observed at pH 1.0, however protein exhibited ∼80% loss of secondary structure which does not comply with the characteristics of a typical MG-state. In contrast at pH 1.5, chymopapain retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii and nearly 30-fold increase in ANS fluorescence with respect to the native state, indicating that MG-state exists at pH 1.5 and not at pH 1.0. ITC measurements revealed that ANS molecules bound to chymopapain via hydrophobic interaction were more at pH 1.5 than at pH 1.0. However, a large number of ANS molecules were also involved in electrostatic interaction with protein at pH 1.0 which, together with hydrophobically interacted molecules, may be responsible for maximum ANS fluorescence. We conclude that maximum ANS-fluorescence alone may not be the criteria for determining the MG of chymopapain. Hence a comprehensive structural analysis of the intermediate is essentially required.     

Characterization of the photoproducts of protoporphyrin IX bound to human serum albumin and immunoglobulin G

Clinically useful photosensitisers (PSs) are likely bound to subcellular and molecular targets during phototherapy. Binding to a macromolecule has the potential to change the photophysical and photochemical characteristics of the PSs that are crucial for their phototoxicity and cell-killing activity. We investigated the effects of binding of a specific PS (protoporphyrin IX or PPIX) to two proteins, human serum albumin (HSA) and a commercially available immunoglobulin (IgG). These two proteins provide two different environments for PPIX. The albumin binds PPIX in hydrophobic binding sites located in subdomain IIA and IIIA, conversely IgG leaves PPIX exposed to the solvent. We show that photophysical parameters such as emission maxima and fluorescence lifetime depend on the binding site. Our results indicate that the different binding site yields very different rates of formation of photoproducts (more than three times higher for PPIX bound to HSA than to IgG) and that different mechanisms of formation may be occurring. Our characterization shows the relevance of protein binding for the photochemistry and ultimately the phototoxicity of PSs.     

Calcium binding of troponin C. I. A potentiometric titration study.

Structural changes of troponin C on calcium binding were studied by hydrogen ion titration, circular dichroism, and fluorescence measurements. The potentiometric titration curves in the carboxyl region are shifted towards lower pH with calcium binding. The intrinsic pK of the carboxyl groups at the calcium binding sites decreases by 0.8 pK unit on calcium binding; on the other hand, magnesium ions have little effect on the intrinsic pK of the carboxyl groups. The intrinsic pK of the imidazole group is not affected by calcium binding. The value of w, an electrostatic interaction factor, is identical for calcium-free and calcium-bound troponin C and is about half of the value calculated assuming a compact sphere. The results of difference titration on the calcium binding indicate that the pH of troponin C solution increases on addition of CaCl2 up to 2 mol of Ca2+ per mol of troponin C and then decreases on further addition of CaCl2. The pH increase is depressed in the presence of MgCl2, in the low pH region, or at high ionic strength. The pH increase is also observed on addition of MgCl2. The ellipticity at 222 nm was measured under the same conditions as the difference titration measurements, and the relation between the pH change and the conformational change of troponin C on calcium binding is discussed based on the results obtained. The number of calcium binding sites and the binding constants estimated by analysis of these difference titration curves were in agreement with the results of Potter and Gergely (22). No magnesium binding site was observed. The tyrosine fluorescence measurements indicated that the binding site near tyrosine-109 is one of the high affinity sites.     

Fluorescence-based assay of estrogen receptor using 12-oxo-9(11)-dehydroestradiol-17 beta

