Circular Dicroism Research Articles

Evaluation of the Binding Properties of A New Phenylurea Appended Carbazole Compound to Pepsin/Trypsin by Computational and Multi-Spectral Analysis.

A novel carbazole compound, named 1-(9-ethyl-9H-carbazol-3-yl)-3-phenylurea (Cpu) was synthesized and its binding properties with protease enzymes (pepsin and trypsin) has been examined by steady-state fluorescence measurements, UV/vis absorption, infrared (FT-IR) and circular dicroism (CD) spectroscopies and also computational methods. The fluorescence experimental results indicated that the quenching mechanism of enzyme by Cpu is static process. The thermodynamic parameters (both negative ΔH/ΔS) and molecular docking results suggested that the binding of Cpu to pepsin/trypsin were driven by hydrogen bonds and van der Waals forces. Based on Förster's theory, the binding distance (r) between pepsin/trypsin and Cpu was calculated to be 3.072/2.784nm, which implies that non-radiative energy transfer occurs from enzyme to Cpu. Furthermore, absorption, CD, and FT-IR spectral analysis provided an evidence that the presence of Cpu induced notable changes in the secondary structures and microenvironmental of both pepsin and trypsin, supporting its significant influence on these enzymes.

New Tricholidic Acid Triterpenoids from the Mushroom Tricholoma ustaloides Collected in an Italian Beech Wood.

The secondary metabolites produced by Tricholoma ustaloides Romagn., a mushroom species belonging to the large Tricholoma genus (Basidiomycota, Tricholomataceae), are unknown. Therefore, encouraged by the interesting results obtained in our previous chemical analyses of a few Tricholoma species collected in Italian woods, we aimed to investigate the secondary metabolites of Tricholoma ustaloides. The chemical analysis involved the isolation and characterization of secondary metabolites through an extensive chromatographic study. The structures of isolated metabolites, including the absolute configuration, were established based on a detailed analysis of MS, NMR spectroscopic, optical rotation, and circular dicroism data, and on comparison with those of related compounds reported in the literature. Two novel lanostane triterpenoids, named tricholidic acids B and C, together with triglycerides, a mixture of free fatty acids, five unidentified metabolites, and the known rare saponaceolides F and J, tricholidic acid, and tricholomenyn C, were isolated from an EtOAc extract of fruiting bodies of Tricholoma ustaloides that were collected in an Italian beech wood. This is the second example of isolation of tricholidic acid derivatives from a natural source. Saponaceolides F and J exhibited high cytotoxicity (IC50 values ≤ 10 μM) against a panel of five human cancer cell lines. The toxicity against myeloid leukemia (HL-60), lung cancer (A-549), hepatocellular cancer (HepG2), renal cancer (Caki-1), and breast cancer (MCF-7) cells was higher than that shown by the very well-known cytotoxic drug cisplatin.

A Study on Ball-Types Phthalocyanines Substituted for Carboxyl Groups: Spectroscopic, Photophysical and Photochemical Properties

The synthesis of ball-type binuclear metal-free (H2BPc) and metallic (magnesium (MgBPc), cobalt (CoBPc), and zinc (ZnBPc)) phthalocyanine derivatives containing four benzoic acid substituents at the peripheral positions is described. The new compounds were fully characterized using elemental analysis, mass spectrometry, fourier-transform infrared, 1H nuclear magnetic resonance and electronic spectroscopies. The Q band absorptions in DMSO were observed at 675/702 for H2BPc, at 668/698 for MgBPc, 672 nm for CoBPc and 681 nm for ZnBPc. Considering their potential application as photosensitisers in photodynamic therapy, the fluorescence quantum yield, triplet quantum yield, and singlet oxygen quantum yield of the diamagnetic derivatives was determined, and photodegredation quantum yields and magnetic circular dicroism measurements were performed for all complexes. The triplet lifetime value of the MgBPc was found to be larger than that of the metal-free and zinc counterparts, which were 60 µs, 130 µs and 60 µs, respectively. H2BPc, MgBPc and ZnBPc were obtained singlet oxygen quantum yield of 0.03, 0.06 and 0.40 while the tiplet quantum yield were 0.84, 0.91 and 0.93 in DMSO, respectively.

