Dynamical and structural changes of an anesthetic analogue in chemical and biological nanocavities

We report on photophysical studies of the interaction between an anesthetic analogue, methyl 2-amino-4,5-dimethoxybenzoate (ADMB), with the human serum albumin (HSA) protein and the normal micelle of n-octyl-beta-D-glucopyranoside (OG) in water solutions. We used steady-state and picosecond time-resolved emission spectroscopy to follow the dynamical and structural changes due to their hydrophobicity and confinement on the photophysical behavior of ADMB. The formed 1:1 complex with the protein is robust with an equilibrium constant of 9.6 x 10(4) M(-1) at 293 K. The fluorescence lifetimes of the 1:1 entity become longer (up to approximately 10 ns), and the emission transients show complex behavior due to the heterogeneity of the media. Rotational time (45 ns) from picosecond anisotropy measurements clearly indicates strong confinement in the robust ADMB:HSA complex. For the ADMB:OG one, the anisotropy decays give time constants of 50 and 980 ps, assigned to free and restricted rotors within the micelle, respectively. The process of energy transfer from the excited tryptophan 214 (Trp214) of HSA to the trapped ADMB occurs with an efficiency of 50%, and the calculated distance between both chromophores is 19 A. We believe that these results are important for a better understanding of processes occurring in encapsulated drugs and thus should be relevant to nanopharmacodynamics.     

Effect of cyclodextrin nanocavity confinement on the photorelaxation of the cardiotonic drug milrinone

We report on steady-state UV-visible absorption, emission, and picosecond emission studies of milrinone (MIR) drug in neutral water and complexed to cyclodextrins (alpha-, beta-, gamma-CD and dimethyl-beta-CD (DM-beta-CD)). The results reveal that MIR forms a 1:1 inclusion complex with CD. Upon encapsulation the emission intensity increases and the fluorescence lifetime changes from approximately 65 ps to 240-350 ps, indicating a confinement effect of the nanocages on the photophysical behavior of the drug. Due to its methyl groups, the DM-beta-CD complex shows the largest effect. The time-anisotropy experiments support the formation of 1:1 inclusion complexes and indicate motion of the drug inside the nanocavity. Furthermore, results of PM3 calculations combined with spectral and dynamical data show that the drug is not fully embedded into the cavities, and the conformation of the included complex explains the relatively short lifetimes and low emission quantum yields of these entities.     

Femtosecond to second studies of a water-soluble porphyrin derivative in chemical and biological nanocavities

The interactions of 5,10,15,20-tetrakis(4-sulfonatophenyl)-porphyrin (TSPP) with a quaternary ammonium modified β-cyclodextrin (QA-β-CD) and human serum albumin (HSA) protein in aqueous solutions at pH 7 were studied using steady-state, stopped-flow, and femtosecond to millisecond spectroscopy. TSPP forms 1:1 and 1:2 complexes with QA-β-CD (K(1) = 1.9 × 10(5) M(-1) and K(2) = 7 × 10(3) M(-1)) at 293 K, whereas with the HSA protein only 1:1 complex (K(1) = 1.7 × 10(6) M(-1)) has been found. The chemical and biological nanocavities have notable effects on the fluorescence lifetimes of the Q(x) state (from 9.3 to 11.1 ns in QA-β-CD and 11.6 ns in HSA). Furthermore, the rotational times (400 ps for the free TSPP, 1.6 and 19 ns for QA-β-CD and HSA protein complexes, respectively) clearly indicate the robustness of the formed entities. The confined environment does not affect much the fs dynamics (0.1-0.2 ps) of the encapsulated molecule. However, it clearly affect the ps one (1-2 ps (H(2)O) and 5-10 ps (QA-β-CD and HSA)). The effect of O(2) on the relaxation of the triplet state of the free and encapsulated TSPP is also studied and the obtained results are discussed in light of the shielding effect provided by the chemical and biological cavities. The observed difference, longer triplet lifetime upon encapsulation, might be relevant to the efficiency of this porphyrin in photodynamic therapy. The presteady-state kinetics of the TSPP:HSA has been studied by the stopped-flow spectrometer, and a two-step model was proposed for the complexation processes. The results show the importance of the initial association step for the overall ligand recognition process. This first step occurs with rate constant of ~4 × 10(5) M(-1) s(-1), which is about 5 orders of magnitude larger than the rate constant of the consecutive relaxation processes. We believe that our observations of molecular interaction between TSPP, QA-β-CD, and HSA protein from femtosecond to second at both ground and electronically first excited state give detailed information to improve our understanding of this kind of system and thus for a better design of drug delivery nanocarriers.     

