Adsorption behavior of bovine serum albumin on lowly activated anionic exchangers suggests a new strategy for solid-phase proteomics

Diluted solutions of bovine serum albumin (BSA) (e.g., 0.1 mg /mL) do not form detectable protein large aggregates. Using gel-filtration experiments, we determined that a diluted solution of BSA is 97% monomeric BSA and 3% dimeric. The adsorption of this diluted BSA on highly activated anionic exchangers (e,g., having 40 micromol/wet g) keeps this mainly monomeric form. When supports activated with 2 micromol/wet g are used, only dimers become adsorbed to the support, accounting for 100% of the offered BSA. When the diluted BSA solution is offered to very mildly activated anionic exchangers (even only 0.125 micromol/wet g), an unexpected adsorption of most of the BSA on the support was also observed. These very slightly activated supports are only able to adsorb very large proteins or very large protein-protein complexes, larger than BSA dimers. In fact, a rapid cross-linking of the adsorbed BSA with dextran-aldehyde reveals the formation of very large BSA-BSA complexes with molecular mass higher than 500 000 Da, complexes that may be observed for soluble BSA with very high concentrations but are not detectable at 0.1 mg/mL. Moreover, the size of the aggregates strongly depends on the concentration of the ionized groups on the support: the less activated the supports are, the higher the sizes of the complexes. It seems that the interaction of the BSA molecules on the margins of the BSA aggregate with the groups on the support may stabilize the whole protein aggregate, although some components are not interacting with the support. Aggregates could account for more than 40% of the BSA in the solution after 50 h of incubation. However, only these large BSA aggregates were adsorbed in the support.     

The hyaluronan–protein complexes at low ionic strength: How the hyaluronidase activity is controlled by the bovine serum albumin

Hyaluronan (HA) hydrolysis catalysed by hyaluronidase (HAase) is strongly inhibited when performed at low HAase over HA concentration ratio and under low ionic strength conditions. The reason is the ability of long HA chains to form electrostatic and non-catalytic complexes with HAase. For a given HA concentration, low HAase concentrations lead to very low hydrolysis rates because all the HAase molecules are sequestered by HA, whilst high HAase concentrations lead to high hydrolysis rates because the excess of HAase molecules remains free and active. At pH 4, non-catalytic proteins like bovine serum albumin (BSA) are able to compete with HAase to form electrostatic complexes with HA, liberating HAase which recovers its catalytic activity. The general scheme for the BSA-dependency is thus characterised by four domains delimited by three noticeable points corresponding to constant BSA over HA concentration ratios. The existence of HA–protein complexes explains the atypical kinetic behaviour of the HA / HAase system. We also show that HAase recovers the Michaelis–Menten type behaviour when the HA molecule complexed with BSA in a constant complexion state, i.e. with the same BSA over HA ratio, is considered for substrate. When the ternary HA / HAase / BSA system is concerned, the stoichiometries of the HA–HAase and HA–BSA complexes are close to 10 protein molecules per HA molecule for a native HA of 1 MDa molar mass. Finally, we show that the behaviour of the system is similar at pH 5.25, although the efficiency of BSA is less.     

Enzymes incorporated in polyelectrolyte complexes. Effect of matrix conformational changes and phase transitions in solutions on catalytic properties

