Interactions of the lanthanide- and hapten-binding sites in the Fv fragment from the myeloma protein MOPC 315.

1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with the Fv fragment of the mouse myeloma protein MOPC 315 were investigated by the techniques of fluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclear magnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenched by the haptens (fluorescence enhancement, epsilon=0.15) and enhanced by Gd(III) (epsilon=1.14) and other lanthanides. The binding of the haptens studied here is insensitive to pH in the range 5.5-7.0 (dissociation constant KH=0.3-1.0 muM) and shows 1:1 stoicheiometry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent; the binding constant (KM) varies from 10 muM at pH7.0 to 700 muM at pH4.8. La(III) binding is less sensitive to pH. The pH-dependences of the metal-binding constants imply that a group in the protein with pKa greater than or equal to 6.2 is involved in the binding, and probably also other groups with lower pKa values. 3. The apparent binding of the haptens is weakened about 20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complex with an interaction between the two binding sites is derived in Appendix I to explain the experimental results at two pH values. 4. Time-dependent fluorescence changes are observed in the presence of Gd(III) at pH5.5. A two-state kinetic scheme involving a 'slow' conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour. 5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on the subsequent addition of lanthanides are consistent with the binding scheme for haptens and lanthanides proposed from the fluorescence studies. A difference between the limiting quenching of the e.s.r. signal from the bound haptens in the presence of saturating concentrations of Gd(III) and La(III) is attributed to dipolar interactions between bound Gd(III) and the nitroxide moiety of the bound hapten. The residual quenching with Gd(III) allows an estimate of 1.2nm to be made for the distance between the two paramagnetic centres. 6. The 270 MHz proton difference spectrum of the Fv fragment resulting from the addition of La(III) suggests that any metal-induced conformational changes are small and involve relatively few amino acid residues on the Fv fragment...     

Binding of lanthanum and gadolinium ions to concanavalin A studied calorimetrically at 25 degrees C

The interaction between Concanavalin A (ConA) and the lanthanide ions La3+ and Gd3+ has been studied calorimetrically at 25 degrees C. The measurements were carried out at a pH of 4.5, where the protein exists prevailingly as a dimer. Calorimetry allows the direct determination of the binding enthalpy and the evaluation of both the apparent association constant, and the apparent free energy and entropy. Three groups of data were collected. The first concerns the interaction of the 'native' protein, i.e., fully metallized with Mn2+ and Ca2+, with the lanthanides. The second concerns the interaction of the completely demetallized protein with La3+ and Gd3+. Finally, the affinity of each complex was tested for the specific sugar alpha-methylmannopyranoside. The analysis of the thermodynamic parameters obtained, led to the following conclusions: 1) a specific site, named S3, exists on the protein for the lanthanides, distinct from the S1 site of the transition metal and from the S2 site, specific for calcium. There is only one S3 site per protomer when the protein has Mn2+ in S1 and Ca2+ in S2. Moreover, there is no appreciable competition for S1 and S2 from the lanthanides. The 'native' protein, metallized with La3+ or Gd3+, is a fully functional protein. 2) The demetallized protein (ApoCon A) has at least two sites per protomer for the lanthanides. The hypothesis is that, besides the S3 site, the lanthanides, in the absence of Mn2+, can also occupy the S1, but not the S2, site. The protein metallized only with gadolinium ion is completely inactive toward the interaction with the mannoside. The same happens when, along with gadolinium, only calcium or manganese is present. Hence, in the absence of the transition metal in S1 or of calcium in S2, the protein is not in the conformation suitable to interact with its specific substrate.     

NMR studies on the surface accessibility of the archaeal protein Sso7d by using TEMPOL and Gd(III)(DTPA-BMA) as paramagnetic probes

Understanding how proteins are approached by surrounding molecules is fundamental to increase our knowledge of life at atomic resolution. Here, the surface accessibility of a multifunctional small protein, the archaeal protein Sso7d from Sulfolobus solfataricus, has been investigated by using TEMPOL and Gd(III)(DTPA-BMA) as paramagnetic probes. The DNA binding domain of Sso7d appears very accessible both to TEMPOL and Gd(III)(DTPA-BMA). Differences in paramagnetic attenuation profiles of (1)H-(15)N HSQC protein backbone amide correlations, observed in the presence of the latter paramagnetic probes, are consistent with the hydrogen bond acceptor capability of the N-oxyl moiety of TEMPOL to surface exposed Sso7d amide groups. By using the gadolinium complex as a paramagnetic probe a better agreement between Sso7d structural features and attenuation profile is achieved. It is interesting to note that the protein P-loop region, in spite of the high surface exposure predicted by the available protein structures, is not approached by TEMPOL and only partially by Gd(III)(DTPA-BMA).     