12-Oxo-9(11)-dehydroestradiol-17 beta (12-oxo-E2) was used to assay estrogen receptor binding in uterine cytosol preparations by an indirect fluorescence assay. In alkaline solution, 12-oxo-E2 has a fluorescence excitation maximum at 402 nm (epsilon = 24,000) and an emission maximum at 480 nm (phi f = 0.57), and its fluorescence can be observed down to 5 X 10(-11) M. The minimum detection limit of 12-oxo-E2 is 25 fmol by spectrofluorometry and 5 fmol by HPLC-fluorometry. Although this compound is not appreciably fluorescent at neutral pH (i.e., at conditions under which it binds to the estrogen receptor), receptor binding by fluorometry can be measured indirectly: After equilibration of 12-oxo-E2 with the receptor preparation and removal of excess free ligand, the receptor-12-oxo-E2 complex is disrupted, and fluorescence measurements are made on the dissociated 12-oxo-E2 in alkaline medium. This fluorometric assay was validated quantitatively by performing simultaneously, on the same receptor preparation, radiometric and fluorometric assays with [3H]E2 and [3H]-12-oxo-E2. The radiometric determinations with both compounds gave nearly equivalent estimates of receptor site concentrations, but the fluorometric estimate of binding site concentration was somewhat less (70-85%) than that expected on the basis of the [3H]E2 radiometric assay. The use of 12-oxo-E2 in an indirect spectro- or HPLC-fluorometric assay provides a means for assaying estrogen receptor concentrations by fluorescence with a sensitivity approaching that of radiometric techniques.     

A comparison study of the interaction between β-lactoglobulin and retinol at two different conditions: spectroscopic and molecular modeling approaches

The interaction between bovin β-Lactoglobulin (β-LG) and retinol at two different pH values was investigated by multispectroscopic, zeta potential, molecular modeling, and conductometry measurements. The steady state and polarization fluorescence spectroscopy revealed that complex formation at two different pH values could occur through a remarkable static quenching. According to fluorescence quenching, one set of binding site at pH 2 and two sets of binding sites at pH 7 were introduced for binding of retinol to β-LG that show the enhancement of saturation score of β-LG to retinol in dimmer condition. The polarization fluorescence analysis represented that there is more affinity between β-LG and retinol at pH 7 rather than at pH 2. The effect of retinol on β-LG was studied by UV-visible, circular dichroism (CD), and synchronous fluorescence, which indicated that retinol induced more structural changes on β-LG at pH 7. β-LG–retinol complex formation at two different pH values was recorded via applying resonance light scattering (RLS) and zeta potential. Conductometry and RLS showed two different behaviors of interaction between β-LG and retinol at two different pH values; therefore, dimmer formation played important roles in different behaviors of interaction between β-LG and retinol. The zeta potential was the implied combination of electrostatic and hydrophobic forces which are involved in β-LG–retinol complex at two different pH values, and the hydrophobic interactions play a dominant role in complex formation. Molecular modeling was approved by all experimental results. The acquired results suggested that monomer and dimmer states of β-LG can be induced by retinol with different behaviors.     

pH-dependent changes in the tryptophan and porphyrin fluorescence of the apomyoglobin complex with protoporphyrin IX and methemoglobin

The fluorescence of protoporphyrin IX (PPIX) complexed with sperm whale apomyoglobin as well as the tryptophan fluorescence of this complex and of metmyoglobin within the pH range of 3.5-13 was studied. It was shown that an increase in pH from 5.3 to 10.8 does not influence the fluorescence of PPIX in the complex and causes no essential changes in the fluorescence of Trp residues, which occur at more acidic and, correspondingly, alkaline pH values simultaneously with the protein denaturation. This is accompanied by a sharp increase in the quantum yield of tryptophan fluorescence due to dissociation of PPIX from the complex. Similar changes are observed in metMb at pH less than 4.3 and greater than 12 which is concomitant with absorption changes in the Soret band, thus indicating a higher stability of metMb towards the acid and alkaline denaturation as compared to the complex. In both cases, a slight alteration in the shape of the tryptophan fluorescence spectrum is observed, which precedes alkaline denaturation of the Mb molecule and is probably due to changes in the conformation of the N-terminal region caused by the break of the salt bridges stabilizing the native structure of the protein.     

The interaction of serum albumins with various drugs in aqueous solution: Gel permeation, calorimetric, and fluorescence data

Thermodynamic data relative to the reversible interaction between human or bovine serum albumin and some organic ligands (S-and R-warfarin, d-and l-oxazepam hemisuccinate, phenyl-butazone, fluorescein) in dilute aqueous solution were determined by means of gel permeation chromatography and microcalorimetric measurements. From an analysis of these data and on the basis of fluorescence titrations the identity of the “primary” binding site on the proteins for some ligands was evidenced, while in other cases a cooperative binding of two different ligands to different binding sites could be discerned.     