Immobilization of enzymes (penicillin amidase and alpha-chymotrypsin) in water-soluble nonstoichiometric polyeloctrolyte complexes (PEC) formed by poly(4-vinyl-N-ethylpyridinium bromide) (polycation) and polymethacrylic acid (polyanion) was carried out. Particles of these PEC consist of a nucleus formed by sequences of salt bonds between the units of oppositely charged polyelectrolytes and the hydrophylic shell formed by ionized groups of polyanions which is in excess in PEC. Such a structure of PEC particles results in a cooperative phase transitions of these systems at slight variations of pH and ionic strength. The work demonstrates phase diagrams of PEC solutions. The values of pH and ionic strength at which phase transitions in solutions of different PEC occur were elucidated. The decrease of pH value from 6.1 to 5.7 leads to reversible phase transition followed by a saltatory increase of Km for immobilized penicillin amidase by 5-10 fold depending on substrate used. The phase transition induced by ionic strength increase up to 0,27 M NaCl doesn't change significantly the Km-value of enzymic reaction. The phenomenon observed can be accounted for by the different structure of PEC particles. The catalytic properties of immobilized chymotrypsin were shown to depend on the loci of enzyme attachment. If the enzyme is bound to polyanion, neither conformational changes of the matrix nor phase transition in solution influence its accessibility for the protein inhibitor, but rather change the binding constant. If the enzyme is attached to polycation, i.e. included in the polycomplex nucleus, two fractions of enzymes accessible and inaccessible for protein inhibitor appear.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of structural-chemical characteristics of water-soluble polyelectrolyte complexes of ovalbumin on their immunological properties

Immunogenicity of soluble protein antigens in the complexes with synthetic polyions may be regarded as depending both on the nature of polymer carrier and the structure of the protein-polyelectrolyte complex. The immunogenicity of stable soluble complexes of ovalbumin (OA) with polycation - quaternized poly-4-vinylpyridine (C-1) and copolymer of acrylic acid and 2-methyl-5-vinylpyridine (C-2) have been evaluated. Immunization of mice by C-1 have induced a vigorous formation of the anti-OA IgG antibodies and IgE homocytotropic antibodies, while immunogenicity of OA in C-2 was comparable with that of OA alone. The analysis of the structural-chemical features of the complexes investigated has shown that enhanced immunogenicity of C-1 may be due to (1) the non-homogeneous distribution of protein globulae among polycation macromolecules and to (2) the formation of complex with an asymmetrical structure, to (3) the high ability of C-1 to adsorb on a surface of the lymphoid cells and to induce a formation of intercellular aggregates. An enhancing of a stability and a size of C-2 in the presence of Cu2+ shows no influence on a immunogenicity of OA. An immunogenicity of both types of complexes does not depend upon the access of determinants of OA to antibodies so far as it has been shown that complex formation in both cases are not accompanied by an alteration of antigenicity and allergenicity of OA.     

Selective activation of murine V beta 8.2 bearing T cells by Pseudomonas exotoxin A.

We have determined that Pseudomonas aeruginosa exotoxin A (PE) can selectively stimulate the proliferation of V beta bearing T lymphocytes. Murine thymocytes were fractionated by selective agglutination with peanut agglutinin (PNA) and the PNA- thymocytes, which represent mature thymocytes, were shown to be responsive to PE stimulation. In addition, mature peripheral T lymphocytes (nylon wool nonadherent splenocytes) were also observed to respond to PE stimulation. Both CD4+ and CD8+ splenic T lymphocyte populations proliferated in response to PE. Flow microfluorimetry analysis of PNA- thymocytes stimulated with PE indicated that V beta 8.2 bearing T cells were preferentially expanded. Thus, our data indicate that PE represents a microbial super antigen which stimulates murine thymocytes which bear the V beta 8.2 element of the T cell receptor.     

Effect of polycation length on its complexation with DNA and with poly(oxyethylene-block-sodium methacrylate)