Molecular dynamics study of the inclusion of cholesterol into cyclodextrins

The interaction of three cyclodextrins (CDs), viz. beta-CD, heptakis (2,6-di-O-methyl)-beta-CD (DM-beta-CD), and 2-hydroxypropyl-beta-CD (HP-beta-CD), with cholesterol was investigated using molecular dynamics (MD) simulations. The free energy along the reaction pathway delineating the inclusion of cholesterol into each CD was computed using the adaptive biasing force method. The association constant and the corresponding association free energy were derived by integrating the potential of mean force (PMF) over a representative ordering parameter. The results show that the free energy profiles possess two local minima corresponding to roughly equally probable binding modes. Among the three CDs, DM-beta-CD exhibits the highest propensity to associate with cholesterol. Ranking for binding cholesterol, viz. DM-beta-CD > HP-beta-CD > beta-CD, agrees nicely with experiment. Partitioning of the PMF into free energy components illuminates that entering of cholesterol into the CD cavity is driven mainly by electrostatic interactions, whereas deeper inclusion results from van der Waals forces and solvation effects. Additional MD simulations were performed to investigate the structural stability of the host-guest complexes near the free energy minima. The present results demonstrate that association of cholesterol and CDs follows two possible binding modes. Although the latter are thermodynamically favorable for all CDs, one of the two inclusion complexes appears to be preferred kinetically in the case of DM-beta-CD.     

Fluorescence of 1,3-Di(1-pyrenyl)propane probe incorporated into human serum albumin protein enforced conformations of the probe

 Steady-state and time-resolved fluorescence spectra of 1,3-di(1-pyrenyl)propane (1Py-(3)-1Py) incorporated into macromolecules of human serum albumin (HSA), into micelles of dodecyltrimethylammo-nium chloride (DTAC), and dissolved in 1,4-dioxane were compared. The steady-state fluorescence spectra indicated that in all the mentioned environments, upon excitation of 1Py-(3)-1Py, light was emitted from the single pyrene chromophores (1Py^*) and from the 1Py, 1Py^* excimers. The time-resolved fluo-rescence emission registered at 480 nm (excimer emission) for 1Py-(3)-1Py in the DTAC micelles and dissolved in 1,4-dioxane allowed to monitor formation of excimer with time constant τ₁=40.0 ns and 9.6 ns, for 1Py-(3)-1Py in the DTAC micelles and in 1,4-dioxane, respectively. However, when the 1Py-(3)-1Py probe was located inside of the macromolecules of HSA, only the decay of emission was observed for excimer with our set-up (t>2 ns after excitation). The instantaneous formation of excimer, unrelated to the decay of monomer excitation, indicates that the considerable fraction of 1Py-(3)-1Py in the hydrophobic pockets of HSA is present as the ground state dimer. The red shift (Δλ=8 nm) and broadening of UV absorption for 1Py-(3)-1Py in HSA (when compared with absorption 1Py-(3)-1Py in 1,4-dioxane) and comparison of exci-tation spectra of 1Py-(3)-1Py in HSA and in 1,4-dioxane also indicate that label molecules bound to some sites of HSA are in the ground state in the dimer conformation. Moreover, the close values of the ratios of intensities of monomer emission to excimer emission, registered 2 ns (5 ns gate) after excitation pulse with duration 300 ps and at the steady-state conditions, indicate that the interconversion between conformers of 1Py-(3)-1Py inside of the macro-molecules of HSA is slow in comparison with the decay time of Py chromophore in the excited state in HSA (two-exponential decay with decay times τ₁=2.41 ns, τ₂=69.0 ns). Thus, ratios of the intensities of monomer and excimer emissions of 1Py-(3)-1Py in HSA do not allow to obtain any information on the local microfluidity inside of the protein macromolecules but could be used for discrimination between different conformations of the probe, possibly located in different protein pockets. Received: 29 April 1996 Accepted: 15 August 1996     

