Unfolding thermodynamics of the Delta-domain in the prohead I subunit of phage HK97: determination by factor analysis of Raman spectra

An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Δ-domain secondary structure and to quantify the thermodynamic parameters of Δ-domain unfolding. The results are compared for the Δ-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the α-helicity (∼ 70%), median melting temperature (T_m = 58 °C), enthalpy (ΔH_m = 50 ± 5 kcal mol^− 1), entropy (ΔS_m = 150 ± 10 cal mol^− 1 K^− 1), and average cooperative melting unit (〈n_c〉 ∼ 3.5) of the in situ Δ-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Δ-domain, despite an enhanced helical secondary structure (∼ 90% α-helix), exhibits diminished thermostability (T_m = 40 °C; ΔH_m = 27 ± 2 kcal mol^− 1; ΔS_m = 86 ± 6 cal mol^− 1 K^− 1) and noncooperative unfolding (〈n_c〉 ∼ 1) vis-à-vis the in situ Δ-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Δ-domains. The present results clarify the key role of the gp5 Δ-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.     

Conformational stability of α-amylase from malted sorghum (Sorghum bicolor): Reversible unfolding by denaturants

α-Amylase from Sorghum bicolor, is reversibly unfolded by chemical denaturants at pH 7.0 in 50 mM Hepes containing 13.6 mM calcium and 15 mM DTT. The isothermal equilibrium unfolding at 27 °C is characterized by two state transition with ΔG (H₂O) of 16.5 kJ mol⁻¹ and 22 kJ mol⁻¹, respectively, at pH 4.8 and pH 7.0 for GuHCl and ΔG (H₂O) of 25.2 kJ mol⁻¹ at pH 4.8 for urea. The conformational stability indicators such as the change in excess heat capacity (ΔC_p), the unfolding enthalpy (H_g) and the temperature at ΔG = 0 (T_g) are 17.9 ± 0.7 kJ mol⁻¹ K⁻¹, 501.2 ± 18.2 kJ mol⁻¹ and 337.3 ± 6.9 K at pH 4.8 and 14.3 ± 0.5 kJ mol⁻¹ K⁻¹, 509.3 ± 21.7 kJ mol⁻¹ and 345.4 ± 4.8 K at pH 7.0, respectively. The reactivity of the conserved cysteine residues, during unfolding, indicates that unfolding starts from the ‘B’ domain of the enzyme. The oxidation of cysteine residues, during unfolding, can be prevented by the addition of DTT. The conserved cysteine residues are essential for enzyme activity but not for the secondary and tertiary fold acquired during refolding of the denatured enzyme. The pH dependent stability described by ΔG (H₂O) and the effect of salt on urea induced unfolding confirm the role of electrostatic interactions in enzyme stability.     

Kinetic study of dissociation of Eu(III) complex with H8dotp (H8dotp = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylphosphonic acid))

The dissociation kinetics of the europium(III) complex with H₈dotp ligand was studied by means of molecular absorption spectroscopy in UV region at ionic strength 3.0 mol dm⁻³ (Na,H)ClO₄ and in temperature region 25-60 °C. Time-resolved laser-induced fluorescence spectroscopy (TRLIFS) was employed in order to determine the number of water molecules in the first coordination sphere of the europium(III) reaction intermediates and the final products. This technique was also utilized to deduce the composition of reaction intermediates in course of dissociation reaction simultaneously with calculation of rate constants and it demonstrates the elucidation of intimate reaction mechanism. The thermodynamic parameters for the formation of kinetic intermediate (ΔH⁰ = 11 ± 3 kJ mol⁻¹, ΔS⁰ = 41 ± 11 J K⁻¹ mol⁻¹) and the activation parameters (E_a = 69 ± 8 kJ mol⁻¹, ΔH^≠ = 67 ± 8 kJ mol⁻¹, ΔS^≠ = −83 ± 24 J K⁻¹ mol⁻¹) for the rate-determining step describing the complex dissociation were determined. The mechanism of proton-assisted reaction was proposed on the basis of the experimental data.     