Polyelectrolyte complexes of a synthetic polycation with either a genomic DNA or a synthetic poly(oxyethylene-block-sodium methacrylate), POE-b-PMANa, have been studied in aqueous solutions as a function of cation:anion ratio, the degree of polymerization of the polycation, the ionic strength, and temperature using dynamic light scattering and turbidity measurements. The polycation was a copolymer of methacryl oxyethyl trimethylammonium chloride and poly(oxyethylene) monomethyl ether monomethacrylate with 4-5 oxyethylene repeating units, PMOTAC-g-POE. The molar masses of the polycations in a homological series were 0.3, 0.9, and 2.1 x 10(6) g/ mol. The amount of comonomers with poly(oxyethylene) tails in the copolymers was 15 mol %. The molar mass of the POE-b-PMANa was 75000 g/mol and that of the POE-block was 5000 g/mol. The molar mass of the polycation was shown to have a dramatic effect on the stability and size of the complexes formed by either of the polyanions. An increase in the polycation molar mass shifts the cloud point toward the lower polycation content in the complexes, and a macro phase separation occurs in the solutions with the cation to anion molar ratios much below than 1:1. Increasing the ionic strength has a similar effect. Further addition of salt to turbid and phase-separated solutions results in dissociation of the complexes, and the polyions dissolve as individual macromolecules. The effect of POE on the stability of polyelectrolyte complexes is discussed as well.     

Further studies on cold insoluble circulating immune complexes in rabbits immunized with bovine albumin.

Cold insoluble circulating immune complexes of BSA and anti-BSA antibody are formed in vivo while immune catabolism of antigen is occurring. The effects of temperature, rate of precipitation and redissolvability of the cold insoluble antigen were studied in this model. Circulating BSA is soluble at 37 degrees, but may precipitate when the temperature is reduced. The solubility of antigen decreases at 24 degrees and below. Complete precipitation occurs in 5-7 days. The antigen in the cryoprecipitate is difficult to redissolve unless low pH citrate or glycine buffers are used.     

Preparation and characterization of beta 1-bungarotoxin bispecific monoclonal antibody.

A hybrid hybridoma (tetradoma) that produces bispecific monoclonal antibodies (mAbs)2, which recognize two different epitopes on the A chain of beta 1-bungarotoxin (beta 1-bgt) at peptide sequences 46-51 and 100-106, has been obtained by fusing two hybridoma cell lines. The bispecific mAb were observed to inhibit 98% of the enzymatic activity of beta 1-bgt and neutralize its lethal toxicity completely. The avidity between the bispecific mAb and beta 1-bgt was noted to be 4.5 x 10(10) (liter/nmol), which is about 45-150 folds higher than the avidity of its two parental mAbs. All the soluble complexes, obtained from bispecific mAb and beta 1-bgt with different molar ratios, emerged in the void volume of size exclusion chromatography column, indicating multiple complexes of beta 1-bgt and bispecific mAb were formed. Based on these results, it indicated that the binding of bispecific mAb with its two epitopes on beta 1-bgt, which facilitates the immuno-complex formation and enhances the avidity, also highly neutralizes the biological activity of beta 1-bgt.     

The influence of charge density of chitosan in the compaction of the polyanions DNA and xanthan

In this study the relative importance of valence and charge density of the polycation chitosan on the compaction process of DNA and xanthan is investigated. Chitosans with approximately equal valence but differing in their charge density were employed to form polyelectrolyte complexes with the two polyanions. The resulting structures (toroids, rods, and globules) have been visualized by AFM. For DNA-chitosan the complexation process was additionally studied by utilizing the fluorescent probe ethidium bromide. The results show that not only the total charge per chitosan molecule (valence), but also the charge density is important in determining the association with polyanions such as DNA and xanthan. Furthermore, it is demonstrated that the pH at which the complexation takes place is an important parameter in the complexation process, influencing the structures formed.     

Biological and polymeric self-assembled hybrid systems: Structure and properties of thylakoid/polyelectrolyte complexes

