Binding of hypocrellin B to human serum albumin and photo-induced interactions

Molecular binding of hypocrellins to human serum albumin (HSA) needs to be further clarified considering the phototherapeutic potentials of hypocrellins to vascular diseases. In the current work, it was estimated that the binding constant of hypocrellin B (HB) to HSA was 2.28 x 10(4) M(-1). Furthermore, based on the fluorescence responses for both HB and the tryptophan of HSA, it was suggested that the binding of HB to HSA should be more specific rather than distributed randomly on the surface of HSA, which was also confirmed by photobleaching of the tryptophan via photosensitization of HB. Besides, it was found that both of the photo-bleaching of the tryptophan and the photo-oxidation of HB were principally oxygen-dependent, suggesting reactive oxygen species generated via the photosensitization of HB, instead of the free radicals of the photosensitizer (HB*-), play the most important role in photodynamic processes.     

Synchrotron vacuum ultraviolet (VUV) photo-induced fragmentation of cyclic dipeptides radical cations

Cyclic dipeptides, due to their chemical properties and various bioactivities, are very attractive for medicinal chemistry. Fragmentations of three simple cyclic dipeptides including cyclo(Gly-Gly), cyclo(Ala-Ala) and cyclo(Gly-Val) in the gas-phase are determined with synchrotron vacuum ultraviolet (VUV) photoionization mass spectrometry (VUV PIMS) and theoretical calculations. Cyclo(Gly-Gly) and cyclo(Ala-Ala) show the similar fragmentation pathways. The primary decomposition reactions of cyclo(Gly-Gly) and cyclo(Ala-Ala) radical cations are found to be HNCO loss and CO elimination. The appearance energies (AEs) of fragment ions [CH2NHCOCH2]+? and [CH3CHNHCOCHCH3]+? are measured to be 10.21 and 9.66±0.05 eV, respectively, which are formed from cyclo(Gly-Gly) and cyclo(Ala-Ala) radical cations with HNCO elimination. Due to the stabilization of the radical cation of cyclo(Gly-Val) with isopropyl side group, the dominant fragment ion m/z 114 assigned as [C4H6N2O2]+? is produced by γ-H migration and i cleavage to lose propylene. The ionization energies (IEs) of three cyclic dipeptides decrease in the order cyclo(Gly-Gly) (9.33±0.05 eV)>cyclo(Ala-Ala) (9.21±0.05 eV)>cyclo(Gly-Val) (9.09±0.05 eV) from measurements of photoionization efficiency spectra. It implies that IEs of cyclic dipeptides are affected by substituent groups and symmetrical characterization of molecular structures. These observations of the chemical properties of cyclic dipeptides radical ion (M+?) may be important for understanding gas-phase molecular reactivity of 2,5-diketopiperazines and guiding diketopiperazine-based drug design.     

Thermodynamic and spectroscopic study of Cu(II) and Ni(II) binding to bovine serum albumin

The thermodynamics of Cu(II) and Ni(II) binding to bovine serum albumin (BSA) have been studied by isothermal titration calorimetry (ITC). The Cu(II) binding affinity of the N-terminal protein site is quantitatively higher when the single free thiol, Cys-34, is reduced (mercaptalbumin), compared to when it is oxidized or derivatized with N-ethylmaleimide. This increased affinity is due predominantly to entropic factors. At higher pH (approximately 9), when the protein is in the basic (B) form, a second Cu(II) binds with high affinity to albumin with reduced Cys-34. The Cu(II) coordination has been characterized by UV-vis absorption, CD, and EPR spectroscopy, and the spectral data are consistent with thiolate coordination to a tetragonal Cu(II), indicating this is a type 2 copper site with thiolate ligation. Nickel(II) binding to the N-terminal site of BSA is also modulated by the redox/ligation state of Cys-34, with higher Ni(II) affinity for mercaptalbumin, the predominant circulating form of the protein.     

