Epsilon Max Research Articles

A Lower Bound for the Canonical Height Associated to a Drinfeld Module

Denis associated to each Drinfeld module M over a global function function field L a canonical height function, which plays a role analogous to that of the Neron-Tate height in the context of elliptic curves. We prove that there exist constants \epsilon>0 and C, depending only on the number of places at which M has bad reduction, such that either x in M is a torsion point of bounded order, or else the canonical height of x is bound below by \epsilon max{h(j_M), deg(D_M)}, where j_M is a certain invariant of the isomorphism class of M, and D_M is the minimal discriminant of M. As an application, we make some observations about specializations of one-parameter families of Drinfeld modules.

Synthesis and Optical Properties of all‐trans‐Oligodiacetylenes

A new series of pure and highly soluble oligodiacetylenes (ODAs) was synthesized in high yield and on a multi-milligram scale by a sequence of Sonogashira reactions with a strongly reduced level of homocoupling. The lambda max and epsilon max of these ODAs show an increase with both chain elongation and solvent polarity. A plot of lambda max absorption versus 1/CL (CL=conjugation length) was shown to be linear. The lambda max converges to 435 nm for the longest members of the series at micromolar concentration. This reveals that the longest wavelength absorption observed for PDA chains (lambda max up to 700 nm) is due to aggregation effects. The fluorescence quantum yield increased from monomer to trimer and decreased for longer ODAs. A similar trend is found for the lifetime of fluorescence with a maximum of 600 ps for the trimer. The observed linearity of the rotational correlation time with the oligomer length implies that the ODA chains in solution lack significant geometrical changes. This implies that the ODAs in solution are fully stretched molecular rods of up to 4 nm in length.

Two Classes of Alongside Charge-Transfer Interactions Defined in One [2]Catenane

A [2]catenane, which incorporates hydroquinone (HQ) and a sterically bulky tetrathiafulvalene (TTF) into a bismacrocycle, has been designed to probe the alongside charge-transfer (CT) interactions taking place between a TTF unit and one of the bipyridinium moieties in the tetracationic cyclophane cyclobis(paraquat-p-phenylene) (CBPQT4+). A template-directed strategy employs the HQ unit as the primary template for formation of the tetracationic cyclophane CBPQT4+, affording the desired [2]catenane structure but as an uncharacteristic green solid. The X-ray crystal structure and detailed 13C NMR assignments have identified a stereoselective preference for catenation about the cis isomer. The 1H NMR spectroscopy, electrochemistry, and X-ray crystallography all confirm that the CBPQT4+ cyclophane encircles the HQ unit, thereby defining a structure which would normally determine a red color. The visible-NIR region of the absorption spectrum displays a band at approximately 740 nm that is unambiguously assigned to a TTF --> CBPQT4+ CT transition on the basis of resonance Raman spectroscopy using 785 nm excitation. The profile of the CT band changes depending on the ratio of the cis- to trans-TTF isomers in the [2]catenane for which the molar absorptivity of each isomer is estimated to be significantly different at epsilon max = 380 and 3690 M-1 cm-1, respectively. Molecular modeling studies confirmed that the observed difference in the absorption spectroscopic profile can be accounted for by both a better overlap of the HOMO(TTF) and LUMO+1(CBPQT4+) as well as a more stable face-to-face (pi...pi) conformation in the trans isomer compared to the edge-to-face cis isomer of the [2]catenane. The latter is arranged for pi-orbital overlap through the sulfur atoms of the TTF unit, thereby defining an [Spi...pi] interaction.