Relationship of rapid light curves of variable fluorescence to photoacclimation and non-photochemical quenching in a benthic diatom

The response of rapid light–response curves (RLCs) of variable fluorescence to changes in short- and long-term photoacclimation status was studied in an estuarine benthic diatom. The diatom Nitzschia palea was grown under low- (LL, 20 μmol m⁻² s⁻¹) and high-light (HL, 400 μmol m⁻² s⁻¹) conditions, with the purpose of characterising the effects of long-term photoacclimation on (i) steady-state light–response curves (LC) of relative electron transport rate, rETR, (ii) the response of RLCs to changes in ambient irradiance (E, the irradiance to which the sample is acclimated to immediately before the RLCs), (iii) the relationship of RLCs to LC parameters and non-photochemical quenching (NPQ). Photoacclimation to LL and HL conditions induced distinct light–response patterns of rETR and NPQ. Higher growth light resulted in rETR vs. E curves with lower initial slopes (α, 0.591 μmol⁻¹ m² s vs. 0.661 μmol⁻¹ m² s, for HL and LL, respectively) and markedly higher maximum rates (rETR_m, 95.9 vs. 29.3), reached under higher E levels (higher light-saturation coefficient, E_k: 162.4 μmol m⁻² s⁻¹ vs. 44.3 μmol m⁻² s⁻¹). Acclimation to HL induced bi-phasic NPQ vs. E curves, with minimum values reached under low E levels (15–25 μmol m⁻² s⁻¹) and not on dark-acclimated samples. The response of RLCs to changes in ambient irradiance varied with the long-term photoacclimation status of the samples. The initial slope, α_RLC, decreased monotonically with E in LL cultures, from 0.68 to 0.25 μmol⁻¹ m² s, while varied bi-phasically in HL-acclimated samples. Typically, α_RLC of HL cultures increased under low E, reaching a maximum of 0.61 μmol⁻¹ m² s under 25–55 μmol m⁻² s⁻¹, and decreased gradually under higher E levels to 0.25 μmol⁻¹ m² s. RLC maximum rETR, rETR_m,RLC, and saturation coefficient E_k,RLC, increased with E following a saturation-like pattern, with the HL cultures presenting markedly higher values for all the E range (maximum rETR_m,RLC values were 108.6 and 33.4 for HL and LL cultures, respectively). An inverse relationship was consistently found between α_RLC and NPQ, both on LL and HL cultures, causing strong correlations (P < 0.001 in all cases) between NPQ and the high light-induced decrease of α_RLC, Δα_RLC. RLCs were confirmed to also provide information on the long-term photoacclimation status, as significant correlations (P < 0.001 both for HL and LL cultures) were verified between E_k and an index based on RLC parameters, Ê_k, both for LL and HL cultures. These results reinforce the usefulness of RLCs as a tool for inferring on the short- and long-term photoacclimation status of samples with different long-term light histories, through the estimation of LC parameters and the monitoring of NPQ levels.     

Cyanide and chloride exchange on homoleptic gold(III) square-planar complexes: variable pressure kinetic investigation by heteronuclear NMR