A novel hybrid system composed of biological components and synthetic polymer, thylakoid/polycation complex, has been formed and studied. Effects of complex formation on the structure, electrostatics and functioning of thylakoid membranes have been examined. Thylakoids from bean leaves were used to form complexes with polycation polyallylamine hydrochloride (PAAH) in two systems: (i) thylakoid/polycation complexes formed in an aqueous bulk phase, and (ii) immobilized thylakoid/polycation planar complexes. Immobilized on a solid substrate surface, thylakoid/polycation complexes were prepared using layer-by-layer stepwise alternate adsorption technique, i.e., via the sequential alternate adsorption of thylakoids and polycation molecules. The morphology of built up structures was investigated by scanning electron microscopy. Light-induced electron transport in chloroplasts was studied by the electron paramagnetic resonance (EPR) method. Spin probe technique was employed to study the structural and electrostatic characteristics of thylakoid membranes. We have found that efficiency of light-induced electron transport in thylakoid membranes and membrane structure were not changed noticeably by PAAH binding to thylakoids in a wide range of PAAH concentrations. The data obtained indicate the physiologically-soft character of polycation interactions with thylakoid membranes and demonstrate effectiveness of interfacial self-assembly approach to fabrication of complex planar functional nanostructures from biological components and synthetic polymers.     

Biological and polymeric self-assembled hybrid systems: structure and properties of thylakoid/polyelectrolyte complexes

A novel hybrid system composed of biological components and synthetic polymer, thylakoid/polycation complex, has been formed and studied. Effects of complex formation on the structure, electrostatics and functioning of thylakoid membranes have been examined. Thylakoids from bean leaves were used to form complexes with polycation polyallylamine hydrochloride (PAAH) in two systems: (i) thylakoid/polycation complexes formed in an aqueous bulk phase, and (ii) immobilized thylakoid/polycation planar complexes. Immobilized on a solid substrate surface, thylakoid/polycation complexes were prepared using layer-by-layer stepwise alternate adsorption technique, i.e., via the sequential alternate adsorption of thylakoids and polycation molecules. The morphology of built up structures was investigated by scanning electron microscopy. Light-induced electron transport in chloroplasts was studied by the electron paramagnetic resonance (EPR) method. Spin probe technique was employed to study the structural and electrostatic characteristics of thylakoid membranes. We have found that efficiency of light-induced electron transport in thylakoid membranes and membrane structure were not changed noticeably by PAAH binding to thylakoids in a wide range of PAAH concentrations. The data obtained indicate the physiologically-soft character of polycation interactions with thylakoid membranes and demonstrate effectiveness of interfacial self-assembly approach to fabrication of complex planar functional nanostructures from biological components and synthetic polymers.     

Mechanism of binding of soluble immune complexes to lymphocytes

Soluble immune complexes prepared with either (a) ¹²⁵I BSA and mouse antiserum to BSA in the presence of fresh mouse serum (AgAbC) or with (b) ¹²⁵I BSA and a 7S fraction of the mouse antiserum to BSA (AgAb) bind to a certain proportion of mouse lymph node and spleen lymphocytes (but not to thymocytes). The efficiency of binding is greater when complexes are prepared at defined antigen/antibody ratios and when the incubation with lymphocytes is performed at 37 °C. However, a significant degree of binding occurs at lower temperatures and even at 0 °C. Cells which bind soluble complexes overlap extensively with complement receptor lymphocytes (CRL) (B lymphocytes) since the specific elimination of CRL also depletes the population of cells which can bind soluble complexes. Two types of interactions are involved in the binding: one mediated by antibody which has been aggregated by antigen and another by complement (probably C3). They can be operationally distinguished because (1) after treatment of the lymphocytes with trypsin, the binding of AgAbC (but not of AgAb) is strongly inhibited; (2) the binding of AgAb (but not of AgAbC) is inhibited by heat-aggregated Ig; (3) the binding of AgAbC (but not of AgAb) to lymphocytes inhibits their subsequent interaction and rosette formation with erythrocytes sensitized by antibody and complement components; and (4) cobra venom factor markedly alters the binding of AgAbC to lymphocytes.     