Dynein Adaptors Such as Drosophila Bicaudal (Dm BicD) Recognize Cargo and are Required to Activate Dynein for Processive Transport

In the absence of cargo, auto-inhibited BicD cannot recruit dynein, but the underlying mechanism of cargo-induced activation is elusive. Our single-molecule processivity assays show that auto-inhibition is abolished in the full-length Bicaudal mutant F684I, which activates dynein motility even in the absence of cargo. To investigate the structural basis for activation, we determined the X-ray structure of the C-terminal cargo-binding domain (CTD) of Dm BicD-CTD/F684I at 2.4 Å resolution. The structure revealed that the mutant has a homotypic coiled-coil registry, in which the two helices of the homodimer are aligned, whereas the wild-type structure has an asymmetric registry, in which the two chains are vertically shifted by ∼1 helical turn in a portion of the coiled-coil. In the mutant, N- terminal ∼20-residue region is disordered for one of the two chains. MD simulation and CD spectroscopy data suggested the structural flexibility of N-terminal region of Dm BicD-CTD/F684I and confirmed the intact protein is in the crystal and alpha-helical. We propose that a coiled-coil registry shift activates BicD for dynein recruitment. The registry shift could either be induced by the F684I mutation or by cargo-binding. Also, the human homolog of the BicD-CTD/F684I mutant (BicD2-CTD/F743I) showed diminished binding to its cargo Nup358. Thus, we propose that in mammalia, a coiled-coil registry shift modulates cargo selection for BicD2-dependent transport pathways, which are important for brain development and chromosome segregation. Furthermore, we investigated the mechanism for cargo recognition by BicD2. Our data from Circular Dicroism spectroscopy and small-angle X-ray scattering suggest that the BicD2 binding domain of Nup358, which is intrinsically disordered on its own, takes on a helical structure when binding to BicD2. This alpha-helix of Nup358 could be a structural feature for cargo recognition by BicD2.

Aptamer BC 007 - Efficient binder of spreading-crucial SARS-CoV-2 proteins

Corona virus disease 2019 (COVID-19) is a respiratory disease caused by a new coronavirus (SARS-CoV-2) which causes significant morbidity and mortality. The emergence of this novel and highly pathogenic SARS-CoV-2 and its rapid international spread poses a serious global public health emergency. To date 32,174,627 cases, of which 962,613 (2.99%) have died, have been reported (https://www.who.int/westernpacific/health-topics/coronavirus, accessed 23 Sep 2020). The outbreak was declared a Public Health Emergency of International Concern on 30 January 2020.There are still not many SARS-CoV-2-specific and effective treatments or vaccines available. A second round of infection is obviously unavoidable.Aptamers had already been at the centre of interest in the fight against viruses before now. The selection and development of a new aptamer is, however, a time-consuming process. We therefore checked whether a clinically developed aptamer, BC 007, which is currently in phase 2 of clinical testing for a different indication, would also be able to efficiently bind DNA-susceptible peptide structures from SARS-CoV-2-spreading crucial proteins, such as the receptor binding domain (RBD) of the spike protein and the RNA dependent RNA polymerase of SARS-CoV-2 (re-purposing). Indeed, several such sequence-sections have been identified. In particular for two of these sequences, BC 007 showed specific binding in a therapy-relevant concentration range, as shown in Nuclear magnetic resonance (NMR)- and Circular dicroism (CD)-spectroscopy and isothermal titration calorimetry (ITC). The excellent clinical toxicity and tolerability profile of this substance opens up an opportunity for rapid clinical testing of its COVID-19 effectiveness.

