Fluorescence Spectral Results Research Articles

Multispectroscopic Investigation of the Interaction Between two Ruthenium(II) Arene Complexes of Curcumin Analogs and Human Serum Albumin.

The interaction between two ruthenium(II) arene complexes of curcumin analogs and human serum albumin (HSA) was systematically investigated by multispectroscopic techniques. The fluorescence spectral results indicated that two complexes quenched the intrinsic fluorescence of HSA through static quenching mode. The quenching constants and the corresponding thermodynamic parameters at different temperatures were calculated. The binding interactions of two complexes with HSA resulted in the complex formation of complex-HSA, and the van der Waals interactions and hydrogen bond interactions played major roles in the complex stabilization. The distances between HSA and two complexes were obtained according to fluorescence resonance energy transfer theory. The site competitive replacement experiments illustrated that two complexes mainly bounded with HSA on site I. The results of synchronous fluorescence spectra, three-dimensional fluorescence spectra, FT-IR spectra, and circular dichroism spectra indicated that the secondary structure of HSA was changed at the present of two complexes. The results of mass spectrometry further validated the binding interaction and the binding number between two complexes and HSA.

Positron annihilation and other experimental studies on polycarbonate/MPDMAPP nanocomposite

ABSTRACTThe composite films of polycarbonate (PC) filled with 1‐(4‐methylphenyl)‐3‐(4‐N,N‐dimethylaminophenyl)‐2‐propen‐1‐one (MPDMAPP) were prepared by solution casting. The FT‐IR results of the prepared films confirmed the hydrophobic and dipole‐dipole interaction between PC and MPDMAPP, which is a major driving force for the formation of charge transfer complex (CTC). UV–Vis absorption spectra showed three peaks and the optical band gap Eg for pure PC is 4.31 eV which decreased to 4.1 eV after 15 wt % doping. The fluorescence spectral results show a strong emission and quenching in the wavelength region 510 to 550 nm for 408 nm excitation due to increase in amorphousness, which is observed in X‐ray diffraction (XRD) results. The decrease in ortho‐positronium (o‐Ps) lifetime τ3 and corresponding intensity I3 from positron annihilation lifetime spectroscopy (PALS) and the S‐parameter from Doppler broadening measurements show both inhibition and quenching of Ps formation in the PC/MPDMAPP composite due to the presence of dimethylamino N(CH3)2 group. The mechanical properties such as Young's modulus, tensile strength, and stiffness increase with doping concentration and confirmed that the composite films are mechanically stable. The growth of nanostructures of MPDMAPP within PC films is studied with SEM and TEM images and confirms the uniform dispersion and interaction between the functional groups of PC and MPDMAPP. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 42053.

Targeting chemical and thermal stability of ovalbumin by simulated honey sugar cocktail

Effect of simulated honey sugar cocktail (SHSC) on chemical and thermal stability of ovalbumin (OVA) was investigated using multiple-spectroscopic techniques. Urea-induced denaturation of OVA produced a transition, characterized by the start-, the mid- and the end-points at 3.2M, 5.9/5.6M and 8.5/8.0M urea, respectively, when studied by MRE222nm and tryptophan fluorescence measurements. Presence of 10% or 20% (w/v) SHSC in the incubation mixture shifted the transition curve towards higher urea concentration in a concentration dependent manner. A comparison of far- and near-UV CD, UV-difference, ANS fluorescence and 3-D fluorescence spectral results of native OVA and 5.9M urea-denatured OVA (U-OVA), obtained in the absence and the presence of 20% (w/v) SHSC suggested SHSC-induced stabilization of U-OVA. Furthermore, a significant shift towards higher denaturant concentration was also noticed in the GdnHCl and thermal transition curves of OVA in the presence of 20% (w/v) SHSC. Taken together, all these results suggested stabilization of OVA against chemical and thermal denaturations by SHSC.

Quest for the binding mode of malachite green with humic acid

The association of malachite green (MG) with humic acid (HA) was investigated by using fluorescence, UV–vis spectroscopy and molecular Modelling method. The fluorescence spectral results indicated that the binding between MG and HA occurred by mainly hydrophobic and electrostatic forces with association constants of KA (298K)=6.24×105L/mol and KA (310K)=10.20×105L/mol. There were more than one binding sites on HA to bind with MG. The binding sites of MG with HA primarily located at the aromatic rings of HA. MG could enter into the hydrophobic cavities of HA to quench the fluorescence of HA. On the contrary, HA binding caused MG to a coplanar conformation with more extended π bond distribution by π–π stacking interactions. The experiment and calculation data both showed that the hydrophobic binding cavities in HA played a key role in its binding with MG.