Photophysics of Hypericin and Hypocrellin A in Complex with Subcellular Components: Interactions with Human Serum Albumin

Time-resolved fluorescence and absorption measurements are performed on hypericin complexed with human serum albumin, HSA (1:4, 1:1 and approximately 5:1 hypericin: HSA complexes). Detailed comparisons with hypocrellin A/HSA complexes (1:4 and 1:1) are made. Our results are consistent with the conclusions of previous studies indicating that hypericin binds to HSA by means of a specific hydrogen-bonded interaction between its carbonyl oxygen and the N1-H of the tryptophan residue in the IIA subdomain of HSA. (They also indicate that some hypericin binds nonspecifically to the surface of the protein.) A single-exponential rotational diffusion time of 31 ns is measured for hypericin bound to HSA, indicating that it is very rigidly held. Energy transfer from the tryptophan residue of HSA to hypericin is very efficient and is characterized by a critical distance of 94 A, from which we estimate a time constant for energy transfer of approximately 3 x 10(-15) s. Although it is tightly bound to HSA, hypericin is still capable of executing excited-state intramolecular proton (or hydrogen atom) transfer in the approximately 5:1 complex, albeit to a lesser extent than when it is free in solution. It appears that the proton transfer process is completely impeded in the 1:1 complex. The implications of these results for hypericin (and hypocrellin A) are discussed in terms of the mechanism of intramolecular excited-state proton transfer, the mode of binding to HSA and the light-induced antiviral and antitumor activity.     

Axial halogen ligand effect on photophysics and optical power limiting of some indium naphthalocyanines

Three axially substituted complexes, 2,3-octa(3,5-di-tert-butylphenoxy)-2,3-naphthalocyaninato indium chloride (1a), 2,3-octa(3,5-di-tert-butylphenoxy)-2,3-naphthalocyaninato indium bromide (1b), and 2,3-octa(3,5-di-tert-butylphenoxy)-2,3-naphthalocyaninato indium iodide (1c) have been synthesized and their photophysical properties have been investigated. Optical power limiting of nanosecond (ns) and picosecond (ps) laser pulses at 532 nm using these complexes has been demonstrated. All complexes display strong Q(0,0) absorption and measurable emission in the near-infrared region and exhibit strong excited-state absorption in the range of 470-700 nm upon ns laser excitation. The different axial ligands show negligible effect on the linear absorption, emission, and transient difference absorption spectra. However, the excited-state lifetime, triplet excited-state quantum yield, and efficiency to generate singlet oxygen are affected significantly by the heavier axial ligand. Brominated and iodinated complexes 1b and 1c show higher triplet excited-state quantum yield, while chlorinated complex 1a has longer excited-state lifetime and is more efficient in generating singlet oxygen. The iodinated complex 1c displayed the best optical limiting due to the higher ratio of excited-state absorption cross section to ground state absorption cross section (sigma(eff)/sigma(0)).     

