D2O Buffer Solution Research Articles

Controlled drug release: On the evolution of physically entrapped drug inside the electrospun poly(lactic-co-glycolic acid) matrix

The drug loading and releasing properties of poly(lactic-co-glycolic acid) (PLGA) were approached with the application of neutron techniques. The neutron reflection (NR) study on the response of PLGA material to vapor and to bulk water revealed that the hydration of PLGA origins from the molecular compatibility between water and PLGA. Hydration is reversible with regard to the change in humidity and temperature. Capecitabine as drug was embedded in the electrospun PLGA fibers. Small angle neutron scattering (SANS) was able to disclose the domain of entrapped drug inside the fibers and trace its evolution over time when the electrospun membrane was incubated in D2O buffer solution. The evolution of drug domains is discussed in terms of the concentration dependence, the temperature dependence, and the relevance between the drug diffusion inside the polymer matrix and the drug release out to the medium. It was observed that, at 20 °C the drug-related domains are relatively small (~ 100 Å) and relax extremely slow while at 37 °C the drug-related domains are relatively larger (~ 200 Å) and relax faster. These behaviors can be related to the glassy property of structural material. The transportation of drug through the polymer matrix relies on the global relaxation of PLGA chains. The variation of fiber diameter vs. incubation time was followed by ultra-small angle neutron scattering (USANS). The bi-phasic or tri-phasic release kinetics from a series of fibers with different drug loading (2%, 5%, 10%, 20%, 30%, 40%, 50%) were discussed based on the SANS and USANS discovery.

2nd coordination sphere controlled electron transfer of iron hangman complexes on electrodes probed by surface enhanced vibrational spectroscopy.

Iron hangman complexes exhibit improved catalytic properties regarding O2 and H2O2 reduction, which are attributed to the presence of a proton donating group in defined vicinity of the catalytic metal centre. Surface enhanced resonance Raman (SERR) and IR (SEIRA) spectro-electrochemistry has been applied concomitantly for the first time to analyse such iron hangman porphyrin complexes attached to electrodes in aqueous solution. While the SERR spectra yield information about the redox state of the central iron, the SEIRA spectra show protonation and deprotonation events of the 2nd coordination sphere. To investigate the influence of a proton active hanging group on the heterogeneous electron transfer between the iron porphyrin and the electrode, two hangman complexes with either an acid or ester functional group were compared. Using time resolved SERR spectroscopy the electron transfer rates of both complexes were determined. Complexes with an acid group showed a slow electron transfer rate at neutral pH that increased significantly at pH 4, while complexes with an ester group exhibited a much faster, but pH independent rate. SEIRA measurements were able to determine directly for the first time a pKa value of 3.4 of a carboxylic hanging group in the immobilized state that shifted to 5.2 in D2O buffer solution. The kinetic data showed an increase of the heterogeneous electron transfer rate with the protonation degree of the acid groups. From these results, we propose a PCET which is strongly modulated by the protonation state of the acid hanging group via hydrogen bond interactions.

Crystal Growth of Lysozyme Controlled by Laser Trapping

We demonstrate the growth of a tetragonal crystal of hen egg-white lysozyme (HEWL) in D2O buffer solution controlled by laser trapping with a focused continuous-wave (CW) near-infrared (NIR) laser beam. The focal spot was located at 10 μm away from the edge of the target crystal that was generated spontaneously, and the crystal growth was observed although the focal spot size was much smaller than the distance. The growth rate of (101) and {110} faces of the tetragonal crystal was examined with various laser powers and polarizations. The rate observed under the irradiation was much different from those in spontaneous growth, namely, the growth rate of the {110} face showed a large decrease or increase depending on the irradiation time. The dynamics and mechanism of this unusual crystal growth behavior is discussed from the viewpoint of a large stable domain formation of the HEWL liquidlike clusters through liquid nucleation and growth and by considering the anisotropy of the cluster domain.

