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W D Horrocks

Publications and source records attributed to W D Horrocks.

At least 37 records · Page 2Linked to original sources
PubMed1989

Physical characterization of and ionophore-mediated europium(III) transport through unilamellar phosphatidylcholine vesicles. A laser-induced europium(III) luminescence spectroscopy study.

A continuation of the study of phospholipid bilayer vesicles as model membrane systems by laser-induced europium(III) luminescence spectroscopy is presented here (B.M. Cader and W. DeW. Horrocks, Jr, Biophys. Chem. 32 (1988) 97). This spectroscopic technique was used to characterize further the physical properties of small and large vesicles composed of dipalmitoylphosphatidylcholine and egg phosphatidylcholine, respectively. Unilamellar preparations were confirmed and internal aqueous volumes were calculated. The calcium-binding carboxylic ionophores, lasalocid A and A23187, were incorporated into the lipid bilayers of these vesicles for the purpose of modeling the mobile carrier mechanism of ion transport across cell membranes. Spectroscopic data implicate the presence of 1:1 and 1:2 europium(III)/lasalocid A complexes within the hydrophobic region, both capable of efficient transport and containing no water molecules in the inner sphere of europium(III). First-order rate constants for lasalocid A-mediated europium(III) transport were determined at 37 and 62 degrees C (0.018 and 0.11 min-1, respectively) using EGTA as a 'flag' to bind and detect the post-transported metal ion.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1988

An optical method for the determination of the isotopic composition of 1H2O/2H2O mixtures: Eu3+ luminescence lifetimes.

A spectroscopic method employing pulsed dye laser instrumentation is described for the determination of the 1H2O/2H2O composition of aqueous solutions by the measurement of reciprocal excited state lifetimes of EuEDTA-. The reciprocal lifetimes, gamma-1, of the 1H2O/2H2O mixtures increase linearly with the mole fraction of 1H2O. For EuEDTA- the relationship between gamma-1 and the mole fraction, chi H, of 1H2O in 1H2O/2H2O mixtures is expressed by the equation chi H = 0.37 gamma-1-0.152, with a sensitivity in chi H of +/- 0.02. The reciprocal lifetimes are independent of pH in the range 5.1 to 10.5, changes in ionic strength, and the type of buffer used in EuEDTA- containing solutions.

Deuterium↗
PubMed1988

Time-resolved europium(III) luminescence excitation spectroscopy: characterization of calcium-binding sites of calmodulin.

Pulsed-dye laser excitation and lifetime spectroscopy of the 7F0----5D0 transition of Eu3+ reveals details of the binding of this ion to the calcium-binding sites of calmodulin (labeled I-IV, starting at the N-terminus). For 10 microM calmodulin Eu3+ binds quantitatively at sites I and II and more weakly at sites III and IV with Kd values of approximately 0.5 microM and 1.0 microM at the latter sites. In D2O solution the time course of luminescence emission of Eu3+-loaded calmodulin can be separated into three exponential components with lifetimes of 2.50 (sites I and II) and 1.70 and 0.63 ms (sites III and IV). This finding permits the time resolution of the excitation spectrum by determination of the amplitudes of the three components as the excitation wavelength is scanned across the spectral profile in 0.1-nm increments. The amplitudes (intensities at time t = O) are plotted as a function of wavelength and the results fitted to three Lorentzian peaks centered at 579.20, 579.40, and 579.32 nm in order of decreasing lifetimes. In H2O solution only two exponential luminescence decay components are resolvable with lifetimes of 0.41 and 0.27 ms, corresponding to sites I and II and sites III and IV, respectively. These results indicate that two water molecules are coordinated to the Eu3+ ions at sites I and II and at either site III or site IV, with three water molecules at the remaining site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

A laser-induced europium (III) ion luminescence study of the interaction of this ion with phospholipid bilayer vesicles above and below the gel to liquid-crystalline phase transition temperature.

Laser-induced europium(III) luminescence spectroscopy was used to investigate the formation and integrity of phospholipid bilayer vesicles produced by ultrasonication and detergent dialysis. Eu(III) ion interactions with these model biological membrane systems were explored. 7F0----5D0 spectral and excited-state lifetime data reveal two distinct, temperature-dependent binding sites, one involving a weak, 'superficial' interaction with the phosphate moiety of the phosphatidylcholine head group, the other involving a more tightly bound ion in a relatively dehydrated region of the head group. This latter 'sequestered' species appeared only at temperatures equal to or below that of the gel----liquid-crystalline phase transition. Systems containing various amounts of cholesterol showed a decrease in the formation of the sequestered species, indicative of a decrease in ion permeability. The results of this study demonstrate that the Eu(III) luminescence technique is useful for detecting major phase alterations in phospholipid bilayer vesicles.

