Temperature and pressure effects on structural and conformational properties of POPC/SM/cholesterol model raft mixtures—a FT-IR, SAXS, DSC, PPC and Laurdan fluorescence spectroscopy study

We report on the effects of temperature and pressure on the structure, conformation and phase behavior of aqueous dispersions of the model lipid “raft” mixture palmitoyloleoylphosphatidylcholine (POPC)/bovine brain sphingomyelin (SM)/cholesterol (Chol) (1:1:1). We investigated interchain interactions, hydrogen bonding, conformational and structural properties as well as phase transformations of this system using Fourier transform-infrared (FT-IR) spectroscopy, small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), and Laurdan fluorescence spectroscopy. The IR spectral parameters in combination with the scattering patterns from the SAXS measurements were used to detect structural and conformational transformations upon changes of pressure up to 7-9 kbar and temperature in the range from 1 to about 80 °C. The generalized polarization function (GP) values, obtained from the Laurdan fluorescence spectroscopy studies also reveal temperature and pressure dependent phase changes. DSC and PPC were used to detect thermodynamic properties accompanying the temperature-dependent phase changes. In combination with literature fluorescence spectroscopy and microscopy data, a tentative p,T stability diagram of the mixture has been established. The data reveal a broad liquid-order/solid-ordered (l_o + s_o) two-phase coexistence region below 8 ± 2 °C at ambient pressure. With increasing temperature, a l_o + l_d + s_o three-phase region is formed, which extends up to ∼27 °C, where a liquid-ordered/liquid-disordered (l_o + l_d) immiscibility region is formed. Finally, above 48 ± 2 °C, the POPC/SM/Chol (1:1:1) mixture becomes completely fluid-like (liquid-disordered, l_d). With increasing pressure, all phase transition lines shift to higher temperatures. Notably, the l_o + l_d (+s_o) phase coexistence region, mimicking raft-like lateral phase separation in natural membranes, extends over a rather wide temperature range of about 40 °C, and a pressure range, which extends up to about 2 kbar for T = 37 °C. Interestingly, in this pressure range, ceasing of membrane protein function in natural membrane environments has been observed for a variety of systems.     

Application of pressure perturbation calorimetry to lipid bilayers

Pressure perturbation calorimetry (PPC) is a new method that measures the heat consumed or released by a sample after a sudden pressure jump. The heat change can be used to derive the thermal volume expansion coefficient, alpha(V), as a function of temperature and, in the case of phase transitions, the volume change, DeltaV, occurring at the phase transition. Here we present the first report on the application of PPC to determine these quantities for lipid bilayers. We measure the volume changes of the pretransition and main transition of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the thermal expansivity of the fluid phase of DMPC and of two unsaturated lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine. The high sensitivity of PPC instrumentation gives accurate data for alpha(V) and DeltaV even upon the application of relatively low pressures of approximately 5 bar.     

Erythrocyte morphological states, phases, transitions and trajectories

Morphological response (MR) of red blood cells represents a triphasic sequence of spontaneously occurring shape transformation between different shape states upon transfer the cells into isotonic sucrose solution in the order: S₀ (initial discoid shape in physiological saline) → S₁ (echinocytic shape at the beginning of MR, phase 1) → S₂ (intermediate discoid shape, phase 2) → S₃ (final stomatocytic shape, phase 3). In this paper, the dynamics of cell shape changes was investigated by non-invasive light fluctuation method and optical microscopy. Among 12 possible transitions between four main shape states, we experimentally demonstrate here an existence of nine transitions between neighbour or remote states in this sequence. Based on these findings and data from the literature, we may conclude that red blood cells are able to change their shape through direct transitions between four main states except transition S₁ → S₀, which has not been identified yet. Some shape transitions and their temporal sequence are in accord with predictions of bilayer couple concept, whereas others for example transitions between remote states S₃ → S₁, S₁ → S₃ and S₃ → S₀ are difficult to explain based solely on the difference in relative surface areas of both leaflets of membrane suggesting more complex mechanisms involved. Our data show that MR could represents a phenomenon in which the major role can play pH and chloride-sensitive sensor and switching mechanisms coupled with transmembrane signaling thus involving both cytoskeleton and membrane in coordinated shape response on changes in cell ionic environment.     