Spectroscopic and thermodynamic studies on the binding of gadolinium(III) to human serum transferrin

A wide variety of thermodynamic, kinetic, and spectroscopic studies have demonstrated differences between the two metal-binding sites of transferrin. In the present investigation, we have further assessed these differences with respect to the binding of gadolinium, evaluated by UV difference spectrophotometry, electron paramagnetic resonance (EPR) titration, EPR difference spectroscopy in conjunction with urea gel electrophoresis, and equilibrium dialysis. Combinations of these studies establish that only one site of the protein binds Gd(III) sufficiently firmly to be characterized. In order to reveal which of the two sites accepts Gd(III), we made use of monoferric transferrins preferentially loaded with Fe(III) at either site in EPR spectroscopic studies. Because of the overlap of signals, difference spectroscopy was required to distinguish resonances arising from Fe(III) and Gd(III) specifically complexed to the protein. When iron is bound to the C-terminal site, leaving the N-terminal site free for binding of gadolinium, the difference spectrum shows no evidence of specific binding. However, when iron is bound to the N-terminal site, the difference spectrum shows a resonance line at g' = 4.1 indicative of specific binding, thus implicating the C-terminal site in the binding of Gd(III). The effective stability constant for the binding of Gd(III) to this site of transferrin at pH 7.4 and ambient pCO2 is 6.8 X 10(6) M-1. At physiological pCO2, the formation of nonbinding carbonato complexes of Gd(III) precludes a substantial role for transferrin in the transport of the lanthanide in vivo.     

Gd(III) complexes as contrast agents for magnetic resonance imaging: a proton relaxation enhancement study of the interaction with human serum albumin

 The non-covalent interaction between human serum albumin (HSA) and DOTA-like Gd(III) complexes containing hydrophobic benzyloxymethyl (BOM) substituents has been thoroughly investigated by measuring the solvent proton relaxation rates of their aqueous solutions. The binding association constants (K _A) to HSA are directly related to the number of hydrophobic substituents present on the surface of the complexes. Furthermore, an estimation of ΔH° and ΔS° has been obtained by the temperature dependence of K _A. Assays performed with the competitor probes warfarin and ibuprofen established that the complexes interact with HSA through two nearly equivalent binding sites located in the subdomains IIA and IIIA of the protein. Strong relaxation enhancements, promoted by the formation of slowly tumbling paramagnetic adducts, have been measured at 20 MHz for complexes containing two and three hydrophobic substituents. The macromolecular adduct with the latter species has a relaxivity of 53.2±0.7 mM^–1 s^–1, which represents the highest value so far reported for a Gd(III) complex. The temperature dependence of the relaxivity for the paramagnetic adducts with HSA indicates long exchange lifetimes for the water molecules dipolarly interacting with the paramagnetic centre. This is likely to be related to the formation, upon hydrophobic interaction of the complexes with HSA, of a clathrate-like, second-coordination-sphere arrangement of water molecules. Besides affecting the dissociative pathway of the coordinated water molecule, this water arrangement may itself significantly contribute to enhancement of the bulk solvent relaxation rate. Received: 6 November 1995 / Accepted: 17 April 1996     

Synthesis and characterization of new porphyrazine-Gd(III) conjugates as multimodal MR contrast agents

Magnetic resonance imaging (MRI) has long been used clinically and experimentally as a diagnostic tool to obtain three-dimensional, high-resolution images of deep tissues. These images are enhanced by the administration of contrast agents such as paramagnetic Gd(III) complexes. Herein, we describe the preparation of a series of multimodal imaging agents in which paramagnetic Gd(III) complexes are conjugated to a fluorescent tetrapyrrole, namely, a porphyrazine (pz). Zinc metalated pzs conjugated to one, four, or eight paramagnetic Gd(III) complexes are reported. Among these conjugates, Zn-Pz-8Gd(III) exhibits an ionic relaxivity four times that of the monomeric Gd(III) agent, presumably because of increased molecular weight and a molecular relaxivity that is approximately thirty times larger, while retaining the intense electronic absorption and emission of the unmodified pz. Unlike current clinical MR agents, Zn-Pz-1Gd(III) is taken up by cells. This probe demonstrates intracellular fluorescence by confocal microscopy and provides significant contrast enhancement in MR images, as well as marked phototoxicity in assays of cellular viability. These results suggest that pz agents possess a new potential for use in cancer imaging by both MRI and near-infrared (NIR) fluorescence, while acting as a platform for photodynamic therapy.     