Polarization Analysis with 3He Spin Filters for Separating Coherent from Incoherent Scattering in Soft Matter Studies

In soft matter small angle neutron scattering (SANS) studies at large Q values, incoherent scattering becomes the dominant signal. In the Q-range of interest to this work, from 0.2 A −1 to about 1.0 A −1 , the coherent scattering from the typical protein or polymer in a D2 Ob uffer solution inevitably drops one to two orders of magnitude or more below the total scattering. Even after careful and accurate subtraction of the measured D2 Ob uffer scattering, the remaining corrected, i.e. sample-only, signal will still be dominated by diffuse incoherent scattering from hydrogen in the sample itself. This is the exact region of interest when one wishes to probe the structural changes in ”living” proteins caused by interactions and motions related to function. To further complicate the problem, there is strong motivation to measure this Q-regime at very low concentrations because it has been shown with wide angle X-ray scattering that proteins can undergo concentration-dependent structural changes that rapidly increase below concentrations of about 5% [1] motivating the study of protein solutions at ever lower concentrations. In this case the signal from the protein will inevitably become much less than the scattering of the D2 Ob uffer solution it is contained in. Polarization analysis offers the opportunity to separate the weak coherent signal from the larger incoherent signal and perhaps enable measurements under the conditions described above. This paper will address the issues associated with the correct separation of coherent and incoherent scattering for soft matter samples. We have performed tests measurements on KWS2 which show the viability of the method on a protonated α-lactalbumin solution at 2.5% (1 mm thick) and 0.25% (2 mm thick) concentrations in a D2 Ob uffer solution. Additionally describe a the method of implementation using 3 He spin filters, some practical considerations, and future plans for a dedicated device at the JCNS. c � 2012 for authors transferring the copyright to Elsevier use the following: Elsevier BV. Selection and/or peer-review under responsibility of the organizing committee for PNCMI 2012.

Heat-induced structural transitions of alpha-crystallin studied by small-angle neutron scattering

Alpha-crystallin from the bovine eye lens was studied by small-angle neutron scattering (SANS) in 90% D2O buffer solution at 20, 50, 60, 65, 75, 85 and 95°C. The temperature points for this study were specified on the basis of differential scanning calorimetric analysis of alpha-crystallin solutions which has shown two endothermic transitions with midpoints at 64.5 and 86°C. The SANS study revealed no significant alpha-crystallin quaternary structure alterations at 50°C as compared with 20°C. At 60-65°C the SANS data confirmed substantial alpha-crystallin quaternary structure rearrangements which resulted in the formation of alpha-crystallin oligomers with a similar shape but approximately twofold increased molecular weight as compared to the native state at 20°C. At higher temperatures (75, 85 and 95°C) the SANS patterns were very similar and were consistent with the scattering by rod-like particles with a cross-section radius of gyration ∼55 This transformation of alpha-crystallin to the rod-like particles was evidently irreversible as these particles remained in solution after cooling to 20°C. Ab initio shape models of the native and high-temperature alpha-crystallin were retrieved with DAMMIN and DAMAVER software. Schematic model of alpha-crystallin heat-induced quaternary structure transitions was considered.

Interface Polymerization in a Polymer Micelle: An NMR Study of the Radical Polymerization of Methyl Methacrylate at the Core−Shell Interface of Polystyrene-block-poly(methacrylic acid) Micelles

Systems containing poly(styrene)-block-poly(methacrylic acid) micelles (1.67 g/L), methyl methacrylate (1.88−3.76 g/L), and initiator (polymerization) or inhibitor (pure solubilization) in D2O buffer solution were studied by 1H NMR. The proton NMR signals of MMA dissolved in the micellar system have the same chemical shifts as those in the corresponding D2O buffer solution but exhibit a characteristically broadened and asymmetric shape. Separate signals with different chemical shifts and signal shapes were observed for MMA absorbed into the poly(styrene) core. From the shape analysis of the signals of the water-dissolved MMA in the micellar system, it can be deduced that the monomer is almost exclusively accumulated near the core−shell interface. Its radial distribution can be approximated by a Gaussian function with the maximum at the core radius R and half-width b of 1.65R and 1.93R for the MMA concentrations 2 and 4 g/L, respectively. An increase in temperature from 295 to 330 K leads to a faster self-...