Assessment of ischemic myocardium by strain-rate imaging during adenosine stress echocardiography

Strain rate (SR) provides a quantitative segmental analysis of myocardial function. However, the use of SR with stress echocardiography to determine the ischemic myocardium has not been completely investigated. The present study aimed to determine the changes in systolic function of the ischemic myocardium by strain-rate imaging (SRI) with adenosine stress echocardiography. Stenosis and complete occlusion of coronary arteries were produced in 11 canine models by constricting the left anterior descending coronary artery (LAD). Myocardial longitudinal strain with adenosine was measured at baseline and during ischemia and infarction. Strain and SR did not differ during ischemia and infarction as compared with that at baseline in non-LAD segments or after adenosine treatment. As compared with baseline, during ischemia, LAD segments showed significantly decreased peak systolic SR (SR(peak sys)) (P < 0.05) and significantly increased ratio of postsystolic strain (epsilon(ps)) to strain during ejection time (epsilon(et)) (epsilon(ps)/epsilon(et)) (P < 0.05); epsilon(max) and epsilon(et) were reduced slightly, epsilon(ps) and the ratio of epsilon(ps) to maximal systolic strain (epsilon(max))(epsilon(ps)/epsilon(max)) were increased minimally, but had no significance(P > 0.05). During infarction, the epsilon(ps) and the ratios of epsilon(ps)/epsilon(max) and epsilon(ps)/epsilon(et) were increased markedly (P < 0.01) and epsilon(et) and SR(peak sys) decreased as compared with that at baseline and during ischemia, whereas epsilon(max) was reduced only with at baseline (P < 0.01). After adenosine treatment, in the non-LAD segments, the values of strain and SR did not change at baseline or during ischemia and infarction and in LAD segments, values did not change at baseline and during infarction. However, during ischemia, SR(peak sys) and epsilon(et) were significantly reduced (P < 0.05), whereas epsilon(ps), epsilon(ps)/epsilon(max) and epsilon(ps)/epsilon(et) were increased (P < 0.05 and < 0.01, respectively). Combined with adenosine stress echocardiography, SRI can quantitatively differentiate the ischemic from non-ischemic myocardium. epsilon(ps)/epsilon(max) and epsilon(ps)/epsilon(et) can be used as objective indices to identify the ischemic myocardium.

Effect of stress ratio on the fatigue behaviour of compact bone

During the fatigue process of bone, cracks generally initiate from the inherent defects existing in the bone. The fatigue lives of bone specimens at different stress levels as well as at different stress ratios R were evaluated using a computer simulation in which the crack propagation behaviour initiated from the inherent defects in the bone are herein considered. The S-N curves as well as the distributions of fatigue lives obtained by the simulations accurately conform with the experimental results. With the strain threshold epsilon(max) representing fatigue failure of the bone specimen, the values of 1500 microepsilon for R = -1, 2500 microepsilon for R = 0.1 and 4000 microepsilon for R = 10 were extrapolated from the simulations. These values conform with experimental values reported in the literature. Such conformity indicates that the strain threshold for fatigue failure is associated with the threshold value for crack propagation.

Aminopyridine iron catecholate complexes as models for intradiol catechol dioxygenases. Synthesis, structure, reactivity, and spectroscopic studies.