Developing a structure-function model for the cryptophyte phycoerythrin 545 using ultrahigh resolution crystallography and ultrafast laser spectroscopy

Cryptophyte algae differ from cyanobacteria and red algae in the architecture of their photosynthetic light harvesting systems, even though all three are evolutionarily related. Central to cryptophyte light harvesting is the soluble antenna protein phycoerythrin 545 (PE545). The ultrahigh resolution crystal structure of PE545, isolated from a unicellular cryptophyte Rhodomonas CS24, is reported at both 1.1A and 0.97A resolution, revealing details of the conformation and environments of the chromophores. Absorption, emission and polarized steady state spectroscopy (298K, 77K), as well as ultrafast (20fs time resolution) measurements of population dynamics are reported. Coupled with complementary quantum chemical calculations of electronic transitions of the bilins, these enable assignment of spectral absorption characteristics to each chromophore in the structure. Spectral differences between the tetrapyrrole pigments due to chemical differences between bilins, as well as their binding and interaction with the local protein environment are described. Based on these assignments, and considering customized optical properties such as strong coupling, a model for light harvesting by PE545 is developed which explains the fast, directional harvesting of excitation energy. The excitation energy is funnelled from four peripheral pigments (beta158,beta82) into a central chromophore dimer (beta50/beta61) in approximately 1ps. Those chromophores, in turn, transfer the excitation energy to the red absorbing molecules located at the periphery of the complex in approximately 4ps. A final resonance energy transfer step sensitizes just one of the alpha19 bilins on a time scale of 22ps. Furthermore, it is concluded that binding of PE545 to the thylakoid membrane is not essential for efficient energy transfer to the integral membrane chlorophyll a-containing complexes associated with PS-II.     

Metal-nucleotide structural characteristics during catalysis by beef heart mitochondrial F1

This study examined the nature of the metal-nucleotide complexes which serve as substrates, products, and intermediates in the beef heart mitochondrial ATPase reaction. The two methods employed involved the use of phosphorothioate ATP analogs as substrates in the presence of Mg2+ or Cd2+ and the use of substitution inert Cr X ATP complexes (the isolated diastereomers of the bidentate complexes) along with the newly synthesized Cr X ITP complexes as inhibitors of both the F1-ATPase and F1-ITPase activities. Little stereoselectivity was observed in the inhibition of F1-ATPase and F1-ITPase activities by the isolated diastereomers of beta,gamma-bidentate CrATP, while the inhibition by the delta,alpha,beta-bidentate CrADP diastereomer was greater than that of the lambda epimer. gamma-Monodentate CrITP was a weak inhibitor of both the ATPase and ITPase activities, whereas beta,gamma-bidentate CrITP failed to show any inhibition at all up to a concentration of 3.2 mM. When adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) was used as the substrate, (VmSp]/(Vm(Rp] with Mg2+ present was 2.7 at 31 degrees C and 3.5 at 13 degrees C. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Mg2+ present were 15.3 at 31 degrees C and 73.3 at 13 degrees C. With Cd2+ present, the (Vm(Sp]/(Vm(Rp] ratios were 0.81 and 0.65 at 31 and 13 degrees C, respectively. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Cd2+ present were 1.17 at 31 degrees C and 1.34 at 13 degrees C. The large activation energy observed for the isomers of CdATP beta S was not observed for MgATP beta S, MgATP, or CdATP. The Vm for Cd adenosine 5'-O-thiotriphosphate (ATP gamma S) hydrolysis was the largest of all the metal-phosphorothioate nucleotide complexes, while that for MgATP gamma S was the smallest. The results are interpreted in terms of a catalytic model for F1-catalyzed nucleotide hydrolysis describing metal-nucleotide chelation during the reaction.     