Europium↗
PubMed1985

Characterization of lanthanide (III) ion binding to calmodulin using luminescence spectroscopy.

Pulsed dye laser excitation spectroscopy of the 7F0----5D0 transition of Eu(III) reveals only a single peak as this ion is titrated into apocalmodulin. A titration based on the intensity of this transition shows that the first two Eu(III) ions bind quantitatively to two tight sites, followed by weaker binding (Kd = 2 microM) to two additional sites under conditions of high ionic strength (0.5 M KC1). This excitation experiment is also shown to be a general method for measuring contaminating levels of EDTA down to 0.2 microM in proton solutions. Experiments with Tb(III) using both direct laser excitation and indirect sensitization of Tb(III) luminescence through tyrosine residues in calmodulin also give evidence for two tight and two weaker binding sites (Kd = 2-3 microM). The indirect sensitization results primarily upon binding to the two weaker sites, implying that Tb(III) binds first to domains I and II, which are remote from tyrosine-containing domains III and IV. The 7F0----5D0 excitation signal of Eu(III) was used to measure the relative overall affinities of the tripositive lanthanide ions, Ln(III), across the series. Ln(III) ions at the end of the series are found to bind more weakly than those at the beginning and middle of the series. Eu(III) excited-state lifetime measurements in H2O and D2O reveal that two water molecules are coordinated to the Eu(III) at each of the four metal ion binding sites. Measurements of Förster-type nonradiative energy-transfer efficiencies between Eu(III) and Nd(III) in the two tight sites were carried out by monitoring the excited-state lifetimes of Eu(III) in the presence and absence of the energy acceptor ion Nd(III).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Spectroscopic studies of metal ion binding to a tryptophan-containing parvalbumin.

The binding of Ca(II) and members of the trivalent lanthanide ion, Ln(III), series to apoparvalbumin (isotype pI = 4.75) from codfish (Gadus callarius L) results in the development of a distinctive sharp feature in the UV absorption spectrum at about 290 nm. Titration curves obtained by monitoring the spectral change in this region reveal a change in slope after the addition of 1 equiv of metal ion and no further rise after 2 equiv has been added, consistent with sequential binding to the principal EF and CD sites. Laser-induced luminescence excitation spectra of the 7F0----5D0 transition of bound Eu(III) demonstrate the quantitative binding of this ion to the principal sites and disclose the presence of a subsidiary site at pH values greater than 6. Metal ion competition experiments monitored by means of this excitation transition show that the early members of the Ln(III) ion series bind more tightly than those at the end. Tryptophan-sensitized Tb(III) luminescence reveals that this ion binds sequentially to the EF and CD sites, in that order. The intrinsic tryptophan fluorescence of apoparvalbumin is increased in a stepwise fashion as Ca(II) or Ln(III) ions bind sequentially, with the exceptions of Eu(III) and Yb(III). The binding of the latter two ions causes quenching of the protein fluorescence via an energy-transfer process which involves low-lying charge-transfer bands. The distance dependences of the tryptophan to Tb(III) and tryptophan to Eu(III) energy-transfer processes are observed to be identical, consistent with a Förster-type mechanism in both cases.

Animals↗
PubMed1985

Stopped-flow kinetic studies of metal ion dissociation or exchange in a tryptophan-containing parvalbumin.

The rates of dissociation of 2 equiv of various metal ions [Ca(II), Cd(II), Pr(III), Nd(III), Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Yb(III), and Lu(III)] from the primary CD and EF metal ion binding sites of parvalbumin (isotype pI = 4.75) from codfish (Gadus callarius L) were measured by stopped-flow techniques. The removal or replacement of metal ions was monitored by changes in sensitized Tb(III) luminescence or in intrinsic protein tryptophan fluorescence as quenching ions [Eu(III) or Yb(III)] were bound or removed or as the apoprotein was formed. In experiments wherein the bound metal ions were removed by mixing the parvalbumin with an excess of 1,2-diaminocyclohexanetetraacetic acid (DCTA), the kinetic traces were best fit by a double exponential with koff rate constants of 1.07 and 5.91 s-1 for Ca(II), 1.54 and 10.5 s-1 for Cd(II), and approximately 0.05 and approximately 0.5 s-1 for all of the trivalent lanthanide ions. In experiments wherein the bound metal ions were exchanged with an excess of a different metal ion, pseudo-first-order rate constants were proportional to the concentration of excess attacking metal ion for both the fast and slow processes in most experiments. In these cases, extrapolation of the rate constants to zero concentration of attacking metal ion gave values which agree well with the DCTA scavenging results. This finding demonstrates that the off rate constants do not depend on the occupancy of the neighboring site and therefore implies that there is no significant cooperativity in metal ion binding between the two sites in parvalbumin.