Four new Fe(III) catecholate complexes, [(bispicMe2en)FeIII(DBC)]+, [(bispicCl2Me2en)FeIII(DBC)]+, [(trispicMeen)FeIII(DBC)]+, and [(BQPA)FeIII(DBC)]+, which all contain aminopyridine ligands, were synthesized. The structure of [(bispicMe2en)FeIII(DBC)]+ was determined by X-ray diffraction. It crystallizes in the triclinic space group P1 with a = 10.666(3) A, b = 13.467(5) A, c = 17.685(2) A, alpha = 93.46(2) degrees, beta = 93.68(2) degrees, gamma = 109.0(3) degrees, V = 2387.4 A3, and Z = 2. All of these complexes were found to be active toward oxidation of catechol by O2 in DMF at 20 degrees C to afford intradiol cleavage products. The catechol was quantitatively oxidized, mainly (90%) into 3,5-di-tert-butyl-5-(carboxymethyl)-2-furanone. Reaction rates were measured, and for the first three (topologically similar) complexes, a correlation of the second-order kinetic constants k with the optical parameters of the two LMCT O(DBC)-->Fe(III) bands was found. In particular, k increases with the epsilon max of the charge-transfer bands. The k value of the complex [(BQPA)FeIII(DBC)]+, containing a tripodal ligand, is smaller than expected on the basis of these correlations. This discrepancy could be related to steric hindrance induced by the BQPA ligand. However, the much lower activity of the bispicen-Fe(III)-type complexes compared to that of the [(TPA)FeIII(DBC)]+ complex synthesized by Jang et al. (J. Am. Chem. Soc. 1991, 113, 9200-9204), despite similar epsilon max values, shows that a knowledge of optical and NMR parameters values is not sufficient to explain the dioxygenase activity rate. In their study of protocatechuate 3,4-dioxygenase, Orville et al. (Biochemistry 1997, 36, 10052-10066) suggested that asymmetric chelation of the catecholate to Fe(III) is of great importance in the efficiency of the intradiol dioxygenase reaction. Indeed, a comparison of the X-ray structures of [(TPA)FeIII(DBC)]+ and [(bispicMe2en)FeIII(DBC)]+ shows that the Fe(III)-O bonds differ by 0.019 A in the former and are identical in the latter. Asymmetry could also play a role in the model complexes. An alternative explanation is the possible existence of a low-spin state for [(TPA)FeIII(DBC)]+, as recently identified in [(TPA)FeIII(cat)]+ by Simaan et al.

Pulse radiolysis study on the rutin and quercetin anions formation and their behaviours in N2-saturated ethanol solution

pulse radiolysis and gamma-radiolysis. The results show that solvated electron can add to rutin and quercetin and produce corresponding radical anions which show a maximum UV absorbance band at 430 and 420 nm. The epsilon(max) of radical anions of rutin and quercetin are 3.5 x 10(3) mol(-1) dm(3) cm(-1) and 5.5 x 10(3) mol(-1) dm(3) cm(-1), respectively. The rate constants of build-up of radical anions of rutin and quercetin are 7.5(+/-0.9) x 10(9) mol(-1) dm(3) s(-1) and 7.7(+/-0.6) x 10(9) mol(-1) dm(3) s(-1), respectively. The decay of radical anions is induced by the protonation which contains a pseudo-first-order reaction and a second-order reaction. For the radical anion of rutin, the rate constants of decay are 1.1(+/-0.1) x 10(4) s(-1) and 2.7(+/-0.2) x 10(9) mol(-1) dm(3) s(-1), respectively. For the radical anion of quercetin, the rate constants of decay are 1.5(+/-0.1) x 10(4) s(-1) and 4.0(+/-0.5) x 10(9) mol(-1) dm(3) s(-1), respectively. The electron transfer between radical anions and acetaldehyde can occur at the rate constants of 8.9(+/-1.2) x 10(7) mol(-1) dm(3) s(-1) for radical anion of rutin and 1.2(+/-0.1) x 10(8) mol(-1) dm(3) s(-1) for radical anion of quercetin. The rate constants of their corresponding counter-reactions are 5.7(+/-0.8) x 10(7) mol(-1) dm(3) s(-1) and 2.0(+/-0.6) x 10(7) mol(-1) dm(3) s(-1), respectively. The equilibrium constants of electron transfer reactions are 1.6 and 6.0, respectively. The standard electrode potential of radical anions of rutin and quercetin are obtained to be -1.94 and -1.98 V. (C) 1999 Elsevier Science Ltd. All rights reserved.