Combining magnetic resonance spectroscopies, mass spectrometry, and molecular dynamics: investigation of chiral recognition by 2,6-di-O-methyl-beta-cyclodextrin

EPR spectroscopy has been employed to investigate the formation of complexes between heptakis-(2,6-O-dimethyl)-beta-cyclodextrin (DM-beta-CD) and different enantiomeric pairs of chiral nitroxides of general structure PhCH2NO.CH(R)R'. Accurate equilibrium measurements of the concentrations of free and included radicals afforded the binding constant values for these nitroxides. The relationship between the stereochemistry of the DM-beta-CD complexes and the thermodynamics of complexation was elucidated by correlating EPR data with 1H-1H NOE measurements carried out on the complexes between DM-beta-CD and the structurally related amine precursors of nitroxides. NOE data suggested that inclusion of the stereogenic center in the DM-beta-CD cavity occurs only when the R substituent linked to the chiral carbon contains an aromatic ring. For these types of complexes, molecular dynamics simulation indicated that the depth of penetration of the stereogenic center into the cyclodextrin cavity is determined by the nature of the second substituent (R') at the asymmetric carbon and is responsible for the observed chiral selectivity. Analysis of mass spectra showed that, for the presently investigated amines, electrostatic external adducts of CDs with protonated amines are detected by ESI-MS.     

Investigation of non-covalent interactions between paramagnetic complexes and human serum albumin by electrospray mass spectrometry

Stable gadolinium(III) chelates are nowadays routinely used as contrast agents for magnetic resonance imaging (MRI). Their non-covalent binding to human serum albumin (HSA) has shown to improve their efficacy. Non-covalent interactions lead to complex formation that can be quantified by several techniques that are mostly tedious and time-consuming. In this study, electrospray ionization mass spectrometry (ESI-MS) was used to investigate the interaction between HSA and several gadolinium(III) complexes. The results were compared with those obtained in the liquid phase. Four gadolinium complexes were investigated: Gd-DTPA 1, Gd-C(4)Me-DTPA 2, Gd-EOB-DTPA 3, and MP-2269 4. Relaxometry studies show that complexes 1 and 2 have no significant affinity for HSA, while complexes 3 and 4 have increasing affinities for the protein. 1:1 and 1:2 complexes between HSA and MP-2269 were detected by ESI-MS for a twofold excess of the contrast agent, whereas a ligand/protein molar ratio of 4:1 was necessary to observe a 1:1 stoichiometry for Gd-EOB-DTPA, an observation that is in good agreement with the known weaker affinity of the contrast agent for the protein. At a fourfold molar excess, no supramolecular complex was observed for Gd-DTPA 1 and Gd-C(4)Me-DTPA 2; a tenfold molar excess was necessary to detect a 1:1 complex, confirming the very weak affinity of these contrast agents for HSA.     

Investigation of heptakis(2,6-di-O-methyl)-beta-cyclodextrin inclusion complexes with flavonoid glycosides by electrospray ionization mass spectrometry

The non-covalent complexes between three flavonoid glycosides (quercitrin, hyperoside and rutin) and heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD) were investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). The 1:1 complexation of each flavonoid glycoside (guest) to the DM-beta-CD (host) was monitored in the negative ion mode by mixing each guest with an up to 30-fold molar excess of the host. The binding constants for all complexes were calculated by a linear equation in the order: DM-beta-CD:quercitrin > DM-beta-CD:rutin > DM-beta-CD:hyperoside. A binding model for the complexes has also been proposed based on the binding constants and tandem mass spectrometric data of these complexes.     

Inclusion of Paracetamol into β-cyclodextrin nanocavities in solution and in the solid state

We report on steady-state UV-visible absorption and emission characteristics of Paracetamol, drug used as antipyretic agent, in water and within cyclodextrins (CDs): β-CD, 2-hydroxypropyl-β-CD (HP-β-CD) and 2,6-dimethyl-β-CD (Me-β-CD). The results reveal that Paracetamol forms a 1:1 inclusion complex with CD. Upon encapsulation, the emission intensity enhances, indicating a confinement effect of the nanocages on the photophysical behavior of the drug. Due to its methyl groups, the Me-β-CD shows the largest effect for the drug. The observed binding constant showing the following trend: Me-β-CD>HP-β-CD>β-CD. The less complexing effectiveness of HP-β-CD is due to the steric effect of the hydroxypropyl-substituents, which can hamper the inclusion of the guest molecules. The solid state inclusion complex was prepared by co-precipitation method and its characterization was investigated by Fourier transform infrared spectroscopy, 1H NMR and X-ray diffractometry. These approaches indicated that Paracetamol was able to form an inclusion complex with CDs, and the inclusion compounds exhibited different spectroscopic features and properties from Paracetamol.     

