Edward R. Birnbaum

Many-body nonradiative energy transfer in a crystalline europium (III) EDTA complex

A pulsed, tunable dye laser has been used to excite the 7F0→ 5D0 electronic transition of the europium (III) ion in single crystals of Na[Eu(EDTA)(H2O)4]doped with varying concentrations of gadolinium. since this electronic transition cannot be split by crystal fields exerted by the ligands surrounding the metal ion, the noticeably broadened and asymmetric excitation spectra observed for this electronic transition at high europium concentrations have been ascribed to interactions between excited europium (III) ions and adjacent, unexcited europium (III) ions, resulting in energy transfer between ions. The lifetimes of these Na[Eu(EDTA)(H2O)4]crystals have been measured and a linear relationship was found between the energy transfer probability (Pda) and the square of the europium (III) concentration. The many-body nonradiative energy transfer process proposed by Fong and Diestler has been used to interpret the observed lifetime data, and the corresponding mechanism has been verified to be due to an electric dipole-dipole interaction. This model is consistent with one excited europium (III) ion transferring energy to two adjacent, unexcited europium (III) ions. As expected, the nonradiative energy transfer disappears if the % europium is decreased in the single crystal by doping small amounts of europium (< 0.3%) into a gadolinium matrix. The lifetime, excitation and emission spectra of a series of complexes containing different percentages of europium will be discussed in terms of nonradiative energy transfer theory.

Distance measurements between the metal-binding sites in thermolysin using terbium ion as a fluorescent probe

Abstract A single terbium ion has been introduced into thermolysin replacing two of the four calcium ions, and the fluorescence properties of the protein-bound terbium have been studied. The fluorescence of Tb+3 is tremendously enhanced (∼7 × 103) upon binding and is significantly quenched when divalent cobalt is substituted for the zinc ion normally found in the enzyme. By use of the Forster equation for energy transfer the distance between the protein-bound Tb+3 and Co+2 in the active site was calculated to be 13.6±0.5 A. This agrees closely with the value of 13.9 A obtained from the crystal structure and suggests that energy transfer between the two metal ions bound to the protein takes place by a dipole-dipole mechanism.

Synthesis of a new chelating gel: Removal of Ca2+ ions from parvalbumin

The synthesis of a chelating gel which contains the effective metal chelating agent ethylenediaminetetraacetic acid covalently linked to amino-agarose is described. This gel is shown to be a rapid and extremely effective material for the removal of tightly bound, but labile metal ions from proteins without introducing contaminants into the biological system. The synthesis involves the formation of an amide linkage between the dangling carboxylate arm of the [Co(EDTA)Cl]2-complex and amino-agarose using a standard carbodiimide coupling reaction. The chelating gel is shown to remove approximately 98.5% of the calcium from fully bound Ca2-parvalbumin and over 99% of the europium from Eu2-parvalbumin.

Investigation of Eu(III) Binding Sites on Datura innoxia Using Eu(III) Luminescence

A pulsed tunable dye laser has been used to obtain excitation spectra and fluorescence decay curves of solid Eu(III)-Datura innoxia and from a series of Eu(III)-containing complexes. Carboxyl and sulfate groups have been demonstrated to be the dominant functional groups for forming binding sites on the cell wall of Datura innoxia at high (≥4) and low (≤3) pH conditions, respectively. The excitation spectra associated with the 7F0 → 5D0 electronic transition of Eu(III) luminescence have been used to provide a measure of the electronic structure factors contributing to the interaction between Eu(III) ions and the binding sites on the cell wall of D. innoxia. The noticeably broadened and asymmetric excitation spectra obtained at high pH conditions are ascribed to multiple binding sites. The corresponding lifetime decay curves exhibited a bi-exponential decay. A pK of 4.5 was determined for the binding of Eu(III) to the cell wall at pH ≥4. Kinetic and thermodynamic studies were also undertaken.

Photoredox reactions of metal ions for photochemical solar energy conversion

Abstract Solar energy conversion to chemical potential energy is thermodynamically feasible by many routes. One possible route is the photochemical reaction of metal ions in water to produce hydrogen and an oxidizer. The photooxidation of several low-valent transition metal ions, including europium(II), vanadium(II), and copper(I) complexes, proceeds in aqueous acidic media according to: Quantum yields in 1.0 M hydrochloric or perchloric acid at 313 nm are: Φ Eu(III) = 0.16, Φ V(III) = 0.15, and Φ Cu(II) = 0.34. This reaction proceeds in visible light with a minimum of photochemical complications for Eu(II) and Cu(I) salts, and since the oxidation of copper(I) halo-complexes is endergic and hence potentially useful for energy storage, the mechanism of photooxidation has been studied. The product quantum yield is strongly affected by the acidity, irradiation wavelength and H-atom scavengers. Photoredox reactions of a number of metal ions and the requirements for using such in a solar energy scheme are discussed.