Binding of anticancer drug daunomycin to parallel G-quadruplex DNA [d-(TTGGGGT)]4 leads to thermal stabilization: A multispectroscopic investigation

Guanine rich DNA sequences form four stranded G-quadruplex structures found in telomeric DNA and oncogene promoters. Small molecules binding and stabilizing the G-quadruplex disrupt telomere maintenance and gene regulation, thereby limiting the proliferation of cancer cells. The anti-cancer drug daunomycin binds to both G-quadruplex DNA and duplex DNA. We have undertaken a study of interaction of daunomycin to [d-(TTGGGGT)]4 comprising telomeric DNA sequence from Tetrahymena thermophilia to understand the mechanisms of its action. Absorbance, fluorescence and circular dicroism spectra show significant change on interaction with no change in wavelength maxima. The daunomycin dimers present in free state in solution are disrupted on binding. Presence of all sequential short inter proton distance contacts in nuclear magnetic resonance spectra confirm external binding. The observed inter molecular nuclear Overhauser enhancements, changes in chemical shift and molecular docking studies establish well defined binding of daunomycin at two different sites of G-quadruplex DNA. Thermal stabilization of [d-(TTGGGGT)]4 by 10–15 °C upon daunomycin binding is expected to reduce access of telomerase to its functional site at telomeres. The present studies on mode of action pave the way for alternate derivatives/analogues by chemical modification of anthracyclines to arrive at a more potent telomerase inhibitor.

A synthetic BMP-2 mimicking peptide induces glioblastoma stem cell differentiation

BackgroundGlioblastoma (GBM) is the most aggressive type of primary brain tumor, characterized by the intrinsic resistance to chemotherapy due to the presence of a highly aggressive Cancer Stem Cell (CSC) sub-population. In this context, Bone Morphogenetic Proteins (BMPs) have been demonstrated to induce CSC differentiation and to sensitize GBM cells to treatments. MethodsThe BMP-2 mimicking peptide, named GBMP1a, was synthesized on solid-phase by Fmoc chemistry. Structural characterization and prediction of receptor binding were obtained by Circular Dicroism (CD) and NRM analyses. Activation of BMP signalling was evaluated by a luciferase reporter assay and western blot. Pro-differentiating effects of GBMP1a were verified by immunostaining and neurosphere assay in primary glioblastoma cultures. ResultsCD and NMR showed that GBMP1a correctly folds into expected tridimensional structures and predicted its binding to BMPR-IA to the same epitope as in the native complex. Reporter analysis disclosed that GBMP1a is able to activate BMP signalling in GBM cells. Moreover, BMP-signalling activation was specifically dependent on smad1/5/8 phosphorylation. Finally, we confirmed that GBMP1a treatment is sufficient to enhance osteogenic differentiation of Mesenchymal Stem Cells and to induce astroglial differentiation of glioma stem cells (GSCs) in vitro. ConclusionsGBMP1a was demonstrated to be a good inducer of GSC differentiation, thus being considered a potential anti-cancer tool to be further developed for GBM treatment. General significanceThese data highlight the role of BMP-mimicking peptides as potential anti-cancer agents against GBM and stimulate the further development of GBMP1a-based structures in order to enhance its stability and activity.

Copper(II) interaction with the Human Prion 103–112 fragment – Coordination and oxidation

The prion protein (PrP) is a membrane-anchored cell surface glycoprotein containing 231 amino acids. It has been associated with a group of neurodegenerative disorders. Copper(II) interaction with the Human Prion 103–112 fragment and its mutants has been studied with various techniques. The studied human prion fragment contains both histidine and methionine residues, while methionine residues are systematically replaced or displaced in the studied mutants. pH-potentiometric, UV–Vis and circular dicroism spectroscopic techniques were applied to study the stoichiometry, stability and structure of the copper(II) complexes, while HPLC-MS and MS/MS were used for identifying the products of copper(II) catalyzed oxidation. The complex formation reactions of the studied ligands are rather similar; only 1:1 complexes are formed, where the imidazole nitrogen of the histidine residue is the main binding site beside the amide nitrogens of the peptide chain. The only difference is, that in the peptides which contain methionine in position 109, in addition to the (Nim,N−,N−) coordination mode, a weak interaction of thioether sulfur atoms can be supposed. The mutant peptide which does not contain methionine did not undergo oxidation, only the fragmentation of the peptide chain was perceived. However, in the case of methionine containing peptides, the peptide chain was not cleaved; but the oxidation of methionine to methionine sulfoxide occurred.

