RESUMO
The reactions of radicals with human serum albumin (HSA) under reductive stress conditions were studied using pulse radiolysis and photochemical methods. It was proved that irradiation of HSA solutions under reductive stress conditions results in the formation of stable protein aggregates. HSA aggregates induced by ionizing radiation are characterized by unique emission, different from the UV emission of non-irradiated solutions. The comparison of transient absorption spectra and the reactivity of hydrated electrons (eaq-) with amino acids or HSA suggests that electron attachment to disulfide bonds is responsible for the transient spectrum recorded in the case of albumin solutions. The reactions of eaq- and CO2â¢- with HSA lead to the formation of the same products. Recombination of sulfur-centered radicals plays a crucial role in the generation of HSA nanoparticles, which are stabilized by intermolecular disulfide bonds. The process of creating disulfide bridges under the influence of ionizing radiation is a promising method for the synthesis of biocompatible protein nanostructures for medical applications. Our Raman spectroscopy studies indicate strong modification of disulfide bonds and confirm the aggregation of albumins as well. Low-temperature measurements indicate the possibility of electron tunneling through the HSA protein structure to specific CyS-SCy bridges. The current study showed that the efficiency of HSA aggregation depends on two main factors: dose rate (number of pulses per unit time in the case of pulse radiolysis) and the temperature of the irradiated solution.
Assuntos
Agregados Proteicos , Albumina Sérica Humana , Humanos , Albumina Sérica Humana/química , Albumina Sérica Humana/metabolismo , Dissulfetos/química , Oxirredução , Análise Espectral Raman , Radicais Livres/química , Radiólise de ImpulsoRESUMO
The purpose of this study was to investigate whether Human Serum Albumin (HSA) can bind N-methylated analogs of hot spots of native insulin. Three N-methylated derivatives of the A13-A19 fragment of native insulin were used: L(N-Me)YQLENY (1), LYQ(N-Me)LENY (2), and L(N-Me)YQ(N-Me)LENY (3). The studied N-methylated insulin fragments possess inhibiting potential against hormone aggregation. A variety of research techniques, including spectroscopic methods and microscopy assays, were used to study the interaction of HSA with the N-methylated insulin fragments. Based on spectroscopic measurements with Congo Red and Thioflavin T, all the analyzed N-methylated peptides were able to interact with the HSA surface. The CD spectrum registered for HSA in the presence of L(N-Me)YQLENY showed the smallest content of α-helix conformation, indicating the most compact HSA structure. Based on the results of MST, the dissociation constants (Kd) for complexes of HSA and peptides 1-3 were 19.2 nM (complex 1), 15.6 nM (complex 2), and 8.07 nM (complex 3). Microscopy assays, dynamic light scattering measurements as well as computer simulation of protein-ligand interaction also confirmed the possibility of docking the N-methylated inhibitors within HSA.
Assuntos
Insulina , Albumina Sérica Humana , Humanos , Albumina Sérica Humana/química , Insulina/metabolismo , Sítios de Ligação , Ligação Proteica , Simulação por Computador , Simulação de Acoplamento Molecular , Termodinâmica , Espectrometria de Fluorescência , Dicroísmo CircularRESUMO
We have shown that many proteins and enzymes (ovalbumin, ß-lactoglobulin, lysozyme, insulin, histone, papain) undergo concentration-dependent reversible aggregation as a result of the interaction of the studied biomolecules. Moreover, irradiation of those protein or enzyme solutions under oxidative stress conditions results in the formation of stable soluble protein aggregates. We assume that protein dimers are mainly formed. A pulse radiolysis study has been made to investigate the early stages of protein oxidation by N3⢠or â¢OH radicals. Reactions of the N3⢠radical with the studied proteins lead to the generation of aggregates stabilized by covalent bonds between tyrosine residues. The high reactivity of the â¢OH with amino acids contained within proteins is responsible for the formation of various covalent bonds (including C-C or C-O-C) between adjacent protein molecules. In the analysis of the formation of protein aggregates, intramolecular electron transfer from the tyrosine moiety to Trp⢠radical should be taken into account. Steady-state spectroscopic measurements with a detection of emission and absorbance, together with measurements of the dynamic scattering of laser light, made it possible to characterize the obtained aggregates. The identification of protein nanostructures generated by ionizing radiation using spectroscopic methods is difficult due to the spontaneous formation of protein aggregates before irradiation. The commonly used fluorescence detection of dityrosyl cross-linking (DT) as a marker of protein modification under the influence of ionizing radiation requires modification in the case of the tested objects. A precise photochemical lifetime measurement of the excited states of radiation-generated aggregates is useful in characterizing their structure. Resonance light scattering (RLS) has proven to be an extremely sensitive and useful technique to detect protein aggregates.