The solid-liquid phase diagrams of binary mixtures of consecutive, even saturated fatty acids: differing by four carbon atoms

The complete solid-liquid phase diagrams for four binary mixtures of saturated fatty acids are presented, for the first time, in this work. These mixtures are formed by caprylic acid (C_8:0) + lauric acid (C_12:0), capric acid (C_10:0) + myristic acid (C_14:0), lauric acid (C_12:0) + palmitic acid (C_16:0) and myristic acid (C_14:0) + stearic acid (C_18:0). The phase diagrams were obtained by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). FT-Raman spectrometry and polarized light microscopy were used to complement the characterization for a complete understanding of the phase diagram. All of the phase diagrams here reported show the same global behavior that is far more complex than previously accepted. They present not only peritectic and eutectic reactions, but also metatectic reactions, due to solid-solid phase transitions common in fatty acids, and regions of solid solution not previously reported. This work contributes to the elucidation of the phase behavior of these important biochemical molecules with implications in various industrial applications.     

Structure and phase behavior of O-stearoylethanolamine: A combined calorimetric, spectroscopic and X-ray diffraction study

Recent studies show that O-acylethanolamines (OAEs), structural isomers of the putative stress-fighting lipids, namely N-acylethanolamines (NAEs), can be derived from NAEs and are present in biological membranes under physiological conditions. In view of this, we have synthesized O-stearoylethanolamine (OSEA) as a representative OAE and investigated its phase behavior and crystal structure. The thermotropic phase transitions of OSEA dispersed in water and in 150 mM NaCl were characterized using calorimetric, spectroscopic, turbidimetric and X-ray diffraction studies. These studies have revealed that when dispersed in water OSEA undergoes a cooperative phase transition centered at 53.8 °C from an ordered gel phase to a micellar structure whereas in presence of 150 mM NaCl the transition temperature increases to 55.8 °C and most likely the bilayer structure is retained above the phase transition. O-Stearoylethanolamine crystallized in the orthorhombic space group P2₁2₁2₁ with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that OSEA molecules adopt a linear structure with all-trans conformation in the acyl chain region. The molecules are organized in a tail-to-tail fashion, similar to the arrangement in a bilayer membrane. These studies are relevant to understanding the role of salt on the phase properties of this new class of lipids.     

Efficiently measuring complex sessile epibenthic organisms using a novel photogrammetric technique

This paper describes a stereo photogrammetry method that allows accurate measurements of volume for sessile epibenthic organisms. It represents a novel approach based on multiple views (five stereo image pairs) from a purpose built stereo digital still camera system combined with a three-dimensional reconstruction software program (CAM). The bias, accuracy, precision and efficiency of the method were assessed in the laboratory using models with three levels of morphological complexity (simple, moderate and complex morphologies) and two sizes (large and small). The technique did not show any biases to observer experience, with no significant difference among observers (p > 0.05). Volume measurements made with CAM were very accurate when compared with the water displacement volume of each model, with an overall mean error of about − 3% (S.E. ± 1%). The CAM volume measurements were more accurate on complex models and moderately complex models than on models with simple morphologies. Also, large models had a higher accuracy than small models. Volume measurements made with CAM were also highly precise with the lowest precision observed being ± 2% of the volume estimate. The time required for a volume estimation using the CAM method was also highly efficient, and the longest time taken for a volume estimation was on average 1 h and 36 min, making it the fastest reported three-dimensional reconstruction method. In field applications, volume estimations of four sponges were all within the observed accuracy, precision and efficiency established during laboratory trials. The accuracy, precision and efficiency demonstrated by the CAM method make this technique highly suitable for routine measurements of the volume of sessile epibenthic organisms.     