Neolignan and megastigmane glucosides from the aerial parts of Isodon japonicus with cell protective effects on BaP-induced cytotoxicity

Six neolignan glucosides, named isodonosides I–VI, and a megastigmane glucoside named isodonmegastigmane I, were isolated together with 15 known compounds from the methanolic extract of aerial parts of Isodon japonicus cultivated in Tokushima, Japan. The chemical structures of the compounds were elucidated based on their MS and NMR spectroscopic analysis. The absolute configurations of the neolignan and megastigmane glucosides were determined by derivatizations, by ECD (electronic circular dicroism) Cotton effect approximation, and by the modified Mosher's method. In addition, a significant cell protective effects of neolignan glucosides on benzo[a]pyrene-induced cytotoxicity was found.

Stabilizing two IgG1 monoclonal antibodies by surfactants: Balance between aggregation prevention and structure perturbation

Surfactants are widely used as stabilizers in the biopharmaceutical formulations to minimize protein aggregation. Under a fixed stress condition, the protecting and destabilizing effects of surfactants are hypothesized to be highly dependent on the species and concentrations of surfactants and mAb. Therefore, we here studied the aggregation-prevention and structure-perturbation effects of eight commonly used surfactants (Tw20, Tw80, Brij35, Chaps, TrX-100, SDS, Pluronic F68 and F127) on two IgG1 solution formulations under agitation, using analytical methodologies including visual inspection, OD350 measurement, HPLC-SEC, circular dicroism, fluorescence spectroscopy and differential scanning calorimetry. We found that: (1) With concentrations range from 0.02 to 2mg/mL, nonionic surfactants were found to offer efficient aggregation-prevention effect, which is superior than the ionic surfactants; and higher surfactant concentration prevented mAb aggregation better especially under prolonged stability test under stress conditions. (2) The surfactant induced structure-perturbation emerged when even higher surfactant concentration (≥2mg/mL) was used, and such effect was surfactant-property dependent; and (3) the two IgG1 demonstrated different aggregation mechanisms and surfactant dependency, especially at high mAb concentrations. In conclusion, surfactants usage in mAb formulations, including the types and concentrations, should strike an optimal balance between the desirable aggregation-prevention and the detrimental structure-perturbation effects, while the consideration of mAb aggregation mechanism and concentration is also required for surfactant assessment.

Electrostatic Interactions between Tau Peptides and Model Biological Membranes: A Folding-Upon-Binding Mechanism

Found in neuronal cells of the central nervous system, tau proteins primarily function in cytoskeletal stability by associating with microtubules. During Alzheimer's disease and other tauopathies, misfolded tau proteins form neurofibrillary tangles (NFTs), thus impairing neuronal function and causing various neurodegenerative effects. The microtubule binding domain of tau consists of four repeat segments (R1-R4), and aggregation of these segments has previously been observed within NFTs. Previous research has also found misfolded tau components associated with the Golgi membrane, but not the plasma membrane. Here, we have used circular dicroism (CD) and isothermal titration calorimetry (ITC) to examine interactions between repeat segments R1-R4 and lipid bilayers of varying charge (DOPC and DOPC/DOPG 80:20). Our data indicate that significant structural changes are induced by charged membrane mimics and an entropy-driven binding process occurs during this interaction. Indeed, binding of tau occurs concomitantly with a classical hydrophobic effect. Thus, we propose a folding-upon-binding event occurring between negatively-charged membranes and the more promiscuous tau repeat segments. Results from calcein leakage assays and membrane fusion FRET measurements will also be presented to further probe the mechanism of bilayer disruption. These results will help explain a possible mechanism of tau misfolding during Alzheimer's disease, and are potentially beneficial to design strategies that prevent or reverse the aggregation process.