Kinetic studies of X⁻ exchange on [AuX₄]⁻ square-planar complexes (where X=Cl⁻ and CN⁻) were performed at acidic pH in the case of chloride system and as a function of pH for the cyanide one. Chloride NMR study (330-365 K) gives a second-order rate law on [AuCl₄]⁻ with the kinetic parameters: (k₂^Au,Cl)²⁹⁸=0.56±0.03 s⁻¹ mol⁻¹ kg; ΔH₂^‡ Au,Cl=65.1±1 kJ mol⁻¹; ΔS₂^‡ Au,Cl=−31.3±3 J mol⁻¹ K⁻¹ and ΔV₂^‡ Au,Cl=−14±2 cm³ mol⁻¹. The variable pressure data clearly indicate the operation of an I_a or A mechanism for this exchange pathway. The proton exchange on HCN was determined by ¹³C NMR as a function of pH and the rate constant of the three reaction pathways involving H₂O, OH⁻ and CN⁻ were determined: k₀^HCN,H=113±17 s⁻¹, k₁^HCN,H=(2.9±0.7)×10⁹ s⁻¹ mol⁻¹ kg and k₂^HCN,H=(0.6±0.2)×10⁶ s⁻¹ mol⁻¹ kg at 298.1 K. The rate law of the cyanide exchange on [Au(CN)₄]⁻ was found to be second order with the following kinetic parameters: (k₂^Au,CN)²⁹⁸=6240±85 s⁻¹ mol⁻¹ kg, ΔH₂^‡ Au,CN=40.0±0.8 kJ mol⁻¹, ΔS₂^‡ Au,CN=−37.8±3 J mol⁻¹ K⁻¹ and ΔV₂^‡ Au,CN=+2±1 cm³ mol⁻¹. The rate constant observed varies about nine orders of magnitude depending on the pH and HCN does not act as a nucleophile. The observed rate constant of X⁻ exchange on [AuX₄]⁻ are two or three orders of magnitude faster than the Pt(II) analogue.     

Mechanistic studies on monodentate-ligand substitution of five-coordinate trigonal-bipyramidal platinum (II) complexes with tris[2-(diphenylphosphino)ethyl]phosphine

Reaction of the five-coordinate trigonal-bipyramidal platinum(II) complex, [Pt(pt)(pp₃)](BF₄) (pt = 1-propanethiolate, pp₃ = tris[2-(diphenylphosphino)ethyl]phosphine), with I⁻ in chloroform gave the five-coordinate square-pyramidal complex with a dissociated terminal phosphino group and an apically coordinated iodide ion in equilibrium. The thermodynamic parameters for the equilibrium between the trigonal-bipyramidal and square-pyramidal geometries, [Pt(pt)(pp₃)]⁺ + I⁻ ? [PtI(pt) (pp₃)], and the kinetic parameters for the chemical exchange were obtained as follows: , ΔH⁰ = − 10 ± 2.4 kJ mol⁻¹, ΔS⁰ = − 36 ± 10 J K⁻¹ mol⁻¹, , ΔH^‡ = 34 ± 4.7 kJ mol⁻¹, ΔS^‡ = − 50 ± 21 J K⁻¹ mol⁻¹. The square-planar trinuclear platinum(II) complex was formed by bridging reaction of one of the terminal phosphino groups of trigonal-bipyramidal [PtCl(pp₃)]Cl with trans-[PtCl₂(NCC₆H₅)₂] in chloroform. From these facts, ligand substitution reactions of [PtX(pp₃)]⁺ (X = monodentate anion) are expected to proceed via an intermediate with a dissociated phosphino group. The rate constants for the chloro-ligand substitution reactions of [PtCl(pp₃)]⁺ with Br⁻ and I⁻ in chloroform approached the respective limiting values as concentrations of the entering halide ions are increased. These kinetic results confirmed the preassociation mechanism in which the square pyramidal intermediate with a dissociated phosphino group and an apically coordinated halide ion is present in the rapid pre-equilibrium.     

Structural studies of trans-N2S2 copper macrocycles

The X-ray crystal structures of two related trans-N₂S₂ copper macrocycles are reported. One was isolated with the copper in the divalent form and the other with copper in its univalent form affording a valuable insight into the changes of geometry and metrical parameters that occur during redox processes in macrocyclic copper complexes. A variable temperature NMR study of the copper(I) complex is reported, indicative of a chair-boat conformational change within the alkyl chain backbone of the macrocycle. It was possible to extract the relevant kinetic and thermodynamic parameters (ΔG^‡, 57.8 kJ mol⁻¹; ΔH^‡, 52.1 kJ mol⁻¹; ΔS^‡, −19.2 J K⁻¹ mol⁻¹) for this process at 298 K. DFT molecular orbital calculations were used to confirm these observations and to calculate the energy difference (26.2 kJmol⁻¹) between the copper(I) macrocycle in a planar and a distorted tetrahedral disposition.     