Low-dimensional compounds containing bioactive ligands. V: Synthesis and characterization of novel anticancer Pd(II) ionic compounds with quinolin-8-ol halogen derivatives

Three novel palladium(II) complexes, NH2(CH3)2[PdCl2(CQ)] (1) (CQ=5-chloro-7-iodo-quinolin-8-ol), NH2(CH3)2[PdCl2(dClQ)] (2) (dClQ=5,7-dichloro-quinolin-8-ol) and NH2(CH3)2[PdCl2(dBrQ)] (3) (dBrQ=5,7-dibromo-quinolin-8-ol) have been prepared and characterized. Their structures contain square-planar [PdCl2(XQ)]− complex anions in which deprotonated XQ ligands are coordinated to the Pd atoms via the pyridine nitrogen and the phenolato oxygen atoms, other two cis-positions are occupied by two chlorido ligands. Negative charges of these anions are balanced by uncoordinated dimethylammonium cations. Coordination of the XQ ligands to Pd(II) atom was confirmed by the differences in the stretching ν(OH) and ν(CN) vibrations in the IR spectra of ligands and prepared complexes while bands of aliphatic CH and NH stretching vibrations observed in the spectra of 1–3 confirm the presence of dimethylammonium cations in the complexes. The binding of complexes 1–3 to calf thymus DNA was investigated using UV–visible and fluorescence emission spectrophotometry. The fluorescence spectral results indicate that the complexes can bind to DNA through an intercalative mode. The Stern–Volmer quenching constants obtained from the linear quenching plot are in the 1.04×104 to 4.35×104M−1 range. The complexes exhibit significant anticancer activity tested on A2780 cells and cisplatin resistant cell line A2780/CP.

Studies on the interaction between vincamine and human serum albumin: a spectroscopic approach

The interaction between vincamine (VCM) and human serum albumin (HSA) has been studied using a fluorescence quenching technique in combination with UV/vis absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling under conditions similar to human physiological conditions. VCM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding constants were calculated from the fluorescence data. Thermodynamic analysis by Van't Hoff equation revealed enthalpy change (ΔH) and entropy change (ΔS) were -4.57 kJ/mol and 76.26 J/mol/K, respectively, which indicated that the binding process was spontaneous and the hydrophobic interaction was the predominant force. The distance r between the donor (HSA) and acceptor (VCM) was obtained according to the Förster's theory of non-radiative energy transfer and found to be 4.41 nm. Metal ions, viz., Na(+), K(+), Li(+), Ni(2+), Ca(2+), Zn(2+) and Al(3+) were found to influence binding of the drug to protein. The 3D fluorescence, FT-IR and CD spectral results revealed changes in the secondary structure of the protein upon interaction with VCM. Furthermore, molecular modeling indicated that VCM could bind to the subdomain IIA (site I) of HSA.

Interaction between toxic azo dye C.I. Acid Red 88 and serum albumins

Serum albumin-toxic dye interaction studies will be of paramount importance in the field of toxicology due to its relation towards the distribution and transportation of dye in blood. In this regard, the binding between C.I. Acid Red 88 (AR88) and serum albumins (HSA and BSA) was investigated by using combination of spectroscopic and molecular modeling methods. The fluorescence results revealed that AR88 interact with serum albumins through the combination of static and dynamic quenching mechanism. The distance “r” between serum albumin and AR88 was obtained according to the Forster resonance energy transfer (FRET) theory. Synchronous fluorescence and CD spectral results showed alterations in the microenvironment and conformation of serum albumins. The molecular docking method is also employed to understand the interaction of AR88 with serum albumins. All these studies confirm that BSA has more affinity towards AR88 than that of HSA which suggests that AR88 is more easily transported in the body of bovid than human and so it is more hazardous to bovids.

Combined multispectroscopic and molecular docking investigation on the interaction between strictosamide and human serum albumin

The interaction between strictosamide (STM) and human serum albumin (HSA) was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy, circular dichroism spectroscopy and molecular modeling under physiological pH 7.4. STM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding site number n and apparent binding constant K(a) were determined at different temperatures by fluorescence quenching. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated as -3.01 kJ/mol and 77.75 J/mol per K, respectively, which suggested that the hydrophobic force played major roles in stabilizing the HSA-STM complex. The distance r between donor and acceptor was obtained to be 4.10 nm according to Förster's theory. After the addition of STM, the synchronous fluorescence and three-dimensional fluorescence spectral results showed that the hydrophobicity of amino acid residues increased and the circular dichroism spectral results showed that the α-helix content of HSA decreased (from 61.48% to 57.73%). These revealed that the microenvironment and conformation of HSA were changed in the binding reaction. Furthermore, the study of molecular modeling indicated that STM could bind to site I of HSA and the hydrophobic interaction was the major acting force, which was in agreement with the binding mode study.