Article

The energy and asymmetry of the optical absorption spectrum of solvated electrons, es- , change in a nonlinear fashion on changing the solvent through the series HOH, CH3OH, CH3CH3OH, (CH3)2CHOH, (CH3)3COH. The ultimate, quantum-statistical mechanical, interpretation of solvated electron spectra is needed to describe the solvent dependence. The previously reported optical spectrum of es- in tert-butanol was somewhat inaccurate, due to a small amount of water in the alcohol and to limitations of the infrared light detector. The present note records the remeasured spectrum and its temperature dependence. The value of the energy at the absorption maximum (EAmax) is 155 zJ (0.97 eV) at 299 K and 112 zJ (0.70 eV) at 338 K; the corresponding values of G epsilon max (10-22 m2 aJ-1) are 1.06 and 0.74. These unusually large changes are attributed to the abnormally rapid decrease of dielectric permittivity of tert-butanol with increasing temperature. The band asymmetry at 299 K is Wb/Wr = 1.8.Key words: optical absorption spectrum, solvated electron, solvent effects, tert-butanol, temperature dependence.

Tensile properties of human tendo achillis: Effect of donor age and strain rate

Two sets of tensile tests were conducted on tendo Achillis taken from 16 embalmed cadavers (donor ages, 36 to 100 years). From the stress, sigma-versus-strain, epsilon results, values of the following properties were obtained: linear stiffness, ST (which is defined as the slope of the linear zone of the post-toe region of the load-versus-extension plot), ultimate tensile strength [UTS], the strain at the UTS point (herein called the ultimate tensile strain) [epsilon max], and the tangent modulus of elasticity, [ET] (which is the slope of the linear zone of the post-toe region of the sigma-epsilon curve). In the first set of tests, the loading rate was 10%/sec. The results are thus: linear stiffness: 685 +/- 262 N/mm.; UTS = 59 +/- 18 MPa; epsilon max = 22 +/- 7%; and ET = 375 +/- 102 MPa. It was found that donor age exerts an insignificant effect on linear stiffness, a marked effect on UTS, an insignificant effect on epsilon max, and a moderate effect on ET (Student's t-test; p < 0.05). In the second set of tests, the test tendons were taken from the cadavers of "middle-aged" donors (52 to 67 years), and the strain rate was 100%/sec. By combining the results of the first and second sets of tests for a subset of this group (i.e., those with matched counterparts), it was found that strain rate has a statistically significant effect on ST and ET but not on UTS and epsilon max (paired Student's t-test; p < 0.05). Based on all results and findings, donor age is suggested as one of the considerations when selecting tendo Achillis to be used as allografts for the repair of severely damaged or ruptured tendo Achillis or anterior cruciate ligament.

Fluorescein-Conjugated Lysine Monomers for Solid Phase Synthesis of Fluorescent Peptides and PNA Oligomers

Fluorescein ethyl ester, I, was used to prepare the fluorescent mixed ester/ether 6-O-(carboxymethyl)-fluorescein ethyl ester, III. Conjugation of III to the epsilon-amino group of alpha-N-Boc-L-lysine, via the N-hydroxysuccinimde ester, IV, gave the Boc-protected fluorescein-conjugated lysine monomer V. Removal of the Boc group, followed by reaction with Fmoc chloride, gave the Fmoc-protected monomer, VI (Figure 1). These Boc- and Fmoc-protected fluorescein-conjugated lysines were readily incorporated into peptides and PNA oligomers during solid phase synthesis to give fluorescent products. Mass spectroscopy and UV studies showed that the fluorophore remains unchanged during solid phase synthesis. In contrast to fluorescein, the photophysical properties of these derivatives are pH independent from pH 3 to 8, with a molar absorption coefficient, epsilon max 456, of 2.9 x 10(4) M-1 cm-1 and fluorescence quantum yield, phi f, of 0.18.