Spectroscopic investigations on partial intercalative binding behaviour of terpyridine based copper(II) complexes with DNA

Copper based metal complexes have been studied extensively towards DNA interaction aspects, the possible interactions being at the major or minor grooves, intercalation between base pairs, etc. The nature of the ligand decides the binding mode of the complexes thereby exerting different biological significance. Based on this, we have synthesized two mixed ligand copper(II) complexes, [Cu(meFtpy)(bpy)](NO3)2.2(H2O) (1) and [Cu(meFtpy)(phen)](NO3)2.(H2O) (2) based on new furfuryl substituted tertiary pyridine ligand (meFtpy) and ancillary ligands (phen, bpy). They are characterized by UV Visible, FT-IR, 1H &13C NMR and mass spectroscopic techniques. The structures of both the complexes are confirmed by single crystal XRD revealing triclinic crystal system showing penta coordination possessing distorted square pyramidal geometry. The binding ability of the complexes has been explored based on the results of DNA binding studies assessed by different spectroscopic techniques like UV absorption titration, fluorescence displacement assay and circular dicroism and found to show partial intercalative behaviour. The binding constant (Kb) values as obtained from UV absorption titration are found to be 1.29×104M−1 for 1 and 1.46×104M−1 for 2 and are compared with the values obtained for doxorubicin, a partial intercalator drug. The binding affinities obtained from absorption titration are found in the order as Kb(doxo)>Kb(2)>Kb(1)>Kb(meFtpy).

Biophysical characterization of the interaction between human serum albumin and n-dodecyl β-d-maltoside: A multi-technique approach

We have studied the effect of biocompatible sugar based surfactant n-dodecyl β-d-maltoside (DDM) on the conformation of human serum albumin (HSA). A multi-technique approach was applied in order to understand the type of interaction and effect of DDM on the secondary and tertiary structure of HSA. Surface tension measurement showed that HSA shifted the critical micelle concentration (cmc) of the surfactant to the higher side that clarifies the complex formation between DDM and HSA which was also confirmed by UV absorption spectroscopy. Fluorescence quenching measurements showed that fluorescence of HSA was quenched by the addition of DDM with a prominent blue shift indicative of the involvement of hydrophobic interaction which was further confirmed by extrinsic fluorescence of organic dye 1-anilino-8-naphthalenesulfonate. Synchronous fluorescence measurement trends suggested that the hydrophobicity increases near the tryptophan residue while an increase in the polarity was observed near tyrosine residues. A collective information obtained by circular dicroism (CD) and Fourier-transform infra-red (FTIR) spectroscopies along with dynamic light scattering revealed the partial unfolding of the protein.

(DIS)Assembly and Structural Stability of mtHsp60 and its Precursor NaÏve Form

Heat shock protein 60kDa is a molecular chaperone (GroEL human homolog) that assists protein folding in mitochondria (mtHsp60). It is synthesized in the cell cytoplasm as a higher molecular weight precursor form (p-mtHsp60) containing a N-terminal targeting sequence, that is cleaved after import into the mitochondrial matrix [1, 2].It has been established, and demonstrated by various techniques, Hsp60 can accumulate in the cytosol, in various pathological conditions (i.e., cancer and chronic inflammatory diseases). The cytosolical Hsp60 accumulation mechanism may occur with or without mitochondrial release concomitantly, so that in the cytosol the two types of 60 kDa chaperonin proteins, (mtHsp60 and its precursor naive form, p-mtHsp60) could coexist [3]. It has been recently observed that in a wide range of concentration, the precursor naive Hsp60 is able to assemble in both heptamers and tetradecamers [4].Key questions still unanswered pertain to the differences in structure-function features that might exist between the well-studied prokaryotic GroEL and the largely unexplored eukaryotic Hsp60 proteins. Moreover, studies on human Hsp60 structure and oligomeric state in vitro could help to validate its role in physiological or pathological cases. In order to pursue this goal, we investigated the (dis)assembly and thermal stability of mtHsp60, p-mtHsp60 and GroEL in vitro, by means of Differential Scanning Calorimetry (DSC) and Isothermal Titration Calorimetry (ITC). Complementar Circular Dicroism (CD) measurements were done to follow the change in the secondary structure due to unfolding.[1] Ellis RJ, 2007, Adv Exp Med Biol., 594: 1-13.[2] Jonathan D, 2000, JHC., 48(1): 45-56.[3] D. Chandra, 2007, J. Biol. Chem., 282: 31289-31301.[4] Vilasi S. et al, 2014, Plos One., 9(5): e97657.