RESUMO
Ionizing radiation (IR) can pass through the human body easily, potentially causing severe damage to all biocomponents, which is associated with increasing oxidative stress. IR is employed in radiotherapy; however, in order to increase safety, it is necessary to minimize side effects through the use of radioprotectors. Water-soluble derivatives of fullerene exhibit antiradical and antioxidant properties, and these compounds are regarded as potential candidates for radioprotectors. We examined the ability of fullerenol C60(OH)36 to protect human erythrocytes, including the protection of the erythrocytal antioxidant system against high-energy electrons. Human erythrocytes irradiated with high-energy [6 MeV] electrons were treated with C60(OH)36 (150 µg/mL), incubated and haemolyzed. The radioprotective properties of fullerenol were determined by examining the antioxidant enzymes activity in the hemolysate, the concentration of -SH groups, as well as by determining erythrocyte microviscosity. The irradiation of erythrocytes (650 and 1300 Gy) reduces the number of thiol groups; however, an attenuation of this harmful effect is observed (p < 0.05) in the presence of C60(OH)36. Although no significant effect of fullerenol was recorded on catalase activity, which was preserved in both control and test samples, a more active protection of other enzymes was evident. An irradiation-induced decrease in the activity of glutathione peroxidase and glutathione reductase became an increase in the activity of those two enzymes in samples irradiated in the presence of C60(OH)36 (p < 0.05 and p < 0.05, respectively). The fourth studied enzyme, glutathione transferase, decreased (p < 0.05) its activity in the irradiated hemolysate treated with C60(OH)36, thus, indicating a lower level of ROS in the system. However, the interaction of fullerenol with the active centre of the enzyme cannot be excluded. We also noticed that radiation caused a dose-dependent decrease in the erythrocyte microviscosity, and the presence of C60(OH)36 reduced this effect (p < 0.05). Overall, we point to the radioprotective effect of C60(OH)36 manifested as the protection of the antioxidant enzymes of human erythrocytes against IR-induced damage, which has not been the subject of intense research so far.
Assuntos
Fulerenos , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Catalase/farmacologia , Elétrons , Eritrócitos/metabolismo , Fulerenos/farmacologia , Glutationa Peroxidase/metabolismo , Glutationa Redutase , Glutationa Transferase , Humanos , Estresse Oxidativo , Espécies Reativas de Oxigênio/farmacologia , Compostos de Sulfidrila/farmacologia , Água/farmacologiaRESUMO
The use of spectroscopic techniques has shown that human serum albumin (HSA) undergoes reversible self-aggregation through protein−protein interactions. It ensures the subsequent overlapping of electron clouds along with the stiffening of the conformation of the interpenetrating network of amino acids of adjacent HSA molecules. The HSA oxidation process related to the transfer of one electron was investigated by pulse radiolysis and photochemical methods. It has been shown that the irradiation of HSA solutions under oxidative stress conditions results in the formation of stable protein aggregates. The HSA aggregates induced by ionizing radiation are characterized by specific fluorescence compared to the emission of non-irradiated solutions. We assume that HSA dimers are mainly responsible for the new emission. Dityrosine produced by the intermolecular recombination of protein tyrosine radicals as a result of radiolysis of an aqueous solution of the protein is the main cause of HSA aggregation by cross-linking. Analysis of the oxidation process of HSA confirmed that the reaction of mild oxidants (Br2â¢−, N3â¢, SO4â¢−) with albumin leads to the formation of covalent bonds between tyrosine residues. In the case of â¢OH radicals and partly, Cl2â¢−, species other than DT are formed. The light emission of this species is similar to the emission of self-associated HSA.