Measurement and global analysis of the absorbance changes in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila

The photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila was examined by time-resolved difference absorption spectroscopy in the wavelength range of 300-600 nm. Both time-gated spectra and single wavelength traces were measured. Global analysis of the data established that in the time domain between 5 ns and 2 s only two intermediates are involved in the room temperature photocycle of PYP, as has been proposed before (Meyer T.E., E. Yakali, M. A. Cusanovich, and G. Tollin. 1987. Biochemistry. 26:418-423; Meyer, T. E., G. Tollin, T. P. Causgrove, P. Cheng, and R. E. Blankenship. 1991. Biophys. J. 59:988-991). The first, red-shifted intermediate decays biexponentially (60% with tau = 0.25 ms and 40% with tau = 1.2 ms) to a blue-shifted intermediate. The last step of the photocycle is the biexponential (93% with tau = 0.15 s and 7% with tau = 2.0 s) recovery to the ground state of the protein. Reconstruction of the absolute spectra of these photointermediates yielded absorbance maxima of about 465 and 355 nm for the red- and blue-shifted intermediate with an epsilon max at about 50% and 40% relative to the epsilon max of the ground state. The quantitative analysis of the photocycle in PYP described here paves the way to a detailed biophysical analysis of the processes occurring in this photoreceptor molecule.

Utilization of conformational flexibility in enzyme action-linkage between binding, isomerization, and catalysis.

An intimate relationship between protein conformational changes and catalysis has often been suggested. The present study employs ligand-induced ultraviolet difference spectra and kinetic parameters determined for Escherichia coli ornithine transcarbamoylase and its site-specific mutants to evaluate the linkage between binding, isomerization, and reaction rate. For the wild-type enzyme, the lead substrate carbamoyl phosphate introduces a large difference absorbance in the enzyme upon binding (delta epsilon max approximately 1,800 M-1 cm-1; Miller, A. W., and Kuo, L. C. (1990) J. Biol. Chem. 265, 15023-15027). The spectrum is the same in lineshape as that produced by the bisubstrate analog N-(phosphonacetyl)-L-ornithine and is 80% as intense. Both substrate and analog cause gross protein conformational rearrangements as evident by swift and severe cracking of enzyme crystals in their presence. For the mutants, the difference spectra actuated by the substrate are the same in lineshape as that of the wild type but vary in intensity. A wide range of substrate affinity and steady-state kinetic constants are also observed for the mutants. When the binding energy of carbamoyl phosphate and the activation energy for transcarbamoylation are calculated for the wild-type and mutant enzymes, they are found to be inversely correlated to the intensity of protein difference absorbance elicited by the lead substrate. Together with analyses of steady-state kinetic parameters derived for various plausible reaction schemes, the experimental data suggest that carbamoyl phosphate induces the committed isomerization in ornithine transcarbamoylase for transition state binding. Our results provide a unique demonstration that an induced-fit isomerization, triggered by binding, either controls or contributes significantly to the rate of an enzyme-catalyzed reaction.

Role of tyrosine 129 in the active site of spinach glycolate oxidase.

The enzymatic properties and the three-dimensional structure of spinach glycolate oxidase which has the active-site Tyr129 replaced by Phe (Y129F glycolate oxidase) has been studied. The structure of the mutant is unperturbed which facilitates interpretation of the biochemical data. Y129F glycolate oxidase has an absorbance spectrum with maxima at 364 and 450 nm (epsilon max = 11400 M-1 cm-1). The spectrum indicates that the flavin is in its normal protonated form, i.e. the Y129F mutant does not lower the pKa of the N(3) of oxidized flavin as does the wild-type enzyme [Macheroux, P., Massey, V., Thiele, D. J., and Volokita, M. (1991) Biochemistry 30, 4612-4619]. This was confirmed by a pH titration of Y129F glycolate oxidase which showed that the pKa is above pH 9. In contrast to wild-type glycolate oxidase, oxalate does not perturb the absorbance spectrum of Y129F glycolate oxidase. Moreover oxalate does not inhibit the enzymatic activity of the mutant enzyme. Typical features of wild-type glycolate oxidase that are related to a positively charged lysine side chain near the flavin N(1)-C(2 = O), such as stabilization of the anionic flavin semiquinone and formation of tight N(5)-sulfite adducts, are all conserved in the Y129F mutant protein. Y129F glycolate oxidase exhibited about 3.5% of the wild-type activity. The lower turnover number for the mutant of 0.74 s-1 versus 20 s-1 for the wild-type enzyme amounts to an increase of the energy of the transition state of about 7.8 kJ/mol. Steady-state analysis gave Km values of 1.5 mM and 7 microM for glycolate and oxygen, respectively. The Km for glycolate is slightly higher than that found for wild-type glycolate oxidase (1 mM) whereas the Km for oxygen is much lower. As was the case for wild-type glycolate oxidase, reduction was found to be the rate-limiting step in catalysis, with a rate of 0.63 s-1. The kinetic properties of Y129F glycolate oxidase provide evidence that the main function of the hydroxyl group of Tyr129 is the stabilization of the transition state.