Electrostatic interactions between hyaluronan and proteins at pH 4: how do they modulate hyaluronidase activity

Hyaluronan (HA) hydrolysis catalyzed by hyaluronidase (HAase) is inhibited at low HAase over HA ratio and low ionic strength, because HA forms electrostatic complexes with HAase, which is unable to catalyze hydrolysis. Bovine serum albumin (BSA) was used as a model to study the HA-protein electrostatic complexes at pH 4. At low ionic strength, there is formation of (i) neutral insoluble complexes at the phase separation and (ii) small positively-charged or large negatively-charged soluble complexes whether BSA or HA is in excess. According to the ionic strength, different types of complex are formed. Assays for HA and BSA led to the determination of the stoichiometry of these complexes. HAase was also shown to form the various types of complex with HA at low ionic strength. Finally, we showed that at 0 and 150 mmol L(-1) NaCl, BSA competes with HAase in forming complexes with HA and thus induces HAase release resulting in a large increase in the hydrolysis rate. These results, in addition to data in the literature, show that HA-protein complexes, which can exist under numerous and varied conditions of pH, ionic strength and protein over HA ratio, might control the in vivo HAase activity.     

Spectroscopic study of TPPS4 nanostructures in the presence of bovine serum albumin.

The influence of bovine serum albumin (BSA) on the formation of J-aggregates of meso-tetra(4-sulfonatophenyl)porphine (TPPS4) in aqueous acid solution (pH 1.3) has been investigated by means of absorption and fluorescence spectroscopy. TPPS4 concentration was kept constant at 2 microM while BSA concentration was varied to get TPPS4 : BSA molar ratios from 1 : 0.005 to 1 : 20. In the presence of protein at all used concentrations the intensity of J-aggregates absorption band was higher than that in the pure solution. Spectral changes indicated that the dynamic equilibrium of the aggregated TPPS4 species was highly dependent on the molar ratio between TPPS4 and BSA. Small relative concentrations of BSA (TPPS4 : BSA, 1 : 0.005-1 : 0.1) had a stimulating effect on formation of J-aggregates. Several fractions of J-aggregates located in protein and aqueous moieties were detected in mixed solutions at intermediate BSA concentrations (TPPS4 : BSA, 1 : 0.5-1 : 8), when the absorbance intensity of the J-aggregates was the highest. At the highest used BSA concentrations (TPPS4 : BSA, 1 : 10-1 : 20) the spectral properties of the remaining J-aggregates were similar to those typical for pure porphyrin solution. Additionally, the split of the Soret band into two with peaks at 440 nm and 423 nm was followed by the simultaneous appearance of Q bands and reflected the formation of TPPS4-BSA complexes including both protonated and deprotonated TPPS4 forms.     

Chemical modification of wheat protein-based natural polymers: grafting and cross-linking reactions with poly(ethylene oxide) diglycidyl ether and ethyl diamine

Mobile poly(ethylene oxide) diglycidyl ether (PEODGE) segments were chemically grafted onto a soluble wheat protein (WP), and different network structures were formed via coupling reactions with ethyl diamine (EDA) in different PEODGE/EDA (PE) ratios. When the PE ratio was 1:1, linear PEs were the predominant segments grafted onto WP chains and the whole WP-PEODGE-EDA (WPE) system was still soluble with an increased molecular weight. Reducing the amount of EDA in the systems produced insoluble cross-linked WPE networks. The broad distribution of network structures and chain mobility resulted in a broad glass transition for the WPE materials. However, the glass transition started at lower temperatures, and the materials became flexible at room temperature. The PE segments were present in all rigid, intermediate, and mobile phases in WPE networks, while the proportion of mobile WP chains was increased as a result of the plasticization effect from the mobile PE segments. The mobility of the most mobile component lipid was also restricted to some extent when forming the cross-linked WPE networks. The study demonstrated that the formation of different network structures with PE segments could significantly improve the flexibility of WP materials, vary the solubility, and modify the mechanical performance of WP-based natural polymer materials.     

