Intrinsic Protein Fluorescence Research Articles

Isothermal Chemical Denaturation: Data Analysis, Error Detection, and Correction by PARAFAC2.

Characterization of a protein's conformational stability is a key step in the development of biotherapeutics, where protein unfolding leads to adverse properties, such as aggregation and loss of efficacy. Isothermal chemical denaturation (ICD) can be applied to determine chemical stability, aiming to identify the optimal solvent conditions, in terms of pH, salt concentration, and added excipients. For seven monoclonal antibodies, this study investigates the observed intrinsic protein fluorescence emission spectra as a function of denaturant concentration. Protein formulations are screened in two experimental series. We show how the peak shapes of folded and unfolded proteins are preserved under added salt (0-140 mM NaCl) and added excipients concentrations, as typically found in biotherapeutic formulations and that only minor effects in tryptophan fluorescence peak tailing are observed over a large pH range (5.5-9.0). The data of seven mAbs, where GuHCl was a suitable denaturant, are modeled using PARAFAC2. PARAFAC2, a linear decomposition method, is well suited for the data and yields robust, valid, and automated models that allow for the detection of erroneous measurements. Analysis of the errors show correlation with the well-based experimental setup, and differences in observed errors between the two experimental series. We additionally show a correction method for these outliers based on PARAFAC2 model scores, such that full transition curves can be retrieved, increasing the accuracy of any subsequent analysis.

Amino Acid Substitution at Phe80 of Mammalian Phenylalanine Hydroxylase Destabilizes Both Resting‐State and Activated Conformations Increasing the Population of Intermediates.

Phenylalanine hydroxylase (PAH) is an allosteric enzyme responsible for maintaining phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria (PKU). PAH equilibrates among long‐lived conformations, including resting‐state (RS‐PAH) and activated (A‐PAH), whose equilibrium position depends upon allosteric Phe binding to the A‐PAH conformation. The RS‐PAH conformation contains a stabilizing cation‐pi sandwich between Phe80, Arg123, and Arg240 (PDB entry 5DEN), which cannot exist in the A‐PAH conformation. Intrinsic protein fluorescence, enzyme kinetic analysis, native PAGE, size exclusion chromatography, limited proteolysis, and behavior on ion exchange resin are reported for F80A, F80D, F80L, and F80R, many as a function of [Phe]. These data indicate that amino acid substitutions at Phe80 destabilizes both the RS‐PAH and A‐PAH conformations so that intermediate, on‐pathway conformations are longer lived. The addition of Phe allows stabilization of the A‐PAH conformation. Kinetic characterization of F80A and F80D reflects allosteric activation while F80L and F80R are constitutively active. The reaction rates of all Phe80 variants suggest relief of a rate determining conformational change present in the wild type protein. Limited proteolysis of WT rPAH in the absence of Phe reveals facile cleavage within a central C‐terminal 4‐helix bundle, reflecting dynamic dissociation of the PAH tetramer. Under these conditions, the Phe80 variants show proteolytic hypersensitity in a linker region that repositions in the RS‐PAH to A‐PAH conformational interchange; this is protected by addition of Phe. We conclude that manipulation of Phe80 dramatically affects the conformational space sampled by PAH, increasing the population of intermediates between RS‐PAH and A‐PAH.Support or Funding InformationNIH 5R01‐NS100081; NIH P30 CA006927

Removal of Chromium (VI) by Escherichia coli Cells Expressing Cytoplasmic or Surface-Displayed ChrB: a Comparative Study.

Various genetically engineered microorganisms have been developed for the removal of heavy metal contaminants. Metal biosorption by whole-cell biosorbents can be enhanced by overproduction of metal-binding proteins/peptides in the cytoplasm or on the cell surface. However, few studies have compared the biosorption capacity of whole cells expressing intracellular or surface-displayed metal-adsorbing proteins. In this study, several constructs were prepared for expressing intracellular and surface-displayed Ochrobactrum tritici 5bvl1 ChrB in Escherichia coli BL21(DE3) cells. E. coli cells expressing surface-displayed ChrB removed more Cr(VI) from aqueous solutions than cells with cytoplasmic ChrB under the same conditions. However, intracellular ChrB was less susceptible to variation in extracellular conditions (pH and ionic strength), and more effectively removed Cr(VI) from industrial wastewater than the surface-displayed ChrB at low pH (<3). An adsorptiondesorption experiment demonstrated that compared with intracellular accumulation, cell-surface adsorption is reversible, which allows easy desorption of the adsorbed metal ions and regeneration of the bioadsorbent. In addition, an intrinsic ChrB protein fluorescence assay suggested that pH and salinity may influence the Cr(VI) adsorption capacity of ChrB-expressing E. coli cells by modulating the ChrB protein conformation. Although the characteristics of ChrB may not be universal for all metal-binding proteins, our study provides new insights into different engineering strategies for whole-cell biosorbents for removing heavy metals from industrial effluents.