Pressure-induced structural rearrangements of bovine pancreatic trypsin inhibitor studied by FTIR spectroscopy.

Fourier transform infrared (FTIR) spectroscopy combined with resolution enhancement techniques, second-derivative and difference spectroscopies, have been used to characterize pressure-induced changes in the structural rearrangements of bovine pancreatic trypsin inhibitor (BPTI) in D2O solution at 25.0 degrees C. According to the observed changes in the amide I' band up to 550 MPa, the secondary structure elements of BPTI, such as the alpha-helix, 3(10)-helix, beta-sheet, and beta-turn, are scarcely rearranged except for the loop structure of residues of 9-17 and 36-43. The polypeptide backbone is not extensively unfolded up to 550 MPa. The minor pressure-induced structural rearrangements are completely reversible. A further increase in pressure above 1000 MPa associated with the precipitation of BPTI in D2O buffer solution induces the partial structural rearrangements of the alpha-helix, beta-turn and/or 3(10)-helix, and beta-sheet. The polypeptide backbone of BPTI is not fully unfolded even above 1000 MPa. Most of the protected backbone amide protons involved in the beta-sheet remain intact in the pressure range where BPTI is not precipitated, while those involved in the alpha-helix and beta-turn and/or 3(10)-helix are exchanged with solvent deuterons. The protected backbone amide protons located near the surface regions are more easily exchanged with solvent deuterons by application of high pressure than those involved in the core.

The polymerization of actin: Study by small angle neutron scattering

We report measurements of small angle neutron scattering from solutions of rabbit muscle G-actin at 3.00 mg/mL in D2O buffer solution, with [Ca2+]=0.52 mM and with [KCl]=15, 8.9, and 5.4 mM. We observe the onset of the polymerization of G-actin to F-actin as the temperature is increased. The polymerization takes place on a time scale of 30–45 min for each temperature jump of 2 °C–3 °C. As the temperature is increased further, the average size of the polymers increases, and the characteristic length scale (or correlation length), ξ, of the F-actin in the dilute solution grows: ξ is about 10 Å below Tp, and about 70 Å a few degrees above Tp. The transition is sharper for lower concentrations of KCl. For the sample with [KCl]=8.9 mM, we observe a peak in ξ at about 2 °C above Tp, which indicates a crossover into the semidilute regime. The transition is essentially reversible, but shows evidence of incomplete depolymerization on cycling. We are unable to apply the available theoretical model for reversible polymerization to rabbit muscle actin because of a lack of information on the enthalpy and entropy of polymerization. However, our observations for rabbit muscle actin are quite analogous to observations on the equilibrium polymerization of α-methylstyrene [A. P. Andrews, K. P. Andrews, S. C. Greer, F. Boué, and P. Pfeuty, Macromolecules 27, 3902 (1994)].

Pressure‐induced structural rearrangements of bovine pancreatic trypsin inhibitor studied by FTIR spectroscopy

Fourier transform infrared (FTIR) spectroscopy combined with resolution enhancement techniques, second-derivative and difference spectroscopies, have been used to characterize pressure-induced changes in the structural rearrangements of bovine pancreatic trypsin inhibitor (BPTI) in D2O solution at 25.0°C. According to the observed changes in the amide I′ band up to 550 MPa, the secondary structure elements of BPTI, such as the α-helix, 310-helix, β-sheet, and β-turn, are scarcely rearranged except for the loop structure of residues of 9–17 and 36–43. The polypeptide backbone is not extensively unfolded up to 550 MPa. The minor pressure-induced structural rearrangements are completely reversible. A further increase in pressure above 1000 MPa associated with the precipitation of BPTI in D2O buffer solution induces the partial structural rearrangements of the α-helix, β-turn and/or 310-helix, and β-sheet. The polypeptide backbone of BPTI is not fully unfolded even above 1000 MPa. Most of the protected backbone amide protons involved in the β-sheet remain intact in the pressure range where BPTI is not precipitated, while those involved in the α-helix and β-turn and/or 310-helix are exchanged with solvent deuterons. The protected backbone amide protons located near the surface regions are more easily exchanged with solvent deuterons by application of high pressure than those involved in the core. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: 209–216, 1998

