Interaction of Cu(II) 3,5-diisopropylsalicylate with human serum albumin - an evaluation of spectroscopic data

The copper(II) complex of 3,5-diisopropylsalicylate is a lipophilic water-insoluble binuclear complex, Cu(II) (3,5-DIPS) , that has attracted interest because of a wide range of pharmacological activities. This study was undertaken to examine bonding interactions between the complex and human serum albumin (HSA) to help elucidate the mode of transport of the complex in vivo. Electron paramagnetic resonance, numerical magnetic resonance and UV-visible absorption spectroscopic studies were performed using 200 M aqueous solutions (pH 7.5) of HSA to which had been added up to three molar equivalents of CuCl , CuSO , or Cu(II) (3,5-DIPS). Both EPR and UV-visible spectra demonstrated the presence of more than one copper bonding site on HSA, and proton NMR spectra showed that the 3,5-DIPS ligand is also bonded to HSA. These results indicate that there is no observable direct coordination of the ligand to copper in the presence of HSA, and that the majority of the copper and 3,5-DIPS bond to HSA at separate sites. Addition of solid Cu(II) (3,5-DIPS) to HSA at pH 7.5 similarly resulted in spectra that suggest that there are no ternary Cu(II)(3,5-DIPS), Cu(II)(3,5-DIPS) , or Cu(II) (3,5-DIPS) complexes formed with HSA. It is concluded that any ternary complexes formed in the presence of HSA are below the spectroscopic detection limits and represent less than 5% of the total copper. © Rapid Science 1998.     

Short peptides are not reliable models of thermodynamic and kinetic properties of the N-terminal metal binding site in serum albumin.

A comparative study of thermodynamic and kinetic aspects of Cu(II) and Ni(II) binding at the N-terminal binding site of human and bovine serum albumins (HSA and BSA, respectively) and short peptide analogues was performed using potentiometry and spectroscopic techniques. It was found that while qualitative aspects of interaction (spectra and structures of complexes, order of reactions) could be reproduced, the quantitative parameters (stability and rate constants) could not. The N-terminal site in HSA is much more similar to BSA than to short peptides reproducing the HSA sequence. A very strong influence of phosphate ions on the kinetics of Ni(II) interaction was found. This study demonstrates the limitations of short peptide modelling of Cu(II) and Ni(II) transport by albumins.     

Palladium(II) complexes of biorelevant ligands. Synthesis,structures, cytotoxicity and rich DNA/HSA interaction studies

In this work, a pair of new palladium(II) complexes, [Pd(Gly)(Phe)] and [Pd(Gly)(Tyr)], (where Gly is glycine, Phe is phenylalanine, and Tyr is tyrosine) were synthesized and characterized by UV–Vis, FT-IR, elemental analysis, ¹H-NMR, and conductivity measurements. The detailed ¹H NMR and infrared spectral studies of these Pd(II) complexes ascertain the mode of binding of amino acids to palladium through nitrogen of -NH₂ and oxygen of -COO^? groups as bidentate chelates. The Pd(II) complexes have been tested for in vitro cytotoxicity activities against cancer cell line of K₅₆₂. Interactions of these Pd(II) complexes with CT-DNA and human serum albumin were identified through absorption/emission titrations and gel electrophoresis which indicated significant binding proficiency. The binding distance (r) between these synthesized complexes and HSA based on Forster?s theory of non-radiation energy transfer were calculated. Alterations of HSA secondary structure induced by complexes were confirmed by FT-IR measurements. The results of emission quenching at three temperatures have revealed that the quenching mechanism of these Pd(II) complexes with CT-DNA and HSA were the static and dynamic quenching mechanism, respectively. Binding constants (K_b), binding site number (n), and the corresponding thermodynamic parameters were calculated and revealed that the hydrogen binding and hydrophobic forces played a major role when Pd(II) complexes interacted with DNA and HSA, respectively. We bid that [Pd(Gly)(Phe)] and [Pd(Gly)(Tyr)] complexes exhibit the groove binding with CT-DNA and interact with the main binding pocket of HSA. The complexes follow the binding affinity order of [Pd(Gly)(Tyr)] > [Pd(Gly)(Phe)] with CT-DNA- and HSA-binding.     