Kinetics of the cis-to-planar interconversion of cis-diaqua(1,4,7,11-tetraazacyclotetradecane)nickel(II) ion

In order to examine the effects of coordinated hydroxide ion and free hydroxide ion in configurational conversion of a tetraamine macrocyclic ligand complex, the kinetics of the cis-to-planar interconversion of cis-[Ni(isocyclam)(H₂O)₂]²⁺ (isocyclam, 1,4,7,11-tetraazacyclotetradecane) has been studied spectrophotometrically in basic aqueous solution. The interconversion requires the inversion of one sec-NH center of the folded cis-complex to have the planar species. Kinetic data are satisfactorily fitted by the rate law, R = k_OH[OH⁻][cis-[Ni(isocyclam)(H₂O)₂]²⁺], where k_OH = 3.84 × 10³ dm³ mol⁻¹ s⁻¹ at 25.0 ± 0.1 °C with I = 0.10 mol dm⁻³ (NaClO₄). The large ΔH^≠, 61.7 ± 3.2 kJ mol⁻¹, and the large positive ΔS^≠, 30.2 ± 10.8 J K⁻¹ mol⁻¹, strongly support a free-base-catalyzed mechanism for the reaction.     

A batch decolorization and kinetic study of Reactive Black 5 by a bacterial strain Enterobacter sp. GY-1

An Enterobacter strain (GY-1) with high activity of decolorization of Reactive Black 5 (RB 5) was isolated from textile wastewater treating sludge. The kinetic characteristics of dye decolorization by the strain GY-1 were determined quantitatively using the diazo dye, RB 5. Effects of different operation parameters (inoculum size, pH, temperature and salinity) and various electron donors on decolorization of the azo dye by GY-1 were systematically investigated to reveal the primary factors that determine the performance of the azo dye decolorization. The decolorization of RB 5 was attributed to extracellular enzymes. A kinetic model was established giving the dependence of decolorization rate on cell mass concentration (first order). Decolorization rate increased with increasing temperature from 20 to 35 °C, which can be predicted by Arrhenius equation with the activation energy (E_a) of 8.50 kcal mol⁻¹ and the frequency factor (A₀) of 6.28 × 10⁷ mg l g MLSS⁻¹ h⁻¹. Michaelis-Menten kinetics and Eadie-Hofstee plot were used to determine V_max, 1.05 mg l⁻¹ h⁻¹ and K_m, 24.06 mg l⁻¹.     

Formation of a wrapped DNA-protein interface: experimental characterization and analysis of the large contributions of ions and water to the thermodynamics of binding IHF to H' DNA