Micro- and macrorheological properties of isotropically cross-linked actin networks

Cells make use of semiflexible biopolymers such as actin or intermediate filaments to control their local viscoelastic response by dynamically adjusting the concentration and type of cross-linking molecules. The microstructure of the resulting networks mainly determines their mechanical properties. It remains an important challenge to relate structural transitions to both the molecular properties of the cross-linking molecules and the mechanical response of the network. This can be achieved best by well defined in vitro model systems in combination with microscopic techniques. Here, we show that with increasing concentrations of the cross-linker heavy meromyosin, a transition in the mechanical network response occurs. At low cross-linker densities the network elasticity is dominated by the entanglement length l_e of the polymer, whereas at high heavy meromyosin densities the cross-linker distance l_c determines the elastic behavior. Using microrheology the formation of heterogeneous networks is observed at low cross-linker concentrations. Micro- and macrorheology both report the same transition to a homogeneous cross-linked phase. This transition is set by a constant average cross-linker distance l_c ≈ 15 μm. Thus, the micro- and macromechanical properties of isotropically cross-linked in vitro actin networks are determined by only one intrinsic network parameter.     

Polyproline fold—In imparting kinetic stability to an alkaline serine endopeptidase

Polyproline II (PPII) fold, an unusual structural element was detected in the serine protease from Nocardiopsis sp. NCIM 5124 (NprotI) based on far UV circular dichroism spectrum, structural transitions of the enzyme in presence of GdnHCl and a distinct isodichroic point in chemical and thermal denaturation. The functional activity and conformational transitions of the enzyme were studied under various denaturing conditions. Enzymatic activity of NprotI was stable in the vicinity of GdnHCl upto 6.0 M concentration, organic solvents viz. methanol, ethanol, propanol (all 90% v/v), acetonitrile (75% v/v) and proteases such as trypsin, chymotrypsin and proteinase K (NprotI:protease 10:1). NprotI seems to be a kinetically stable protease with a high energy barrier between folded and unfolded states. Also, an enhancement in the activity of the enzyme was observed in 1 M GdnHCl upto 8 h, in organic solvents (75% v/v) for 72 h and in presence of proteolytic enzymes. The polyproline fold remained unaltered or became more prominent under the above mentioned conditions. However, it diminished gradually during thermal denaturation above 60 °C. Thermal transition studies by differential scanning calorimetry (DSC) showed scan rate dependence as well as irreversibility of denaturation, the properties characteristic of kinetically stable proteins. This is the first report of PPII helix being the global conformation of a non structural protein, an alkaline serine protease, from a microbial source, imparting kinetic stability to the protein.     

Pressure perturbation and differential scanning calorimetric studies of bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

Differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) were used to characterize thermal phase transitions, membrane packing, and volumetric properties in multilamellar vesicles (MLVs) composed of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at different temperatures. For PLFE MLVs derived from cells grown at 78 degrees C, the first DSC heating scan exhibits an endothermic transition at 46.7 degrees C, a small hump near 60 degrees C, and a broad exothermic transition at 78.5 degrees C, whereas the PPC scan reveals two transitions at approximately 45 degrees C and 60 degrees C. The endothermic peak at 46.7 degrees C is attributed to a lamellar-to-lamellar phase transition and has an unusually low DeltaH (3.5 kJ/mol) and DeltaV/V (0.1%) value, as compared to those for the main phase transitions of saturated diacyl monopolar diester lipids. This result may arise from the restricted trans-gauche conformational changes in the dibiphytanyl chain due to the presence of cyclopentane rings and branched methyl groups and due to the spanning of the lipid molecules over the whole membrane. The exothermic peak at 78.5 degrees C probably corresponds to a lamellar-to-cubic phase transition and exhibits a large and negative DeltaH value (-23.2 kJ/mol), which is uncommon for normal lamellar-to-cubic phospholipid phase transformations. This exothermic transition disappears in the subsequent heating scans and thus may involve a metastable phase, which is irreversible at the scan rate used. Further, there is no distinct peak in the plot of the thermal expansion coefficient alpha versus temperature near 78.5 degrees C, indicating that this lamellar-to-cubic phase transition is not accompanied by any significant volume change. For PLFE MLVs derived from cells grown at 65 degrees C, similar DSC and PPC profiles and thermal history responses were obtained. However, the lower growth temperature yields a higher DeltaV/V ( approximately 0.25%) and DeltaH (14 kJ/mol) value for the lamellar-to-lamellar phase transition measured at the same pH (2.1). A lower growth temperature also generates a less negative temperature dependence of alpha. The changes in DeltaV/V, DeltaH, and the temperature dependence of alpha can be attributed to the decrease in the number of cyclopentane rings in PLFE at the lower growth temperature. The relatively low DeltaV/V and small DeltaH involved in the phase transitions help to explain why PLFE liposomes are remarkably thermally stable and also echo the proposal that PLFE liposomes are generally rigid and tightly packed. These results help us to understand why, despite the occurrence of thermal-induced phase transitions, PLFE liposomes exhibit a remarkably low temperature sensitivity of proton permeation and dye leakage.     