Assuntos
Corantes Fluorescentes , Albumina Sérica Humana , Humanos , Oxirredução , Radiação Ionizante , Albumina Sérica Humana/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
The rate of formation of dichloride anions (Cl2â¢-) in dilute aqueous solutions of HCl (2-100 mmol·kg-1) was measured by the technique of pulse radiolysis over the temperature range of 288-373 K. The obtained Arrhenius dependence shows a concentration averaged activation energy of 7.3 ± 1.8 kJ·mol-1, being half of that expected from the mechanism assuming the â¢OHCl- intermediate and supporting the ionic equilibrium-based mechanism, i.e., the formation of Cl2â¢- in the reaction of â¢OH with a hydronium-chloride (Cl-·H3O+) contact ion pair. Assuming diffusion-controlled encounter of the hydronium and chloride ions and including the effect of the ionic atmosphere, we showed that the reciprocal of τ, the lifetime of (Cl-·H3O+), follows an Arrhenius dependence with an activation energy of 23 ± 4 kJ·mol-1, independent of the acid concentration. This result indicates that the contact pair is stabilized by hydrogen bonding interaction of the solvent molecules. We also found that at a fixed temperature, τ is noticeably increased in less-concentrated solutions (mHCl < 0.01 m). Since this concentration effect is particularly pronounced at near ambient temperatures, the increasing pair lifetime may result from the solvent cage effect enhanced by the presence of large supramolecular structures (patches) formed by continuously connected four-bonded water molecules.
RESUMO
Radiation crosslinking was employed to obtain nanocarriers based on poly(acrylic acid)-PAA-for targeted delivery of radioactive isotopes. These nanocarriers are internally crosslinked hydrophilic macromolecules-nanogels-bearing carboxylic groups to facilitate functionalization. PAA nanogels were conjugated with an engineered bombesin-derivative-oligopeptide combined with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate chelating moiety, aimed to provide selective radioligand transport. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) toluene-4-sulfonate was used as the coupling agent. After tests on a model amine-p-toluidine-both commercial and home-synthesized DOTA-bombesin were successfully coupled to the nanogels and the obtained products were characterized. The radiolabeling efficiency of nanocarriers with 177Lu, was chromatographically tested. The results provide a proof of concept for the synthesis of radiation-synthesized nanogel-based radioisotope nanocarriers for theranostic applications.
RESUMO
The purpose of this study was to investigate whether Human Serum Albumin (HSA) can bind native human insulin and its A13-A19 and B12-B17 fragments, which are responsible for the aggregation of the whole hormone. To label the hormone and both hot spots, so that their binding positions within the HSA could be identified, 4-(1-pyrenyl)butyric acid was used as a fluorophore. Triazine coupling reagent was used to attach the 4-(1-pyrenyl)butyric acid to the N-terminus of the peptides. When attached to the peptides, the fluorophore showed extended fluorescence lifetimes in the excited state in the presence of HSA, compared to the samples in buffer solution. We also analyzed the interactions of unlabeled native insulin and its hot spots with HSA, using circular dichroism (CD), the microscale thermophoresis technique (MST), and three independent methods recommended for aggregating peptides. The CD spectra indicated increased amounts of the α-helical secondary structure in all analyzed samples after incubation. Moreover, for each of the two unlabeled hot spots, it was possible to determine the dissociation constant in the presence of HSA, as 14.4 µM (A13-A19) and 246 nM (B12-B17). Congo Red, Thioflavin T, and microscopy assays revealed significant differences between typical amyloids formed by the native hormone or its hot-spots and the secondary structures formed by the complexes of HSA with insulin and A13-A19 and B12-B17 fragments. All results show that the tested peptide-probe conjugates and their unlabeled analogues interact with HSA, which inhibits their aggregation.