Heating of Milk Before or After Homogenization Changes its Coagulation Behaviour During Acidification

Diffusing wave spectroscopy and rheology were employed to investigate the acid coagulation behaviour of heated-homogenized milk. As heating before or after homogenization would cause differences in the protein adsorption at the interface, the order of processing was investigated. Tween 20 was added to the homogenized samples to displace most of the proteins from the interface, and the effect of the fat globules on structure formation was compared to that of the original fat globules covered of milk proteins. In both homogenized samples the fat globules participated in the formation of the network, but there were differences in the aggregates present at the interface and in the serum, and this difference affected the coagulation behaviour of the milk. Milk heated before homogenization showed aggregation at a higher pH than milk heated after homogenization. In both cases, the fat globules showed destabilization at pH of about 5.8, but only in the case of the milk heated before homogenization gelation occurred at this pH. This was attributed to the higher amount of soluble whey protein complexes interacting with the protein loaded fat globules, in the case of milk heated before homogenization.     

A spectroscopic study of the reaction of NAMI, a novel ruthenium(III)anti-neoplastic complex, with bovine serum albumin.

The reaction of Na[transRuCl4Me2SO(Im)] (NAMI; where Im is imidazole), a novel anti-neoplastic ruthenium(III) complex, with BSA, was studied in detail by various physico-chemical techniques. It is shown that NAMI, following chloride hydrolysis, binds bovine serum albumin tightly; spectrophotometric and atomic absorption data point out that up to five ruthenium ions are bound per albumin molecule when BSA is incubated for 24 h with an eightfold excess of NAMI. CD and electronic absorption results show that the various ruthenium centers bound to albumin exhibit well distinct spectroscopic features. The first ruthenium equivalent produces a characteristic positive CD band at 415 nm whereas the following NAMI equivalents produce less specific and less marked spectral effects. At high NAMI/BSA molar ratios a broad negative CD band develops at 590 nm. Evidence is provided that the bound ruthenium centers remain in the oxidation state +3. By analogy with the case of transferrins it is proposed that the BSA-bound ruthenium ions are ligated to surface histidines of the protein; results from chemical modification experiments with diethylpyrocarbonate seem to favor this view. Spectral patterns similar to those shown by NAMI are observed when BSA is reacted with two strictly related ruthenium(III) complexes Na[transRuCl4(Me2SO)2] and H(Im)[transRuCl4(Im)2] (ICR), implying a similar mechanism of interaction in all cases. It is suggested that the described NAMI-BSA adducts may form in vivo and may be relevant for the biological properties of this complex; alternatively NAMI/BSA adducts may be tested as specific carriers of the ruthenium complex to cancer cells. Implications of these findings for the mechanism of action of NAMI and of related ruthenium(III) complexes are discussed.     

Reactions of gold(III) complexes with serum albumin.

The reactions of a few representative gold(III) complexes -[Au(ethylenediamine)2]Cl3, [Au(diethylentriamine)Cl]Cl2, [Au(1,4,8,11-tetraazacyclotetradecane)](ClO4)2Cl, [Au(2,2',2'-terpyridine)Cl]Cl2, [Au(2,2'-bipyridine)(OH)2][PF6] and the organometallic compound [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine-H)(OH)][PF6]- with BSA were investigated by the joint use of various spectroscopic methods and separation techniques. Weak metal-protein interactions were revealed for the [Au(ethylenediamine)2]3+ and [Au(1,4,8,11-tetraazacyclotetradecane)]3+ species, whereas progressive reduction of the gold(III) centre was observed in the cases of [Au(2,2'-bipyridine)(OH)2]+ and [Au(2,2',2'-terpyridine)Cl]2+. In contrast, tight metal-protein adducts are formed when BSA is reacted with either [Au(diethylentriamine)Cl]2+ and [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine-H)(OH)]+. Notably, binding of the latter complex to serum albumin results in the appearance of characteristic CD bands in the visible spectrum. It is suggested that adduct formation for both of these gold(III) complexes occurs through coordination at the level of surface histidines. Stability of these gold(III) complexes/serum albumin adducts was tested under physiologically relevant conditions and found to be appreciable. Metal binding to the protein is tight; complete detachment of the metal from the protein has been achieved only after the addition of excess potassium cyanide. The implications of the present results for the pharmacological activity of these novel cytotoxic agents are discussed.