To characterize driving forces and driven processes in formation of a large-interface, wrapped protein-DNA complex analogous to the nucleosome, we have investigated the thermodynamics of binding the 34-base pair (bp) H′ DNA sequence to the Escherichia coli DNA-remodeling protein integration host factor (IHF). Isothermal titration calorimetry and fluorescence resonance energy transfer are applied to determine effects of salt concentration [KCl, KF, K glutamate (KGlu)] and of the excluded solute glycine betaine (GB) on the binding thermodynamics at 20 °C. Both the binding constant K_obs and enthalpy ΔH°_obs depend strongly on [salt] and anion identity. Formation of the wrapped complex is enthalpy driven, especially at low [salt] (e.g., ΔH^o_obs = − 20.2 kcal·mol^− 1 in 0.04 M KCl). ΔH°_obs increases linearly with [salt] with a slope (dΔH°_obs/d[salt]), which is much larger in KCl (38 ± 3 kcal·mol^− 1 M^− 1) than in KF or KGlu (11 ± 2 kcal·mol^− 1 M^− 1). At 0.33 M [salt], K_obs is approximately 30-fold larger in KGlu or KF than in KCl, and the [salt] derivative SK_obs = dlnK_obs/dln[salt] is almost twice as large in magnitude in KCl (− 8.8 ± 0.7) as in KF or KGlu (− 4.7 ± 0.6).A novel analysis of the large effects of anion identity on K_obs, SK_obs and on ΔH°_obs dissects coulombic, Hofmeister, and osmotic contributions to these quantities. This analysis attributes anion-specific differences in K_obs, SK_obs, and ΔH°_obs to (i) displacement of a large number of water molecules of hydration [estimated to be 1.0(± 0.2) × 10³] from the 5340 Å² of IHF and H′ DNA surface buried in complex formation, and (ii) significant local exclusion of F⁻ and Glu⁻ from this hydration water, relative to the situation with Cl⁻, which we propose is randomly distributed. To quantify net water release from anionic surface (22% of the surface buried in complexation, mostly from DNA phosphates), we determined the stabilizing effect of GB on K_obs: dlnK_obs/d[GB]  = 2.7 ± 0.4 at constant KCl activity, indicating the net release of ca. 150 H₂O molecules from anionic surface.     

Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles

Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue α-helical membrane protein that is involved in transporting Cu⁺ throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with ΔG_w° = 12.9 kJ mol^− 1, m = 4.1 kJ mol^− 1 M^− 1, C_m = 3 M, and ΔCp_w° = 0.93 kJ mol^− 1 K^− 1. These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures.     

A kinetic investigation of the oxidative addition reactions of the dimeric Bu4N[Ir2(μ-Dcbp)(cod)2] complex with iodomethane

The kinetic results of the oxidative addition of iodomethane to Bu₄N[Ir₂(μ-Dcbp)(cod)₂] (Dcbp = 3,5-dicarboxylatepyrazolate anion) show that oxidative addition can occur via a direct equilibrium pathway (K₁ = 88(22) acetone, 51(3) 1,2-dichloroethane, 55(4) dichloromethane, 52(12) acetonitrile and 43(5) M⁻¹ chloroform) or a solvent-assisted pathway (k₂, k₃). Oxidative addition occurs mainly along the direct pathway, which is a factor 10-40 faster than the solvent-assisted pathway. The observed solvent effect cannot be attributed to the donosity or polarity of the solvents. The fairly negative ΔS^≠ value (−110(7) J K⁻¹ mol⁻¹) and the positive ΔH^≠ value (+47(2) kJ mol⁻¹) for the oxidative addition step are indicative of an associative process.     

Horizontal or vertical photobioreactors? How to improve microalgae photosynthetic efficiency

The productivity of a vertical outdoor photobioreactor was quantitatively assessed and compared to a horizontal reactor. Daily light cycles in southern Spain were simulated and applied to grow the microalgae Chlorella sorokiniana in a flat panel photobioreactor.The maximal irradiance around noon differs from 400 μmol photons m⁻² s⁻¹ in the vertical position to 1800 μmol photons m⁻² s⁻¹ in the horizontal position. The highest volumetric productivity was achieved in the simulated horizontal position, 4 g kg culture⁻¹ d⁻¹. The highest photosynthetic efficiency was found for the vertical simulation, 1.3 g of biomass produced per mol of PAR photons supplied, which compares favorably to the horizontal position (0.85 g mol⁻¹) and to the theoretical maximal yield (1.8 g mol⁻¹). These results prove that productivity per unit of ground area could be greatly enhanced by placing the photobioreactors vertically.     

Luminostat operation: a tool to maximize microalgae photosynthetic efficiency in photobioreactors during the daily light cycle?