Animals↗
PubMed1985

Comparative study of glutamine synthetase bound lanthanide(III) ions using NMR relaxation and lanthanide(III) luminescence techniques.

Changes in the intrinsic fluorescence intensity of glutamine synthetase induced by lanthanide(III) ion binding demonstrate the existence of three types of sites for these ions. The sites are populated sequentially during titrations of the enzyme, and the first two have a stoichiometry of 1 per enzyme subunit. The number of water molecules coordinated to Eu(III) bound to the first site was determined by luminescence lifetime techniques to be 4.1 +/- 0.5. The hydration of Gd(III) bound to the same site was studied by magnetic field dependent water proton longitudinal relaxation rate measurements, and by water proton and deuteron relaxation measurements of one sample at single magnetic fields. The magnetic resonance techniques also yield a value of 4 for the hydration number.

Binding Sites↗
PubMed1985

Catalytic Ca2+-binding site of pancreatic phospholipase A2: laser-induced Eu3+ luminescence study.

7F0----5D0 excitation spectroscopy of Eu3+ has been used to study the catalytic Ca2+-binding site of pancreatic phospholipases A2. Eu3+ binds competitively with Ca2+ to the enzyme with retention of about 5% of the activity found with Ca2+. The dissociation constants for the Eu3+-enzyme complexes of bovine phospholipase A2 and porcine isophospholipase A2 are 0.22 mM and 0.16 mM, respectively. Results obtained with the porcine phospholipase A2 at neutral pH indicate aggregation of this enzyme at protein concentrations above 0.18 mM. The Eu3+ bound at the catalytic site of pancreatic phospholipase A2 is coordinated to four or five water molecules, which, in conjunction with binding constant data, suggests the involvement of two or three protein ligands. Addition of a monomeric substrate analogue to the enzyme-Eu3+ complex results in the loss of an additional water molecule from the first coordination sphere of the bound Eu3+. This result suggests an interaction between the negative charge of the polar head group of the substrate analogue and the Eu3+. Binding of the enzyme-Eu3+ complex to micelles results in a nearly complete dehydration of the Eu3+ bound to the catalytic center. In the phospholipase A2-Eu3+-micelle complex, only one H2O molecule is coordinated to Eu3+. This dehydration at the active site of phospholipase A2 in the protein-lipid complex can be an important reason for the enhanced activity of this enzyme at lipid-water interfaces.

Animals↗
PubMed1984

Characterization of ATP complexes with lanthanide (III) ions.

Several spectroscopic techniques are used to investigate the stoichiometry and properties of ATP complexes with lanthanide(III) (Ln(III)], ions. The ATP2-lanthanide(III) complex predominates at millimolar ATP levels and dissociates to the 1:1 complex with a Kd of 300 +/- 50 microM for the Eu(III) case. Two independent techniques, viz. field-dependent water proton relaxation for the Gd(III) complex and metal ion luminescence lifetime measurements for the Eu(III) complex, yield a value of approximately 2 for the number of water molecules coordinated to the metal ion. The latter technique yields an approximate metal-ion hydration number of 4 for the 1:1 complex. Dynamic properties of the Gd(III) X ATP2 complex including the temperature dependence of correlation times describing rotation of the complex and ATP exchange have been studied by field-dependent water-proton relaxation and by temperature-dependent 31P NMR relaxation studies. These data are consistent with formation of a 2:1 ATP-lanthanide complex at millimolar ATP concentrations. Other types of complexes are detected under conditions in which there is insufficient ATP to satisfy the 1:2 metal:nucleotide stoichiometry.

Adenosine Triphosphate↗
PubMed1984

Laser-induced lanthanide luminescence as a probe of metal ion-binding sites of human Factor Xa.