A spectroscopic study of nickel(II)-bovine serum albumin binding and reactivity

The pH dependence of the uv/visible and CD spectra of the 1:1 Ni(BSA) complex in aqueous solutions is interpreted in terms of a major square-planar form and an octahedral form. At pH 7.4, the two forms, respectively, account for ca. 70% and 30% of the total Ni(II). The two forms are in rapid equilibrium with each other and so both probably involve Ni(II) binding to the N-terminal region of the albumin protein. The kinetics of the equilibrium reaction of Ni(BSA) with His were studied at 37 degrees C in buffered media of pH 7.4 and 9.3. In line with predictions, the two Ni(BSA) forms show markedly different reactivities, with the square-planar form being the more thermodynamically stable and the less reactive. The octahedral form reacts with an observed zero-order dependence on His concentration while the square-planar form shows both zero-order and first-order dependence, the latter being the more dominant. The significance of the slow equilibrium rate at pH 7.4 to the possible physiological role of Ni-albumin in blood serum is discussed.     

Effect of cis-, trans-diamminedichloroplatinum(II) and DBP on human serum albumin

Both isomers of diamminedichloroplatinum(II) bind to albumin and induce the formation of the albumin dimer (MW approximately 140 kDa). The trans isomer exhibits a much greater tendency to induce a protein dimerization than the cis isomer. Under similar experimental conditions, the phosphonic derivative of diammineplatinum(II) (DBP) does not induce any dimer formation. The amount of bound complex per mol of human serum albumin (HSA, for an incubation time of 7 days) was found to be 6, 10.5 and 1 mol for cis-, trans-DDP and DBP, respectively. The relative fluorescence intensity of platinum-bound HSA decreases to about 55% for cis-DDP, 45% for trans-DDP and to 85% for DBP when compared to the complex-free protein, suggesting that the binding occurs in the proximity of the Trp214 residue. The structural studies (CD) have shown that only DDP-isomers cause the distinct modification of HSA native structure (alpha-helical content). Pt(II) complexes binding to HSA affect the affinity of HSA towards heme and bilirubin. High excess of DDP prevents the heme and bilirubin binding, while DBP affects this binding much less effectively due to the low amount of the protein-bound complex. Reactions of platinum complexes with albumin are believed to play an important role in the metabolism of this anticancer drug. The minor effect of DBP on HSA may indicate that the toxicity of the phosphonate analog is much lower than toxicities of DDP isomers, most likely due to kinetic reasons.     

Multi-spectroscopic study on interaction of bovine serum albumin with lomefloxacin-copper(II) complex

The binding reactions of lomefloxacin-copper(II) complex (LMF-Cu) or LMF to bovine serum albumin (BSA) in physiological solution were investigated by multi-spectroscopy. The binding constant, the number of binding sites and the binding distance between LMF-Cu or LMF and BSA were obtained by a fluorescence quenching method and according to the mechanism of Forster-type dipole-dipole non-radioactive energy-transfer, respectively. Enthalpy and entropy changes for two systems were calculated to be -7.970 kJ mol(-1) and 47.438 J mol(-1)K(-1) for LMF-BSA, -12.469 kJ mol(-1) and 33.542 J mol(-1)K(-1) for LMF-Cu-BSA, respectively. The highly positive values observed for the entropy give evidence for a strong interaction. The values of DeltaH and DeltaS in two systems are similar, indicating that electrostatic interactions in two systems play major role. The effect of LMF-Cu or LMF on the conformation of BSA was also analyzed by synchronous fluorescence, three-dimensional fluorescence and circular dichroism spectra. The results showed that the presence of Cu ion in LMF-Cu can affect the conformation of BSA to some degree. All the results revealed that the addition of copper ion promotes the interaction of lomefloxacin with bovine serum albumin.     