Synthesis of biological active thiosemicarbazone and characterization of the interaction with human serum albumin

The synthesis of a new biological active reagent, 2-((1,4-dihydroxy)-9,10-anthraquinone) aldehyde thiosemicarbazone (DHAQTS), was designed. The interaction between DHAQTS and HSA was studied by fluorescence spectroscopy in combination with molecular modeling under simulation of physiological conditions. According to the results of fluorescence measurements, the quenching mechanism was suggested to be static. The thermodynamic parameters are calculated by van't Hoff equation, which demonstrated that hydrophobic interactions are the predominant intermolecular forces stabilizing the complex. The number of binding sites (n) was calculated. Through the site marker competitive experiment, DHAQTS was confirmed to be located in site I of HSA. The binding distance r=2.83nm between the donor HSA and acceptor DHAQTS was obtained according to Förster's non-radiative energy transfer theory. The three-dimensional fluorescence spectral results showed the conformation and microenvironment of HSA changed in the presence of DHAQTS. The effects of common ions on the binding of DHAQTS to HSA were also evaluated. The experimental results were in agreement with the results obtained via a molecular docking study.

Probing the Interaction Between a Surfactant–Cobalt(III) Complex and Bovine Serum Albumin

The mechanism of binding of the surfactant–cobalt(III) complex, cis-[Co(phen)2(C14H29NH2)Cl](ClO4)2⋅3H2O (phen = 1,10-phenanthroline, C14H29NH2 = tetradecylamine) with bovine serum albumin (BSA) was investigated by UV–vis absorption, circular dichroism (CD) and fluorescence spectroscopic techniques. The results of fluorescence titration revealed that the surfactant–cobalt(III) complex quenched the intrinsic fluorescence of BSA through a combination of static and dynamic quenching. The apparent binding constant (K a) and number of binding sites (n) were calculated below and above the critical micelle concentration (CMC). The thermodynamic parameters determined by the van’t Hoff analysis of the constants (ΔH ∘=14.87 kJ⋅mol−1; ΔS ∘=152.88 J⋅mol−1⋅K−1 below the CMC and 25.70 kJ⋅mol−1 and 243.14 J⋅mol−1⋅K−1, respectively, above the CMC) clearly indicate that the binding is entropy-driven and enthalpically disfavored. Based on Förster’s theory of non-radiation energy transfer, the binding distance, r, between donor (BSA) and the acceptor (surfactant–cobalt(III) complex) was evaluated. UV–vis, CD and synchronous fluorescence spectral results showed that the binding of the surfactant–cobalt(III) complex to BSA induced conformational changes in BSA.

Identification and characterization of a cytochrome b559 Synechocystis 6803 mutant spontaneously generated from DCMU-inhibited photoheterotrophical growth conditions

We identified a spontaneously generated mutant from Synechocystis sp. PCC6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 μM DCMU. This mutant carries an R7L mutation on the α-subunit of cyt b559 in photosystem II (PSII). In the recent 2.9 Å PSII crystal structural model, the side chain of this arginine residue is in close contact with the heme propionates of cyt b559. We called this mutant WR7Lα cyt b559. This mutant grew at about the same rate as wild-type cells under photoautotrophical conditions but grew faster than wild-type cells under photoheterotrophical conditions. In addition, 77 K fluorescence and 295 K chlorophyll a fluorescence spectral results indicated that the energy delivery from phycobilisomes to PSII reaction centers was partially inhibited or uncoupled in this mutant. Moreover, WR7Lα cyt b559 mutant cells were more susceptible to photoinhibition than wild-type cells under high light conditions. Furthermore, our EPR results indicated that in a significant fraction of mutant reaction centers, the R7Lα cyt b559 mutation induced the displacement of one of the axial histidine ligands to the heme of cyt b559. On the basis of these results, we propose that the Arg7Leu mutation on the α-subunit of cyt b559 alters the interaction between the APC core complex and PSII reaction centers, which reduces energy delivery from the antenna to the reaction center and thus protects mutant cells from DCMU-induced photo-oxidative stress.