Ruthenium (II) thiacrown complexes as hydrogen-transfer reduction catalysts

An investigation into the potential of a series of Ruthenium (II) thiacrown complexes with general formula [Ru([9]aneS₃)Cl₂(L)] and [Ru([n]aneS₄)Cl(L)]⁺, where L = DMSO or Ph₃P, n = 12, 14, or 16, as hydrogen transfer reduction catalysts is reported. As part of these studies two new complexes incorporating [Ru([12]aneS₄)] and [Ru([16]aneS₄)] metal centres have been synthesised and fully characterised. The X-ray structure of one of these complexes is also reported. The UV/Vis spectra of these complexes are dominated by π → π* transitions, with weaker d → d transitions also being apparent. Using these eight structurally related complexes, studies on the transfer hydrogenation of acetophenone using propan-2-ol as the hydride source were carried out. This work revealed that the complexes displayed catalytic activity in the presence of a base promoter. Although the activity of these complexes were considerably lower than that of structurally related mixed-donor ligand systems, there was some evidence that the flexibility of the ligand did have an effect on initial catalytic activity.     

Determination of the γ-secretase inhibitor MK-0752 in human plasma by online extraction and electrospray tandem mass spectrometry (HTLC–ESI-MS/MS)

A sensitive and rapid HTLC–ESI-MS/MS method with an advanced online sample preparation was developed for determination of the γ-secretase inhibitor MK-0752 in human plasma using an internal standard. Plasma samples (100 μL) were diluted and injected directly onto an online extraction column (Cohesive Cyclone MAX 0.5 mm × 50 mm, >30 μm), the sample matrix was washed out with an aqueous solution, and retained analytes were eluted out and transferred directly to the analytical column (Phenomenex Gemini 3μ C18 110A, 50 mm × 2.0 mm at 50 °C) for separation using a gradient mobile phase. The eluted analytes were then detected on an API-3000 LC–MS/MS System with ESI and a negative multiple reaction monitoring mode. The monitored ion transitions were m/z 441 → 175 for MK-0752 and 496 → 175 for the internal standard. Online extraction recoveries were 81%. The method was validated and was linear in the range of 0.05–50 μg/mL. Within-day and between-day precisions were < 8.6%, and accuracies were 0.7 and 7.1%. This method was applied to the measurement of plasma MK-0752 levels in a Phase I study of pediatric patients with recurrent or refractory brain tumors.     

Atomic force microscopy and force spectroscopy study of Langmuir-Blodgett films formed by heteroacid phospholipids of biological interest