The simultaneous binding of lanthanide and N-acetylglucosamine inhibitors to hen egg-white lysozyme in solution by 1H and 13C nuclear magnetic resonance.

Lanthanide ions and the N-acetylglucosamine (GlcNAc) sugars are able to bind simultaneously to hen egg-white lysozyme (EC 3.2.1.17). The present study characterizes the properties of the ternary complexes with lysozyme, which involve up to seven paramagnetic lanthanides and two diamagnetic lanthanides, together with alpha GlcNAc, beta GlcNAc, alpha MeGlcNAc and beta MeGlcNAc. pH titrations and binding titrations of the GlcNAc sugars with lysozyme-La(III) complexes show that the GlcNAc sugars bind to at least two independent sites and that one of them competes with La(III) for binding to lysozyme. Given the known binding site of lanthanides at Asp-52 and Glu-35, the competitive binding site of GlcNAc is identified as subsite E. A simple analysis of the paramagnetic-lanthanide-induced shifts shows that the GlcNAc sugar binds in subsite C, in accordance with crystallographic results [Perkins, Johnson, Machin & Phillips (1979) Biochem. J. 181, 21-36]. This finding was refined by several computer analyses of the lanthanide-induced shifts of 17 proton and carbon resonances of beta MeGlcNAc. Good fits were obtained for all the signals, except for two that were affected by exchange broadening phenomena. No distinction could be made between a fit for a two-position model of Ln(III) binding with axial symmetry to lysozyme, according to the crystallographic result, or a one-position model with axial symmetry where the Ln(III) is positioned mid-way between Asp-52 and Glu-35. Although this work establishes the feasibility of lanthanide shift reagents for study of protein-ligand complexes, further work is required to establish the manner in which lanthanides bind to lysozyme in solution.     

MD and NMR studies of alpha-bungarotoxin surface accessibility

Protein surface accessibility represents a dimension of structural biology which has not been discussed in details so far, in spite of its fundamental role in controlling the molecular recognition process. In the present report the surface accessibility of alpha-bungarotoxin, a small and well characterized protein, has been investigated by analyzing its interaction with solvent and paramagnetic molecules in an integrated way. The presence of strong hydration sites, identified by a combined analysis of MD simulation and NMR results, seems to prevent the access of Gd(III)DTPA-BMA to the protein surface. On the contrary, the limited hydration of the alpha-bungarotoxin active site favors frequent encounters between the paramagnetic probe and the protein in the latter region. All the data obtained here for alpha-bungarotoxin suggest that shape and stability of the solvation shell control its surface accessibility and, hence, intermolecular interactions in a way which could be common to many other proteins.     

Ternary Gd(III)L-HSA adducts: evidence for the replacement of inner-sphere water molecules by coordinating groups of the protein. Implications for the design of contrast agents for MRI

Two novel gadolinium(III) chelates based on the structure of the heptadentate macrocyclic 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) ligand have been synthesized and their relaxometric and luminescent properties thoroughly investigated. They contain two water molecules in the inner coordination sphere in fast exchange with the bulk solvent and bear either a p-bromobenzyl or a p-phosphonatomethylbenzanilido substituent for promoting further interaction with macromolecular substrates. Upon interaction with human serum albumin the expected relaxation enhancement is not observed owing to displacement of the two inner-sphere water molecules of the complexes by a donor atom (likely from a carboxylate group) on the protein and possibly the phosphate anion of the buffered solution, respectively. We modeled the observed behavior by measuring the decrease of the relaxation rate of the water protons upon addition of malonate anion to aqueous solutions of the complexes. Conversely, no change in the hydratation state of the Gd(III) center for both complexes has been observed when the substrate for the formation of the macromolecular adduct is represented by poly-beta-cyclodextrin.     