Interaction of N′‐(1‐Carboxyethylidene)salicylhydrazide with Bovine Serum Albumin

Abstract Under the simulated physiological condition of an animal body, the interaction between N′‐(1‐carboxyethylidene)salicylhydrazide (CESH) and bovine serum albumin (BSA) was investigated by fluorescence spectra, UV‐Vis absorption spectra and circular dichroism (CD) spectra. The experiment results showed that the fluorescence of BSA was strongly quenched by CESH because of the formation of a CESH‐BSA complex, indicating a static quenching mechanism in the binding process. The modified Stern‐Volmer quenching constants (Ka) were 11.23×104, 7.103×104, 4.934×104 and 2.495×104 L·mol−1 at 292, 298, 304 and 310 K, respectively. The thermodynamic parameters ΔG, ΔH and ΔS at the four temperatures were calculated according to van't Hoff equation and the results indicated that hydrogen bonds and van der Waals force played major roles in stabilizing the CESH‐BSA complex. The distance r=4.26 nm between the donor BSA and acceptor CESH was obtained according to F?rster's non‐radiative energy transfer theory. The synchronous fluorescence spectral results indicated that the hydrophobicity of tyrosine residue increased while the microenvironment around tryptophan residue had no change. The CD and three‐dimensional fluorescence spectral results showed that in the presence of CESH, the α‐helix content of BSA decreased and the microenvironment and conformation of BSA changed.

Studies of the interaction between Sudan I and bovine serum albumin by spectroscopic methods

The interaction of Sudan I (SD) and bovine serum albumin (BSA) under physiological condition was investigated by the methods of fluorescence, UV–vis absorbance and circular dichroism (CD) spectroscopy. Fluorescence data revealed that the fluorescence quenching of BSA by SD was the result of the formation of BSA–SD complex, and the effective quenching constants ( K a) were 4.457 × 10 4, 4.113 × 10 4, 3.642 × 10 4 and 3.488 × 10 4 L mol −1 at 292, 298, 304 and 310 K, respectively. The thermodynamic parameters, enthalpy change (Δ H) and entropy change (Δ S) for the reaction were calculated to be −10.76 kJ mol −1 and 53.97 J mol −1 K −1 according to van’t Hoff equation. The results indicated that the hydrophobic force was the dominant intermolecular force in stabilizing the complex. The distance r between donor (BSA) and acceptor (SD) was obtained to be 4.59 nm according to Förster’s non-radioactive energy transfer theory. After the addition of SD, the synchronous fluorescence spectral results showed that the hydrophobicity of amino acid residues increased and the CD spectral results showed that the α-helix content of BSA decreased (from 63.60% to 50.17%). These revealed that the microenvironment and conformation of BSA were changed in the binding reaction.

Fluorescence Study on the Interaction of Bovine Serum Albumin with P-Aminoazobenzene

In this paper, the interaction between p-aminoazobenzene (PAAB) and BSA was investigated mainly by fluorescence quenching spectra, circular dichroism (CD) and three-dimensional fluorescence spectra under simulative physiological conditions. It was proved that the fluorescence quenching of BSA by PAAB was mainly a result of the formation of a PAAB-BSA complex. The modified Stern-Volmer quenching constant K(a) and the corresponding thermodynamic parameters DeltaH, DeltaG and DeltaS at different temperatures were calculated. The results indicated that van der Waals interactions and hydrogen bonds were the predominant intermolecular forces in stabilizing the complex. The distance r=4.33 nm between the donor (BSA) and acceptor (PAAB) was obtained according to Förster's non-radioactive energy transfer theory. The synchronous fluorescence, CD and three-dimensional fluorescence spectral results showed that the hydrophobicity of amino acid residues increased and the losing of alpha-helix content (from 63.57 to 51.83%) in the presence of PAAB. These revealed that the microenvironment and conformation of BSA were changed in the binding reaction.

Spectroscopic studies of the interaction between methylene blue and G-quadruplex

G-rich single-stranged DNA (5′-TTAG-GG-3′) adopted a G-quadruplex structure in buffer containing potassium ions. The spectroscopic feature and the interaction between methylene blue and G-qadruplex have been investigated by circular dichroism, and nuclear magnetic resonance spectroscopy. The UV-Vis absorption and fluorescence spectral results show that the fluorescence behavior of MB by single-stranded DNA fits Stern-Volmer static quenching equation very well and they formed 1: 1 complexes; dimeric G-quadruplexes were bound to MB with 1: 1 or 2: 1, and their equilibrium constants were 1.047×105 and 8.79×104 L/mol, respectively. Based on the above results and 1H-NMR spectral data, one may conclude that MB stacked either the terminal tetrads to form 1: 1 complexes or between two terminal tetrads of G-quadruplexes to form 1: 2 sandwich complexes with G-qudruplexes.