A calorimetric investigation of the interaction of the lac repressor with inducer

A calorimetric study has been made of the interaction between the lac repressor and isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The buffer-corrected enthalpy of reaction at 25 degrees C was found to be -15.6, -24.7, -4.6 kJ/mol of bound IPTG at pH 7.0, pH 8.1, and pH 9.0, respectively. This large range of enthalpy values is in contrast to a maximum difference in the free energy of the reaction of only 1.5 kJ/mol of bound IPTG between these pH values. The reaction was found by calorimetric measurements in different buffers to be accompanied by an uptake of 0.29 mol of protons/mol of bound IPTG at pH 8.1. The pH dependency of the reaction enthalpy suggests differences in the extent of protonation of the binding site and the involvement of H bonding with IPTG. The lack of strong hydrophobic contributions in the IPTG binding process is revealed by the absence of any determinable heat capacity change for the reaction at pH 7.0. The presence of phosphate buffer significantly alters the enthalpy of IPTG binding at higher pH values, but has little effect upon the binding constant. This implies that highly negative phosphate species change the nature of the IPTG binding site without any displacement of phosphate upon IPTG binding.     

Plastocyanin conformation. The effect of nitrotyrosine modification and pH

Plastocyanin isolated from several species including spinach, poplar, and lettuce showed conformational changes both upon reduction and upon lowering the pH as determined by near-ultraviolet absorption and fluorescence measurements. The fluorescence excitation maximum was at 278 nm for all species of plastocyanin measured. In the case of spinach, the emission maximum was at 310–312 nm, similar to a tyrosine residue in solution. The fluorescence intensity increased 22% upon reduction of plastocyanin at pH 7.0. In poplar plastocyanin, the emission maximum was shifted to 335 nm and increased only 10% upon reduction. The 335 nm emission peak observed in poplar plastocyanin is attributed to Tyr 80 which is hydrogen bonded to a carbonyl group on the protein backbone. Tyr 83 was also shown to undergo fluorescence changes upon reduction since the redox state-dependent fluorescence changes decreased for a nitrotyrosine (nitrotyrosine-plastocyanin) derivative of this residue. These results show that the east face of the molecule, which contains both Tyr 80 and 83 as well as a possible binding site [1,2], undergoes conformational changes upon reduction. These conformational changes may be involved in promoting smooth electron transport between plastocyanin and its reaction partners. Both the absorption and fluorescence were found to be pH dependent. The quantum yield for fluorescence increased sharply below pH 6 for both oxidized and reduced spinach plastocyanin. This may be related to the appearance of a redox-inactive form of reduced plastocyanin [3]. The conformational changes observed at low pH may provide a mechanism for control of electron transport by the proton gradient. Low concentrations of CaCl₂ (10 mM) had no effect on plastocyanin fluorescence. However, addition of 2.7 M (NH₄)₂SO₄ eliminated the redox-dependent fluorescence changes.     

Reversible unfolding of bovine beta-lactoglobulin mutants without a free thiol group

Bovine beta-lactoglobulin (beta-lg) has been used extensively as a model for studying protein folding. One of the problems preventing clarification of the folding mechanism is the incomplete reversibility from the unfolded state, probably caused by the thiol-disulfide exchange between a free thiol at Cys-121 and two disulfide bonds. We constructed and expressed three beta-lg subtype A mutants in which Cys-121 was replaced by Ala, Ser, or Val (i.e. C121A, C121S, and C121V). We studied the reversibilities of these mutants from urea denaturation using circular dichroism, tryptophan fluorescence, reversed-phase and gel-filtration high performance liquid chromatographies, and SDS-PAGE. The folded structure of each mutant was similar to that of wild-type beta-lg. Urea-induced unfolding at pH 7.0 and 3.0 showed that although the C121S mutation notably decreases the stability, the destabilizing effects of the C121A and C121V mutations are less severe. For all of the mutants, complete refolding from the unfolded state in 8 M urea at both pH 7.0 and 3.0 was observed. Kinetics of the formation of the irreversibly unfolded species of wild-type beta-lg in 8 M urea at pH 7.0 indicated that, first, an intramolecular thiol-disulfide exchange occurs to produce a mixture of species with non-native disulfide bonds followed by the intermolecular thiol-disulfide exchange producing the oligomers. These results indicate that intramolecular and intermolecular thiol-disulfide exchange reactions cause the low reversibility of wild-type beta-lg especially at neutral pH and that the mutation of Cys-121 improves the reversibility, enabling us to study the folding of beta-lg more exactly under various conditions.     

