Hydrogen peroxide biosensor based on microperoxidase-11 entrapped in lipid membrane

A highly catalytic activity microperoxidase-11 (MP-11) biosensor for H2O2 was developed to immobilizing the heme peptide in didodecyldimethylammonium bromide (DDAB) lipid membrane. The enzyme electrode thus obtained responded to H2O2 without electron mediator or promoter, at a potential of +0.10 V versus Ag \ AgCl. A linear calibration curve is obtained over the range from 2.0 x 10(-5) to 2.4 x 10(-3) M. The biosensor responds to hydrogen peroxide in 15 s and has a detection limit of 8 x 10(-7) M (S/N = 3) Providing a natural environment with lipid membrane for protein immobilization and maintenance of protein functions is a suitable option for the design of biosensors.

Promoter effect of La3+ on the electrochemical reaction of microperoxidase-11

The effect of La3+ on the electrochemical behavior and structure of heme undecapeptide-microperoxidase-11 (MP-11)-in the aqueous solution was investigated using cyclic voltammetry, circular dichroism (CD) and UV-vis absorption spectrometry. It was found for the first time that La3+ would promote the electrochemical reaction of MP-11 at the glassy carbon (GC) electrode. This is mainly due to the fact that La3+ would induce more beta-turn and alpha-helical conformations from the random coil conformation of MP-11 and increase the non-planarity of the heme.

Surface-enhanced Raman spectroscopy of microperoxidase-11 on roughed silver electrodes

The conformation of microperoxidase-11 (MP-11) adsorbed on roughened silver electrodes was studied using surface-enhanced Fourier transform Raman spectroscopy. The results demonstrate that MP-11 was initially adsorbed via its polypeptide chain with a alpha-helix conformation, as indicated by the enhancement of the characteristic bands related to the amides I and III. The weak resonance effect of the porphyrin macrocycle in the near IR region contributes to the spectrum of the heme group. The presence of imidazole as the sixth ligand to the heme iron influences the conformation of the polypeptide chain of MP-11 on the electrode surface. Evaporation of solvent water results in an opened conformation of the adsorbed MP-11. which allows the heme group to contact the electrode surface directly.

Resonance Raman and surface-enhanced Raman spectroscopic study of microperoxidase-11

The electron transfer and structure of microperoxidase-11(MP-11) in solution and at electrode/solution interface were studied by electrochemical, resonance Raman and surface-enhanced Raman spectroscopic techniques. Results show that the central iron in heme group was six-coordinated in solution, whereas it was converted to five-coordinated state as MP-11 was adsorbed on the surface of a roughened silver electrode, due to the reorientation of MP-11 molecules. The electrochemical properties of MP-11 were directly affected by the coordination state of heme iron.

Electrochemistry and spectroscopy study on the interaction of microperoxidase-11 with lipid membrane

The interaction of microperoxidase-11 (MP11) with cationic lipid vesicles of didodecyldimethylammonium bromide (DDAB) induces an alpha -helical conformation from random coil conformations in solution and this change then makes heme macrocycle more distorted. DDAB-induced MP11 conformations were investigated by cyclic votammetry (CV), circular dichroism (CD) and UV-vis spectrometry. All results indicate that the binding of MP11 in solution to DDAB vesicles and the ordered structure formation are driven by mostly electrostatic interaction between negatively charged residues in the undecapeptide and positively charged lipid headgroups on the membrane surface. Upon binding to DDAB, its half-peak potential was also changed. The mechanism of the interaction between MP11 and DDAB was also discussed. (C) 2001 Elsevier Science B.V. All rights reserved.

The direct electrochemistry behavior of Cyt c on the modified glassy carbon electrode by SBA-15 with a high-redox potential

It is discovered that SBA-15 (santa barbara amorphous) can provide the favorable microenvironments and optimal direct electron-transfer tunnels (DETT) of immobilizing cytochrome c (Cyt c) by the preferred orientation on it. A high-redox potential (254 mV vs. Ag/AgCl) was obtained on glassy carbon (GC) electrode modified by immobilizing Cyt c on rod-like SBA-15. With ultraviolet-visible (UV-vis), circular dichroism (CD), FTIR and cyclic voltammetry, it was demonstrated that immobilization made Cyt c exhibits stable and ideal electrochemical characteristics while the biological activity of immobilized Cyt c is retained as usual.