7F0 ---- 5D0 excitation spectroscopy of Eu(III) has shown that human Factor Xa has two high affinity lanthanide ion-binding sites. The deuterium isotope effect on the reciprocal lifetime (tau-1) of excited Eu(III) in human Factor Xa has indicated that 2 to 3 water molecules remain on Eu(III) after being complexed by Factor Xa, suggesting that 3-6 ligand atoms are provided by the protein, probably through two or three gamma-carboxyglutamic acids (GLA). F orster -type interlanthanide energy transfer has been utilized to measure the distance between the high affinity metal ion-binding sites of human Factor Xa using Tb(III) as an energy donor and Nd(III), Ho(III), or Er(III) as energy acceptors. Tau-1 values of Tb(III) in the presence of the acceptor ions Nd(III), Ho(III), and Er(III) were 1.90, 1.66, and 1.76 ms-1, respectively, which compared to 1.31 ms-1 in the presence of the nonacceptor ion Gd(III), yield energy transfer efficiencies of 0.29, 0.20, and 0.24, respectively. From these efficiencies and published critical distances (R0) ( Horrocks , W. DeW ., Jr., Rhee , M-J., Snyder, A. P., and Sudnick , D. R. (1980) J. Am. Chem. Soc. 102, 3650-3652), the distance between two high affinity sites is estimated to be 10.7 A. Based on these data, we propose that the two high affinity sites of human Factor Xa consist of two paired GLA residues; GLA-19, GLA-20 and GLA-25, GLA-26 together with one of the remaining single GLA residues for each site.

1-Carboxyglutamic Acid↗
PubMed1982

Laser-induced europium(III) luminescence as a probe of the metal ion mediated association of human prothrombin with phospholipid.

7F0 leads to 5D0 excitation spectroscopy of Eu(III) has been used to investigate the Eu(III) and phospholipid binding properties of human prothrombin. The results indicate that human prothrombin contains four high-affinity Eu(III) binding sites which are distributed into two classes of binding sites. When 4 equiv of Eu(III) is bound to prothrombin, the prothrombin is capable of binding to phospholipid vesicles. The deuterium isotope effect on the lifetime of the Eu(III)-prothrombin complex and the Eu(III)-prothrombin-phospholipid complex was used to determine the number of water molecules coordinated to the Eu(III). In both complexes, each of the Eu(III)'s coordinated to 2.5 +/- 0.5 water molecules. These results indicate that the binding of the Eu(III)-prothrombin complex to the phospholipid does not require the formation of a prothrombin-Eu(III)-phospholipid bridge.

Calcium↗
PubMed1982

Interresidue distance measurements in proteins. Fluorescent energy transfer between tryptophans and a Ru(III)(NH3)5-histidine complex in alpha-lytic protease and lysozyme.

The mechanism by which the intrinsic fluorescence of tryptophan residues in alpha-lytic protease and lysozyme are quenched by a complex formed between the single histidine residue in each protein and Ru(III)(NH3)5 was investigated. The R0 values for alpha-lytic protease and lysozyme were 15.5 and 11.8 A, respectively. Good agreement between the efficiency of energy transfer measured experimentally and that calculated from the X-ray data, assuming the Förster dipole-dipole mechanism, demonstrates that this mechanism is appropriate. The ease with which the ruthenium-labeled enzymes can be synthesized and purified suggests that the Ru(III)(NH3)5-His complex may have general utility in structural studies of proteins in solution.

Ammonia↗
PubMed1981

Lanthanide ion luminescence probes. Characterization of metal ion binding sites and intermetal energy transfer distance measurements in calcium-binding proteins. 1. Parvalbumin.

Eu(III) laser excitation spectroscopy of the 7F0 leads to 5D0 transition reveals spectral features characteristic of the occupation of the CD and EF Ca(II)-binding sites of parvalbumin by Eu(III) ions. In addition, at pH 6.5, a signal attributable to binding at a third site is observed. This feature is abolished upon lowering the pH to 3.8. At pH 6.5, the appearance of this feature correlates with the decrease in luminescence intensity during titrations of parvalbumin with Eu(III) or Tb(III) after more than approximately 1.8 equiv of either of these ions has been added. Eu(III) ions in the primary sites coordinate zero to two H2O molecules while the third site involves about three coordinated H2O molecules. Parvalbumins in which mixed pairs of lanthanide ions, Ln(III), occupy the CD and EF sites were prepared. Nonradiative energy transfer between Eu(III) and Tb(III) acting as luminescent donors and various other Ln(III) ions serving as acceptors was observed by monitoring the excited-state lifetimes of the donor ions using a pulsed dye laser apparatus. With the assumption of a Förster-type dipole--dipole mechanism, inter-binding-site distance estimates were made from our measurements and are in reasonable agreement with the distance (11.8 A) obtained by X-ray crystallography, especially when Eu(III) is the donor. R0 values (critical distances for 50% energy transfer) in H2O solution range from 9.2 A for the Tb(III)--Ho(III) donor-acceptor pair down to 5.7 A for the Eu(III)--Ho(III) pair.