Observation of aqueous Cm(III)/Eu(III) and UO 22+ nanoparticulates at concentrations approaching solubility limit by laser-induced fluorescence spectroscopy

Eu(III), Cm(III) and the uranyl ion display intense fluorescence spectra in the visible range and the spectroscopic characteristics are dependent on the composition and structure of the individual metal complexes. In this work, we demonstrate the application of laser-induced time-resolved fluorescence spectroscopy in identification of nanoparticles of (i) Eu(III) and Cm(III) in basic solutions (pH > 10) in the presence of organic chelates including EDTA, HEDTA, NTA and oxalate and (ii) sodium uranyl phosphate after equilibration with synthetic sodium uranyl phosphate suspensions. Fluorescence spectral and SEM results indicate that Eu(III) and Cm(III) can exist as colloidal nanoparticles in filtered 0.1 M NaOH solutions. Such nanoparticles, which display largely red-shifted fluorescence spectra as compared with the aqueous complexes and unusually short fluorescence lifetimes, contribute to the measured concentrations of Eu(III)/Cm(III) in the aqueous solutions. Similarly, uranyl spectroscopic signatures indicate that the determination of the solubility of uranium phosphate minerals is prone to the presence of uranyl phosphate nanoparticles. Due to the presence of such nanoparticles, the common solubility measurements may only indicate an upper limit of the “true” solubility.

Photoinduced amino–imino tautomerization reaction in 2-aminopyrimidine and its methyl derivatives with acetic acid

The electronic absorption and fluorescence spectra of 2-aminopyrimidine (2APM), 2-amino-4-methylpyrimidine (2A4MPM), and 2-amino-4,6-dimethylpyrimidine (2ADMPM) with acetic acid (AcOH) were measured in isooctane (2,2,4-trimethylpentane) at room temperature. From the absorption spectra, a hydrogen-bonded complex formation of the 2APM/AcOH, 2A4MPM/AcOH, and 2ADMPM/AcOH systems was recognized in isooctane. The enthalpy changes (−Δ H) for the complex formation were estimated to be ca. 41.2–45.1 kJ mol −1 and increased in proportion to the numbers of the methyl group introduced into the 2APM. The −Δ H values refer to the formation of the hydrogen-bonded 1:1 complex between the ring nitrogen atom and NH 2 group of the aminopyrimidine and the OH and C O groups of AcOH, respectively. In the 2A4MPM/AcOH double hydrogen-bonded complex the OH group of AcOH is thought to be linked to the ring nitrogen at the 1-postion of 2A4MPM. The fluorescence spectral results indicate that the double proton transfer reaction takes place during the excited state, and gives rise to an imino-tautomer vibration emission, from analogy with the fluorescences of 1-methyl-2(1H)-pyrimidinimine (MPMI), 1,4-dimethyl-2(1H)-pyrimidinimine (DMPMI), and 1,4,6-trimethyl-2(1H)-pyrimidinimine (TMPMI). The fluorescence quantum yields of the imino-tautomers also increased in proportion to the numbers of the methyl group introduced into the 2APM.

Fluorescence and FT-IR spectroscopic studies of Suwannee river fulvic acid complexation with aluminum, terbium and calcium