Interaction between La3+ and MP-11 in the physiological solution

In this paper, the interaction between La3+ and microperoxidase-11 (MP-11) in the imitated physiological solution was investigated with the electrochemical method, circular dichroism (CD) and ultraviolet-visible (UV-vis) absorption spectroscopy. It was found that the interaction ways between La3+ and MP-11 are different with increasing the molar ratio of La3+ and MP-11. When the molar ratio of La3+ and MP-11 is less than 2, La3+ mainly interacts with the metacetonic acid group of the heme group in the MP-11 molecules, causing the increase in the non-planarity of the porphyrin cycle in the heme group and the decrease in the content of the random coil conformation of MP-11. These structural changes would increase the exposure extent of the electrochemical active center of MP-11 and thus, La3+ can promote the electrochemical reaction of MP-11 and its electrocatalytic activity for the reduction of H2O2 at the glassy carbon (GC) electrode. However, when the molar ratio of La3+ and MP-11 is larger than 3, except binding to the carbonyl oxygen of the metacetonic acid group in the heme group, La3+ interacts also with the oxygen-containing groups of the amides in the polypeptide chains of the MP-11 molecules, leading to the increase in the contents of the random coil conformation in the peptide of the MP-11 molecule, comparing with that for the molar ratio of less than 2.

基质辅助激光解吸电离飞行时间质谱表征细胞色素C

A matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF—MS) technique was used for analysis of moleculear weight of cytochrome C.The effects of three kinds of matrix,such as 2,5-dihydroxybenzoic acid(DHB),a-cyano-4-hydroxycinnamic acid(a-CHC) and sinapinic acid(SA),were used to compared and a suitable a-CHC was found.Experimental data showed that this method was properable to analysis of the congeneric biochemical samples.

pH-dependent conformational changes of ferricytochrome c induced by electrode surface microstructure

pH-dependent processes of bovine heart ferricytochrome c have been investigated by electronic absorption and circular dichroism (CD) spectra at functionalized single-wall carbon 'nanotubes (SWNTs) modified glass carbon electrode (SWNTs/ GCE) using a long optical path thin layer cell. These methods enabled the pH-dependent conformational changes arising from the heme structure change to be monitored. The spectra obtained at functionalized SWNTs/GCE reflect electrode surface microstructure-dependent changes for pH-induced protein conformation, pK(a) of alkaline transition and structural microenvironment of the ferricytochrome c heme. pH-dependent conformational distribution curves of ferricytochrome c obtained by analysis of in situ CD spectra using singular value decomposition least square (SVDLS) method show that the functionalized SWNTs can retain native conformational stability of ferricytochrome c during alkaline transition.

细胞色素c在纳米氧化铝模板修饰电极上的直接电化学

细胞色素c(Cytochrome c, Cyt c)是生物体中最常见的氧化-还原蛋白质, 研究其在电极上的直接电化学, 对于理解和认识生命体内的电子转移机制具有重要意义[1]. Cyt c与裸固体电极表面的直接接触通常会使其失去生物活性, 因此, Cyt c的电化学研究常借助于媒介体以实现其与电极之间的电子转移[2]. 纳米金属氧化物模板的表面积大且化学和光化学性质稳定, 被广泛应用于太阳能电池[3]和金属沉积[4]等领域. 本文研究氧化铝(AAO)模板对4,4′-二硫二吡啶存在下Cyt c直接电化学促进作用.

Membrane Deformability and Membrane Tension of Single Isolated Mitochondria

Mitochondria dynamics is crucial to many biological processes such as mitochondria fusion and fission, which is highly correlated to the mechanics of single mitochondria. However, the mechanobiological coupling of mitochondria has been poorly understood. Here membrane deformability and membrane tension of individual mitochondria isolated from MtDsRed labeled human embryonic T-Rex-293 kidney cells were measured using a micropipette aspiration assay. The results demonstrated that membrane deformation of isolated mitochondria exhibited an elastic transition phase followed by an equilibrium phase, and mitochondrial membrane tension was proportional to the area compressibility. It was also indicated that mitochondrial membrane deformability was significantly affected by physical chemical factors such as osmotic pressure or pH value, and was further correlated to mitochondrial functionality in different respiratory states and Ca2+ regulation. These findings provide a new insight into understanding the mechanical regulation of mitochondrial physiology.