Complex approach for analysis of snake venom \u03b1-neurotoxins binding to HAP, the high-affinity peptide

Snake venom α-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor. The combinatorial high-affinity peptide (HAP), homologous to the C-loop of α1 and α7 nAChR subunits, binds biotinylated α-bungarotoxin (αBgt) with nanomolar affinity and might be a protection against snake-bites. Since there are no data on HAP interaction with other toxins, we checked its binding of α-cobratoxin (αCtx), similar to αBgt in action on nAChRs. Using radioiodinated αBgt, we confirmed a high affinity of HAP for αBgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with αCtx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAP-αBgt binding constant directly (29 nM). These methods also confirmed weak HAP interaction with αCtx (>10000 nM). We attempted to enhance it by modification of HAP structure relying on the known structures of α-neurotoxins with various targets and applying molecular dynamics. A series of HAP analogues have been synthesized, HAP[L9E] analogue being considerably more potent than HAP in αCtx binding (7000 nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions.

Multinuclear Ir-BODIPY complexes: Synthesis and binding studies

The use of fluorescent based metal complexes is a flourishing area of research particularly in the field of chemical biology. Herein, we synthesized and fully characterized a series of three new multinuclear iridium based-BODIPY metal complexes in high yield. The free ligands and their metal complexes are highly emissive due to the presence of the BODIPY chromophore. Owing to the growing popularity of metal complexes as bioactive drug candidates, we performed their binding studies with DNA and protein, in vitro. Their dose dependent tendency to interact with DNA and ability to quench the intrinsic fluorescence of BSA protein confirmed their high bioactivity. With significant hyperchromic and red shifts observed with dsDNA, and prominent decline in the spectra of ssDNA, complex Ir3L3 showed the highest electrostatic interaction with genomic DNA among all complexes. Likewise, Ir3L3 also showed maximum dose-dependent quenching of the BSA protein in both emission as well as absorbance spectra, indicating its ability to interact with protein.

Correction: Thioamide quenching of intrinsic protein fluorescence.

Correction for 'Thioamide quenching of intrinsic protein fluorescence' by Jacob M. Goldberg et al., Chem. Commun., 2012, 48, 1550-1552.

Determination of thermodynamic binding parameters and type of interaction between the А/Bangkok/1/1979 (Н3N2) influenza virus hemagglutinin and phosphatidylcholine liposome

The study of interaction between surface viral proteins and model phospholipids is important for learning more details about the mechanisms of viral penetration into cells during infection. In this context, liposomes represent suitable systems for modeling a cell membrane. The binding of hemagglutinin (HA) of influenza virus with phosphatidylcholine liposomes was studied by equilibrium adsorption. It was interesting elucidate changes occurring in the structure of a protein during its translocation from the surface into the interior part of the membrane. In this work, we have studied characteristics of the protein-lipid interaction during HA complex formation with phospholipids including adsorption of HA on a phospholipid bilayer. Using the Scatchard equation and the Gibbs-Helmholtz equation at pH 4.0 and pH 6.0 thermodynamic parameters were determined. The results concluded the hydrophobic type of interaction between viral protein and liposomes. The additional confirmation of hydrophobic protein-lipid interaction presence was determination of HA distribution constants in two-phase systems: dextran-polyethylene glycol (K1) and dextran-polyethylene glycol esterified with palmitic acid (K2). The presence of hydrophobic interaction between HA and the liposome membrane was also confirmed using the quenching method of intrinsic protein fluorescence by a neutral quencher with acrylamide. At pH 4.0, an increase in the Stern-Volmer quenching constant was observed for the HA+liposome from phosphatidylcholine system, which is caused by structural changes in HA upon incorporation into the liposome bilayer. The fluorescence quenching rate constants calculated using the Stern-Volmer equation indicate a static quenching mechanism in which the quencher interacts with fluophors of a stationary protein molecule. The obtained results are interesting for not only studying virus and cell fusion theoretically, but also have practical applications. Using values of the protein-bilayer binding constant and free energy constant, it is possible to select the optimal phospholipid composition of liposomes or virosomes to obtain a stronger complex with various viral proteins. With two-phase systems, it is possible to determine the presence of hydrophobic sites on the viral protein surface, which can be used for evaluation both protein-lipid and protein-protein interaction.

