Rapid SERS Detection of Botulinum Neurotoxin Type A

Surface-enhanced Raman scattering (SERS) is a powerful technique for decoding of 2-5-component mixes of analytes. Low concentrations of analytes and complex biological media are usually non-decodable with SERS. Recognition molecules, such as antibodies and aptamers, provide an opportunity for a specific binding of ultra-low contents of analyte dissolved in complex biological media. Different approaches have been proposed to provide changes in SERS intensity of an external label upon binding of ultra-low contents of the analytes. In this paper, we propose a SERS-based sensor for the rapid and sensitive detection of botulinum toxin type A. The silver nanoisland SERS substrate was functionalized using an aptamer conjugated with a Raman label. The binding of the target affects the orientation of the label, providing changes in an analytical signal. This trick allowed detecting botulinum toxin type A in a one-stage manner without additional staining with a monotonous dose dependence and a limit of detection of 2.4 ng/mL. The proposed sensor architecture is consistent with the multiarray detection systems for multiplex analyses

Direct Interaction of Mitochondrial Cytochrome c Oxidase with Thyroid Hormones: Evidence for Two Binding Sites

Thyroid hormones regulate tissue metabolism to establish an energy balance in the cell, in particular, by affecting oxidative phosphorylation. Their long-term impact is mainly associated with changes in gene expression, while the short-term effects may differ in their mechanisms. Our work was devoted to studying the short-term effects of hormones T2, T3 and T4 on mitochondrial cytochrome c oxidase (CcO) mediated by direct contact with the enzyme. The data obtained indicate the existence of two separate sites of CcO interaction with thyroid hormones, differing in their location, affinity and specificity to hormone binding. First, we show that T3 and T4 but not T2 inhibit the oxidase activity of CcO in solution and on membrane preparations with Ki ≈ 100–200 μM. In solution, T3 and T4 compete in a 1:1 ratio with the detergent dodecyl-maltoside to bind to the enzyme. The peroxidase and catalase partial activities of CcO are not sensitive to hormones, but electron transfer from heme a to the oxidized binuclear center is affected. We believe that T3 and T4 could be ligands of the bile acid-binding site found in the 3D structure of CcO by Ferguson-Miller’s group, and hormone-induced inhibition is associated with dysfunction of the K-proton channel. A possible role of this interaction in the physiological regulation of the enzyme is discussed. Second, we find that T2, T3, and T4 inhibit superoxide generation by oxidized CcO in the presence of excess H2O2. Inhibition is characterized by Ki values of 0.3–5 μM and apparently affects the formation of O2●− at the protein surface. The second binding site for thyroid hormones presumably coincides with the point of tight T2 binding on the Va subunit described in the literature

Interaction of Terminal Oxidases with Amphipathic Molecules

The review focuses on recent advances regarding the effects of natural and artificial amphipathic compounds on terminal oxidases. Terminal oxidases are fascinating biomolecular devices which couple the oxidation of respiratory substrates with generation of a proton motive force used by the cell for ATP production and other needs. The role of endogenous lipids in the enzyme structure and function is highlighted. The main regularities of the interaction between the most popular detergents and terminal oxidases of various types are described. A hypothesis about the physiological regulation of mitochondrial-type enzymes by lipid-soluble ligands is considered

Interaction of Amphipathic Peptide from Influenza Virus M1 Protein with Mitochondrial Cytochrome Oxidase

The Bile Acid Binding Site (BABS) of cytochrome oxidase (CcO) binds numerous amphipathic ligands. To determine which of the BABS-lining residues are critical for interaction, we used the peptide P4 and its derivatives A1-A4. P4 is composed of two flexibly bound modified α-helices from the M1 protein of the influenza virus, each containing a cholesterol-recognizing CRAC motif. The effect of the peptides on the activity of CcO was studied in solution and in membranes. The secondary structure of the peptides was examined by molecular dynamics, circular dichroism spectroscopy, and testing the ability to form membrane pores. P4 was found to suppress the oxidase but not the peroxidase activity of solubilized CcO. The Ki(app) is linearly dependent on the dodecyl-maltoside (DM) concentration, indicating that DM and P4 compete in a 1:1 ratio. The true Ki is 3 μM. The deoxycholate-induced increase in Ki(app) points to a competition between P4 and deoxycholate. A1 and A4 inhibit solubilized CcO with Ki(app)~20 μM at 1 mM DM. A2 and A3 hardly inhibit CcO either in solution or in membranes. The mitochondrial membrane-bound CcO retains sensitivity to P4 and A4 but acquires resistance to A1. We associate the inhibitory effect of P4 with its binding to BABS and dysfunction of the proton channel K. Trp residue is critical for inhibition. The resistance of the membrane-bound enzyme to inhibition may be due to the disordered secondary structure of the inhibitory peptide

Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties

Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve this problem, a series of mutants were constructed with substitutions of Lys residues in the universal binding site (UBS) which interact electrostatically with negatively charged Asp and Glu residues at the binding sites of CytC partners, bc1 complex and CcO. The aim of this study was to select a mutant that had lost its function as an electron carrier in the ETC, retaining the structure and ability to quench radicals. It was shown that a mutant CytC with substitutions of five (8Mut) and four (5Mut) Lys residues in the UBS was almost inactive toward CcO. However, all mutant proteins kept their antioxidant activity sufficiently with respect to the superoxide radical. Mutations shifted the dipole moment of the CytC molecule due to seriously changed electrostatics on the surface of the protein. In addition, a decrease in the redox potential of the protein as revealed by the redox titrations of 8Mut was detected. Nevertheless, the CD spectrum and dynamic light scattering suggested no significant changes in the secondary structure or aggregation of the molecules of CytC 8Mut. Thus, a variant 8Mut with multiple mutations in the UBS which lost its ability to electron transfer and saved most of its physico-chemical properties can be effectively used as a detector of superoxide generation both in mitochondria and in other systems