An electron paramagnetic resonance study of bovine alpha-lactalbumin-metal ion complexes

alpha-lactalbumin has at least three distinct cation binding regions: a Ca(II)-Gd(III) site, a Cu(II)-Zn(II) site and a VO2+ site as observed from electron paramagnetic resonance (EPR) studies of complexes with the bovine protein. Gadolinium, which bound to the calcium site of the protein with a subnanomolar dissociation constant, yielded EPR spectra at 9.5 GHz (X-band) that exhibited features from g = 8 to g = 2. At 35 GHz (Q-band) the central fine structure transition (Ms = 1/2----Ms = -1/2) gave a well-defined powder pattern. The zero-field splitting was large, as reflected in the second-order splitting of the central fine structure transition of about 1 kG. There was also evidence for additional, low affinity binding site(s) for Gd(III). Addition of either Zn(II) or Al(III) did not affect the amplitudes or positions of the bound Gd(III) EPR spectrum. The Cu(II)-alpha-lactalbumin complex gave a typical axially symmetric spectrum (g parallel = 2.260, g perpendicular = 2.056, A parallel = 171 G) with a partially resolved superhyperfine interaction attributable to at least one directly coordinated nitrogen ligand. Addition of Cu(II) to Gd(III)-alpha-lactalbumin gave an EPR spectrum that was a superposition of signals from the individual Gd(III)- and Cu(II)-alpha-LA spectra. The absence of any magnetic interactions in the Gd(III)-Cu(II)-alpha-lactalbumin species indicated that the two cation sites were more than 10 A apart. On the other hand, addition of Zn(II) to Cu(II)-alpha-lactalbumin gave a set of EPR lines due to free or loosely bound Cu(II), confirming that the Cu(II) was displaced by zinc.(ABSTRACT TRUNCATED AT 250 WORDS)     

Towards MRI contrast agents of improved efficacy. NMR relaxometric investigations of the binding interaction to HSA of a novel heptadentate macrocyclic triphosphonate Gd(III)-complex

 A novel heptacoordinating ligand consisting of a thirteen-membered tetraazamacrocycle containing the pyridine ring and bearing three methylenephosphonate groups (PCTP-[13]) has been synthesized. Its Gd(III) complex displays a remarkably high longitudinal water proton relaxivity (7.7 mM^–1 s^–1 at 25  °C, 20 MHz and pH 7.5) which has been accounted for in terms of contributions arising from (1) one water molecule bound to the metal ion, (2) hydrogen-bonded water molecules in the second coordination sphere, or (3) water molecules diffusing near the paramagnetic chelate. Variable-temperature ¹⁷O-NMR transverse relaxation data indicate that the residence lifetime of the metal-bound water molecule is very short (8.0 ns at 25  °C) with respect to the Gd(III) complexes currently considered as contrast agents for magnetic resonance imaging. Furthermore, GdPCTP-[13] interacts with human serum albumin (HSA), likely through electrostatic forces. By comparing water proton relaxivity data for the GdPCTP-[13]-HSA adduct, measured as a function of temperature and magnetic field strength, with those for the analogous adduct with GdDOTP (a twelve-membered tetraaza macrocyclic tetramethylenephosphonate complex lacking a metal-bound water molecule), it has been possible to propose a general picture accounting for the main determinants of the relaxation enhancement observed when a paramagnetic Gd(III) complex is bound to HSA. Basically, the relaxation enhancement in these systems arises from (1) water molecules in the hydration shell of the macromolecule and protein exchangeable protons which lie close to the interaction site of the paramagnetic complex and (2) the metal bound water molecule(s). As far as the latter contribution is concerned, the interaction with the protein causes an elongation of the residence lifetime of the metal-bound water molecule, which limits, to some extent, the potential relaxivity enhancement expected upon the binding of the paramagnetic complex to HSA. Received: 27 January 1997 / Accepted: 12 May 1997     

Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis

Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein–ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond. Both protein-tag adducts form stable lanthanide complexes, of which the binding affinities and paramagnetic tensors are tunable with respect to the 6-methyl group in pyridine. Paramagnetic relaxation enhancement (PRE) effects of Gd(III) complex on protein-tag adducts were evaluated in comparison with pseudocontact shift (PCS), and the results indicated that both 4PS-PyDTTA and 4PS-6M-PyDTTA tags are rigid and present high-quality PREs that are crucially important in elucidation of the dynamics and interactions of proteins and protein-ligand complexes. We also show that these two tags are suitable for in-situ protein NMR analysis.     

The identification of cation-binding domains on the surface of microsomal cytochrome b5 using 1H-NMR paramagnetic difference spectroscopy.