Biochemical characterization of unusual meso-2,3-butanediol dehydrogenase from a strain of Bacillus subtilis

meso-2,3-Butanediol dehydrogenase (BDH) catalyzes the redox reaction between (R)-acetoin and meso-2,3-butanediol (meso-BD). The BDHs isolated from bacteria are generally characterized as homo-tetramers. In the present work, an isolated and identified high acetoin (AC)-yielding (41.63g/L) bacteria strain belonging to Bacillus subtilis but without 2,3-butanediol (BD) byproduct during its fermentation was selected. To understand the characteristics of BDH activity from this high AC-yielding strain, we cloned, purified and compared the BDH from Enterobacter aerogenes (CICC10293) (E. a-BDH) with the BDH from this high AC-yielding strain B. subtilis. Sequence alignments indicate a non-conservative amino acid substitution from Asp to Gly at site 194 on the B. s-BDH compared to that of E. a-BDH. Enzymatic analysis of E. a-BDH and D194G B. s-BDH shows D194G B. s-BDH has almost lost its entire enzymatic activity. Moreover, Isothermal titration calorimetry (ITC) measurements detected the substrate binding for the D194G B. s-BDH but no reaction was detected. Circular dicroism (CD) spectroscopy characterization revealed an identical secondary structure of E. a-BDH and D194G B. s-BDH. Remarkably, D194G B. s-BDH is highly susceptible to protease digestion, suggesting that the aspartic acid to glycine substitution might cause the proteolytic susceptibility of D194G B. s-BDH. Furthermore, by homology modeling with meso-2,3-butanediol dehydrogenase from Klebsiella pneumoniae (K. p-BDH) as a template, Gly194 seems to lose the hydrogen bond interactions with the surrounding residues (Gly206, Gly207 and Thr209), resulting in a putative conformational changes of D194G B. s-BDH which might be responsible for the loss of activity.

Cassane diterpenes from Caesalpinia platyloba

The dichloromethane extract from the leaves of Caesalpinia platyloba provided cassane diterpenes whose structures were determined as (-)-(5S,6R,8S,9S,10R,14R)-6-acetoxyvouacapane (1), (-)-(5S,6R,8S,9S,10R,12Z,14R)-6-acetoxycassa-12,15-diene (3), and (-)-(5S,6R,8S,9S,10R,13E)-6-acetoxycassa-13,15-diene (4). Compound 1 was chemically correlated with (-)-(5S,6R,8S,9S,10R,14R)-6-hydroxyvouacapane (2), (+)-(5S,8S,9S,10R,14R)-6-oxovouacapane (5), and (+)-(5S,6S,8S,9S,10R,14R)-6-acetoxyvouacapane (6), the last one previously isolated from Dipteryx lacunifera. The absolute configurations of all six diterpenes 1-6 were established by comparison of DFT calculated vibrational circular dicroism spectra of 1, 2 and 5 with those obtained experimentally. In addition, several reported chemical shifts for 2 and 5 were reassigned based on two-dimensional NMR measurements.