Characterization of the copper(II)- and nickel(II)-transport site of human serum albumin. Studies of copper(II) and nickel(II) binding to peptide 1-24 of human serum albumin by 13C and 1H NMR spectroscopy

As a basis for understanding the role of albumin in the transport of metal ions, detailed investigations have been carried out to elucidate the structure of Ni(II)- and Cu(II)-binding site of the peptide residue corresponding to the NH2-terminal peptide fragment 1-24 of human serum albumin by 1H and 13C NMR spectroscopy. These studies have been conducted in aqueous medium at different pH values and at different ligand/metal ratios. The results show the following: (i) Diamagnetic Ni(II) complex and paramagnetic Cu(II) complex are in slow exchange NMR time scale. (ii) Titration results of Ni(II)-bound form of peptide 1-24 show the presence of a 1:1 complex in the wide pH range (6.0-11.0), and the same stoichiometry is proposed for Cu(II) as well. (iii) Analysis of the spectra suggests that both Ni(II) and Cu(II) have one specific binding site at the NH2-terminal tripeptide segment (Asp-Ala-His...) involving the Asp alpha-NH2, His N(1) imidazole, two deprotonated peptide nitrogens (Ala NH and His NH), and the Asp COO- group. (iv) Complexation of Ni(II) and Cu(II) causes conformational change near the metal-binding site of the polypeptide chain, but there is no other binding group involved besides those in the first three residues.     

Spectroscopic study on the interaction of bovine serum albumin with zinc(II) phthalocyanine

The interaction between the photosensitive antitumour drug, 2(3),9(10),16(17),23(24)‐tetra‐(((2‐aminoethylamino)methyl)phenoxy)phthalocyaninato‐zinc(II) (ZnPc) and bovine serum albumin (BSA) has been investigated using various spectroscopic methods. This work may provide some useful information for understanding the interaction mechanism of anticancer drug–albumin binding and gain insight into the biological activity and metabolism of the drug in blood. Based on analysis of the fluorescence spectra, ZnPc could quench the intrinsic fluorescence of BSA and the quenching mechanism was static by forming a ground state complex. Meanwhile, the Stern–Volmer quenching constant (K_SV), binding constant (K_b), number of binding sites (n) and thermodynamic parameters were obtained. Results showed that the interaction of ZnPc with BSA occurred spontaneously via hydrogen bond and van der Waal's force. According to Foster's non‐radioactive energy transfer theory, the energy transfer from BSA to ZnPc occurred with high possibility. Synchronous fluorescence and circular dichroism (CD) spectra also demonstrated that ZnPc induced the secondary structure of and conformation changes in BSA, especially α helix. Copyright © 2015 John Wiley & Sons, Ltd.     

Binding of Cu(II) to non-prosthetic sites in ceruloplasmin and bovine serum albumin

The binding of Cu(II) to native human, porcine, bovine and ovine ceruloplasmin (Cp) and to bovine serum albumin (bSA) has been studied at pH 7.4, 30 mM barbital buffer. The results were analyzed for the strength and the number of binding sites using Scatchard plots. Evidence for additional copper binding sites in Cp and bSA was obtained suggesting a role for copper ion in the homeostatic regulation of Cu(II) and other metal ions in the serum. In the binding studies the Cp was freed of exogenous Cu(II) by passing it over a Chelex-100 column. Two flow rates were used, 4 ml/hr and 40 ml/hr, which removed Cu(II) of different affinities. Cp passed at the slower flow rate (Cp4) only contained the prosthetic copper atoms. Cp passed at the faster flow rate (Cp40) contained one additional copper atom with a Ka approximately 10(7) M-1. Another 2-6 Cu(II) ion could be added to the Cp40 with an average affinity of about Ka approximately 10(5) M-1. The Cu(II) ions found in Cp provide two distinguishable classes: (1) the prosthetic copper atoms and (2) the exogenous copper atoms that can be removed by Chelex-100. For bSA one copper atom was bound strongly with a Ka value approaching 10(12) - 10(13) M-1 and was not removed by Chelex-100 at any flow rate. A second copper atom was found with a Ka = 5.2 x 10(6) M-1 and was removed by Chelex-100 at 4 ml/hr. Three additional copper atoms were bound with a Ka = 1.6 x 10(5) M-1; they were readily removed by Chelex-100 at 40 ml/hr but were nondialysable.     