One-dimensional and two-dimensional 1H-NMR methods and paramagnetic difference spectroscopy have defined cation binding domains on the surface of the tryptic fragment of microsomal cytochrome b5. The addition of tris(ethylenediamine) chromium(III) [Cr(en)3(3+)] to solutions of ferricytochrome b5 reveals at least three distinct sites on the surface of the protein to which highly charged cations may bind (20 mM phosphate pH 7.0, T = 300 K). Surprisingly only one of these sites is located close to the haem edge region of the protein, whilst the remaining two sites are more remote. Site I contains the exposed haem C13 propionate and a series of carboxylate residues that includes glutamates 37, 38, 43, 44, and 48. Sites II and III are located away from the haem edge region and are delineated by the broadening of aromatic resonances of histidines 26 and 80. Further investigation of the interaction between Cr(en)3(3+) and cytochrome b5 using two-dimensional double-quantum-filtered correlated spectroscopy shows that resonances assigned to Glu59, Asp60, Glu79, Asp82 and Asp83 are broadened with the distribution of these charged side chains correlating with the relaxation broadening observed from one-dimensional experiments. In a binary complex with ferricytochrome c, Cr(en3(3+) broadens many cytochrome b45 resonances including the haem propionates, His26, Ala54, Thr55 and His80. Although the pattern of line-broadening of resonances at sites II and III is unaltered by complex formation, cytochrome c shields residues at site I, the haem edge site. The results indicate that the interaction between cytochrome b5 and c in a binary complex involves multiple protein configurations.     

A decadentate Gd(III)-coordinating paramagnetic cosolvent for protein relaxation enhancement measurement

Solvent paramagnetic relaxation enhancement (sPRE) arises from random collisions between paramagnetic cosolvent and protein of interest. The sPRE can be readily measured, affording protein structure information. However, lack of an inert cosolvent probe may yield sPRE values that are not consistent with protein structure. Here we synthesized a new sPRE probe, triethylenetetraamine hexaacetate trimethylamide gadolinium, or Gd(III)–TTHA–TMA. With a total of 10 coordination sites, this paramagnetic cosovlent eliminates an inner-sphere water molecule that can otherwise transfer relaxation to protein through exchange. With the metal ion centered, the new probe is largely spherical with a radius of 4.0 Å, permitting accurate back calculation of sPRE. The effectiveness Gd(III)–TTHA–TMA as a sPRE probe was demonstrated on three well-studied protein systems.     

New paramagnetic supramolecular adducts for MRI applications based on non-covalent interactions between Gd(III)-complexes and beta- or gamma-cyclodextrin units anchored to chitosan

Gd(III) complexes are used as magnetic resonance imaging (MRI) contrast agents because they greatly enhance the relaxation rate of water protons of tissues in which they distribute, an effect that is much more marked if the paramagnetic complex is part of a macromolecular system. Furthermore applications in molecular imaging, require that as many units of contrast agent as possible be directed to the site of interest. To this end we synthesised a polymer made of chitosan functionalized with beta- and gamma-cyclodextrins (CDs) that is able to form high-affinity adducts with suitably functionalized Gd(III) complexes. beta- and gamma-CDs were first treated with maleic anhydride to afford 6-monosubstituted derivatives that reacted regioselectively with the amino groups of chitosan. Reaction times and yields were markedly improved by carrying out these reactions under high-intensity ultrasound or microwave irradiation. Compared to the CD monomers, beta- and gamma-CD-chitosan adducts show large increases both in terms of their binding affinity towards Gd(III) complexes and in relaxivity values and they appear promising carriers for the in vivo vehiculation of Gd(III) complexes.     

NMR studies of BPTI aggregation by using paramagnetic relaxation reagents

Paramagnetic probes, whose approach to proteins can be monitored by nuclear magnetic resonance (NMR) studies, have been found of primary relevance for investigating protein surfaces accessibility. Here, paramagnetic probes are also suggested for a systematic investigation on protein aggregation. Bovine pancreatic trypsin inhibitor (BPTI) was used as a model system for aggregation by analyzing its interaction with TEMPOL and Gd(III)DTPA-BMA. Some of the measured paramagnetic relaxation rates of BPTI protons exhibited a reverse dependence on protein concentration, which can be attributed to the formation of transient BPTI aggregates.     