Molecular Interaction of Amino Acid-Based Gemini Surfactant with Human Serum Albumin: Tensiometric, Spectroscopic, and Molecular Docking Study.

Binding effect and interaction of N,N′-dialkyl cystine based gemini surfactant (GS); 2(C12Cys) with human serum albumin (HSA) were systematically investigated by the techniques such as surface tension measurement, UV−visible spectroscopy, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking studies. The surface tension measurement exhibited that HSA shifted the critical micelle concentration of the 2(C12Cys) GS to the higher side that confirms the complex formation among 2(C12Cys) GS and HSA which was also verified by UV–visible, fluorescence, and CD spectroscopy. Increase in the concentration of 2(C12Cys) GS increases the absorption of the HSA protein but has a reverse effect on the fluorescence intensity. The analysis of UV–visible study with the help of a static quenching method showed that the value acquired for the bimolecular quenching constant (kq) quenches the intrinsic fluorescence of the HSA protein. Synchronous fluorescence spectrometry declared that the induced-binding conformational changes in HSA and CD results explained the variations in the secondary arrangement of the protein in presence of 2(C12Cys) GS. The present study revealed that the interaction between 2(C12Cys) GS and HSA is important for the preparation and properties of medicines. Molecular docking study provides insight into the specific binding site of 2(C12Cys) GS into the sites of HSA.

Applications of Differential Scanning Fluorometry and Related Technologies in Characterization of Protein-Ligand Interactions.

Differential scanning fluorometry (DSF) is an efficient and high-throughput method to analyze protein stability, as well as detect ligand interactions through perturbations of the protein's melting temperature. The method monitors protein unfolding by observing the fluorescence changes of a sample, whether through an environmentally sensitive fluorophore or by intrinsic protein fluorescence, while a temperature gradient is applied. Here, we describe in detail how to develop and optimize DSF assays to identify protein-ligand interactions while exploring different buffer and additive conditions. Analysis of the data and further applications of the method are also discussed.

Cycloplatinated(II) Derivatives of Mercaptopurine Capable of Binding Interactions with HSA/DNA.

In this study, two new bis-cyclometalated Pt(II) complexes, [Pt(C^N)(S^N)] [S^N = deprotonated 6-mercaptopurine (6-MP) and C^N = deprotonated 2-phenylpyridine (ppy), 2a; C^N = deprotonated benzo[h]quinoline (bhq), 2b], are synthesized by the reaction of [PtR(SMe2)(C^N)] (R = Me or p-MeC6H4) with 1 equiv of 6-mercaptopurine (6-HMP) at room temperature. The complexes are fully characterized using 1H and 13C NMR spectroscopies, electrospray ionization mass spectrometry, and elemental analysis. Biomolecular interaction of complex 2a with human serum albumin (HSA) is studied by fluorescence, UV-vis, and circular dichroism (CD) spectroscopies. The binding constants (Kb) and number of binding sites (n) are evaluated using the Stern-Volmer equation. The intrinsic fluorescence of protein is quenched by a static quenching mechanism, with a binding constant of Kb ∼ 105 reflecting a high affinity of complex 2a for HSA. The thermodynamic parameters (ΔH°, ΔG°, and ΔS°) indicate that the interaction is a spontaneous process and hydrophobic forces play a main role in the reaction. The displacement experiments demonstrate that the reactive binding sites of HSA to complex 2a are mainly located within its hydrophobic cavity in subdomain IIA (site I). Synchronous fluorescence spectra reveal that complex 2a affected the microenvironment of tryptophan-214 residues in subdomain IIA of HSA. In the case of interaction of complex 2b and HSA, because of overlapping of the emission spectra of complex 2b with HSA, chemometric approaches are applied. The results indicate significant interaction between the tryptophan residue of HSA and complex 2b. Moreover, the binding of Pt(II) complexes 2a and 2b causes a reduction of the α-helix content of HSA, as obtained by far-UV CD spectroscopy. The average binding distance (r) between Pt(II) complexes and HSA is obtained by Förster's resonance energy-transfer theory. Also, a molecular docking simulation reveals that π-π-stacking and hydrophobic interactions between these complexes and HSA are significant. Furthermore, the interactions of platinum complexes, 2, with calf-thymus DNA (CT-DNA) are investigated. The UV-vis results and ethidium bromide competitive studies support an intercalative interaction of both Pt(II) complexes with DNA. The new complexes 2 are also screened for anticancer activities. The results show that complexes 2 exhibit significant anticancer activity against the K562 (chronic myelogenous leukemia) cell line.

