Electrochemical Biosensor for the Detection of Glycated Albumin.

BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia. The process of AD can begin 20 years before any symptom of cognitive loss. Thus, the development of systems for early diagnosis and prevention is very important. The mechanism of AD is still under debate. Nevertheless, higher levels of glycated albumin in cerebrospinal fluid and plasma are observed in AD patients. Therefore, glycated albumin could be a biomarker of AD development. METHODS: Electrochemical biosensor for direct determination of glycated albumin was based on thiol derivative of pentetic acid (DTPA) complex with Cu(II) created on gold electrode surface. His-tagged domains of Receptors for Advanced Glycation End Products (RAGE) were applied as analytical active element for glycated albumin recognition. The binding of glycated albumin by His6- RAGE domains was monitored using Osteryoung square - wave voltammetry. RESULTS: Electrodes modified with His6 - RAGE VC1 natural domain generated decrease of Cu(II) redox currents in the presence of glycated albumin. Human albumin, Aß 1-40 and S100B protein caused negligible influence on biosensors responses towards glycated albumin. The detection limits were: 2.3 pM, 1.1 pM, 2.9 pM and 3.1 pM in the presence of: buffer, buffer + albumin, buffer + S100B, buffer + Aß1-40 , respectively. CONCLUSION: The presented electrochemical biosensor was successfully applied for the determination of glycated albumin. Considering analytical parameters such as good selectivity and sensitivity in pM range, biosensor could be recommended as an analytical tool for medical samples analysis.

An Interplay of S-Nitrosylation and Metal Ion Binding for Astrocytic S100B Protein.

Mammalian S100B protein plays multiple important roles in cellular brain processes. The protein is a clinically used marker for several pathologies including brain injury, neurodegeneration and cancer. High levels of S100B released by astrocytes in Down syndrome patients are responsible for reduced neurogenesis of neural progenitor cells and induction of cell death in neurons. Despite increasing understanding of S100B biology, there are still many questions concerning the detailed molecular mechanisms that determine specific activities of S100B. Elevated overexpression of S100B protein is often synchronized with increased nitric oxide-related activity. In this work we show S100B is a target of exogenous S-nitrosylation in rat brain protein lysate and identify endogenous S-nitrosylation of S100B in a cellular model of astrocytes. Biochemical studies are presented indicating S-nitrosylation tunes the conformation of S100B and modulates its Ca2+ and Zn2+ binding properties. Our in vitro results suggest that the possibility of endogenous S-nitrosylation should be taken into account in the further studies of in vivo S100B protein activity, especially under conditions of increased NO-related activity.

Voltammetric detection of S100B protein using His-tagged receptor domains for advanced glycation end products (RAGE) immobilized onto a gold electrode surface.

In this work we report on an electrochemical biosensor for the determination of the S100B protein. The His-tagged VC1 domains of Receptors for Advanced Glycation End (RAGE) products used as analytically active molecules were covalently immobilized on a monolayer of a thiol derivative of pentetic acid (DPTA) complex with Cu(II) deposited on a gold electrode surface. The recognition processes between the RAGE VC1 domain and the S100B protein results in changes in the redox activity of the DPTA-Cu(II) centres which were measured by Osteryoung square-wave voltammetry (OSWV). In order to verify whether the observed analytical signal originates from the recognition process between the His6-RAGE VC1 domains and the S100B protein, the electrode modified with the His6-RAGE C2 and His6-RAGE VC1 deleted domains which have no ability to bind S100B peptides were applied. The proposed biosensor was quite sensitive, with a detection limit of 0.52 pM recorded in the buffer solution. The presence of diluted human plasma and 10 nM Aß(1-40) have no influence on the biosensor performance.

Post-translational S-nitrosylation is an endogenous factor fine tuning the properties of human S100A1 protein.