In this study fluorescence emission and IR spectroscopy have been used to investigate the interaction of the class A (oxygen seeking ‘hard acid’) metal Al 3+, with Suwannee River fulvic acid. Addition of Al 3+ ion results in a significant enhancement in fulvic acid fluorescence emission (at λ em=424 nm) and significant red shift of the excitation wavelength (from λ ex=324 nm to λ ex=344 nm) at low pH values (pH∼4.0–5.0). At pH 4.0 (0.1 M ionic strength), where the predominant aluminum ion species is the ‘free’ (aquo) ion, the fulvic acid fluorescence reaches 142% of the value in the absence of added metal ion. Analysis of the pH 4.0 and pH 5.0 fluorescence enhancement data with the nonlinear (single site) model of Ryan and Weber indicated binding constants in the range of 4.67·10 4–2.87·10 6 M −1 and concentrations of ligand sites in the range of 18.6·10 −6–24.0·10 −6 M, both consistent with previous studies performed on both aquatic and soil fulvic acids. Companion fluorescence experiments performed on two other class A metal ions, Ca 2+ and Tb 3+, indicated no significant enhancement or quenching with Ca 2+ and only slight quenching with Tb 3+. Comparison of FT-IR spectra collected on fulvic acid alone and fulvic acid in the presence of the three class A metals (Al 3+, Ca 2+ and Tb 3+) provides strong evidence for the involvement of carboxyl carbonyl functions in the binding of all three metal ions, which is not unexpected. The spectra also reveal, however, a very pronounced difference in the 4000–2000 cm −1 IR spectral region between the Al 3+ spectrum and the Ca 2+ and Tb 3+ spectra. The –OH stretch spectral region in the Al 3+ spectrum has a major component shifted to higher energy (compared to fulvic acid alone or to fulvic acid in the presence of Ca 2+ or Tb 3+). Even more striking is the emergence of a pronounced IR band at 2407 cm −1, which is present only in the Al 3+ spectrum. The results of fluorescence and IR experiments with the model compounds salicylic acid and phthalic acid further confirm that both salicylic acid-like sites and phthalic acid-like sites are likely complexation sites for Al 3+ in fulvic acid and are major contributors to the observed spectroscopic changes associated with Al 3+ ion complexation. From a comparison of both the fluorescence and IR spectral results for all three class A metals, differing most strongly in the value of their ionic index, it seems clear that major sources of the deviation in spectral properties between Al 3+ and Ca 2+/Tb 3+ is the unusually high value of its charge density and relatively low propensity for involvement in covalent bonding interactions (very high ionic index and relatively low covalent index in the Nieboer and Richardson classification of environmental metals), as well as affinity for certain functional groups.

Layer-by-layer self-assembled multilayer film of cationic oligo-(phenylene vinylene) and polyelectrolytes based on electrostatic interaction

Self-assembled multilayer film of a cationic oligo( p-phenylene vinylene) derivative, 4,4′-(1,4-phenylene dithenylene)-bis-( N-methyl pyridinium iodide) (OPV), and the anionic poly(sodium 4-styrene sulfonate) (PSS) was fabricated by using electrostatic interactions as driving force. X-ray diffraction patterns (XRD) indicate that this method can effectively eliminate the crystallization of OPV. UV–VIS spectra reveal that OPV molecules tend to form H-aggregate in the multilayer film. Fluorescence spectral results show that the luminescent peak of the OPV in multilayer film underwent a blue shift about 80 nm compared with that of OPV in solid state. The emission intensity of OPV in the multilayer film is much stronger than that of OPV in spin-cast film due to the isolation of OPV in the film by PSS.

Native-like structure and self-association behavior of apolipoprotein A-I in a water/n-propanol solution

The effect of n-propanol on the secondary and tertiary structure of human apolipoprotein A-I (apoA-I), an interfacial protein, was investigated using near and far ultraviolet (UV)-circular dichroism (CD) and fluorescence spectroscopy, as well as limited proteolytic digestion with trypsin, and cross-linking with bis(sulfosuccinimidyl) suberate. The structure of apoA-I in n-propanol (30%, v/v) was compared with that in Tris buffer and in reconstituted, spherical or discoidal, high density lipoproteins (rHDL). Addition of n-propanol to apoA-I in Tris buffer induces major changes in its near and far CD spectra: α-helical content increases by 27% and the near UV-CD spectrum becomes very similar to that of apoA-I in rHDL particles. Fluorescence spectral, lifetime, and polarization results, and quenching by KI confirm that major structural changes occur in the N-terminal half of apoA-I as n-propanol is added: the Trp residues become more exposed to solvent than in buffer alone or in rHDL. Higher concentrations of guanidine hydrochloride or urea are required to denature apoA-I in n-propanol than in buffer alone, but a similar free energy of unfolding is observed. The N-terminus of apoA-I is relatively resistant to trypsin digestion and the C-terminus has equivalent digestion sites for apoA-I in the three states, but the kinetics of digestion are much slower in n-propanol and in rHDL compared to apoA-I in Tris buffer. Cross-linking experiments reveal that dimers of apoA-I exist in n-propanol, in contrast to dimers plus multimeric aggregates in Tris buffer. From these results we conclude that in 30% n-propanol the structure of apoA-I approaches that of ‘native’ lipid-bound apoA-I, in contrast to its structure in the aqueous Tris buffer.