Animals↗
PubMed1981

Lanthanide ion luminescence probes. Characterization of metal ion binding sites and intermetal energy transfer distance measurements in calcium-binding proteins. 2. Thermolysin.

Eu(III) laser excitation spectroscopy of the 7F0 leads to 5D0 transition reveals spectral features characteristic of the occupation of Eu(III) ions in the S(1), S(3), and S(4) Ca-(II)-binding sites of thermolysin. Various hybrid Eu(III) thermolysin species were prepared in which the Eu(III) ion resides in the S(1) or S(3) and S(4) or all three Eu(III)-binding sites of the metalloprotein. The Eu(III) ion in the S(1) site coordinates to one H2O molecule while the other two sites coordinate to about three to four H2O molecules. Thermolysin hybrids in which the donor Ln(III) ion occupies S(1) and acceptor Ln(III) ions occupy sites S(3) and S(4) were prepared. Nonradiative energy transfer between Eu(III) and Tb(III) acting as luminescent donors and various other Ln(III) ions serving as acceptors was observed by monitoring the excited-state lifetimes of the donor ions using a pulsed dye laser apparatus. Virtually all energy transfer occurs between sites S(1) and S(4) while less than 1% of the donor's energy is transferred from S(1) to S(3). With the assumption of a Förster-type dipole--dipole mechanism, the experimental inter-binding-site distance estimates between sites S(1) and S(4) are in good agreement with the distance (11.7 A) obtained by X-ray crystallography. R0 values (critical distances for 50% energy transfer) in H2O solution range from 9.3 A for the Tb-(III)--Ho(III) donor--acceptor pair down to 5.3 A for the Eu-(III)--Ho(III) pair.

Binding Sites↗
PubMed1981

Binding of lanthanide ions to troponin C.

Tb3+ and Eu3+ bound to troponin C were detected by (1) changes in the fluorescence of the tyrosine chromophore of the protein or (2) the luminescence of the ions themselves excited by energy transfer from the protein or by direct excitation using a pulsed laser light source [Horrocks, W. DeW., Jr., & Sudnick, D. R. (1979) Science (Washington, D.C.) 206, 1194-1196]. Titrations carried out in the absence and presence of Ca2+ suggest two classes of binding sites (two sites in each class) for the lanthanides, corresponding to the high- and the low-affinity sites for Ca2+. Computer analysis, assuming competition between Ca2+ and the lanthanide, using the binding constants of Ca2+ for TnC [Potter, J. D., & Gergely, J. (1975( J. Biol. Chem. 250, 4628-4633] yields 5.2 X 10(8) M-1 and 9.7 X 10(6) M-1 for Tb3+ and 4.7 X 10(9) M-1 and 5.3 X 10(7) M-1 for Eu3+, for the high- and the low-affinity sites, respectively. From lifetimes of laser-induced luminescence in H2O and in D2O, the number of water molecules coordinated to Eu3+ was two at the high and three at the low affinity sites.

Animals↗
PubMed1980

Structural and electronic mimics of the active site of cobalt(II)-substituted zinc metalloenzymes.

Complexes of cobalt(II) and zinc(II) which involve monodentate coordination of two alkyl carboxylate and two imidazole ligands in a slightly distorted tetrahedral fashion have visible and magnetic circular dichroism spectra remarkably similar to the cobalt(II)-substituted proteolytic enzymes thermolysin and carboxypeptidase A. Single crystal x-ray structure determinations on [Co(C2H5COO)2Im2], Im = imidazole, and its zinc counterpart reveal only minor structural differences between the cobalt and zinc species. Electron paramagnetic resonance spectra of cobalt(II) doped into zinc(II) complexes with known structures demonstrate the extreme sensitivity of the g-values to minor structural differences.

Binding Sites↗