Design of a helix-bundle cross-link: NMR and UV-visible spectroscopic analyses and molecular modeling of ring-oxidized retinals

In order to generate potential chemical cross-links for studying the chromophore binding site of bacteriorhodopsin and related helix-bundle proteins, MnO2 was used to oxidize all-trans-retinal's ring moiety. The structures and solution conformations of three ring-oxidized retinal analogues have been determined by using UV-visible absorption and 1H and 13C NMR spectroscopies, primarily with regard to (i) the introduction of a functional group at the ring end of the chromophore, (ii) the retention of the all-trans geometry of the polyenal side chain, and (iii) the torsional angle of the ring-polyenal bond. Analyses of their UV-visible absorption spectral parameters (lambda max, epsilon max, and vibrational fine structure) and NMR spectral parameters (1H-1H coupling constants, 1H and 13C NMR chemical shifts, and 1H homonuclear Overhauser effects) indicated the 4-oxo and the 2,3-dehydro-4-oxo derivatives both possess the twisted 6-s-cis conformation adopted by most six-membered ring analogues of retinal in solution or crystal. However, the alpha-dioxocyclopentenyl analogue exists in solution predominantly (70-80%) as the planar 6-s-trans conformer, similar to violerythrine chromophore analogues. In order to identify the minor solution forms, molecular modeling and geometry optimizations using the semiempirical molecular orbital method AM1 defined two additional symmetry-related minima at +/- 30-40 degrees in its C6-C7 torsional energy profile. Because the chromophores of bacterio- and halorhodopsins and sensory rhodopsins are bound as the 6-s-trans conformer [Harbison, G.S., Smith, S.O., Pardoen, J.A., Courtin, J.M.L., Lugtenburg, J., Herzfeld, J., Mathies, R.A., & Griffin, R.G. (1985) Biochemistry 24, 6955-6962; Baselt, D.R., Fodor, S.P.A., van der Steen, R., Lugtenburg, J., Bogomolni, R.A., & Mathies, R.A. (1989) Biophys. J. 55, 193-196], we suggest that the cyclopentenyl analogue's alpha-diketo function may be favorably positioned within the binding pocket and sufficiently reactive toward nucleophilic attack to cross-link an arginine located in or near the ring end of the chromophore cavity: Arg134 according to the current model of bacteriorhodopsin's tertiary structure [Henderson, R., Baldwin, J.M., Ceska, T.A., Zemlin, F., Beckmann, E., & Downing, K.H. (1990) J. Mol. Biol. 213, 899-929] or Arg82 as postulated from an alternate model constructed primarily to accommodate the external point charge contribution to bacteriorhodopsin's opsin shift.

Synthesis and copper(II)-binding properties of the N-terminal peptide of human α-fetoprotein