Langmuir-Blodgett (LB) films of two heteroacid phospholipids of biological interest 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), as well as a mixed monolayer with χ_POPC = 0.4, were transferred onto mica in order to investigate by a combination of atomic force microscopy (AFM) and force spectroscopy (FS) their height, and particularly, their nanomechanical properties. AFM images of such monolayers extracted at 30 mN m^− 1 revealed a smooth and defect-free topography except for the POPE monolayer. Since scratching such soft monolayers in contact mode was proved unsuccessful, their molecular height was measured by means of the width of the jump present in the respective force-extension curves. While for pure POPC a small jump occurs near zero force, for the mixed monolayer with χ_POPC = 0.4 the jump occurs at ∼ 800 pN. Widths of ∼ 2 nm could be established for POPC and χ_POPC = 0.4, but not for POPE monolayer at this extracting pressure. Such different mechanical stability allowed us to directly measure the threshold area/lipid range value needed to induce mechanical stability to the monolayers. AFM imaging and FS were next applied to get further structural and mechanical insight into the POPE phase transition (LC-LC′) occurring at pressures > 36.5 mN m^− 1. This phase transition was intimately related to a sudden decrease in the area/molecule value, resulting in a jump in the force curve occurring at high force (∼ 1.72 nN). FS reveals to be the unique experimental technique able to unveil structural and nanomechanical properties for such soft phospholipid monolayers. The biological implications of the nanomechanical properties of the systems under investigation are discussed considering that the annular phospholipids region of some transmembrane proteins is enriched in POPE.     

Pressure perturbation calorimetry of lipoproteins reveals an endothermic transition without detectable volume changes. Implications for adsorption of apolipoprotein to a phospholipid surface

Plasma lipoproteins are assemblies of lipids and apolipoproteins that mediate lipid transport and metabolism. High-density lipoproteins (HDL) remove excess cell cholesterol and provide protection against atherosclerosis. Important aspects of metabolic HDL remodeling, including apolipoprotein dissociation and lipoprotein fusion, are mimicked in thermal denaturation. We report the first study of the protein-lipid complexes by pressure perturbation calorimetry (PPC) beyond 100 °C. In PPC, volume expansion coefficient α(v)(T) is measured during heating; in proteins, α(v)(T) is dominated by hydration. Calorimetric studies of reconstituted HDL and of human high-density, low-density, and very low-density lipoproteins reveal that apolipoprotein unfolding, dissociation, and lipoprotein fusion are endothermic transitions without detectable volume changes. This may result from the limited applicability of PPC to slow kinetically controlled transitions such as thermal remodeling of lipoproteins and/or from the possibility that this remodeling causes no significant changes in the solvent structure and, hence, may not involve large transient solvent exposure of apolar moieties. Another conclusion is that apolipoprotein A-I in solution adsorbs to the phospholipid surface; protein hydration is preserved upon such adsorption. We posit that adsorption to a phospholipid surface helps recruit free apolipoprotein to the plasma membrane and facilitate HDL biogenesis.     

An oriented adsorption strategy for efficient solid phase PEGylation of recombinant staphylokinase by immobilized metal-ion affinity chromatography

Conjugation of truncated recombinant staphylokinase (trSak) with polyethylene glycol (PEG) is an effective way to overcome its short plasma half-life and enhance its therapeutic potential. However, conventional amine directed PEGylation chemistry inevitably led to modification at its functionally important N terminus, which resulted in a significantly reduced bioactivity of trSak. In this study, a novel solid phase PEGylation process was developed to shield the N-terminal region of the protein from PEGylation. The process was achieved by oriented adsorption of an N-terminally His-tagged trSak (His-trSak) onto an immobilized metal-ion affinity chromatography (IMAC). His-trSak was efficiently separated and retained on IMAC media before reaction with succinimidyl carbonate mPEG (SC-mPEG, 5, 10 or 20 kDa). The IMAC derived mono-PEGylated His-trSak showed structural and stability properties similar to the liquid phase derived conjugate. However, isoelectric focusing electrophoresis analysis revealed that mono-PEGylated His-trSaks via solid phase PEGylation were more homogeneous than those from liquid phase PEGylation. Moreover, tryptic peptide mapping analysis suggested that a complete N-terminal blockage of IMAC bound His-trSak from PEGylation with 10 kDa- and 20 kDa-SC-mPEG. In contrast, only partial protection of the N-terminal region was obtained for 5 kDa-SC-mPEG. Bioactivities of 10 kDa- and 20 kDa-PEG-His-trSak conjugates without N-terminal PEGylation were significantly higher than those of randomly PEGylated products. This further demonstrated the advantage of our new on-column PEGylation strategy.     