Assuntos
Insulina/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos/química , Albumina Sérica Humana/metabolismo , Sítios de Ligação/genética , Dicroísmo Circular , Fluorescência , Humanos , Insulina/química , Insulina/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Peptídeos/genética , Agregação Patológica de Proteínas/tratamento farmacológico , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Ligação Proteica/genética , Estrutura Secundária de Proteína , Albumina Sérica Humana/química , Albumina Sérica Humana/genéticaRESUMO
A new mechanism for the dichloride radical anion (Cl2â¢-) formation in diluted acidic chloride solutions is proposed on the grounds of pulse radiolysis measurements of the optical absorption growth at 340 nm and the density functional theory and Hartree-Fock computations. We show that the rate of â¢OH conversion into Cl2â¢- is determined by the equilibrium concentration of the ionic pair H3O+·Cl-. According to the proposed mechanism, the diffusional encounter of â¢OH and H3O+·Cl- is followed by fast concerted charge/proton transfer ( k(25 °C) = 6.2 × 1012 s-1) to yield Clâ¢, which then reacts with Cl- to produce Cl2â¢-. The mechanism has been confirmed by the observed first-order growth of the Cl2â¢- absorption and a direct proportionality of the rate constant to the activities of H3O+ and Cl- ions. The salt effect on the rate of Cl2â¢- formation is due to the ionic strength effect on the equilibrium H3O+ + Cl- â H3O+·Cl-.
RESUMO
Fullerenols (polyhydroxylated fullerene C60) are nanomaterial with potentially broad applicability in biomedical sciences with high antioxidant ability, thus, we investigated the radioprotecting potential of fullerenol C60(OH)36 on human erythrocytes irradiated by high-energy electrons of 6â¯MeV. The results demonstrate that C60(OH)36 at concentration of 150⯵g/mL protects the erythrocytes against the radiation-induced hemolysis (comparing to non-protected cells, we observed 30% and 39% protection for 0.65 and 1.3â¯kGy irradiation doses, respectively). The protecting effect was confirmed by 32% decreased release of potassium cations comparing to the cells irradiated without C60(OH)36. Measurements of the amount of lactate dehydrogenase (LDH) released from the irradiated erythrocytes showed that the size of the pores formed by irradiation was not sufficient to release LDH across the erythrocyte membranes. We also report a significant decrease of the affinity of acetylcholinesterase (AChE) for the substrate in the presence of fullerenol, indicating the relatively strong adsorption of C60(OH)36 to components of plasma membrane. Changes in membrane fluidity detected by fluorescence spectroscopy and conformational changes in membrane proteins detected by spin labeling suggest the dose-dependent formation of disulfide groups as an effect of oxidation and this process was inhibited by C60(OH)36. We suppose that scavenging the ROS as well as adsorption of fullerenol to membrane proteins and steric protection of -SH groups against oxidation are responsible for the observed effects.
Assuntos
Membrana Eritrocítica/metabolismo , Fulerenos/metabolismo , Hemólise/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Radiação Ionizante , Acetilcolinesterase/metabolismo , Células Cultivadas , Elétrons , Membrana Eritrocítica/química , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Fulerenos/química , Fulerenos/farmacologia , Hemólise/efeitos da radiação , Humanos , L-Lactato Desidrogenase/metabolismo , Fluidez de Membrana/efeitos dos fármacos , Fluidez de Membrana/efeitos da radiação , Potássio/metabolismo , Substâncias Protetoras/síntese química , Substâncias Protetoras/metabolismoRESUMO
Physicochemical studies on drug interactions with human serum albumin (HSA) are relevant for elucidation, at the molecular level, of the processes occurring in vivo. In this work using optical spectroscopic methods (fluorescence, absorption, circular dichroism), we have investigated aqueous HSA solutions containing pharmaceutically important isoquinoline alkaloids, berberine and palmatine. The primary objective was to verify whether the two compounds are located in the subdomain IIA of the secondary HSA structure as reported in literature. We prove that the excited state of Trp214 residue is not quenched by the alkaloids; all observed changes in fluorescence spectra are due to inner filter effects. Furthermore, differential absorption spectra indicate that the ligands remain in a waterlike microenvironment. We infer that bound alkaloid molecules are located at the protein/water interface. Yet, such binding mode can induce some unfolding of the HSA molecule detectable in the far-UV circular dichroism (CD) spectra. We have also performed, for the first time, pulse radiolysis studies of hydrated electron scavenging in the HSA/alkaloid systems and have measured steady-state absorption spectra of irradiated samples. The results reveal that neither berberine nor palmatine is effectively protected by the protein against one-electron reduction, which is consistent with the aforementioned conclusion.