Characterization of arginine preventive effect on heat-induced aggregation of insulin

Aggregation of proteins can affect their efficacy, and is especially important concerning therapeutic proteins such as insulin. Use of additives such as amino acids can counteract this deleterious process. Heat-induced aggregate formation of human insulin was kinetically studied with the use of various concentrations of the protein, at different temperatures, and in the presence of EDTA by UV–visible spectrophotometry. Effect of arginine, lysine, and histidine was then tested on the process at pH 4.8 and 45 °C. Kinetic parameters of the obtained growth curves (parameters t* and t0.5 characterizing the rate of the nucleation stage and the rate of the stage of aggregate growth respectively) were computed in all these conditions, and structure of aggregates was characterized by spectrofluorimetry, and transmission electron microscopy (TEM). Presence of high concentrations of the chelator EDTA increased aggregation. Among used additives, L-arginine (50 mM) most efficiently suppresses the heat-induced amorphous aggregation of insulin, affecting parameters t0.5 and t* presumably by preserving the protein’s structure, as observed by the protein intrinsic fluorescence and CD spectra, and smaller formed aggregates in TEM images and dynamic light scattering. Docking experiment and subsequent molecular dynamics simulation indicated possible sites of interaction for arginine with the B-chain of insulin.

Sulfolobus acidocaldarius Microvesicles Exhibit Unusually Tight Packing Properties as Revealed by Optical Spectroscopy.

In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sa-MVs). The most abundant proteins in Sa-MVs are the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10–11 mM of the surfactant n-tetradecyl-β-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from S. acidocaldarius (LUVPLFE), LUVs reconstituted from the tetraether lipids extracted from Sa-MVs (LUVMV) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUVMV, and LUVPLFE were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan’s GP and red-edge excitation shift (REES) values in Sa-MVs and LUVMV decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan’s REES values are high (9.3–18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUVMV and LUVPLFE) and low (0.4–5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan’s excited state dipole moment, and “solvent” reorientation around Laurdan’s chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan’s lifetime.

Effects of malondialdehyde‐induced protein oxidation on the structural characteristics of rice protein

SummaryMalondialdehyde (MDA) was selected to represent a secondary by‐product of lipid peroxidation during rice ageing. This study aimed to investigate the effects of MDA modification on the structural characteristics of rice protein. The results showed that as MDA concentration increased, rice protein carbonyl and disulphide groups increased, but sulphydryl content decreased. The blue shift of maximum fluorescence peak, the decrease of rice protein intrinsic fluorescence intensity and the reduction of surface hydrophobicity indicated the formation of protein aggregates caused by MDA oxidative modification. The results of molecular weight distribution and particle size distribution showed that MDA modification resulted in the formation of soluble protein aggregates, and the decrease of rice protein solubility indicated that insoluble protein aggregates were formed. Results of protein electrophoresis showed that MDA modification contributed to rice protein aggregation via non‐disulphide covalent bonds. The results showed that rice protein gradually aggregated with increasing MDA concentration.

Automated High-Throughput Capillary Circular Dichroism and Intrinsic Fluorescence Spectroscopy for Rapid Determination of Protein Structure.

Assessing the physical stability of proteins is one of the most important challenges in the development, manufacture, and formulation of biotherapeutics. Here, we describe a method for combining and automating circular dichroism and intrinsic protein fluorescence spectroscopy. By robotically injecting samples from a 96-well plate into an optically compliant capillary flow cell, complementary information about the secondary and tertiary structural state of a protein can be collected in an unattended manner from considerably reduced volumes of sample compared to conventional techniques. We demonstrate the accuracy and reproducibility of this method. Furthermore, we show how structural screening can be used to monitor unfolding of proteins in two case studies using (i) a chaotropic denaturant (urea) and (ii) low-pH buffers used for monoclonal antibody (mAb) purification during Protein A chromatography.