Interactions of the carrier ligands of antidiabetic metal complexes with human serum albumin: a combined spectroscopic and separation approach with molecular modeling studies

The specific binding of carrier ligands of antidiabetic vanadium(IV) and zinc(II) complexes into drug binding pockets of human serum albumin (HSA) has been investigated via displacement reactions of site markers such as warfarin and dansylglycine by different spectroscopic (fluorescence, circular dichroism, NMR) and separation methods (capillary zone electrophoresis, ultrafiltration-UV). Conditional stability constants of the ligands were calculated for the binding at sites I and II of HSA. Binding site I was found to be the primary binding site for 2,6-pyridine dicarboxylic acid (dipic) and picolinic acid (pic), and site II for 6-methylpicolinic acid (6-Mepic) and maltol, although dipic, 6-Mepic and pic displace both site markers at differing extents. The experimental data is complemented by protein-ligand docking calculations for dipic and 6-Mepic which support the observations.     

Biological evaluations of newly-designed Pt(II) and Pd(II) complexes using spectroscopic and molecular docking approaches

To perform biological evaluations of newly-designed Pt(II) and Pd(II) complexes, the present study was conducted with targeted protein human serum albumin (HSA) and HCT116 cell line as model of human colorectal carcinoma. The binding of Pt(II) and Pd(II) complexes to HSA was analyzed using fluorescence spectroscopy and molecular docking. The thermal stability and alterations in the secondary structure of HSA in the presence of Pt(II) and Pd(II) complexes were investigated using the thermal denaturation method and circular dichroism (CD) spectroscopy. The cytotoxicity of the Pt(II) and Pd(II) complexes was studied against the HCT116 cell line using MTT assay. The binding analysis revealed that the fluorescence findings were well in agreement with docking results such that there is only one binding site for each complex on HSA. Binding constants of 8.7?×?10³ M^?1, 2.65?×?10³ M^?1, 0.3?×?10³ M^?1, and 4.4?×?10³ M^?1 were determined for Pd(II) and Pt(II) complexes (I–IV) at temperature of 25?°C, respectively. Also, binding constants of 1.9?×?10³ M^?1, 15.17?×?10³ M^?1, 1.9?×?10³ M^?1, and 13.1?×?10³ M^?1 were determined for Pd(II) and Pt(II) complexes (I–IV) at temperature of 37?°C, respectively. The results of CD and thermal denaturation showed that the molecular structure of HSA affected by interaction with Pt(II) and Pd(II) complexes is stable. Cytotoxicity studies represented the growth suppression effect of the Pt(II) and Pd(II) complexes toward the human colorectal carcinoma cell line. Therefore, the results suggest that the new designed Pt(II) and Pd(II) complexes are well promising candidates for use in cancer treatment, particularly for human colorectal cancer.

Communicated by Ramaswamy H. Sarma     

Oxindole-Schiff base copper(II) complexes interactions with human serum albumin: Spectroscopic, oxidative damage, and computational studies

CD and EPR were used to characterize interactions of oxindole-Schiff base copper(II) complexes with human serum albumin (HSA). These imine ligands form very stable complexes with copper, and can efficiently compete for this metal ion towards the specific N-terminal binding site of the protein, consisting of the amino acid sequence Asp-Ala-His. Relative stability constants for the corresponding complexes were estimated from CD data, using the protein as competitive ligand, with values of log K_CuL in the range 15.7-18.1, very close to that of [Cu(HSA)] itself, with log K_CuHSA 16.2. Some of the complexes are also able to interfere in the α-helix structure of the protein, while others seem not to affect it. EPR spectra corroborate those results, indicating at least two different metal species in solution, depending on the imine ligand. Oxidative damage to the protein after incubation with these copper(II) complexes, particularly in the presence of hydrogen peroxide, was monitored by carbonyl groups formation, and was observed to be more severe when conformational features of the protein were modified. Complementary EPR spin-trapping data indicated significant formation of hydroxyl and carbon centered radicals, consistent with an oxidative mechanism. Theoretical calculations at density functional theory (DFT) level were employed to evaluate Cu(II)-L binding energies, L → Cu(II) donation, and Cu(II) → L back-donation, by considering the Schiff bases and the N-terminal site of HSA as ligands. These results complement previous studies on cytotoxicity, nuclease and pro-apoptotic properties of this kind of copper(II) complexes, providing additional information about their possibilities of transport and disposition in blood plasma.     

<Emphasis Type="Italic">In silico</Emphasis> analysis of paraoxon binding by human and bovine serum albumin

Albumin is known to be able to cleave ether bonds in organophosphates (OPs). Amino acids responsible for esterase and pseudo-esterase albumin activity towards OPs are not yet finally identified. Presumably, Sudlow’s site I with the Tyr150 residue shows a “true” esterase activity, while Sudlow’s II site with the Tyr411 residue—a pseudo-esterase one. Both human (HSA) and bovine (BSA) serum albumins were used in in vitro studies of albumin (pseudo)esterase activity towards OPs. There is a body of evidence that the efficiency of interaction of different xenobiotics differs for these two proteins. Using paraoxon as an example, the aim of this study was to conduct an in silico study of the OP interaction with the previously identified potential sites of HSA and BSA (pseudo)esterase activity, to estimate the possibility of enzymatic reactions at these sites, to comparatively analyze these proteins from the evolutionary viewpoint, and to assess the possibility of extrapolating the experimental data obtained on BSA to a human organism. Molecular docking of paraoxon into the sites of HSA and BSA potential (pseudo)esterase activity has been performed. Conformational changes occurring in the resultant complexes with time have been studied by molecular dynamics simulation. It has been shown that Sudlow’s site II is less liable to evolutionary changes. Binding of modulators at other sites is not required for productive sorption of OPs and the phosphorylation reaction at Sudlow’s site II. It has been concluded that simi lar results for HSA and BSA could be expected for the irreversible binding of OPs at Sudlow’s site II. Since Sudlow’s site I is less conservative, diff erent binding efficiency could be expected for rigid molecules or optically active compounds. Both for HSA and BSA, productive binding of OPs at Sudlow’s site I is possible only after changes in the albumin molecule structure induced by binding of modulators at other sites.     

Structural interrogation of phosphoproteome identified by mass spectrometry reveals allowed and disallowed regions of phosphoconformation

Background

Many biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is one of the most important transport proteins in the cardiovascular system due to its great binding capacity and high physiological concentration. HSA has a preference for accommodating neutral lipophilic and acidic drug-like ligands, but is also surprisingly able to bind positively charged peptides. Understanding of how short cationic antimicrobial peptides interact with human serum albumin is of importance for developing such compounds into the clinics.

Results

The binding of a selection of short synthetic cationic antimicrobial peptides (CAPs) to human albumin with binding affinities in the μM range is described. Competitive isothermal titration calorimetry (ITC) and NMR WaterLOGSY experiments mapped the binding site of the CAPs to the well-known drug site II within subdomain IIIA of HSA. Thermodynamic and structural analysis revealed that the binding is exclusively driven by interactions with the hydrophobic moieties of the peptides, and is independent of the cationic residues that are vital for antimicrobial activity. Both of the hydrophobic moieties comprising the peptides were detected to interact with drug site II by NMR saturation transfer difference (STD) group epitope mapping (GEM) and INPHARMA experiments. Molecular models of the complexes between the peptides and albumin were constructed using docking experiments, and support the binding hypothesis and confirm the overall binding affinities of the CAPs.

Conclusions

The biophysical and structural characterizations of albumin-peptide complexes reported here provide detailed insight into how albumin can bind short cationic peptides. The hydrophobic elements of the peptides studied here are responsible for the main interaction with HSA. We suggest that albumin binding should be taken into careful consideration in antimicrobial peptide studies, as the systemic distribution can be significantly affected by HSA interactions.     

Metal-catalyzed oxidation of human serum albumin does not alter the interactive binding to the two principal drug binding sites

It is well known that various physiological factors such as pH, endogenous substances or post-translational modifications can affect the conformational state of human serum albumin (HSA). In a previous study, we reported that both pH- and long chain fatty acid-induced conformational changes can alter the interactive binding of ligands to the two principal binding sites of HSA, namely, site I and site II. In the present study, the effect of metal-catalyzed oxidation (MCO) caused by ascorbate/oxygen/trace metals on HSA structure and the interactive binding between dansyl-L-asparagine (DNSA; a site I ligand) and ibuprofen (a site II ligand) at pH 6.5 was investigated. MCO was accompanied by a time-dependent increase in carbonyl content in HSA, suggesting that the HSA was being oxidized. In addition, The MCO of HSA was accompanied by a change in net charge to a more negative charge and a decrease in thermal stability. SDS-PAGE patterns and α-helical contents of the oxidized HSAs were similar to those of native HSA, indicating that the HSA had not been extensively structurally modified by MCO. MCO also caused a selective decrease in ibuprofen binding. In spite of the changes in the HSA structure and ligand that bind to site II, no change in the interactive binding between DNSA and ibuprofen was observed. These data indicated that amino acid residues in site II are preferentially oxidized by MCO, whereas the spatial relationship between sites I and II (e.g. the distance between sites), the flexibility or space of each binding site are not altered. The present findings provide insights into the structural characteristics of oxidized HSA, and drug binding and drug-drug interactions on oxidized HSA.     

Investigation on the interaction of newly designed potential antibacterial Zn(II) complexes with CT-DNA and HSA

Two Zn(II) complexes of formula [Zn(bpy)(Gly)]NO₃ (I) and [Zn(phen)(Gly)]NO₃ (II) (where bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline and Gly = glycine) were synthesized and characterized by elemental analysis, molar conductance measurements, UV–vis, FT-IR, and ¹H NMR spectra. The interaction ability of these complexes with calf thymus DNA was monitored using spectroscopic methods, including UV–vis absorption spectroscopy, ethidium bromide displacement, Fourier transform infrared, and electrophoretic mobility assay. Further, the human serum albumin interactions of complexes I and II were investigated using UV–vis absorption spectroscopy, fluorescence quenching, circular dichroism, and Fourier transform infrared. The results obtained from these analyses indicated that both complexes interact effectively with CT-DNA and HSA. The binding constant (K_b), the Stern–Volmer constant (K_sv), and the number of binding sites (n) at different temperatures were determined for CT-DNA and HSA. Also, the negative ΔH° and ΔS° values showed that both hydrogen bonds and van der Waals forces played major roles in the association of CT-DNA-Zn(II) and HSA-Zn(II) complex formation. The displacement experiments suggested that Zn(II)-complexes primarily bound to Sudlow’s site II of HSA. The distance between the donor (HSA) and the acceptor (Zn(II) complexes) was estimated on the basis of the Forster resonance energy transfer (FRET) and the alteration of HSA secondary structure induced by the compounds were confirmed by FT-IR spectroscopy. The complexes follow the binding affinity order of I > II with DNA and II > I with HSA. Finally, Antibacterial activity of complexes I and II have been screened against gram positive and gram negative bacteria.     