Assuntos
Alcaloides/metabolismo , Alcaloides de Berberina/metabolismo , Albumina Sérica/metabolismo , Sítios de Ligação , Cátions , Dicroísmo Circular , Humanos , Modelos Moleculares , Espectrometria de FluorescênciaRESUMO
Aqueous solutions of anionic surfactants Cl(C3F6O)nCF2COOX, consisting of n = 2 and 3 perfluoroisopropoxy units and the counterion X = Na+ or NH4+, were studied by the method of fluorescence quenching with the use of (1-pyrenylbutyl)trimethylammonium bromide as a luminophore, and 1,1'-dimethyl-4,4'bipyridinium dichloride (methyl viologen) as a quencher. From the kinetics of fluorescence decay (time-resolved experiments) micellar aggregation numbers, N, and rate constants of the intramicellar quenching were determined for a wide range of surfactant concentrations, on the basis of the model developed by Infelta and Tachiya. The results are discussed in terms of the shape of the aggregates and the degree of counterion binding. The most important conclusions include: (i) a significant increase of N with increasing surfactant concentration suggests that spherical micelles formed at critical micellar concentration (CMC) transform into ellipsoidal aggregates, (ii) the degree of counterion binding to micelles is higher for NH4+ than for Na+, leading to higher N values in the case of the ammonium salt (n = 2), and (iii) at concentrations close to CMC the longer chain surfactant (n = 3) forms loose aggregates suggesting significant permeation with water molecules. An additional finding of this study is that the micelle-bound luminophore and quencher can form a ground-state complex, and for this reason the N values cannot be evaluated properly from the steady-state fluorescence intensity data using the equation proposed by Turro and Yekta.
Assuntos
Ácidos Carboxílicos/química , Éteres/química , Corantes Fluorescentes/química , Fluorocarbonos/química , Cinética , Sensibilidade e Especificidade , Soluções/química , Espectrometria de Fluorescência/métodos , Tensoativos/química , Fatores de Tempo , Água/químicaRESUMO
There is increasing interest concerning the use of natural antioxidants as low toxic antileukemic compounds. Antoksyd S (C/E/XXI), is a novel herbal drug derived in Poland from the powdered roots of Scutellaria baicalensis, and the biological activities of its major components (baicalin and baicalein) were compared on the human leukemia cell line HL-60. On MTT assay, Antoksyd S (C/E/XXI) showed an obvious cytotoxic effect on HL-60 cells, which was compared with those caused by cisplatin and doxorubicin under the same experimental conditions. A comparative assay of the antioxidative and prooxidative capacity of Antoksyd S (C/E/XXI) was also undertaken using two different reactive species: superoxide (O2-) and a transition metal (Cu2+). Antoksyd S (C/E/XXI) has low toxicity, acting as a modifier of HL-60 cells proliferation and as an antioxidant, which could act prooxidatively in the presence of transition metal ions. Taken together, it seems reasonable to suggest that Antoksyd S (C/E/XXI) as compared to baicalin and baicalein, or to the cytostatics cisplatin and doxorubicin, might be an especially good candidate for the future development of new therapeutic techniques, alone or in "combination treatment regimens", to enhance leukemia cell killing.