BACKGROUND: S100A1 protein is a proposed target of molecule-guided therapy for heart failure. RESULTS: S-Nitrosylation of S100A1 is present in cells, increases Ca(2+) binding, and tunes the overall protein conformation. CONCLUSION: Thiol-aromatic molecular switch is responsible for NO-related modification of S100A1 properties. SIGNIFICANCE: Post-translational S-nitrosylation may provide functional diversity and specificity to S100A1 and other S100 protein family members. S100A1 is a member of the Ca(2+)-binding S100 protein family. It is expressed in brain and heart tissue, where it plays a crucial role as a modulator of Ca(2+) homeostasis, energy metabolism, neurotransmitter release, and contractile performance. Biological effects of S100A1 have been attributed to its direct interaction with a variety of target proteins. The (patho)physiological relevance of S100A1 makes it an important molecular target for future therapeutic intervention. S-Nitrosylation is a post-translational modification of proteins, which plays a role in cellular signal transduction under physiological and pathological conditions. In this study, we confirmed that S100A1 protein is endogenously modified by Cys(85) S-nitrosylation in PC12 cells, which are a well established model system for studying S100A1 function. We used isothermal calorimetry to show that S-nitrosylation facilitates the formation of Ca(2+)-loaded S100A1 at physiological ionic strength conditions. To establish the unique influence of the S-nitroso group, our study describes high resolution three-dimensional structures of human apo-S100A1 protein with the Cys(85) thiol group in reduced and S-nitrosylated states. Solution structures of the proteins are based on NMR data obtained at physiological ionic strength. Comparative analysis shows that S-nitrosylation fine tunes the overall architecture of S100A1 protein. Although the typical S100 protein intersubunit four-helix bundle is conserved upon S-nitrosylation, the conformation of S100A1 protein is reorganized at the sites most important for target recognition (i.e. the C-terminal helix and the linker connecting two EF-hand domains). In summary, this study discloses cysteine S-nitrosylation as a new factor responsible for increasing functional diversity of S100A1 and helps explain the role of S100A1 as a Ca(2+) signal transmitter sensitive to NO/redox equilibrium within cells.

Reaction of the XPA zinc finger with S-nitrosoglutathione.

S-Nitrosoglutathione (GSNO) is an intracellular redox signaling molecule, also implicated in nitrosative stress. GSNO actions include modifications of Cys thiols in proteins. In this study, we focused on a GSNO reaction with a Cys4 zinc finger (ZF) sequence of human protein XPA, crucial to the nucleotide excision repair pathway of DNA repair. By using a corresponding synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf) and combining the detection of noncovalent and covalent complexes by ESI-MS with zinc release monitored by the zinc-sensitive chromophore 4-(2-pyridylazo)resorcinol (PAR), we demonstrated that the reaction of XPAzf with GSNO yielded S-nitrosylated intermediates, intrapeptide disulfides, and mixed glutathione disulfides. The reaction started with the formation of a complex of GSNO with ZnXPAzf followed by thiol transnitrosylation reactions and the final formation of disulfides. The results obtained suggest that at low levels/transient exposures, GSNO may act as a reversible regulator of Cys4 ZF activity, whereas transnitrosylation by GSNO, occurring at prolonged exposures, may cause deleterious effects to the functions of Cys 4 ZF proteins. In the case of XPA, this may lead to DNA repair inhibition.

Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: the reaction of XPA zinc finger with H(2)O(2).

Oxidation plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI-MS) is a very useful technique to study products of ZF oxidation, but its application has been limited largely to qualitative analysis of reaction products. On the other hand, ESI-MS has been applied successfully on several occasions to determine binding constants in metalloproteins. We used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI-MS might be used quantitatively to study ZF reaction kinetics. For this purpose, we studied oxidation of the Zn(II) complex of XPAzf (ZnXPAzf) by H(2)O(2) using three techniques in parallel: high-performance liquid chromatography (HPLC) of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI-MS. Single and double intrapeptide disulfides were detected by ESI-MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI-MS may indeed be used in quantitative kinetic studies of ZF reactions. The comparison of experimental information demonstrated that the formation of the Cys5-Cys8 single disulfide was responsible for zinc release.

Redox modifications of the C-terminal cysteine residue cause structural changes in S100A1 and S100B proteins.

S100 is a family of small, acidic, calcium binding proteins involved in the control of a multitude of intra- and extracellular processes, including many pathologies. The application of the analytical methodology based on the combination of RP HPLC and ESI-MS allowed for the characterization of S-nitrosylation and S-glutathionylation in two representative S100 proteins: S100A1 and S100B. The GSNO related S-nitrosylation of the conserved C-terminal cysteine is strongly activated by the binding of Ca(II) to S100A1 and of Ca(II) and Zn(II) to S100B. This modification results in a global alteration of protein structure, as demonstrated by a variety of techniques. The presented results provide a mechanistic basis for further studies of the function of S100 proteins in the control of redox-based and metal-based signal transduction.