AB0086 Characterization of a human monoclonal antibody that neutralizes interleukin-20

BackgroundInterleukin-20 (IL-20) is a member of the IL-10 cytokine family and signals via the IL-20R1/IL-20R2 and IL-20R2/IL-22R1 heterodimer receptor complexes. Elevated amounts of IL-20 have been demonstrated in autoimmune disease...

A novel glucose biosensor based on phosphonic acid-functionalized silica nanoparticles for sensitive detection of glucose in real samples

An effective strategy for preparation amperometric biosensor by using the phosphonic acid-functionalized silica nanoparticles (PFSi NPs) as special modified materials is proposed. In such a strategy, glucose oxidase (GOD) was selected as model protein to fabricate glucose biosensor in the presence of phosphonic acid-functionalized silica nanoparticles (PFSi NPs). The PFSi NPs were first modified on the surface of glassy carbon (GC) electrode, then, GOD was adsorbed onto the PFSi NPs film by drop-coating. The PFSi NPs were characterized by transmission electron microscopy (TEM) and nuclear magnetic resonance (NMR) spectra. The interaction of PFSi NPs with GOD was investigated by the circular dicroism spectroscopy (CD). The results showed PFSi NPs could essentially maintain the native conformation of GOD. The direct electron transfer of GOD on (PFSi NPs)/GCE electrode exhibited excellent electrocatalytic activity for the oxidation of glucose. The proposed biosensor modified with PFSi NPs displayed a fast amperometric response (5s) to glucose, a good linear current–time relation over a wide range of glucose concentrations from 5.00×10−4 to 1.87×10−1M, and a low detection limit of 2.44×10−5M (S/N=3). Moreover, the biosensor can be used for assessment of the concentration of glucose in many real samples (relative error<3%). The GOD biosensor modified with PFSi NPs will have essential meaning and practical application in future that attributed to the simple method of fabrication and good performance.

Direct Electrochemistry and Electrocatalytic Behavior of Hemoglobin Entrapped in Ag@C Nanocables/Gold Nanoparticles Nanocomposites Film

Direct electrochemistry of hemoglobin (Hb) was successfully fabricated by immobilizing Hb on the nanocomposites containing of Ag@C nanocables and Au nanoparticles (AuNPs) modified glassy carbon electrode (GCE). The immobilized Hb retained its biological activity and shown high catalytic activities to the reduction of H2O2 by circular dicroism (CD) spectrum, fourier transform infrared (FT-IR) spectrum and cyclic voltammetry (CV). Experimental conditions such as scan rate and pH Value were studied and optimized. The results indicated that the resulting biosensor are linear to the concentrations of H2O2 in the ranges of 6.67 x 10(-7)-2.40 x 10(5) M, and the detection limit is 2.02 x 10(-7) M. The electrochemical biosensor has also high stability and good reproducibility.

Effect of Temperature and Ionic Strength on Structure and Chaperone Activity of Glycated and Non-Glycated Alpha-Crystallins

As major chaperone of eye lens, alpha-crystallin (α-Crs) is responsible for the transparency and refractive power of this region by preventing denaturation and precipitation of other proteins. As shown previously, cataract formation was positively associated with high salt intake and the elevation of blood sugar level. Here the effect of both temperature and ionic strength were studied on structure and chaperoning function of glycated and non-glycated α-Crs. While chaperone activity of these proteins was increased as function of temperature elevation, in the presence of sodium salt (0- 160 mM), it was significantly decreased. As shown by fluorescence and circular dicroism (CD) instruments, the salt induced structural alteration of α-Crs was accompanied with the exposure of hydrophobic surfaces and a transition from alpha- helical to beta-sheet structures. Moreover, the structural alterations induced by the salt were more pronounced in the case of glycated α-Crs compared to that of non-glycated protein counterpart. Overall this study shows the structural changes accompanied with lose of the chaperone activity of α-Crs induced by sodium chloride. Consequently, the obtained results may provide new evidences for the relationship between high salt intakes and cataract disease induced particularly by prolonged hyperglycemia.