Comparison of the binding behavior of FCCP with HSA and HTF as determined by spectroscopic and molecular modeling techniques

The interaction of carbonylcyanide p‐(trifluoromethoxy) phenylhydrazone (FCCP) with human serum albumin (HSA) and human transferrin (HTF) was investigated using multiple spectroscopy, molecular modeling, zeta‐potential and conductometry measurements of aqueous solutions at pH 7.4. The fluorescence, UV/vis and polarization fluorescence spectroscopy data disclosed that the drug–protein complex formation occurred through a remarkable static quenching. Based on the fluorescence quenching, two sets of binding sites with distinct affinities for FCCP existed in the two proteins. Steady‐state and polarization fluorescence analysis showed that there were more affinities between FCCP and HSA than HTF. Far UV‐CD and synchronous fluorescence studies indicated that FCCP induced more structural changes on HSA. The resonance light scattering (RLS) and zeta‐potential measurements suggested that HTF had a greater resistance to drug aggregation, whereas conductometry measurements expressed the presence of free ions improving the resistance of HSA to aggregation. Thermodynamic measurements implied that a combination of electrostatic and hydrophobic forces was involved in the interaction between FCCP with both proteins. The phase diagram plots indicated that the presence of second binding site on HSA and HTF was due to the existence of intermediate structures. Site marker competitive experiments demonstrated that FCCP had two distinct binding sites in HSA which were located in sub‐domains IIA and IIIA and one binding site in the C‐lobe of HTF as confirmed by molecular modeling. The obtained results suggested that both proteins could act as drug carriers, but that the HSA potentially had a higher capacity for delivering FCCP to cancerous tissues. Copyright © 2013 John Wiley & Sons, Ltd.     

Interaction of virstatin with human serum albumin: spectroscopic analysis and molecular modeling

Virstatin is a small molecule that inhibits Vibrio cholerae virulence regulation, the causative agent for cholera. Here we report the interaction of virstatin with human serum albumin (HSA) using various biophysical methods. The drug binding was monitored using different isomeric forms of HSA (N form ～pH 7.2, B form ～pH 9.0 and F form ～pH 3.5) by absorption and fluorescence spectroscopy. There is a considerable quenching of the intrinsic fluorescence of HSA on binding the drug. The distance (r) between donor (Trp214 in HSA) and acceptor (virstatin), obtained from Forster-type fluorescence resonance energy transfer (FRET), was found to be 3.05 nm. The ITC data revealed that the binding was an enthalpy-driven process and the binding constants K(a) for N and B isomers were found to be 6.09×10(5 )M(-1) and 4.47×10(5) M(-1), respectively. The conformational changes of HSA due to the interaction with the drug were investigated from circular dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. For 1:1 molar ratio of the protein and the drug the far-UV CD spectra showed an increase in α- helicity for all the conformers of HSA, and the protein is stabilized against urea and thermal unfolding. Molecular docking studies revealed possible residues involved in the protein-drug interaction and indicated that virstatin binds to Site I (subdomain IIA), also known as the warfarin binding site.     

Site-site interactions in EF-hand calcium-binding proteins. Laser-excited europium luminescence studies of 9-kDa calbindin, the pig intestinal calcium-binding protein

Europium(III) binding to 9-kDa calbindin from pig intestines was studied by direct excitation of the 7Fo----5Do transition of the ion and by near-ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C-terminal metal-binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N-terminal site (site I) by europium gives excitation spectra that are pH-dependent and show a peak at 579.4 nm (peak 1a) at pH 5 which shifts to 578.7 nm (peak 1b) over the pH range 5-7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration. There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1-calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions.