Modification of the heme·heme oxygenase system with iron and cobalt tetrasulfonated phthalocyanines

Modification of heme·heme oxygenase by iron(III) and cobalt(II) tetrasulfonated phthalocyanines has been performed. New compounds have been isolated and their properties have been investigated by difference spectroscopy, electrophoresis, molecular weight estimation, electron paramagnetic resonance (EPR) and carboxymethylation at histidyl groups. Spectrophotometric titration data indicate the ratio of the reagents in this process to be 1:1. The visible absorption spectra show the main peak at 650 nm for the iron compound and 682 nm for the cobalt one. Electrophoresis and molecular weight estimation show both complexes to be monomers. Cobalt(II) tetrasulfonated phthalocyanine, under aerobic conditions with heme oxygenase protein, undergoes autooxidation to the cobalt(III) complex, as has been proved by EPR and spectroscopic data. Iron and cobalt phthalocyanine modified heme·heme oxygenase with excess dithionite is reduced at the phthalocyanine ligand. In the presence of oxygen, the reduction product transforms into oxygenated Fe(III)Lheme oxygenase or Co(III)heme oxygenase, respectively. Reduction of the iron(III) model complex with ascorbic acid under anaerobic conditions leads to degradation of the phthalocyanine moiety, while Co(III)heme oxygenase with ascorbic acid is reduced to Co(II)Lheme oxygenase. As has been shown by carboxymethylation of the heme oxygenase protein at the histidine residues, the predominant binding site of both phthalocyanine complexes is the heme-binding histidyl residue. There is evidence that there is a second binding site with lower affinity towards Co(II)L on the heme oxygenase protein. Iron and cobalt tetrasulfonated phthalocyanines are not able to displace heme from the heme·heme oxygenase complex. In this reaction the iron complex undergoes degradation and the cobalt one gives a hybrid compound with heme·heme oxygenaseHeme oxygenase protein complexes with iron and cobalt tetrasulfonated phthalocyanines do not exhibit activity in their oxidative degradation.     

Comparison and analysis on the serum‐binding characteristics of aspirin–zinc complex and aspirin

This study was designed to compare the protein‐binding characteristics of aspirin–zinc complex (AZN) with those of aspirin itself. AZN was synthesized and interacted with a model transport protein, human serum albumin (HSA). Three‐dimensional fluorescence, ultraviolet–visible and circular dichroism (CD) spectra were used to characterize the interaction of AZN with HSA under physiological conditions. The interaction mechanism was explored using a fluorescence quenching method and thermodynamic calculation. The binding site and binding locality of AZN on HSA were demonstrated using a fluorescence probe technique and Förster non‐radiation energy transfer theory. Synchronous fluorescence and CD spectra were employed to reveal the effect of AZN on the native conformation of the protein. The HSA‐binding results for AZN were compared with those for aspirin under consistent experimental conditions, and indicated that aspirin acts as a guide in AZN when binding to Sudlow's site I, in subdomain IIA of the HSA molecule. Moreover, compared with aspirin, AZN showed greater observed binding constants with, but smaller changes in the α‐helicity of, HSA, which proved that AZN might be easier to transport and have less toxicity in vivo.     

Effect of cucurbitacins on bilirubin-albumin binding in human plasma

The aim of this study is to investigate the effect of three cucurbitacins (Cuc) E, D and I on the bilirubin-albumin binding, both in human serum albumin (HSA) and in plasma. Bilirubin-HSA solution and plasma free of cucurbitacins were prepared as well as others containing serial concentrations of cucurbitacins. The concentration of unbound bilirubin was determined in bilirubin-HSA solution and the direct and total bilirubin concentrations were measured in plasma (with normal or elevated bilirubinemia) by Jendrassik and Grof method. In the conditions we adopted Cuc E and D (to a lesser extent), decreased the levels of unbound bilirubin in bilirubin-HSA solution and decreased direct bilirubin concentration and total bilirubin concentration in plasma in a dose-dependent manner while Cuc I had no effect. The effect of Cuc is related to the presence of native HSA. Thus, when albumin was absent or has been denatured by heating or by urea, Cuc E did not modify bilirubin levels, suggesting that the native structure of albumin is essential for such activity. The interaction of HSA with Cuc E was investigated by fluorescence spectroscopy. Cuc E increased the intrinsic fluorescence of the protein and the magnitude of fluorescence intensity of bilirubin-albumin complex. We concluded that Cuc E and D produced a rearrangement in the structure of albumin, particularly in the domain-II, resulting in an increase in the binding of bilirubin to albumin regardless to whether it's conjugated to glucuronic acid or unconjugated.     

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.     

Impact of Potential Blockers on Ru(III) Complex Binding to Human Serum Albumin

The effects of aspirin, vitamin B2 and warfarin as potential blockers of the ruthenium binding sites in HSA were investigated through UV/visible, circular dichroism (CD), fluorescence spectroscopy and the inductively coupled plasma-atomic emission spectroscopy ICP(AES). The studies on the interactions of several biologically relevant molecules with HSA have shown that drugs like aspirin or warfarin may strongly influence the interaction of serum protein with anticancer drugs. It can derive from the influence of the drug on protein conformation or binding close to binding site of anticancer drug. Aspirin, vitB2 and warfarin bind to IIA subdomain leading to partial blocking of the ruthenium binding site in HSA.     

Paclitaxel-HSA interaction. Binding sites on HSA molecule

Paclitaxel (trade name Taxol) is one of the world's most effective anticancer drugs. It is used to treat several cancers including tumours of the breast, ovary and lung. In the present work the interaction of paclitaxel with human serum albumin (HSA) in aqueous solution at physiological pH has been investigated through CD, fluorescence spectroscopy and by the antibody precipitate test. Binding of paclitaxel to albumin impact on protein structure and it influences considerably albumin binding of other molecules like warfarin, heme or bilirubin. The paclitaxel-HSA interaction causes the conformational changes with the loss of helical stability of protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the paclitaxel-bound HSA decreased, suggesting that perturbation around the Trp 214 residue took place. This was confirmed by the destabilization of the warfarin binding site, which includes Trp 214, and high affinity bilirubin binding site located in subdomain IIA.     

Determining binding sites of drugs on human serum albumin using FIA-QCM

A simple and effective method was developed for determining binding sites of drugs on human serum albumin (HSA) by independent binding or competitive displacement of bilirubin using flow injection analysis-quartz crystal microbalance (FIA-QCM) system. Both independent and competitive bindings were entirely monitored in real time. Bilirubin as a site I-binding ligand was pre-bound to HSA sensor so as to occupy the drug-binding site I. When the model site II-binding drugs (ibuprofen, ketoprofen and flurbiprofen) were injected into the bilirubin pre-bound HSA system, the frequency continuously decreased by 6Hz, 4Hz and 5Hz, respectively, which was the same as that of their individual binding to HSA sensor. It indicated that the drug binding to site II was independent and did not interfere with bilirubin binding. However, when the model site I-binding drugs (iodipamide and magnesium salicylate) were introduced into the system, the frequency remained unchanged in the initial several minutes and then rapidly decreased by 4Hz for iodipamide and increased by 4Hz for magnesium salicylate. This phenomenon revealed site I-binding drugs competitively bound to HSA against bilirubin and displaced the pre-bound bilirubin. The results demonstrate FIA-QCM can be a valid approach for monitoring the dynamic interaction between drugs and HSA in real time further identifying drug-binding sites without the need of labels.     

Structural studies of iron and cobalt tetrasulfonated phthalocyanine-globin complexes.

The structure of the complexes of iron and cobalt tetrasulfonated phthalocyanines with globin has been investigated by circular dichroism (CD), electron paramagnetic resonance (EPR) and polyacrylamide gel electrophoresis. Electrophoretic investigations and the molecular weight estimation indicates that the model complexes in the solutions are dimers. It is evident from the results of CD measurements that the incorporation of the iron or cobalt tetrasulfonated phthalocyanine into apohemoglobin significantly increases the helical structure of the protein and causes an appearance of the induced Soret and visible Cotton effects. Unlike methemoglobin, several discrete transition energies in the CD Soret band of Fe(III)L-globin are observed which suggest an inequivalence of the subunits within this complex. This suggestion is supported by EPR studies, which show that the iron atoms in Fe(III)L-globin are in two low electronic states. Electronic structures of the cobalt ions in Co(II)L-globin and oxyCo(II)L-globin are similar to those of coboglobin and oxycoboglobin, respectively, as is proved by EPR results. On this basis we conclude that the oxygen adduct of Co(II)L-globin can be described as a superoxide ion corrdinated to a formally cobaltic phthalocyanine compound.     

Modification of isolated α and β chains of human hemoglobin by the metal tetrasulfonated phthalocyanines. Studies on hybrid phthalocyanine-heme intermediates

α and β chains of hemoglobin have been modified with cobalt(II) tetrasulfonated phthalocyanine in place of heme. They display properties very similar to those of iron(II) phthalocyanine modified α and β chains. Mixed together they form tetrameric cobalt(II) phthalocyanine hemoglobin.Incorporation of Co(II)L into α and β globins results in stabilization of the protein structure, which is shown by a marked increase in its helicity content. Cobalt phthalocyanine substituted α and β chains are able to combine reversibly with oxygen giving more stable oxygenated species than their native analogues. The rate of both processes is lower in the case of the modified α chain. Recombination of the phthalocyanine α and β chains with the alternate heme containing chains give tetrameric hybrid hemoglobins. These comprise two phthalocyanine modified subunits and two heme containing subunits. The helicity content of the tetrameric hybrid hemoglobin calculated for one subunit is lower that the arithmetic mean of helicities for its isolated subunits. This suggests a destabilizing chain-chain interaction within the tetramer. Unlike in the separated subunits, oxygen binding by hybrid hemoglobins is irreversible. Deoxygenation by argon bubbling leads to the formation of inactive species which in oxygen atmosphere undergo irreversible oxidation with destruction of the complex.     

The fatty acid analogue 11-(dansylamino)undecanoic acid is a fluorescent probe for the bilirubin-binding sites of albumin and not for the high-affinity fatty acid-binding sites.

1. The fluorescent fatty acid probe 11-(dansylamino)undecanoic acid (DAUDA) binds with high affinity to bovine and human serum albumin (BSA and HSA) at three sites. 2. The Kd of the primary binding site could not be determined; however, the two secondary sites appeared to be equivalent, with an apparent Kd of 8 x 10(-7) M for both BSA and HSA. 3. The spectral characteristics of DAUDA when bound to the primary site of the two albumins were different, with HSA producing a greater fluorescence enhancement and emission maximum at a shorter wavelength (480 nm) than for BSA (495 nm). 4. Displacement studies indicated that the DAUDA-binding sites were not equivalent to the primary long-chain fatty acid-binding sites on albumin, but corresponded to the bilirubin sites. Fatty acyl-CoAs also bind to the bilirubin sites, as do medium-chain fatty acids. 5. The solubility, stability and spectral properties of DAUDA make it an excellent probe for investigating the bilirubin-binding sites of albumin, particularly HSA.     

Paramyxovirus membrane protein enhances antibody production to new antigenic determinants in the actin molecule: a model for virus-induced autoimmunity.

Paramyxovirus membrane (M) protein specifically binds to cellular actin but not to bovine serum albumin or myoglobin, as determined by affinity chromatography and enzyme-linked immunosorbent assay. The binding site for M protein on actin is different from the binding sites for antiactin antibodies. The interaction of M protein with actin resulted in production of antibodies to several new antigenic sites on the actin molecule. Five rabbits immunized with actin alone produced antibodies against the N-terminal sequence (residues 1 to 39). Another five rabbits immunized with a mixture of M protein and actin produced antibodies against a C-terminal fragment and a central region as well as the N-terminal fragment. By immunoblotting with proteolytic fragments of actin, the new antigenic sites were located between amino acid residues 40 to 113, 114 to 226, and 227 to 375. Antisera taken from some patients with recent measles virus infections demonstrated antiactin antibodies to sites other than the N-terminal fragment of actin (amino acids 1 to 39). The interaction of paramyxovirus M protein with actin and the subsequent production of antibodies to new antigenic sites may serve as a model for one of the mechanisms of virus-induced autoimmunity.     

The disaccharide anthracycline MEN 10755 binds human serum albumin to a non-classical drug binding site

The interaction of the novel disaccharide anthracycline MEN 10755 with human serum albumin (HSA) was investigated by visible absorption and fluorescence spectroscopies and by ultrafiltration. Notably, MEN 10755 binds serum albumin far stronger than doxorubicin. Albumin binding results into a drastic quenching of the intrinsic fluorescence of MEN 10755; a binding constant of 1.1 x 10(5) was determined from fluorescence data. To localize the HSA binding site of MEN 10755 competition experiments were carried out with ligands that are selective for the different drug binding sites of the protein. No relevant competition effects were seen in the case of warfarin, diazepam and hemin, known ligands of sites I, II and III, respectively. Modest effects were observed following addition of palmitic acid that targets the several fatty acid binding sites of the protein. In contrast, extensive displacement of the bound anthracycline was achieved upon addition of ethacrinic acid. On the basis of these results, it is proposed that MEN 10755 binds serum albumin tightly to a non-canonical surface binding site for which it competes specifically with ethacrinic acid.     

Studies on the interactions between human serum albumin and imidazolium [trans-tetrachlorobis(imidazol)ruthenate(III)].

The interactions between imidazolium [trans-tetrachlorobis(imidazol) ruthenate(III)] (Ru-im) and human serum albumin (HSA) have been investigated through UV-Vis, CD, fluorescence spectroscopy and by the antibody precipitation test. Binding of Ru(III)-imidazole species to albumin has a strong impact on the protein structure and influences considerably the albumin binding of other molecules such as warfarin or heme. The metal complex-HSA interactions cause conformational changes with the loss of helical stability of the protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the ruthenium-bound HSA decreased, suggesting that perturbation around the Trp 214 residue took place. This was confirmed by the destabilisation of the warfarin binding site which includes Trp 214, observed in the metal-bound HSA.     

Probing the interaction of human serum albumin with bilirubin in the presence of aspirin by multi-spectroscopic, molecular modeling and zeta potential techniques: insight on binary and ternary systems

Here, we report on the effect of aspirin (ASA), on the binding parameters with regard to bilirubin (BR) to human serum albumin (HSA). Two different classes of binding sites were detected. Binding to the first and second classes of the binding sites was dominated by hydrophobic forces in the case of HSA-BR, whereas in the case of the ternary system, binding to the first and second classes of the binding sites was achieved by electrostatic interaction. The binding constant (K(a)) and number of binding site (n) obtained were 1.6 × 10(6)M(-1) and 0.98, respectively, for the primary binding site in the case of HSA-BR, and 3.7 × 10(6)M(-1) and 0.84, respectively, in the presence of ASA (ternary complex) at λ(ex)= 280 nm. The progressive quenching of the protein fluorescence as the BR concentration increased indicated an arrangement of the domain IIA in HSA. Changes in the environment of the aromatic residues were also observed by synchronous fluorescence spectroscopy (SFS). Changes of the secondary structure of HSA involving a decrease of α-helical and β-sheet contents and increased amounts of turns and unordered conformations were mainly found at high concentrations of BR. For the first time, the relationship between the structural parameters of HSA-BR by RLS for determining the critical induced aggregation concentration (C(CIAC)) of BR in the absence and presence of ASA was investigated, and there was a more significant enhancement in the case of the ternary mixture as opposed to the binary one. Changes in the zeta potential of HSA and the HSA-ASA complex in the presence of BR demonstrated a hydrophobic adsorption of this anionic ligand onto the surface of HSA in the binary system as well as both electrostatic and hydrophobic adsorption in the case of the ternary complex. By performing docking experiments, it was found that the acting forces between BR and HSA were mainly hydrophobic > hydrogen bonding > electrostatic interactions, and consequently BR had a long storage time in blood plasma, especially in the presence of ASA. This was due to the electrostatic interaction force between the BR and HSA being stronger in (HSA-ASA) BR than in the HSA-BR complex. In addition, it was demonstrated that, in the presence of ASA, the first binding site of BR on HSA was altered, but the parameters of binding did not become significantly modified, and thus the affinity of BR barely changed with and without ASA.     

Investigations with spectroscopy, zeta potential and molecular modeling of the non-cooperative behaviour between cyclophosphamide hydrochloride and aspirin upon interaction with human serum albumin: binary and ternary systems from multi-drug therapy

The interaction between cyclophosphamide hydrochloride (CYC) and aspirin (ASA) with human serum albumin (HSA) was studied by various kind of spectroscopic, ζ potential and molecular modeling under physiological conditions. The fluorescence data showed that the binding of drugs to proteins caused strong static fluorescence quenching. The analysis of the fluorescence quenching of HSA in the binary and ternary systems displayed that ASA was affected by the complex formed between CYC and HSA. Moreover, CYC was influenced by the HSA-ASA complex. The inherent binding information, including the quenching mechanism, binding constants, number of binding sites, effective quenching constant, fraction of the initial fluorescence and thermodynamic parameters were measured by the fluorescence quenching technique at various temperatures. In addition, according to the synchronous fluorescence spectra of HSA, the results showed that the fluorescence quenching of HSA originated from the Trp and Tyr residues, and indicated a conformational change of HSA with the addition of the drugs. Far-UV CD spectra of HSA were recorded before and after the addition of ASA and CYC as binary and ternary systems. An increase in intensity of the positive CD peak of HSA was observed in the presence of the drugs. The results were interpreted by excited interactions between the aromatic residues of the HSA binding sites and the drugs bound to them. The distance r between donor and acceptor was obtained by the Forster energy according to fluorescence resonance energy transfer (FRET) and found to be 2.35 nm and 1.78 nm for CYC and ASA, respectively. This confirmed the existence of static quenching for proteins in the presence of CYC and ASA. Furthermore, docking studies pointed at a reduction of the affinity of each of the drug compounds to the protein in the presence of the other in meaningful amounts. Pre-binding of any of the said compounds forced the second to bind in a non-optimized location and orientation. The potential at the electrokinetic shear surface of the protein-drug solution were measured at several concentrations of the drugs by the ζ potential technique, which confirmed experimental and theoretical results.     

Interaction of prostaglandins and fatty acids with native and acetaldehyde-alkylated proteins

Using intrinsic and probe fluorescence, microcalorimetry and isotopic methods, the interactions of prostaglandins (PG) E2 and F2 alpha and some fatty acids with native and alkylated proteins (human serum albumin (HSA) and rat liver plasma membrane PG receptors), were studied. The fatty acid and PG interactions with human serum albumin (HSA) resulted in effective quenching of fluorescence of the probe, 1.8-anilinonaphthalene sulfonate (ANS), bound to the protein. Fatty acids competed with ANS for the binding sites; the efficiency of this process increased with an increase in the number of double bonds in the fatty acid molecule. PG induced a weaker fluorescence quenching of HSA-bound ANS and stabilized the protein molecule in a lesser degree compared to fatty acids. The sites of PG E2 and F2 alpha binding did not overlap with the sites of fatty acid binding on the HSA molecule. Nonenzymatic alkylation of HSA by acetaldehyde resulted in the abnormalities of binding sites for fatty acids and PG. Modification of the plasma membrane proteins with acetaldehyde sharply diminished the density of PG E2 binding sites without changing the association constants. Alkylation did not interfere with the parameters of PG F2 alpha binding to liver membrane proteins.     

Water-in-silicone oil emulsion stabilizing surfactants formed from native albumin and alpha,omega-triethoxysilylpropyl-polydimethylsiloxane

Contact with hydrophobic silicones frequently leads to protein denaturation. However, it is demonstrated that albumin in water-in-silicone oil emulsions retains its native structure in the presence of a functional, triethoxysilyl-terminated silicone polymer, TES-PDMS. Both HSA and TES-PDMS were essential for the formation of stable water-in-silicone oil emulsions: attempts to generate stable emulsions using independently either the protein or the functionalized silicone as a surfactant failed. Confocal microscopy indicated that the human serum albumin (HSA) preferentially adsorbed at the oil/water interface, even in the presence of another protein (glucose oxidase). A variety of experiments demonstrated that the hydrolysis of the Si-OEt groups on the functional silicone occurred only to a limited extent, consistent with the absence of a covalent linkage between the silicone and protein, or of cross-linked silicones at the interface. The fluorescence spectra of HSA extracted from the emulsions, front-faced fluorescence experiments on the HSA/silicone emulsion itself, and HSA/salicylate binding studies all demonstrated that the stability of the water/oil interface decreased as the protein began to unfold: unfolding of the protein in the emulsion was slower than in aqueous solution. The experimental evidence indicated that the interaction between HSA and TES-PDMS is not associated with either homomolecular (HSA/HSA; TES-PDMS/TES-PDMS) interactions or with covalent linkage between two the polymers. Rather, the data is consistent with the direct binding of unhydrolyzed Si(OEt) 3 groups to native HSA. The nature of these interactions is discussed.     

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.