cytochrome-c-t and catechol

Dynamic coating of the surface in capillary electrophoresis with chemiluminescence detection (CE-CL) using an off-column coaxial flow interface for the determination of four hemeproteins was developed. This method is based on the luminol-hydrogen peroxide reaction catalyzed by metalloproteins in alkaline medium. The experimental setup of the CE-CL system with the proposed off-column coaxial interface was evaluated by separation and detection of dopamine and catechol based on inhibition of the luminol-potassium ferricyanide reaction. Highly efficient separation of the two model compounds with symmetrical peak shape and satisfactory reproducibility was achieved by using this interface. In addition, in order to obtain a good resolution for hemeproteins, polyvinylpyrrolidone (PVP) combined with sodium dodecyl sulfate (SDS) were introduced as dynamic modifiers to reduce the unwanted adsorption of non-specific protein. Several parameters affecting the CE separation and CL detection were investigated in detail. Under the optimized conditions, a mixture of the four hemeproteins (horseradish peroxidase (HRP), catalase (Cat), myoglobin (Mb) and cytochrome C (Cyt C)) could be well separated within 20 min. The linear ranges of the four proteins were 5.7 × 10(-8) to 1.1 × 10(-6) mol L(-1) for HRP, 4.0 × 10(-8) to 2.0 × 10(-6) mol L(-1) for Cat, 1.1 × 10(-10) to 5.6 × 10(-8) mol L(-1) for Mb, and 3.8 × 10(-7) to 7.7 × 10(-6) mol L(-1) for Cyt C. The limits of detection (LODs) (S/N = 3) for HRP, Cat, Mb and Cyt C were 2.2 × 10(-8) mol L(-1) (104.5 amol), 1.6 × 10(-8) mol L(-1) (74 amol), 5.6 × 10(-11) mol L(-1) (0.26 amol), and 1.95 × 10(-7) mol L(-1) (0.89 fmol), respectively. The proposed method has been successfully applied to the analysis of low-level Mb in a spiked human urine sample and the recoveries were above 97%. Our primary result demonstrated that the proposed CE-CL method has great potential for Mb determination in clinical diagnosis.

Topics: Catalase; Catechols; Cytochromes c; Dopamine; Electrophoresis, Capillary; Ferricyanides; Hemeproteins; Horseradish Peroxidase; Humans; Hydrogen Peroxide; Limit of Detection; Luminescent Measurements; Luminol; Metalloproteins; Myoglobin; Myoglobinuria; Sensitivity and Specificity

Herein, a series of biocompatible, robust, pH/sugar-sensitive, core-cross-linked, polyion complex (PIC) micelles based on phenylboronic acid-catechol interaction were developed for protein intracellular delivery. The rationally designed poly(ethylene glycol)-b-poly(glutamic acid-co-glutamicamidophenylboronic acid) (PEG-b-P(Glu-co-GluPBA)) and poly(ethylene glycol)-b-poly(l-lysine-co-ε-3,4-dihydroxyphenylcarboxyl-L-lysine) (PEG-b-P(Lys-co-LysCA)) copolymers were successfully synthesized and self-assembled under neutral aqueous condition to form uniform micelles. These micelles possessed a distinct core-cross-linked core-shell structure comprised of the PEG outer shell and the PGlu/PLys polyion complex core bearing boronate ester cross-linking bonds. The cross-linked micelles displayed superior physiological stabilities compared with their non-cross-linked counterparts while swelling and disassembling in the presence of excess fructose or at endosomal pH. Notably, either negatively or positively charged proteins can be encapsulated into the micelles efficiently under mild conditions. The in vitro release studies showed that the release of protein cargoes under physiological conditions was minimized, while a burst release occurred in response to excess fructose or endosomal pH. The cytotoxicity of micelles was determined by cck-8 assay in HepG2 cells. The cytochrome C loaded micelles could efficiently delivery proteins into HepG2 cells and exhibited enhanced apoptosis ability. Hence, this type of core-cross-linked PIC micelles has opened a new avenue to intracellular protein delivery.

Topics: Antineoplastic Agents; Apoptosis; Boronic Acids; Catechols; Cell Survival; Cross-Linking Reagents; Cytochromes c; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Fructose; Hep G2 Cells; Humans; Hydrogen-Ion Concentration; Ions; Micelles; Molecular Structure; Polymers; Structure-Activity Relationship

Recent work has highlighted the involvement of a dopamine derivative, 5-S-cysteinyl-dopamine (CysDA), in neurodegeneration and apoptotic cell death. In this paper we study in further detail the apoptotic process activated by this catechol-thioether derivative of dopamine in SH-SY5Y neuroblastoma cells. CysDA activates a cascade of events by an initial perturbation of Calcium homeostasis in the cell. Cell treatment with the catechol-thioether induces an immediate rise in intracellular Ca(2+) concentration, as demonstrated by a shift in the indo-1 dye emission spectrum, and a sustained high calcium concentration at long times of incubation. Fluorescence microscopy data show that the treatment of cells induces mitochondrial transmembrane potential depolarization, a clear evidence of the onset of apoptotic process. Programmed cell death activation is also demonstrated by cytochrome c release from the mitochondria, by an increased activity of both caspase-8 and -9 and by the poly(ADP-ribose)polymerase (PARP-1) cleavage, yielding the typical 86 kDa fragment due to caspase-3 activity. Overall, our data support the hypothesis that CysDA may induce apoptotic death in neuronal cells, via an initial perturbation of calcium homeostasis in the cytosol.

Topics: Apoptosis; Blotting, Western; Calcium; Catechols; Cell Line, Tumor; Cysteinyldopa; Cytochromes c; Humans; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Neuroblastoma; Neurons; Poly(ADP-ribose) Polymerases; Signal Transduction