Spectroscopic studies on the interaction between riboflavin and albumins

The interactions between riboflavin (RF) and human and bovine serum albumin (HSA and BSA) were studied by using absorption and fluorescence spectroscopic methods. Intrinsic fluorescence emission spectra of serum albumin in the presence of RF show that the endogenous photosensitizer acts as a quencher. The decrease of fluorescence intensity at about 350 nm is attributed to changes in the environment of the protein fluorophores caused by the ligand. The quenching mechanisms of albumins by RF were discussed. The binding constants and binding site number were obtained at various temperatures. The distance between albumins and RF in the complexes suggests that the primary binding site for RF is close to tryptophan residue (Trp214) of HSA and Trp212 of BSA. The hydration process of albumins has also been discussed.     

A photo-induced electron transfer study of an organic dye anchored on the surfaces of TiO2 nanotubes and nanoparticles

We report on femtosecond-nanosecond (fs-ns) studies of the triphenylamine organic dye (TPC1) interacting with titania nanoparticles of different sizes, nanotubes and nanorods. We used time-resolved emission and absorption spectroscopy to measure the photoinduced dynamics of forward and back electron transfer processes taking place in TPC1-titania complexes in acetonitrile (ACN) and dichloromethane (DCM) solutions. We observed that the electron injection from the dye to titania occurs in a multi-exponential way with the main contribution of 100 fs from the hot excited charge-transfer state of anchored TPC1. This process competes with the relaxation of the excited state, mainly governed by solvation, that takes place with average time constants of 400 fs in ACN and 1.3 ps in DCM solutions. A minor contribution to the electron injection process takes place with longer time constants of about 1-10 ps from the relaxed excited state of TPC1. The latter times and their contribution do not depend on the size of the nanoparticles, but are substantially smaller in the case of nanotubes (1-3 ps), probably due to the caging effect. The contribution is also smaller in DCM than in ACN. The efficient back recombination takes place also in a multi-exponential way with times of 1 ps, 15 ps and 1 ns, and only 20-30% of the initial injected electrons in the conduction band are left within the first 1 ns after excitation. The faster recombination rates are suggested due to those originating from the free electrons in the conduction band of titania or the electrons in the shallow trap states, while the slower recombination is due to the electrons in the deep trap states. The results reported here should be relevant to a better understanding of the photobehaviour of an organic dye with promising potential for use in solar cells. They should also help to determine the important factors that limit the efficiency of solar cells based on the triphenylamine-based dyes for solar energy conversion.     