Conformational transition pathways explored by Monte Carlo simulation integrated with collective modes

Conformational transitions between open/closed or free/bound states in proteins possess functional importance. We propose a technique in which the collective modes obtained from an anisotropic network model (ANM) are used in conjunction with a Monte Carlo (MC) simulation approach, to investigate conformational transition pathways and pathway intermediates. The ANM-MC technique is applied to adenylate kinase (AK) and hemoglobin. The iterative method, in which normal modes are continuously updated during the simulation, proves successful in accomplishing the transition between open-closed conformations of AK and tense-relaxed forms of hemoglobin (C^α− root mean square deviations between two end structures of 7.13 Å and 3.55 Å, respectively). Target conformations are reached by root mean-square deviations of 2.27 Å and 1.90 Å for AK and hemoglobin, respectively. The intermediate conformations overlap with crystal structures from the AK family within a 3.0-Å root mean-square deviation. In the case of hemoglobin, the transition of tense-to-relaxed passes through the relaxed state. In both cases, the lowest-frequency modes are effective during transitions. The targeted Monte Carlo approach is used without the application of collective modes. Both the ANM-MC and targeted Monte Carlo techniques can explore sequences of events in transition pathways with an efficient yet realistic conformational search.     

An LC-MS/MS method for determination of forsythiaside in rat plasma and application to a pharmacokinetic study

A highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of forsythiaside in rat plasma using epicatechin as internal standard. The analytes were extracted by solid-phase extraction and chromatographied on a C₁₈ column eluted with a gradient mobile phase of acetonitrile and water both containing 0.2% formic acid. The detection was performed by negative ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 623 → 161 and m/z 289 → 109 for forsythiaside and epicatechin, respectively. The assay was linear over the concentration ranges of 2.0–50.0 and 50.0–5000.0 ng/mL with limits of detection and quantification of 0.2 and 1.0 ng/mL, respectively. The precision was <10.8% and the accuracy was >91.9%, and extraction recovery ranged from 81.3% to 85.0%. This method was successfully applied to a pharmacokinetic study of forsythiaside in rats after intravenous (20 mg/kg) and oral (100 mg/kg) administration, and the result showed that the compound was poorly absorbed with an absolute bioavailability being approximately 0.5%.     

Influence of native starch's properties on starch nanocrystals thermal properties

Starch nanocrystals (SNC) are crystalline square-like platelet about 10 nm thick and 50-100 nm equivalent diameters. Depending on the botanic origin of starch these platelets show different features. The aim of the present study was (i) to assess the thermal stability of SNC in different processing conditions (i.e., excess water and dry) and (ii) to investigate the potential influence of botanic origin on thermal stability. The thermal properties of five types of starches (waxy maize, normal maize, high amylose maize, potato and wheat) and their corresponding SNC were characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). SNC revealed two endothermic transitions. No correlation between melting temperature and botanic origin was found. However, a review of starch thermal properties allowed to postulate for the mechanism involved in SNC thermal transitions. It was also found that SNC can be used in wet processes below 100 °C and in dry processes below 150-200 °C to avoid melting.     

Redefining the Dry Molten Globule State of Proteins

Dynamics and function of proteins are governed by the structural and energetic properties of the different states they adopt and the barriers separating them. In earlier work, native-state triplet–triplet energy transfer (TTET) on the villin headpiece subdomain (HP35) revealed an equilibrium between a locked native state and an unlocked native state, which are structurally similar but have different dynamic properties. The locked state is restricted to low amplitude motions, whereas the unlocked state shows increased conformational flexibility and undergoes local unfolding reactions. This classified the unlocked state as a dry molten globule (DMG), which was proposed to represent an expanded native state with loosened side-chain interactions and a solvent-shielded core. To test whether the unlocked state of HP35 is actually expanded compared to the locked state, we performed high-pressure TTET measurements. Increasing pressure shifts the equilibrium from the locked toward the unlocked state, with a small negative reaction volume for unlocking (ΔV⁰ = − 1.6 ± 0.5 cm³/mol). Therefore, rather than being expanded, the unlocked state represents an alternatively packed, compact state, demonstrating that native proteins can exist in several compact folded states, an observation with implications for protein function. The transition state for unlocking/locking, in contrast, has a largely increased volume relative to the locked and unlocked state, with respective activation volumes of 7.1 ± 0.4 cm³/mol and 8.7 ± 0.9 cm³/mol, indicating an expansion of the protein during the locking/unlocking transition. The presented results demonstrate the existence of both compact, low-energy and expanded, high-energy DMGs, prompting a broader definition of this state.     