Population genetic structure of the parasitic nematode Camallanus cotti inferred from DNA sequences of ITS1 rDNA and the mitochondrial COI gene

The population genetic structure of fish parasitic nematode, Camallanus cotti, collected from the Yangtze River, Pearl River and Minjiang River in China was investigated. From these parasites, the similar to 730 bp of the first internal transcribed spacer of ribosomal DNA (ITS1 rDNA) and the 428 bp of mitochondrial cytochrome c oxidase subunit I (COI) gene were sequenced. For the ITS1 rDNA data set, highly significant Fst values and low rates of migration were detected between the Pearl River group and both the Yangtze River (Fst = 0.70, P < 0.00001; Nm = 0.21) and Minjiang River (Fst = 0.73, P < 0.00001; Nm = 0.18) groups, while low Fst value (Fst = 0.018, P > 0.05) and high rate of migration (Nm = 28.42) were found between the Minjiang and the Yangtze rivers. When different host/locality populations (subpopulations) within each river were considered, subpopulations between the Yangtze River and Minjiang River had low Fst values (<= 0.12) and high Nm values (>3.72), while Pearl River subpopulations were significantly different from the Yangtze River and Minjiang River subpopulations (Fst >= 0.59; Nm < 1). The COI gene data set revealed a similar genetic structure. Both phylogenetic analyses and a statistical parsimony network grouped the Pearl River haplotypes into one phylogroup, while the Yangtze River and Minjiang River haplotypes formed a second group. These results suggested that the Yangtze River and Minjiang River subpopulations constituted a single reproductive pool that was distinct from the Pearl River subpopulations. In addition, the present study did not find host-related genetic differentiation occurring in the same drainage. (C) 2009 Published by Elsevier B.V.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Phylogeny of the cuttlerishes (Mollusca : Cephalopoda) based on mitochondrial COI and 16S rRNA gene sequence data

To clarify cuttlefish phylogeny, mitochondrial cytochrome c oxidase subunit 1 (COI) gene and partial 16S rRNA gene are sequenced for 13 cephalopod species. Phylogenetic trees are constructed, with the neighbor-joining method. Coleoids are divided into two main lineages, Decabrachia and Octobrachia. The monophyly of the order Sepioidea, which includes the families Sepiidae, Sepiolidae and Idiosepiidae, is not supported. From the two families of Sepioidea examined, the Sepiolidae are polyphyletic and are excluded from the order. On the basis of 16S rRNA and amino acid of COI gene sequences data, the two genera (Sepiella and Sepia) from the Sepiidae can be distinguished, but do not have a visible boundary using COI gene sequences. The reason is explained. This suggests that the 16S rDNA of cephalopods is a precious tool to analyze taxonomic relationships at the genus level, and COI gene is fitter at a higher taxonomic level (i.e., family).

A preliminary phylogenetic analysis of Metapenaeopsis (Decapoda : Penaeidae) based on mitochondrial dna sequences of selected species from the Indo-West Pacific

Phylogenetic relationships within Metapenaeopsis remain largely unknown. The modern revision of the genus suggests that the shape of the petasma, followed by the presence of a stidulating organ, are the most important distinguishing taxonomic features. In the present study, phylogenetic relationships were studied among seven Metapenaeopsis species from the Indo-West Pacific based on partial sequences of mitochondrial 16S rRNA and cytochrome c oxidase I (COI) genes. Mean sequence divergence was 6.4% for 16S and 15.8% for COI. A strikingly large nucleotide distance (10.0% for 16S and 16.9% for COI) was recorded between M. commensalis, the only Indo-West Pacific species with a one-valved petasma, and the other species with a two-valved petasma. Phylogenetic analyses using neighbor-joining, maximum parsimony, and maximum likelihood generated mostly identical tree topologies in which M. commensalis is distantly related to the other species. Two clades were resolved for the remaining species, one with and the other without a stridulating organ, supporting the main groupings of the recent taxonomic revision. Results of the present study also indicate that the deep-water forms represent a relatively recent radiation in Metapenaeopsis.