Excitation decay pathways of Lhca proteins: a time-resolved fluorescence study

Light-harvesting complex I (LHCI), which serves as a peripheral antenna for photosystem I (PSI) in green plants, consists mainly of four polypeptides, Lhca1-4. We report room temperature emission properties of individual reconstituted monomeric Lhca proteins (Lhca1, -2, -3, and -4) and dimeric Lhca1/4, performed by steady-state and time-resolved fluorescence techniques. The emission quantum yields of the samples are approximately 0.12, 0.085, 0.081, 0.041, and 0.063 for Lhca1, -2, -3, -4, and the -1/4 dimer, respectively, which is considerably lower than the value of 0.22 found for light-harvesting complex II (LHCII), the main peripheral antenna complex of photosystem II in green plants. The decay components of LHCI proteins can be divided in two categories: Lhca1 and Lhca3 have decay times of 1.1-1.6 ns and 3.3-3.6 ns, and Lhca2 and Lhca4 have decay times of 0.7-0.9 ns and 3.1-3.2 ns. These categories seem to correlate with the pigment composition of the samples. All decay times are faster than that observed previously for LHCII. When the absolute emission yields and the lifetimes of the Lhca samples are combined, the overall emission properties of the individual Lhca proteins are expressed in terms of their emitting dipole moment strength. In the samples without extreme red states, that is, Lhca1 and Lhca2, the emitting dipole moment has a value close to unity (relative to monomeric chlorophyll in acetone), which is similar to that for LHCII, whereas, in the samples with the red-most state (F-730), that is, Lhca3, -4, and the -1/4 dimer, the emitting dipole moment has a value less than unity (0.6-0.8), which can be explained by mixing the red-most (exciton) state with a dark charge-transfer state, as suggested in previous PSI red pigment studies. In addition, we find a lifetime component of approximately 50-150 ps in all red-pigment-containing samples, which cannot be due to "slow" energy transfer, but is instead assigned to an unrelaxed state of the pigment-protein, which, on this time-scale, is converted into the final emitting state.     

Investigating 2,2'-bipyridine-3,3'-diol as a microenvironment-sensitive probe: its binding to cyclodextrins and human serum albumin

The 2,2'-bipyridine-3,3'-diol molecule (BP(OH)2) was investigated as a potential photophysical probe in inclusion and biological studies. Binding of BP(OH)2 to cyclodextrins (CDs) and human serum albumin (HSA) was studied by following the changes in its absorption and fluorescence spectra. The stoichiometric ratios and binding constants of the complexes were deduced by fitting the changes in the spectral intensity to binding isotherms. The stoichiometric ratio in the BP(OH)2/(alpha-CD) complex is dominated by 1:2, whereas in all other CDs and in HSA this ratio is 1:1. The structure of the BP(OH)2:(alpha-CD)2 complex, calculated using ab initio methods, indicates that the inclusion of the BP(OH)2 molecule is axial and centered between the two cavities of alpha-CD with van der Waals and electrostatic interactions dominating the binding. Analysis of these results along with the inclusion results of BP(OH)2 in beta-CD, methyl-beta-CD, 2,6-di-O-methyl-beta-CD, and gamma-CD shows that absorption and fluorescence of BP(OH)2 are very sensitive to the change in the cavity size of CD and its hydrophobicity. This change is reflected in the form of a decrease in the intensity of the absorption peaks of the BP(OH)2/water complex in the region 400-450 nm and a red shift in the fluorescence peak as the cavity size decreases and its hydrophobicity increases. Binding of BP(OH)2 as a probe ligand to HSA, a prototype protein, reflects the hydrophobic interior of HSA in a similar manner. The spectral changes indicate that BP(OH)2 binds in the hydrophobic cavity of HSA's subdomain IIA. The results presented here show that BP(OH)2 can be used in binding sites and biological systems as a microenvironment-sensitive probe.     

Excited state dynamics in recombinant water-soluble chlorophyll proteins (WSCP) from cauliflower investigated by transient fluorescence spectroscopy