Spectroscopic and molecular modeling investigations on heat induced behaviour of soy proteins

Soy protein derivatives are widely used in food industry. Because structural properties of proteins vary with the environment conditions, thermal processing might be useful for modulating proteins functionality. The effect of thermal treatment at temperatures ranging from 25 to 100°C on soy proteins behaviour was tested using fluorescence spectroscopy and in silico techniques. Experimental tests were carried out at pH 4.5 and 7.0. Intrinsic fluorescence of proteins was monitored for probing the dynamics of Trp and Tyr microenvironments, such as to estimate the eventual conformation changes. Shifts of the maximum emission were recorded with pH change and temperature increase, and were associated to proteins structure alteration. Synchronous spectra indicated maximum and minimum fluorescence intensity of both chromophores at 70°C and 90°C, respectively, attributed to denaturation of the main soy proteins. Further molecular dynamics simulations, performed at single molecule level to complement the experimental results, indicated higher structural stability at 100oC of glycinin trimer compared to the β-conglycinin trimer.

Novel aminoquinoline-based solvatochromic fluorescence probe: Interaction with albumin, lysozyme and characterization of amyloid fibrils

A novel aminoquinoline derivative (AQ) was synthesized and applied as a solvatochromic fluorescent probe to study proteins and their alterations. AQ is not fluorescent in aqueous solution but has its fluorescence quantum yield significantly increased upon binding to albumin. The generation of an induced circular dichroism signal in AQ confirmed the complexation. The Job's plot method revealed an 1:1 stoichiometry for the host-guest complex. The binding constant was determined by AQ fluorescence increase (2.7 × 105 mol−1 L) and by protein intrinsic fluorescence quenching (5.1 × 105 mol−1 L). The displacement of AQ from albumin by warfarin and ibuprofen showed that Sudlow's drug site-I is the preferential binding site. By applying the Bilot-Kawski solvatochromic model to the spectral shifts of fifteen solvents, the microenvironment dielectric constant at albumin site-I was estimated (ε = 14.8). In agreement, the average fluorescence lifetime of AQ complexed with albumin (6.11 ns) was close to dichloromethane (6.53 ns) and acetone (6.34 ns), which have dielectric constants of 8.9 and 21.0, respectively. Albumin was thermically treated to formation of amyloid fibril aggregates. AQ was able to differentiate the altered and native protein. Sodium dodecyl sulfate-induced aggregation of lysozyme to amyloid fibril was also efficiently detected by the AQ fluorescence increase. AQ was as efficient as the chromogenic bromocresol purple in the quantitative analysis of albumin. In conclusion, AQ can be considered a new solvatochromic fluorescent probe with several potential applications.

Interaction of a Model Hydrophobic Drug Dimethylcurcumin with Albumin Nanoparticles.

The aim of present study was to investigate the binding interactions of a model hydrophobic molecule, dimethylcurcumin (DMC) with nanoparticle form of bovine serum albumin (BSA) using fluorescence spectroscopy techniques. For this, BSA nanoparticles (size = 62.0 ± 3.5nm, molecular weight = 11,243 ± 3445 kD) prepared by thermal denaturation method was mixed with DMC in solution and monitored for fluorescence emission of tryptophan (Trp) residue as well as DMC separately. The emission maximum of DMC in nanoparticles form exhibited more blue sift and quenched the excited state of tryptophan (Trp) by six fold higher than in the native form of BSA. By analyzing Trp fluorescence, the mean binding constant (K) estimated for the interaction of DMC with native and nanoparticles forms of BSA was 2.7 ± 0.4 × 104M-1 and 1.5 ± 0.5 × 105M-1 respectively. Together these results suggested that DMC experienced a more rigid environment in nanoparticles than in native form of BSA. Additionally the above determined K values were in agreement with those reported previously by absorption techniques. Further direct energy transfer was observed between Trp and DMC, using which the distance (r) calculated between them was 28.25 ± 0.27Ǻ in BSA native. Similar analysis involving BSA nanoparticle and DMC revealed a distance of 24.25 ± 1.05Ǻ between the hydrophobic core and the ligand. Finally interaction of DMC with BSA was validated through molecular docking studies, which indicated sub-domain IIA as the binding site of DMC. Thus it is concluded that intrinsic fluorescence of protein can be utilized to study the interaction of its different physical forms with any hydrophobic ligand.