Pressure-induced structural rearrangements of bovine pancreatic trypsin inhibitor studied by FTIR spectroscopy

Fourier transform infrared (FTIR) spectroscopy combined with resolution enhancement techniques, second-derivative and difference spectroscopies, have been used to characterize pressure-induced changes in the structural rearrangements of bovine pancreatic trypsin inhibitor (BPTI) in D2O solution at 25.0°C. According to the observed changes in the amide I′ band up to 550 MPa, the secondary structure elements of BPTI, such as the α-helix, 310-helix, β-sheet, and β-turn, are scarcely rearranged except for the loop structure of residues of 9–17 and 36–43. The polypeptide backbone is not extensively unfolded up to 550 MPa. The minor pressure-induced structural rearrangements are completely reversible. A further increase in pressure above 1000 MPa associated with the precipitation of BPTI in D2O buffer solution induces the partial structural rearrangements of the α-helix, β-turn and/or 310-helix, and β-sheet. The polypeptide backbone of BPTI is not fully unfolded even above 1000 MPa. Most of the protected backbone amide protons involved in the β-sheet remain intact in the pressure range where BPTI is not precipitated, while those involved in the α-helix and β-turn and/or 310-helix are exchanged with solvent deuterons. The protected backbone amide protons located near the surface regions are more easily exchanged with solvent deuterons by application of high pressure than those involved in the core. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: 209–216, 1998

Solution Conformation of the (-)-trans-anti-5-Methylchrysene-dG Adduct opposite dC in a DNA Duplex: DNA Bending Associated with Wedging of the Methyl Group of 5-Methylchrysene to the 3'-Side of the Modification Site

This paper reports on NMR-molecular mechanics structural studies of the (-)-trans-anti-[MC]dG adduct positioned opposite dC in the sequence context of the d(C1-C2-A3-T4-C5-[MC]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17-G18- A19-T20-G21-G22) duplex [designated (-)-trans-anti-[MC]dG.dC 11-mer duplex]. This adduct is derived from the trans addition at C4 of (-)-anti-1(S),2(R)-dihydroxy-3(R),4(S)-epoxy-1,2,3,4-tetrahydro-5-met hylchrysen e [(-)-anti-5-MeCDE] to the N2 position of dG6 in this duplex sequence. The 5-methyl group is located adjacent to the MC(C4) binding site, with these groups juxtaposed in a sterically crowded bay region in the adduct duplex. The 5-methylchrysenyl and the nucleic acid exchangeable and nonexchangeable protons were assigned following analysis of two-dimensional NMR data sets in H2O and D2O buffer solution. The solution structure of the (-)-trans-anti-[MC]dG.dC 11-mer duplex has been determined by incorporating DNA-DNA and carcinogen-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results establish that the [MC]dG6.dC17 base pair and flanking dC5.dG18 and dC7.dG16 base pairs retain Watson-Crick alignments upon adduct formation. The aromatic chrysenyl ring is positioned in the minor groove of a right-handed B-DNA helix and stacks predominantly over the sugar of the dC17 residue across from it on the unmodified complementary strand. The chrysenyl ring points toward the 3'-end of the modified strand with its 5-methyl group inserting between the modified [MC]dG6.dC17 and dC7.dG16 base pairs. The adduct duplex bends by approximately 47 degrees as a result of the wedged insertion of the 5-methyl group from the minor groove face of the duplex. The solution structure of the (-)-trans-anti-[MC] dG.dC 11-mer duplex is compared with that of the corresponding (-)-trans-anti-[BP]dG.dC 11-mer [De los Santos et al. (1992) Biochemistry 31, 5245-5252] in which the [BP]dG adduct is derived from the binding of (-)-anti-BPDE [7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene] to the N2 position in the same DNA sequence context. Although the solution structures of the (-)-trans-anti-stereoisomers of 5-methylchrysenyl-dG and benzo[a]pyrenyl-dG adducts opposite dC exhibit many features in common with each other, the [MC]dG adduct which contains a bay region methyl group bends the DNA helix to a greater extent than in the corresponding [BP]dG adduct, which lacks a bay region methyl group.(ABSTRACT TRUNCATED AT 400 WORDS)