A theoretical study of Ru(II) polypyridyl DNA intercalators: Structure and electronic absorption spectroscopy of [Ru(phen)2(dppz)] and [Ru(tap)2(dppz)] complexes intercalated in guanine-cytosine base pairs

The structural and spectroscopic properties of [Ru(phen)₂(dppz)]²⁺ and [Ru(tap)₂(dppz)]²⁺ (phen = 1,10-phenanthroline; tap = 1,4,5,8-tetraazaphenanthrene; dppz = dipyridophenazine ) have been investigated by means of density functional theory (DFT), time-dependent DFT (TD-DFT) within the polarized continuum model (IEF-PCM) and quantum mechanics/molecular mechanics (QM/MM) calculations. The model of the Δ and Λ enantiomers of Ru(II) intercalated in DNA in the minor and major grooves is limited to the metal complexes intercalated in two guanine-cytosine base pairs. The main experimental spectral features of these complexes reported in DNA or synthetic polynucleotides are better reproduced by the theoretical absorption spectra of the Δ enantiomers regardless of intercalation mode (major or minor groove). This is especially true for [Ru(phen)₂(dppz)]²⁺. The visible absorption of [Ru(tap)₂(dppz)]²⁺ is governed by the MLCT_tap transitions regardless of the environment (water, acetonitrile or bases pair), the visible absorption of [Ru(phen)₂(dppz)]²⁺ is characterized by transitions to metal-to-ligand-charge-transfer MLCT_dppz in water and acetonitrile and to MLCT_phen when intercalated in DNA. The response of the IL_dppz state to the environment is very sensitive. In vacuum, water and acetonitrile these transitions are characterized by significant oscillator strengths and their positions depend significantly on the medium with blue shifts of about 80 nm when going from vacuum to solvent. When the complex is intercalated in the guanine-cytosine base pairs the ¹IL_dppz transition contributes mainly to the band at 370 nm observed in the spectrum of [Ru(phen)₂(dppz)]²⁺ and to the band at 362 nm observed in the spectrum of [Ru(tap)₂(dppz)]²⁺.     

Determination of aconitine,hypaconitine and mesaconitine in urine using hollow fiber liquid-phase microextraction combined with high-performance liquid chromatography

Hollow fiber liquid-phase microextraction (HF-LPME) coupled with high-performance liquid chromatography was used to simultaneously determine three Aconitum alkaloids, including aconitine (AC), hypaconitine (HA) and mesaconitine (MA) in human urine sample. Analytes were extracted from 5 mL urine sample containing 1.0 mmol/L NaOH into 1-octanol membrane phase impregnated in the pores of hollow fiber wall, and then back extracted into acidified aqueous solution in the lumen of the hollow fiber. After extraction, 10 μL of the acceptor phase was analyzed directly by HPLC. In this method, some important extraction parameters, such as organic solvent, extraction time, stirring rate, pH of donor phase and acceptor phase, temperature, and the volume of acceptor phase were optimized. This method provided 98- to 288-fold enrichment factors within 60 min of extraction and good repeatability with RSDs of 0.99–7.22%. The calibration curves were linear over the ranges of 16.0–128.0 μg/L for AC, 11.0–88.0 μg/L for HA and 8.1–64.8 μg/L for MA in human urine sample, with correlation coefficients of 0.9949, 0.9969 and 0.9904, respectively. Limits of detection were from 0.7 to 1.5 μg/L, and recoveries from spiked urine sample varied from 84.4% to 106.2% for AC, 77.3% to 85.6% for HA and 90.1% to 100.8% for MA.