Human serum albumin binding assay based on displacement of a non selective fluorescent inhibitor

In this paper, we describe a fluorescent antibacterial analog, 6, with utility as a competition probe to determine affinities of other antibacterial analogs for human serum albumin (HSA). Analog 6 bound to HSA with an affinity of 400+/-100 nM and the fluorescence was environmentally sensitive. With 370 nm excitation, environmental sensitivity was indicated by a quenching of the 530 nm emission when the probe bound to HSA. Displacement of dansylsarcosine from HSA by 6 indicated it competed with compounds that bound at site II (ibuprofen binding site) on HSA. Analog 6 also shifted the NMR peaks of an HSA bound oleic acid molecule that itself was affected by compounds that bound at site II. In addition to binding at site II, 6 interacted at site I (warfarin binding site) as indicated by displacement of dansylamide and the shifting of NMR peaks of an HSA bound oleic acid molecule affected by warfarin site binding. Additional evidence for multiple site interaction was discovered when a percentage of 6 could be displaced by either ibuprofen or phenylbutazone. A competition assay was established using 6 to determine relative affinities of other antibacterial inhibitors for HSA.     

The binding affinity of human serum albumin and paclitaxel through MMPBSA based on docked complex

The distribution, free concentration and metabolism of drugs can be significantly altered as a result of binding to albumin. At the same time, the conformational of serum albumin was also changed by interaction with low molecular weight drugs. In present work, we first equilibrated HSA in aqueous solution to obtain the solvated-HSA model. Further solvated-HSA was performed molecular docking with paclitaxel to find the binding sites. The two docking HSA-paclitaxel complexes were obtained and further equilibrated by a 12 ns MD simulation. Then, MMPBSA method was used to investigate the binding free energy of them. Finally, we correlated the fluctuations of residues with corresponding changes in the secondary structure by dssp method. Two binding sites of paclitaxel were found on HSA having considered the solvation effect. More hydrogen bonds were formed at site I respected to site II. A larger binding energy for primary binding also indicated that paclitaxel showed higher binding affinity mainly due to the stronger hydrogen bonding interactions. There was a significant difference between the two complexes on structure according to the dssp results. Moreover, structure of the binding sites exhibited more fluctuations after binding paclitaxel compared with other regions. Paclitaxel binding also induced distinct conformational changes in drug binding site even when it was empty and have contributed to a reduced binding capacity of HSA towards adriamycin.     

Surface-enhanced Raman spectroscopy study of the interaction of the antitumoral drug emodin with human serum albumin

Surface-enhanced Raman spectroscopy was employed in this work to study the interaction between the antitumoral drug emodin and human serum albumin (HSA), as well as the influence of fatty acids in this interaction. We demonstrated that the drug/protein interaction can take place through two different binding sites which are probably localized in the IIA and IIIA hydrophobic pockets of HSA and which correspond to Sudlow's I and II binding sites, respectively. The primary interaction site of this drug seems to be site II in the defatted albumin. Fatty acids seem to displace the drug from site II to site I in nondefatted HSA, due to the high affinity of fatty acids for site II. The drug interacts with the protein through its dianionic form in defatted HSA (when placed in the site II) and through its neutral form in the site I of nondefatted albumins.     

In vitro binding of leukotriene B4 (LTB4) to human serum albumin: evidence from spectroscopic, molecular modeling, and competitive displacement studies

Circular dichroism (CD) and UV absorption spectroscopy were utilized for the first time to investigate the interaction between leukotriene B4 (LTB4) and human serum albumin (HSA) in vitro. The weak intrinsic CD signal of LTB4 was enhanced fivefold in the presence of HSA. The red-shifted, hypochromic, and reduced vibrational fine structure of the ligand/protein UV absorption spectrum indicated complexation of the two molecules in solution. Results obtained from CD titration experiments were subjected to non-linear regression analysis to estimate the binding parameters (Ka = 6.7 x 10(4) M(-1), n = 1). Palmitic acid strongly decreased the induced CD signal of the LTB4/HSA complex, suggesting the role of a high-affinity fatty acid HSA binding site in the leukotriene complexation. Molecular modeling calculations based on the crystal structure of HSA predicted that the long-chain fatty acid site that overlaps with drug binding site II in subdomain IIIA was the most likely binding location for LTB4. Using the drug site II-specific marker ligand rac-ibuprofen, this prediction was confirmed with induced-CD displacement measurements. To the authors' knowledge, the current study represents the first demonstration of binding of LTB4 to HSA in vitro and has implications for the biological transport of this important pro-inflammatory mediator in vivo.     