The present study describes the fluorescence emission properties of recombinant water-soluble chlorophyll (Chl) protein (WSCP) complexes reconstituted with either Chl a or Chl b alone (Chl a only or Chl b only WSCP, respectively) or mixtures of both pigments at different stoichiometrical ratios. Detailed investigations were performed with time and space correlated ps fluorescence spectroscopy within the temperature range from 10 to 295 K. The following points were found: (a) The emission spectra at room temperature (295 K) are well characterized by bands with a dominating Lorentzian profile broadened due to phonon scattering and peak positions located at 677, 684 and 693 nm in the case of Chl a only WSCP and at 665, 675 and 689 nm for Chl b only WSCP. In addition, all spectra contain minor bands in the longer wavelength region. (b) The emission spectra at 10 K of samples suspended in buffer containing 50% glycerol are dominated by bands peaking at 668 nm for Chl b only WSCP and at 685 nm for Chl a only WSCP and samples reconstituted with mixtures of Chl a and Chl b. (c) At 10 K and in buffer with 50% glycerol the decay kinetics of WSCP samples with Chl a only are dominated by a component with a time constant of 6.2 (+/-0.2) ns at 685 nm while those of WSCP containing mixtures of Chl a and Chl b are characterized by a slightly shorter value of 6.0 (+/-0.2) ns. WSCP containing Chl b only exhibits a distinctly longer value of 7.0 (+/-0.3) ns at an emission wavelength of 668 nm. (d) The decay associated emission spectra at 10 K of all samples exhibit at least 3 decay components with time constants of 80-120 ps, 2-4 ns and 6-7 ns in 50% glycerol. These results are consistently described within the framework of our previously presented model (J. Phys. Chem. B 2007, 111, No. 46, 13325; J. Phys. Chem. B 2007, 111, No. 35, 10487) , for the structural motifs of chlorophyll binding to the tetrameric protein matrix of WSCP. It is shown that formation of strongly coupled open sandwich dimers does not lead to quenching of 1Chl a* or 1Chl b*.     

Interaction of hypericin with serum albumins: surface-enhanced Raman spectroscopy, resonance Raman spectroscopy and molecular modeling study

Surface-enhanced Raman spectroscopy, resonance Raman spectroscopy and molecular modeling were employed to study the interaction of hypericin (Hyp) with human (HSA), rat (RSA) and bovine (BSA) serum albumins. The identification of the binding site of Hyp in serum albumins as well as the structural model for Hyp/HSA complex are presented. The interactions mainly reflect: (1) a change of the strength of H bonding at the N1-H site of Trp; (2) a change of the Trp side-chain conformation; (3) a change of the hydrophobicity of the Trp environment; and (4) a formation of an H-bond between the carbonyl group of Hyp and a proton donor in HSA and RSA which leads to a protonated-like carbonyl in Hyp. Our results indicate that Hyp is rigidly bound in IIA subdomain of HSA close to Trp214 (distance 5.12 A between the centers of masses). In the model presented the carbonyl group of Hyp is hydrogen bonded to Asn458. Two other candidates for hydrogen bonds have been identified between the bay-region hydroxyl group of Hyp and the carbonyl group of the Trp214 peptidic link and between the peri-region hydroxyl group of Hyp and the Asn458 carbonyl group. It is shown that the structures of the Hyp/HSA and Hyp/RSA complexes are similar to, and in some aspects different from, those found for the Hyp/BSA complex. The role of aminoacid sequence in the IIA subdomains of HSA, RSA and BSA is discussed to explain the observed differences.     

Interaction of 7-hydroxyflavone with human serum albumin: a spectroscopic study

Numerous recent investigations have revealed that various synthetic as well as therapeutically active natural flavonoids possess novel luminescence properties that can serve as highly sensitive monitors for exploring their interactions with relevant physiological targets. Here we report a detailed study on the interactions of the model flavone, 7-hydroxyflavone (7HF) with the plasma protein human serum albumin (HSA), employing electronic absorption, fluorescence (steady state and time resolved) and induced circular dichroism (ICD) spectroscopy. The spectral data indicate that in the protein matrix, the neutral 7HF molecules are predominantly transformed to a conjugate anion (7HFA) by a proton abstraction in the ground state. The protein (HSA) environment induces dramatic enhancements in the fluorescence emission intensity, anisotropy (r) and lifetime (tau) values, as well as pronounced changes in the fluorescence excitation and emission profiles of the fluorophore. Moreover, evidence for efficient F?rster type resonance energy transfer (FRET, from tryptophan to 7HFA) is presented, from which we infer that the binding site of 7HF in HSA is proximal (estimated distance, R=23.6A) to the unique tryptophan - 214 residue present in the inter-domain (between IIA and IIIA domains) loop region of the protein. The binding constant (K=9.44x10(4)M(-1)) and the Gibbs free energy change (DeltaG=-28.33kJ/mol) for 7HFA-HSA interaction have been estimated from the emission data. Finally, the near-UV circular dichroism (CD) studies show that the electronic transitions of 7HF are strongly perturbed on binding to the chiral host (HSA), leading to the appearance of ICD bands. Implications of these results are discussed.     