A study of carboxylic and amino acid complexes of neodymium(III) by difference absorption spectroscopy

Abstract The interaction of neodymium(III) with acetate, alanine, histidine, benzoate, and anthranilate has been studied using changes in the visible absorption spectrum of neodymium(III) upon complexation. At pHs below 6 only the carboxyl group of alanine coordinates to the metal ion, whereas at pHs near 7 the α-amino group begins to coordinate. The carboxyl group alone of histidine coordinates below pH 4. Between pH 4 and 6.5 the imidazole group of histidine coordinates forming a bidentate complex, and above pH 6.5 the α-amino group also begins to coordinate forming a tridentate complex. From a comparison of benzoate and anthranilate complexes it is clear that the amino group of anthranilate does coordinate to form a bidentate complex at a pH very near the pKa of the amino group of anthranilate.

A determination of the relative compactness of the Ca2+-binding sites of a Ca2+-binding fragment of troponin-c and parvalbumin using lanthanide-induced 1H NMR shifts

The calcium regulation of muscle contraction in vertebrate skeletal muscle results from the interaction of calcium with the protein complex troponin [ 11. Troponin is composed of three subunits, of which only TnC appears to bind Ca2‘ [2,3]. The amino acid sequence of TnC has been determined [4,5]. TnC has a strong sequence homology to carp parvalbumin [6,7]. Parvalbumin is roughly half of the size of TnC and binds 2 mol Ca2+ [8,9] compared to 4 mol Ca2+ in TnC [lo]. The crystal structure of parvalbumln from carp has been determined [ 11 ,I 21. The similarity between the primary sequences of TnC and parvalbumin suggests a possible location in the sequence of TnC for the 4 Ca”-binding sites [4,6]. Recently there has been considerable interest in studying the properties of fragments of TnC and parvalbumin containing single Ca”-binding sites. A cyanogen bromide fragment of TnC, labelled CB-9, that binds a single Ca2’ and mimics the conformational change of TnC upon Ca2+ binding has been reported [ 13-151. This fragment contains 52 residues from LYS 84 through homoserine 135, corresponds to the third Ca2+-binding site in the intact protein, and binds Ca2* with an affinity about two orders of magnitude lower than the intact protein. Similarly, parvalbumin has been split into two fragments A and B corresponding to the amino acids l-+75 and 76+108 in the intact molecule [ 16,171. These fragments bind Ca2+ with dissociation constants several orders of magnitude less strongly than intact parvalbumin [9].

The location of the lanthanide ion binding site on bovine trypsin

Summary Using the effect of a paramagnetic probe, Gd +3 , on the NMR relaxation time of inhibitor protons, the metal-inhibitor distances in a bovine trypsin ternary complex has been measured. The decrease due to Gd +3 in the spin-spin and spin-lattice relaxation times of the ortho and methyl protons of the inhibitor, p -toluamidine, has been measured at pH 6. The Solomon-Bloembergen equations were used to calculate distances of 8.8 ± 0.5 A and 10.0 ± 0.5 A from the metal ion to the ortho and methyl protons, respectively. From examination of the crystal structure of the enzyme it appears that the side chains of Asp 194 and Ser 190 are likely ligands for the metal ion.

The calcium ion binding site in bovine chymotrypsin A

Abstract The effect of Gd 3+ on the nuclear magnetic resonance (nmr) relaxation rates, T 1 m −1 and T 2 m −1 , of inhibitor protons in metal-inhibitor-α-chymotrypsin ternary complexes has been measured. The Solomon-Bloembergen equations were used to calculate the distance from the methyl protons of p -toluamidine (a competitive inhibitor) to the Gd 3+ binding site which is 9.2 ± 0.5 A. Calcium ion and gadolinium ion compete for the same binding site on α-chymotrypsin. Distances from the specificity pocket of α-chymotrypsin to the metal binding site have been measured by fluorescence energy transfer experiments. By observing energy transfer between proflavine and Nd 3+ , Pr 3+ , or Ho 3+ , we have been able to calculate a distance of approximately 10 A between the two chromophores. This agrees well with the data obtained by nmr techniques and also gives nearly identical values to those obtained for trypsin (Darnall, D., Abbott, F., Gomez, J. E., and Birnbaum, E. R., Biochemistry 15 , 5017, 1976). This is consistent with the calcium ion binding sites being composed of the same residues in both trypsin and α-chymotrypsin.

Tyrosine fluorescence as a measure of denaturation in thermolysin

The heat and guanidine hydrochloride denaturation of thermolysin has been followed by fluorescence techniques. The native enzyme has a single emission peak which is decreased in intensity and which splits into two clearly resolved peaks upon denaturation. These data are interpreted to indicate that energy transfer from tyrosine to tryptophan occurs in the native enzyme which is lost upon denaturation. Even though zinc is fully bound to thermolysin at 90 degrees C or in the presence of 6 M guanidine hydrochloride, removal of zinc from the denatured enzyme has no effect on the emission spectrum. Removal of Ca2+ from the denatured enzyme. These data indicate that even though the metal ions are bound to the denatured protein, they provide little structural integrity to the protein as measured by energy transfer between tyrosine and tryptophan.