Phenotypic characterization of the binding of tetracycline to human serum albumin

Because of the widely usage of the veterinary drug tetracycline (TC), its residue exist extensively in the environment (e.g., animal food, soils, surface water, and groundwater) and can enter human body, being potential harmful. Human serum albumin (HSA) is a major transporter for endogenous and exogenous compounds in vivo. The aim of this study was to examine the interaction of HSA with TC through spectroscopic and molecular modeling methods. The inner filter effect was eliminated to get accurate binding parameters. The site marker competition experiments revealed that TC binds to site II (subdomain IIIA) of HSA mainly through electrostatic interaction, illustrated by the calculated negative ΔH° and ΔS°. Furthermore, molecular docking was applied to define the specific binding sites, the results of which show that TC mainly interacts with the positively charged amino acid residues Arg 410 and Lys 414 predominately through electrostatic force, in accordance with the conclusion of thermodynamic analysis. The binding of TC can cause conformational and some microenvironmental changes of HSA, revealed by UV-visible absorption, synchronous fluorescence, and circular dichroism (CD) results. The accurate and full basic data in the work is beneficial to clarifying the binding mechanism of TC with HSA in vivo and understanding its effect on protein function during the blood transportation process.     

Isomeric N-alkylpyridylporphyrins and their Zn(II) complexes: inactive as SOD mimics but powerful photosensitizers

The ortho, meta, and para isomers of cationic N-alkylpyridylporphyrins and their Zn(II) complexes were compared in terms of their photodynamic properties. The ortho Zn(II) complex was found to be the most efficient in causing photooxidation of NADH in vitro. In Escherichia coli, however, the para and meta isomers were better photosensitizers than their ortho analogs. The lower potency of the ortho compound in vivo seems to be due to its lower intracellular concentration. All porphyrins tested were more efficient in killing E. coli and in photooxidizing NADH than the hematoporphyrin derivative. Antibiotic resistance did not affect the photokill, which implies that the cationic N-alkylpyridylporphyrins, as their Zn(II) complexes, can be used as bactericidal agents against antibiotic-resistant strains of gram-negative bacteria.     

Studies of copper(II) binding to glycylglycyl-L-tyrosine-N-methyl amide, a peptide mimicking the NH2-terminal copper(II)-binding site of dog serum albumin by analytical potentiometry, spectrophotometry, CD, and NMR spectroscopy

Unlike human serum albumin (HSA), dog serum albumin (DSA) does not possess the characteristics of the specific first binding site for Cu(II). In DSA, the important histidine residue in the third position, responsible for the Cu(II)-binding specificity in HSA, is replaced by a tyrosine residue. In order to study the influence of the tyrosine residue in the third position of DSA, a simple model of the NH2-terminal native sequence tripeptide of DSA, glycylglycyl-L-tyrosine-N-methylamide (GGTNMA) was synthesized and its Cu(II)-binding properties studied by analytical potentiometry, spectrophotometry, CD, and NMR spectroscopy. The species analysis indicated the existence of five mono-complexes at different protonation states: MHA, MA, MH-1A, MH-2A, MH-3A, and only one bis-complex MH-2A-2. The complexing ability of GGTNMA to Cu(II) was found to be weaker than that of the Cu(II) binding peptide models of HSA. The visible absorption spectra of Cu(II)-GGTNMA complexes are similar to those observed in the case of DSA-Cu(II) complexes. The weaker binding and the spectral properties of Cu(II)-GGTNMA complexes are consistent with less specific Cu(II)-binding properties of the peptide of this sequence similar to what was noted with DSA. CD results are in excellent agreement with species analysis and visible spectra where it is clearly evident that Cu(II) binds to GGTNMA starting from the alpha-NH2 group and step by step to deprotonated amide nitrogens as the pH is raised. The absence of any charge transfer band around 400 nm strongly indicates that Cu(II) does not bind to the phenolate group. Furthermore, NMR results are consistent with the noninvolvement of the tyrosine residue of GGTNMA in Cu(II) complexation. Thus, it is clear that the low Cu(II)-binding affinity of DSA is due to the genetic substitution of tyrosine for histidine at the NH2-terminal region of the protein.     