Synthesis of the native copper(II)-transport site of human serum albumin and its copper(II)-binding properties.

A derivative of the native-sequence tripeptide of the specific Cu(II)-transport site of human serum albumin, L-aspartyl-L-alanyl-L-histidine N-methylamide, was synthesized, and its binding to Cu(II) was examined to determine the influence of the side-chain groups on the Cu(II) binding. The equilibria involved in the Cu(II)-L-aspartyl-L-alanyl-L-histidine N-methylamide system were investigated by analytical potentiometry. Three complex species were found in the pH range 4-10. The same species were identified in both the visible and circular-dichroism spectra. The main species present in the physiological pH range is shown to have the same ligands around the square-planar Cu(II) ion as those reported for albumin and tripeptides diglycyl-L-histidine and its N-methylamide derivative. The results obtained from competition experiments showed that this tripeptide has a higher affinity towards Cu(II) than has albumin itself. The overall findings are compared with those from albumin. At neutral pH the side chains do not play any important role in the Cu(II) binding, but at low pH the beta-carboxyl group of the N-terminal aspartic residue becomes important. A possible competition site on albumin for Cu(II) at low pH is discussed.     

Selective separation of human serum albumin with copper(II) chelated poly(hydroxyethyl methacrylate) based nanoparticles

Poly(hydroxyethyl methacrylate) (PHEMA) nanoparticles with an average size of 300 nm in diameter and with a polydispersity index of 1.156 were produced by surfactant free emulsion polymerization. Specific surface area of the PHEMA nanoparticles was found to be 996 m²/g. Metal-chelating ligand 3-(2-imidazoline-1-yl)propyl(triethoxysilane) (IMEO) was covalently attached to the PHEMA nanoparticles. IMEO content was 0.97 mmol IEMO/g. The morphology and properties of these nanoparticles were characterized with scanning electron microscopy, Fourier transform infrared spectroscopy and atomic force microscopy. The Cu²⁺-chelated PHEMA–IMEO nanoparticles were used in the adsorption-elution studies of human serum albumin (HSA) in a batch system. Maximum HSA adsorption amount of the Cu²⁺ chelated nanoparticles was 680 mg HSA/g. The PHEMA–IMEO–Cu²⁺ nanoparticles exhibited a quite high adsorption capacity and fast adsorption rate due to their high specific surface area and the absence of internal diffusion resistance.     

Kinetic studies of mobilization of copper(II) from human serum albumin with chelating agents

The kinetics of the mobilizing reactions of five chelating agents for human serum albumin (HSA)-bound copper(II) [Cu(II)] have been studied spectrophotometrically. The decreasing sequence of reaction rate has been determined to be EDTA greater than DTPA greater than EGTA greater than NTA greater than IDA. A group of mathematical models were established to define the mechanisms of the competitive reactions between low-molecular-weight ligand and macromolecular ligand. All reactions of the five chelating agents follow a process involving the intermediate ternary complexes: (formula; see text) The reactions of DTPA and EDTA were found to be different from those of EGTA, NTA, and IDA. In the former cases, the reactions are likely following an overlapping mechanism in which the rate constant k1 was closed to k2. The reactions involving the other three chelators are different in k1 much greater than k2.     

Fibrillation of human serum albumin shows nonspecific coordination on stoichiometric increment of Copper(II)

Protein aggregation is related to a series of pathological disorders the main cause of which are the fibrillar species generated during the process. Human serum albumin (HSA) undergoes rapid fibrillation in the presence of Cu(II) at pH 7.4 in 60% ethanol after 6-h incubation (～65?°C) followed by room temperature incubation. Here, we have investigated the effect of a stoichiometric variation of Cu(II) on the self-assembly of HSA using Congo red and thioflavin T dye-binding studies, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, electron paramagnetic resonance spectroscopy, fluorescence microscopy and transmission electron microscopy. The simulation of EPR spectra suggests that with the increment in Cu(II) ion concentration, there is a change in ligand field coordination. Kinetic parameters indicate reduced cooperativity that may be related to the nonspecific coordination on increment of Cu(II) concentration. Cu(II) is also able to direct the accumulation of a large number of fibers along with a formation of dense fibrillar network which is evident from microscopic images.     