Influence of paramagnetic ions bound to human serum albumin on water 1HNMR relaxation times

The extent to which various paramagnetic ions (Cu2+, Mn2+ and Gd3+) free and bound to human serum albumin alter the water proton relaxation times at two frequencies has been investigated. NMR relaxation parameters, T1 and T2, were measured at 5 and 10 MHz using a saturation recovery (90 degrees-tau-90 degrees) and a spin-echo (90 degrees-tau-180 degrees) sequence respectively. We found that all three ions enhance their effectiveness in inducing water proton magnetic relaxation when they are bound to human serum albumin and that Gd3+ is the most effective in pure water and Mn2+ in the presence of the protein. Cu2+ has a smaller effect, but it presents an interesting behaviour correlated with the existence of two different binding sites, which is also confirmed by electronic paramagnetic resonance spectra. The results indicate the potential usefulness of large molecular paramagnetic complexes as contrast agents in NMR Imaging.     

The interaction of lanthanides with isolated sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

The interaction of lanthanides with isolated sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle and the effects of lanthanides on 45Ca2+ uptake by the vesicles were studied. 153Gd3+ was taken up by the vesicles in the absence of ATP and oxalate in a time-dependent manner, reaching a maximum total accumulation of 380 nmol 153Gd3+/mg protein after 20 min with 200 microM 153Gd3+. This 153Gd3+ accumulation was not washed out by 1 mM EGTA. The addition of ATP induced the release of 87% of the bound 153Gd3+, leaving behind irreversibly-accumulated 153Gd3+. Pre-incubation of the vesicles with lanthanides in the absence of ATP and oxalate inhibited 45Ca2+ uptake without affecting Ca2+-ATPase activity. The percent inhibition of 45Ca2+ uptake increased with length of pre-incubation of the vesicles with lanthanides, reaching 33% after 20 min of pre-incubation. Increasing the 45Ca2+ concentration or adding ATP or oxalate to the preincubation medium abolished these inhibitory effects on 45Ca2+ uptake.     

The binding of lanthanides to non-immune rabbit immunoglobulin G and its fragments.

The binding of Gd(III) to rabbit IgG (immunoglobulin G) and the Fab (N-terminal half of heavy and light chain), (Bab')2 (N-terminal half of heavy and light chains joined by inter-chain disulphide bond), Fc (C-terminal half of heavy-chain dimer)and pFc' (C-terminal quarter of heavy-chain dimer) fragments was demonstrated by measurements of the enhancement of the solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 there are six specific Gd(III)-binding sites on the IgG. These six sites can be divided into two classes; two very 'tight' sites on the Fc fragment (Kd approx. 5 muM) and two weaker sites on each Fab region (Kd approx. 140 muM). Ca(II) does not apparently compete for these metal-binding sites. The metal-binding parameters for IgG can be explained as the sum of the metal binding to the isolated Fab and Fc fragments, suggesting that there is no apparent interaction between the Fab and Fc regions in the IgG molecule. The binding of Gd(III) to Fab and Fc fragments was also monitored by measuring changes in the electron-spin-resonance spectrum of Gd(III) in the presence of each fragment and also by monitoring the effects of Gd(III) on the protein fluorescence at 340 nm (excitation 295 nm). The fluorescence of Tb(III) solutions of 545 nm (excitation 295 nm) is enhanced slightly on addition of Fab or Fc.     

Calcium binding proteins: optical stopped-flow and proton nuclear magnetic resonance studies of the binding of the lanthanide series of metal ions to parvalbumin

Optical stopped-flow techniques have been used to determine the dissociation rate constants (koff) for the lanthanide(III) ions from carp (pI 4.25) parvalbumin. For most of the 13 different lanthanides studied, the release kinetics were diphasic, composed of both a fast phase (whose rate varied across the series, La3+ leads to Lu3+, between the limits -1.2 less than or equal to log kFAST less than or equal to -0.7) and a slower phase (whose rate varied across the series, La3+ leads to Lu3+, between the limits -1.2 greater than or equal to log kSLOW greater than or equal to -2.9). In addition, the La3+- and Lu3+-induced changes in the 270-MHz proton nuclear magnetic resonance spectrum of parvalbumin were used to calculate the dissociation constants for these specific lanthanides from the two high-affinity Ca2+ binding sites. The KD for one site appears to remain constant across the lanthanide series, determined to be 4.8 X 10(-11) M for both La3+ and Lu3+. The other site, however, is evidently quite sensitive to the nature of the bound Ln3+ ion and shows a strong preference for La3+ (KD,La = 2.0 X 10(-11) M; KD,Lu = 3.6 X 10(-10) M). We conclude from these observations that reports of nearly indistinguishable CD/EF binding site affinities for parvalbumin complexes of the middle-weight lanthanides (i.e., Eu3+, Gd3+, and Tb3+) are quite reasonable in view of the crossover in relative CD/EF site affinities across the lanthanide series.