The luminostat regime has been proposed as a way to maximize light absorption and thus to increase the microalgae photosynthetic efficiency within photobioreactors. In this study, simulated outdoor light conditions were applied to a lab-scale photobioreactor in order to evaluate the luminostat control under varying light conditions. The photon flux density leaving the reactor (PFD_out) was varied from 4 to 20 μmol photons m⁻² s⁻¹and the productivity and photosynthetic efficiency of Chlorella sorokiniana were assessed.Maximal volumetric productivity (1.22 g kg⁻¹ d⁻¹) and biomass yield on PAR photons (400-700 nm) absorbed (1.27 g mol⁻¹) were found when PFD_out was maintained between 4 and 6 μmol photons m⁻² s⁻¹. The resultant photosynthetic efficiency was comparable to that already reported in a chemostat-controlled reactor. A strict luminostat regime could not be maintained under varying light conditions. Further modifications to the luminostat control are required before application under outdoor conditions.     

Adduct formation of [(η-C7H7)Zr(η-C5H5)] with phosphines and N-heterocyclic carbenes: An experimental and theoretical study

The reaction of [(η⁷-C₇H₇)Zr(η⁵-C₅H₅)] with two Lewis bases, tetramethylimidazolin-2-ylidene and PMe₃, is reported and their stability probed via spectroscopic and theoretical methods. The strongly σ-basic N-heterocyclic carbene forms a stable adduct which has been structurally characterised, whilst the PMe₃ ligand coordinates weakly to the metal centre. Variable temperature ³¹P NMR spectroscopy has been used to determine the activation energy for this process (ΔG^‡ = 40.5 ± 1.9 kJ mol⁻¹). DFT calculations have been performed on both complexes and the structures discussed. In addition, the enthalpies for the formation of these compounds have been calculated [ΔH⁰(Zr-IMe) = −56.3 kJ mol⁻¹; ΔH⁰(Zr-PMe₃) = −2.3 kJ mol⁻¹] and show that the N-heterocyclic carbene forms a thermodynamically much more stable adduct than that with PMe₃.     

Kinetics study of the substitution reaction of fac-[Fe(CN)2(CO)3I] with PPh3

Substitution reaction of fac-[Fe^II(CN)₂(CO)₃I]⁻ with triphenylphosphine (PPh₃) produced mono phosphine substituted complex cis-cis-[Fe^II(CN)₂(CO)₂(PPh₃)I]⁻. Crystal structure of the product showed that carbonyl positioned trans- to iodide was replaced by PPh₃. The substitution reaction was monitored by quantitative infrared spectroscopic method, and the rate law for the substitution reaction was determined to be rate = k[[Fe^II(CN)₂(CO)₂(PPh₃)I]⁻][PPh₃]. Transition state enthalpy and entropy changes were obtained from Eyring equation k = (k_BT/h)exp(−ΔH^≠/RT + ΔS^≠/R) with ΔH^≠ = 119(4) kJ mol⁻¹ and ΔS^≠ = 102(10) J mol⁻¹ K⁻¹. Positive transition state entropy change suggests that the substitution reaction went through a dissociative pathway.     

Synthesis, crystal structure and interaction with DNA and HSA of (N,N'-dibenzylethane-1,2-diamine) transition metal complexes