The N-terminal native sequence tripeptide of alpha-fetoprotein, L-threonyl-L-leucyl-L-histidine N-methylamide, was synthesized and its interaction with Cu(II) ions was investigated by potentiometric titration at 25 degrees C in 0.15 M-NaCl and by visible-absorption, e.p.r. and n.m.r. spectroscopy. Analyses of the results in the pH range 4-10 indicated the presence of multiple complex species in solution: MHL, MH-2L, MHL2, ML2 and MH-1L2, where M, H and L represent metal ion, proton and ligand anion respectively. Only the species MH-2L and MH-1L2 are present in significant amounts at physiological pH. The results of the visible-absorption spectroscopy are consistent with the findings of species distribution that MH-2L is the major complex species detected above physiological pH that has the spectral characteristics of lambda max. = 523 nm and epsilon max. = 98 M-1.cm-1. The nine superhyperfine lines in e.p.r. spectra of the major species MH-2L strongly support the co-ordination of four nitrogen atoms by Cu(II). Both 1H- and 13C-n.m.r. studies suggest that the species MH-2L is a square-planar complex. The results from the equilibrium-dialysis experiments showed that this peptide is able to compete with albumin for Cu(II) ions. At equimolar concentrations of albumin and the peptide, about 52% of the Cu(II) was bound to the peptide. The possibility that alpha-fetoprotein plays an important role as the Cu(II)-transport protein in fetal life is discussed.

Gene 32 protein, the single-stranded DNA binding protein from bacteriophage T4, is a zinc metalloprotein.

Gene 32 protein (g32P) isolated from bacteriophage T4-infected Escherichia coli and from an overproduction vector derived from the plasmid pKC30 contains 1 mol of tightly incorporated Zn(II) per mol of protein. A linear incorporation of three molar equivalents of p-hydroxymercuriphenylsulfonate (PMPS) results in a linear release of 1.1 mol of Zn(II) from the protein. Reversal of formation of the g32P-PMPS complex with thiol in the presence of EDTA results in a zinc-free apo-g32P. Cd(II) and Co(II) can be exchanged with the intrinsic Zn(II) ion. The Cd(II) protein shows a charge-transfer band at approximately 250 nm. The Co(II) protein shows a set of absorption bands typical of a tetrahedral Co(II) complex (epsilon max = 660 M-1 X cm-1 at 645 nm), and two intense charge-transfer bands are present at 355 nm (epsilon = 2,250 M-1 X cm-1) and 320 nm (epsilon = 3,175 M-1 X cm-1). These observations are consistent with three cysteines as ligands to the Zn(II) ion in g32P. Zn(II) g32P undergoes precise limited proteolysis by trypsin to produce the small fragments A and B and the core, g32P-(A + B). Under identical conditions, apo-g32P is hydrolyzed rapidly beyond the g32P-(A + B) stage to produce many proteolyzed fragments. Fluorescence quenching experiments show that at low protein concentration apo-g32P has markedly altered binding affinity for poly(dT) relative to native g32P. Three of the four cysteines of g32P are found in a tyrosine-rich sequence corresponding to residues 72-116 and implicated in DNA binding by 1H NMR investigations. Zn(II) appears to provide a conformational element contributing to DNA binding by coordinating the cysteine and possibly histidine side chains in the sequence -Cys-X3-His-X5-Cys-X2-Cys-, residues 77-90, located in the DNA binding domain of g32P.

Pressure and temperature dependence of dielectric properties of Pb(Mg1/2W1/2)O3

The dielectric properties such as the permittivity, epsilon , and the dielectric loss tangent, tan delta , of Pb(Mg1/2W1/2)O3 were measured at various pressures up to 8 kbar. The permittivity epsilon obeys the Curie-Weiss law in the paraelectric phase at constant pressure. With increasing pressure p, the antiferroelectric transition temperature Tn decreases with a pressure coefficient dTn/dp=-3.8+or-0.2K kbar-1, the extrapolated Curie-Weiss temperature T0 increases with a pressure coefficient dT0/dp=1.4+or-0.2K kbar-1, the Curie constant C decreases at a rate of about 3.9% kbar-1, and the maximum value of epsilon , epsilon max, is almost constant at pressures below 4 kbar and then increases. The increase in epsilon max with p is associated with the decrease in the difference Delta T(=Tn-T0) with p. with increasing p, the difference Delta T decreases and extrapolates to zero at the pressure pt of about 18 kbar. At this pressure pt, the change from the antiferroelectric transition to the ferroelectric one is expected. At constant temperature, the pressure dependence of epsilon can be represented by a Curie-Weiss-like relationship expressed in terms of pressure: epsilon = epsilon 0*+C*/(p-p0). Both constants C* and p0 are functions of temperature. The dielectric properties associated with the antiferroelectric transition are interpreted in terms of a phenomenological theory.