Binding of cadmium(II) and zinc(II) to human and dog serum albumins. An equilibrium dialysis and 113Cd-NMR study.

The binding of Cd(II) and Zn(II) to human serum albumin (HSA) and dog serum albumin (DSA) has been studied by equilibrium dialysis and 113Cd(II)-NMR techniques at physiological pH. Scatchard analysis of the equilibrium dialysis data indicate the presence of at least two classes of binding sites for Cd(II) and Zn(II). On analysis of the high-affinity class of sites, HSA is shown to bind 2.08 +/- 0.09 (log K = 5.3 +/- 0.6) and 1.07 +/- 0.12 (log K = 6.4 +/- 0.8) moles of Cd(II) and Zn(II) per mole of protein, respectively. DSA bound 2.02 +/- 0.19 (log K = 5.1 +/- 0.8), and 1.06 +/- 0.15 (log K = 6.0 +/- 0.2) moles of Cd(II) and Zn(II) per mole of protein, respectively. Competition studies indicate the presence of one high-affinity Cd(II) site on both HSA and DSA that is not affected by Zn(II) or Cu(II), and one high-affinity Zn(II) site on both HSA and DSA that is not affected by Cd(II) or Cu(II). 113Cadmium-HSA spectra display three resonances corresponding to three different sites of complexation. In site I, Cd(II) is most probably coordinated to two or three histidyl residues, site II to one histidyl residue and three oxygen ligands (carboxylate), while for the most upfield site III, four oxygens are likely to be involved in the binding of the metal ion. The 113Cd(II)-DSA spectra display only two resonances corresponding to two different sites of complexation. The environment around Cd(II) at sites I and II on DSA is similar to sites I and II, respectively, on HSA. No additional resonances are observed in any of these experiments and in particular in the low field region where sulfur coordination occurs. Overall, our results are consistent with the proposal that the physiologically important high-affinity Zn(II) and Cd(II) binding sites of albumins are located not at the Cu(II)-specific NH2-terminal site, but at internal sites, involving mostly nitrogen and oxygen ligands and no sulphur ligand.     

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.     

Anti-coagulant rodenticide binding properties of human serum albumin: a biochromatographic approach

In this paper, the anti-coagulant rodenticide-human serum albumin (HSA) binding was investigated using a perturbation method to calculate the solute distribution isotherms. It was shown that rodenticide can bound either on the benzodiazepine HSA site with low affinity (site I) or on the warfarin HSA site with high affinity (site II). The thermodynamic parameters of this association were calculated for the two HSA binding sites. For the site II, the rodenticide-HSA association was governed enthalpically whereas for the site I, this one was driven entropically. Moreover, the role of the magnesium (Mg(2+)) and calcium (Ca(2+)) on this association was carried out. It was clearly demonstrated that the rodenticide affinity for the site I was not affected by modifying the bulk solvent surface tension whereas for the site II the association constant increased strongly with the Mg(2+) or the Ca(2+) concentration in the bulk solvent. These results showed that the rodenticide-HSA affinity and thus the rodenticide toxicological effect depends on the Mg(2+) or Ca(2+) concentration.