A new ligand, N,N′-dibenzylethane-1,2-diamine (L) and its four transition metal(II) complexes, ML₂(OAc)₂ · 2H₂O (M = Cu, Ni, Zn, Co), have been synthesized and characterized by elemental analysis, mass spectra, molar conductivity, NMR and IR. Moreover, the crystals structure of Cu(II) and Ni(II) complexes characterized by single crystal X-ray diffraction showed that the complexes have a similar molecular structure. Ni(II) has an regular octahedral coordination environment complexes, but typical Jahn Teller effect influenced Cu(II) in an elongated octahedral environment. The interaction between complexes and calf thymus DNA were studied by UV and fluorescence spectra measure, which showed that the binding mode of complexes with DNA is intercalation. Under physiological pH condition, the effects of Cu(OAc)₂L₂ · 2H₂O and Ni(OAc)₂L₂ · 2H₂O on human serum albumin were examined by fluorescence. The results of spectroscopic measurements suggested that the hydrophobic interaction is the predominant intermolecular force. The enthalpy change ΔH⁰ and the entropy change ΔS⁰ of Cu(OAc)₂L₂ · 2H₂O and Ni(OAc)₂L₂ · 2H₂O were calculated to be −11.533 kJ mol⁻¹ and 46.339 J mol⁻¹ K⁻¹, −11.026 kJ mol⁻¹ and 46.396 J mol⁻¹ K⁻¹, respectively, according to the Scatchard’s equation. The quenching mechanism and the number of binding site (n ≈ 1) were also obtained from fluorescence titration data.     

Evaluation of a lab-scale tidal flow constructed wetland performance: Oxygen transfer capacity, organic matter and ammonium removal

Oxygen transfer capacity and removal of ammonium and organic matter were investigated in this study to evaluate the performance of a lab-scale tidal flow constructed wetland. Average oxygen supply under tidal operation (350 g m⁻² d⁻¹) was much higher than in conventional constructed wetlands (<100 g m⁻² d⁻¹), resulting in enhanced removal of BOD₅ and NH₄⁺. Theoretical oxygen demand from BOD₅ removal and nitrification was approximately matched by the measured oxygen supply, which indicated aerobic consumption of BOD₅ and NH₄⁺ under tidal operation. When BOD₅ removal increased from 148 g m⁻² d⁻¹ to 294 g m⁻² d⁻¹, neither exhausted oxygen from the aggregate matrix during feeding period (111 g m⁻² d⁻¹) nor effluent dissolved oxygen (DO) concentration (2.8 mg/L) changed significantly, demonstrating that the oxygen transfer potential of the treatment system had not been exceeded. However, even though DO had not been exhausted, inhibition of nitrification was observed under high BOD loading. The loss of nitrification was attributed to excessive heterotrophic biofilm growth believed to induce oxygen transfer limitations or oxygen competition in thickened biofilms.     

An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.)

The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500 ± 500 Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5-6.5 and showed maximum activity at 60 ± 0.5 °C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly β-structures. The purified protease is thermostable. The apparent T_m, (mid point of thermal inactivation) was found to be 70 ± 0.5 °C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy (E_a) was found to be 44.0 ± 0.3 kcal mol⁻¹. The activation enthalpy (ΔH^∗), free energy change (ΔG^∗) and entropy (ΔS^∗) were estimated to be 43 ± 4 kcal mol⁻¹, −26 ± 3 kcal mol⁻¹ and 204 ± 10 cal mol⁻¹ K⁻¹, respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P₁ position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.     

Thermodynamic analysis of hydration in human serum heme-albumin

Ferric human serum heme-albumin (heme-HSA) shows a peculiar nuclear magnetic relaxation dispersion (NMRD) behavior that allows to investigate structural and functional properties. Here, we report a thermodynamic analysis of NMRD profiles of heme-HSA between 20 and 60 °C to characterize its hydration. NMRD profiles, all showing two Lorentzian dispersions at 0.3 and 60 MHz, were analyzed in terms of modulation of the zero field splitting tensor for the S = ⁵/₂ manifold. Values of correlation times for tensor fluctuation (τ_v) and chemical exchange of water molecules (τ_M) show the expected temperature dependence, with activation enthalpies of −1.94 and −2.46 ± 0.2 kJ mol⁻¹, respectively. The cluster of water molecules located in the close proximity of the heme is progressively reduced in size by increasing the temperature, with ΔH = 68 ± 28 kJ mol⁻¹ and ΔS = 200 ± 80 J mol⁻¹ K⁻¹. These results highlight the role of the water solvent in heme-HSA structure-function relationships.