Spectroscopic studies on interactions of the tetrakis(acetato)chloridodiruthenium(II,III) complex and the Ru2(II,III)-NSAID-derived metallodrugs of ibuprofen and ketoprofen with human serum albumin

Diruthenium paddlewheel-structured complexes bearing a Ru₂(II,III) multiply bonded core show promising potential in medicinal chemistry. This work reports studies on the interactions of the tetrakis(acetato)chloridodiruthenium(II,III) complex (RuAc), [Ru₂(μ-O₂CCH₃)₄Cl], and the corresponding Ru₂(II,III)-non-steroidal anti-inflammatory drug (NSAID) metallodrugs of the NSAIDs ibuprofen (RuIbp) and ketoprofen (RuKet) with the human serum albumin (HSA). Circular dichroism (CD) studies showed that the three Ru₂ complexes interact with the HSA and induce conformational changes on the secondary structure of the protein. The reaction of the RuAc complex with the protein was monitored and the RuAc/HSA binding constant was estimated on the basis of electronic absorption spectroscopy data. Fluorescence emission spectroscopy studies were performed for all the Ru₂ complex/HSA systems and the Stern–Volmer constants and the thermodynamic parameters were determined for the RuAc/HSA binding. Mass spectrometry data confirmed the presence of the Ru₂ complexes in the protein phase after ultrafiltration. The studies suggest that the nature of the RuAc binding to the HSA is distinct from that of the derived RuIbp and RuKet metallodrugs. Electrostatic forces, accompanied by coordination of the metal to the amino acid side chains of the protein, seem to be the main forces acting in the RuAc/HSA binding, while non-covalent/hydrophobic forces might be predominant in the Ru₂-NSAID metallodrug/protein interactions. The findings suggest that the HSA protein might be a potential carrier in the blood plasma for the Ru₂(II,III)-NSAID metallodrugs.     

Effect of beta-cyclodextrin nanocavity confinement on the photophysics of robinetin

We have studied the confinement of robinetin, a therapeutically active plant flavonol, in cyclodextrin (CDx) nanocavities, using steady state and time resolved fluorescence spectroscopy. Enhanced tautomer emission (arising from excited state intramolecular proton transfer (ESIPT)) as well as dramatically blue shifted (approximately 10 nm in beta-CDx and approximately 33 nm in SHP beta-CDx) normal fluorescence observed upon addition of the beta-CDxs indicate that robinetin readily enters the doughnut-shaped hydrophobic cavity of beta-CDx where the chromone moiety is well shielded from external hydrogen bonding perturbations. Detailed analyses of the fluorescence data (emission profile, anisotropy, decay times) indicate that robinetin forms 1:1 inclusion complexes with both natural and chemically modified beta-cyclodextrins (beta-CDx and SHP beta-CDx) with affinity constant values K=195+/-17 M(-1) and 1055+/-48 M(-1) respectively, indicating the prospective utility of SHP beta-CDx in particular as an effective drug carrier. Unlike beta-CDxs, alpha-CDxs do not form inclusion complexes with robinetin. To further characterize the robinetin/beta-CDxs complexes, circular dichroism (CD) spectroscopic studies have been performed, which reveal that incorporation of robinetin molecules in the chiral environment of the beta-CDxs strongly affects the electronic transitions of robinetin leading to the occurrence of positive induced circular dichroism (ICD) bands in the near ultra-violet (UV) region. Molecular mechanics calculations show that the inclusion complex with the chromone ring inserted into the beta-CDx cavity is most favorable, in agreement with our spectroscopic data.