Solution conformation of the (-)-trans-anti-benzo[c]phenanthrene-dA ([BPh]dA) adduct opposite dT in a DNA duplex: intercalation of the covalently attached benzo[c]phenanthrenyl ring to the 3'-side of the adduct site and comparison with the (+)-trans-anti-[BPh]dA opposite dT stereoisomer.

This paper reports on NMR-molecular mechanics structural studies of the (-)- trans-anti-benzo[c]phenanthrene-dA adduct positioned opposite dT in the sequence context of the d(C1-T2-C3-T4-C5-[BPh]A6-C7-T8-T9-C10-C11).d(G12- G13-A14-A15-G16-T17-G18-A19-G20-A21- G22) duplex (designated as the (-)-trans-anti-[BPh]dA.dT 11-mer duplex). This adduct is derived from the covalent binding of (-)-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-benzo[c]phenanthrene [(-)-anti-BPhDE] to N6 of dA6 in this duplex sequence. The benzo[c]phenanthrenyl and nucleic acid exchangeable and nonexchangeable protons were assigned in the predominant conformation following analysis of two-dimensional NMR data sets in H2O and D2O buffer solution. The solution structure of the (-)-trans-anti-[BPh]dA.dT 11-mer duplex has been determined by incorporating intramolecular and carcinogen-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results show that the [BPh]dA6.dT17 base pair propeller twists and buckles slightly to permit the covalently attached benzo[c]phenanthrenyl ring to intercalate between the [BPh]dA6.dT17 and dC7.dG16 base pairs to the 3'-side of the [BPh]dA6 lesion site without disrupting the Watson-Crick hydrogen bond alignments in the modified duplex. The strain in the highly sterically hindered fjord region of the benzo[c]phenanthrenyl moiety is relieved by the propeller-like nonplanar geometry of the aromatic phenanthrenyl ring system, which stacks predominantly with the dG16 and dT17 bases on the unmodified strand. The benzylic ring adopts a distorted half-chair form, in which the H1 and H2 protons are pseudo-diequatorial and the H3 and H4 protons are pseudodiaxial. The current observation that the (-)-trans-anti-[BPh]dA positioned opposite dT intercalates to the 3'-side of the intact modified base pair contrasts with our previous demonstration that the stereoisomeric (+)-trans-anti-[BPh]dA adduct positioned opposite dT intercalates to the 5'-side of the intact modified base pair [Cosman, M., et al. (1993b) Biochemistry 32, 12488-12497]. These stereochemically induced structural differences between isomeric [BPh]dA lesions derived from the binding of chiral (+)- and (-)-anti-BPhDE enantiomers may in turn profoundly influence the interactions of the carcinogen-modified DNA with repair and replication enzymes in the cell.

Further NMR-Spectroscopic Studies of Interaction of Phospholipid Liposomes with Methacryloyloxydecyl Dihydrogen Phosphate (MDP) in Dental Adhesives

To determine how MDP interacts with liposomes, the chemical shifts of dipalmitoylphosphatidylcholine (DPPC)/MDP and dilauroylphosphatidylethanolamine (DLEA)/cholesterol (CS)/MDP liposomes were studied by NMR spectroscopy using a D2O buffer solution at pH 7.0 as a model for biological membranes. Proton chemical shifts of MDP enhanced shielding in DPPC liposomes together with an increase in the mobility of DPPC. However, MDP signals were not observed in DLEA/CS liposomes due to saturation. It is known that an ionized chemical does not lead to increased permeability of cell membranes composed of a lipid bilayer. However, MDP, which is ionized at pH 7.0, had a large interaction with the liposome systems. This appeared to arise from hydrophobic interaction between deca methylene groups of MDP and acyl chains of phospholipid.