The in vitro glycation of human serum albumin in the presence of Zn(II)

Amino groups of human serum albumin (HSA) can react non-enzymatically with carbonyl groups of reducing sugars to form advanced glycation end products (AGEs). These AGEs contribute to many of the chronic complications of diabetes including atherosclerosis, cataract formation and renal failure. The current study focused on in vitro non-enzymatic reactivity of glyceraldehyde (GA) and methylglyoxal (MG) with HSA and evaluated the rate and extent of AGE formation in the presence of varied concentrations of Zn(II). At normal physiological conditions, GA and MG readily react with HSA. The presence of Zn(II) in HSA-GA or HSA-MG incubation mixtures reduced AGE formation. This finding was confirmed by UV and fluorescence spectrometry, HPLC techniques, and matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF). HPLC studies revealed decreased adduct formation of the glycated protein in the presence of Zn(II). The inhibition of AGE formation was intense at elevated Zn(II) concentrations. The results of this study suggest that Zn(II) may prove to be a potent agent in reducing AGE formation.     

Equilibrium studies of zinc(II) and cobalt(II) binding to tripeptide analogues of the amino terminus of human serum albumin

Serum albumin is known to bind several divalent metal ions at the amino terminus of the protein. Two peptide analogues for the amino terminus of human albumin, L-aspartyl-L-alanyl-L-histidine-N-Methyl amide (AAHNMA) and glycylglycyl-L-histidine-N-methyl amide (GGHNMA) have been synthesized, and their interactions with Zn(II) and Co(II) ions have been studied using analytical potentiometry. The stability constants of the species and their distribution as a function of pH were determined in 0.16-M KNO₃ at 25°. Comparison of the modes of interaction of the Zn(II) and Co(II) with each of the above peptides indicate that, although Co(II) is a valuable tool for the study of Zn(II) interaction with metalloenzymes, it is considerably less useful as a Zn(II) model with small peptide molecules. The potentiometric properties of the two peptide-Zn(II) systems have been compared to the potentiostatic properties of the albumin-Zn(II) system. The results indicate that AAHNMA is a better analogue for the Zn(II)-HSA interaction than is GGHNMA. The findings suggest that the Zn(II)-HSA binding site is best described as a compound site containing both a histidyl and a neighboring carboxyl group.     

Interaction of copper (II) complexes by bovine serum albumin: spectroscopic and calorimetric insights

Serum albumins being the most abundant proteins in the blood and cerebrospinal fluid are significant carriers of essential transition metal ions in the human body. Studies of copper (II) complexes have gained attention because of their potential applications in synthetic, biological, and industrial processes. Study of binding interactions of such bioinorganic complexes with serum albumins improves our understanding of biomolecular recognition process essential for rational drug design. In the present investigation, we have applied quantitative approach to explore interactions of novel synthesized copper (II) complexes viz. [Cu(L¹)(L²)ClO₄] (complex I), [Cu(L²)(L³)]ClO₄] (complex II) and [Cu(L⁴)₂(H₂O)₂] (complex III) with bovine serum albumin (BSA) to evaluate their binding characteristics, site and mode of interaction. The fluorescence quenching of BSA initiated by complexation has been observed to be static in nature. The binding interactions are endothermic driven by entropic factors as confirmed by high sensitivity isothermal titration calorimetry. Changes in secondary and tertiary structure of protein have been studied by circular dichroism and significant reduction in α-helical content of BSA was observed upon binding. Site marking experiments with warfarin and ibuprofen indicated that copper complexes bind at site II of the protein.