The reaction between NADPH and mercuric reductase from Pseudomonas aeruginosa.

The reaction between the FAD-containing enzyme, mercuric reductase, and its reducing substrate, NADPH, has been studied at 5 degrees C, pH 7.3, by rapid-scan and fixed-wavelength stopped-flow techniques with the aim to characterize reaction intermediates. With an excess of NADPH the spectral changes observed in rapid-scan experiments can be described by a simple kinetic model, A----B----C----D, where A, B, C, and D represent distinct spectral species. The first step is virtually complete within the dead time of the apparatus. It is associated with a 16% bleaching of flavin absorbance and the appearance of a very broad charge-transfer band (epsilon max = 1.9 mM-1 cm-1) centered near 600 nm. The second step (k = 43 s-1) involves a further small bleaching of flavin absorbance and intensification of the charge-transfer band (epsilon max = 3.3 mM-1 cm-1) which becomes centered near 580 nm. The third step (k = 8 s-1) involves an intensification and a blue shift of the main FAD absorption band. Concurrently, the charge-transfer band increases in intensity and becomes centered near 530 nm (epsilon 530 = 5.0 mM-1 cm-1). While the first two steps involve 1 mol of NADPH per mol of FAD, the third step requires a second equivalent of NADPH. A 2-electron-reduced enzyme (EH2) can be obtained by treatment of the oxidized enzyme (E) with dithioerythritol. Addition of 1 eq of NADP+ to dithioerythritol-generated EH2 gives rise to a spectral species similar to the kinetic intermediate C, while the addition of 1 eq of NADPH gives rise to a spectral species similar to D. It is proposed that B largely corresponds to an E-NADPH complex, C to an EH2-NADP+ complex, and D to an EH2-NADPH complex.

Serum iron determination using ferene triazine

The synthesis and characterisation of a new colorimetric reagent for iron, 3-(2-pyridyl)-5,6-bis(2-furyl)-1,2,4-triazine are described. Peak assignments and chemical shifts of the high resolution 13C nmr spectrum are given. The reagent forms a tris iron(II) complex with epsilon max of 32000 Lcm-1 mol-1 at 577 nm. The results of precision, linearity and recovery studies for use of the reagent in direct serum iron determinations are reported. Thiourea effectively suppresses copper interference for the serum iron determinations. With the procedure used, the presence of hemolysed cells in the serum had no effect on serum iron levels recorded.

Reactions with the oxidized iron protein of Azotobacter vinelandii nitrogenase: formation of a 2Fe center.

The Fe-S center of oxidized Fe protein from Azotobacter vinelandii nitrogenase is decomposed by alpha,alpha'-dipyridyl in a biphasic process. In the presence of MgATP, 2 Fe are immediately removed by chelation while the additional irons are removed only after several hours. A slower biphasic Fe release also was observed in the presence of chelator alone. MgADP prevented the Fe release by chelator. An intermediate in the reaction was isolated containing 2 Fe. The visible spectrum of the intermediate was similar to that of 2Fe-2S ferredoxins (epsilon max at 325, 416, and 460 nm of 16.1, 11.3, and 9.0 mM-1 cm-1). The 2Fe form was electron paramagnetic resonance (EPR) silent until partially reduced with sodium dithionite. The EPR spectral properties were similar to 2Fe-2S ferredoxins; namely, the Fe center had resonances at g = 2.00, 1.94, and 1.92 which were detectable, essentially unbroadened at 70 K. The results suggest that in the oxidized (2+) state Fe protein can undergo a 4Fe to 2Fe conversion.