BlsA Is a Low to Moderate Temperature Blue Light Photoreceptor in the Human Pathogen Acinetobacter baumannii.

Light is an environmental signal that produces extensive effects on the physiology of the human pathogen Acinetobacter baumannii. Many of the bacterial responses to light depend on BlsA, a bluelight using FAD (BLUF)-type photoreceptor, which also integrates temperature signals. In this work, we disclose novel mechanistic aspects of the function of BlsA. First, we show that light modulation of motility occurs only at temperatures lower than 24°C, a phenotype depending on BlsA. Second, blsA transcript levels were significantly reduced at temperatures higher than 25°C, in agreement with BlsA protein levels in the cell which were undetectable at 26°C and higher temperatures. Also, quantum yield of photo-activation of BlsA (lBlsA) between 14 and 37°C, showed that BlsA photoactivity is greatly compromised at 25°C and absent above 28°C. Fluorescence emission and anisotropy of the cofactor together with the intrinsic protein fluorescence studies suggest that the FAD binding site is more susceptible to structural changes caused by increments in temperature than other regions of the protein. Moreover, BlsA itself gains structural instability and strongly aggregates at temperatures above 30°C. Overall, BlsA is a low to moderate temperature photoreceptor, whose functioning is highly regulated in the cell, with control points at expression of the cognate gene as well as photoactivity.

Responses of catalase and superoxide dismutase to low-dose quantum dots on molecular and cellular levels

With the wider application of cadmium-containing quantum dots (Cd-QDs) in biomedical fields, it is easier for people to be exposed. Studies have suggested that Cd-QDs could release cadmium ion and induce oxidative effects due to the disruption of redox equilibrium. Antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD), play an important role in organisms to resist the negative impact of exogenous substances. Molecular mechanisms of antioxidant enzymes with Cd-QDs remain unclear, however. In this study, structural and functional changes of CAT and SOD have been investigated under low dose Cd-QDs exposure. Cell viability, malondialdehyde (MDA) level, CAT and SOD activities were influenced by Cd-QDs in hepatocytes of mice. To further investigate the responses of CAT and SOD to Cd-QDs, multiple spectroscopic, calorimetric and activity measurements were carried out. Similar interaction patterns were observed that result in interaction force, structural and functional changes: Cd-QDs combine with CAT and SOD through hydrophobic forces; Intrinsic fluorescence of proteins was statically quenched by Cd-QDs and new complexes were formed; Also, the skeleton and secondary structure (with α-helix decrease) of CAT and SOD was influenced. Taken together, we suggest that Cd-QDs chosen in this study induce oxidative stress effects to hepatocytes but have not caused serious oxidative stress damage at concentrations below 10 μg/mL. MPA-CdSe/ZnS QDs caused the lowest level of oxidative stress which is associated with the induction of antioxidant proteins. This paper presents responses of CAT and SOD to low-dose Cd-QDs, and provides a reference for evaluating health damages caused by Cd-QDs.

DEVELOPING A SENSOR SYSTEM FOR IDENTIFICATION OF POLYCYCLIC AROMATIC HYDROCARBONS AS ENVIRONMENTAL TOXICANTS IN AQUEOUS AND PROTEIN MEDIA

Aim: to investigate the possibility of identifying toxic polycyclic aromatic hydrocarbons (PAHs) in aqueous and protein media using a fluorescent sensor system based on serum albumins. Methods: The studies were conducted using fluorescent methods, in particular, by quenching the intrinsic fluorescence of proteins, recording the fluorescence of PAHs and using the pyrene luminescent probe. Results: It was experimentally found that when PAHs were introduced into serum albumin solutions, a general decrease in the fluorescence intensity of proteins was observed, indicating PAHs' binding to protein macromolecules and, probably, the formation of a non-fluorescent serum protein-PAH complex. The analysis of the fluorescence spectra of PAHs in serum albumins was carried out. The dependences of changes in the intensity of the fluorescence spectra maxima of PAHs in albumins with an increase in the concentration of PAHs were linear, which makes it possible to use these systems for analytical purposes to determine toxic PAHs in protein and aqueous media. Conclusions: The results of the luminescent study on the interaction of PAHs with protein molecules can be used for monitoring of environmental toxicants in various media, as well as for the development of the methods for early diagnosis of diseases associated with the effect of various agents on the proteins.