Herb pair of Huangqi-Danggui exerts anti-tumor immunity to breast cancer by upregulating PIK3R1.

BACKGROUND: According to traditional Chinese medicine (TCM), drugs supplementing the vital energy, Qi, can eliminate tumors by restoring host immunity. The objective of this study is to investigate the underlying immune mechanisms of anti-tumor activity associated with Qi-supplementing herbs, specifically the paired use of Huangqi and Danggui. METHODS: Analysis of compatibility regularity was conducted to screen the combination of Qi-supplementing TCMs. Using the MTT assay and a transplanted tumor mice model, the anti-tumor effects of combination TCMs were investigated in vitro and in vivo. High content analysis and flow cytometry were then used to evaluate cellular immunity, followed by network pharmacology and molecular docking to dissect the significant active compounds and potential mechanisms. Finally, the anti-tumor activity and the mechanism of the active ingredients were verified by molecular experiments. RESULTS: There is an optimal combination of Huangqi and Danggui that, administered as an aqueous extract, can activate immunity to suppress tumor and is more effective than each drug on its own in vitro and in vivo. Based on network pharmacology analysis, PIK3R1 is the core target for the anti-tumor immunity activity of combined Huangqi and Danggui. Molecular docking analysis shows 6 components of the combined Danggui and Huangqi extract (quercetin, jaranol, isorhamnetin, kaempferol, calycosin, and suchilactone) that bind to PIK3R1. Jaranol is the most important component against breast cancer. The suchilactone/jaranol combination and, especially, the suchilactone/kaempferol combination are key for immunity enhancement and the anti-tumor effects of the extract. CONCLUSIONS: The combination of Huangqi and Danggui can activate immunity to suppress breast cancer and is more effective than the individual drugs alone.

Methionine redox regulation of actin-interacting proteins primarily governs antioxidative signaling and response to the salvianolic acid B treatment in EA.hy926 cells.

Actin-interacting proteins are important molecules for filament assembly and cytoskeletal signaling within vascular endothelium. Disruption in their interactions causes endothelial pathogenesis through redox imbalance. Actin filament redox regulation remains largely unexplored, in the context of pharmacological treatment. This work focused on the peptidyl methionine (M) redox regulation of actin-interacting proteins, aiming at elucidating its role on governing antioxidative signaling and response. Endothelial EA.hy926 cells were subjected to treatment with salvianolic acid B (Sal B) and tert-butyl-hydroperoxide (tBHP) stimulation. Mass spectrometry was employed to characterize redox status of proteins, including actin, myosin-9, kelch-like erythroid-derived cap-n-collar homology-associated protein 1 (Keap1), plastin-3, prelamin-A/C and vimentin. The protein redox landscape revealed distinct stoichiometric ratios or reaction site transitions mediated by M sulfoxide reductase and reactive oxygen species. In comparison with effects of tBHP stimulation, Sal B treatment prevented oxidation at actin M325, myosin-9 M1489/1565, Keap1 M120, plastin-3 M592, prelamin-A/C M187/371/540 and vimentin M344. For Keap1, reaction site was transitioned within its scaffolding region to the actin ring. These protein M oxidation regulations contributed to the Sal B cytoprotective effects on actin filament. Additionally, regarding the Keap1 homo-dimerization region, Sal B preventive roles against M120 oxidation acted as a primary signal driver to activate nuclear factor erythroid 2-related factor 2 (Nrf2). Transcriptional splicing of non-POU domain-containing octamer-binding protein was validated during the Sal B-mediated overexpression of NAD(P)H dehydrogenase [quinone] 1. This molecular redox regulation of actin-interacting proteins provided valuable insights into the phenolic structures of Sal B analogs, showing potential antioxidative effects on vascular endothelium.

Bidirectional crosstalk of the cAMP/ROS-dependent signaling pathways in inflammatory macrophage: An activation of formononetin.

Bacterial lipopolysaccharide (LPS) is a toxic stimulant to macrophage inflammation. Inflammation intersects cell metabolism and often directs host immunopathogenesis stress. We aim here at pharmacological discovering of formononetin (FMN) action, to which anti-inflammatory signaling spans across immune membrane receptors and second messenger metabolites. In ANA-1 macrophage stimulated by LPS, and simultaneous treatment with FMN, results show the Toll-like receptor 4 (TLR4) and estrogen receptor (ER) signals, in concert with reactive oxygen species (ROS) and cyclic adenosine monophosphate (cAMP), respectively. LPS stimulates inactivation of the ROS-dependent nuclear factor erythroid 2-related factor 2 (Nrf2) by upregulating TLR4, but it does not affect cAMP. However, FMN treatment not only activates Nrf2 signaling by TLR4 inhibition, but also it activates cAMP-dependent protein kinase activities by upregulating ER. The cAMP activity gives rise to phosphorylation (p-) of protein kinase A, liver kinase B1 and 5'-AMP activated protein kinase (AMPK). Moreover, bidirectional signal crosstalk is amplified between p-AMPK and ROS, as FMN combinational validation with AMPK activator/inhibitor/target small-interfering RNA or ROS scavenger. The signal crosstalk is well positioned serving as the 'plug-in' knot for rather long signaling axis, and the immune-to-metabolic circuit via ER/TLR4 signal transduction. Collectively, convergence of the FMN-activated signals drives significant reduction of cyclooxygenase-2, interleukin-6 and NLR family pyrin domain-containing protein 3, in LPS-stimulated cell. Although anti-inflammatory signaling is specifically related to the immune-type macrophage, the p-AMPK antagonizing effect arises from FMN combination with ROS scavenger H-bond donors. Information of our work assists in predictive traits against macrophage inflammatory challenges, using phytoestrogen discoveries.

PGC-1α/NRF1-dependent cardiac mitochondrial biogenesis: A druggable pathway of calycosin against triptolide cardiotoxicity.

Mitochondrion-related cardiotoxicity due to cardiotoxin stimuli is closely linked to abnormal activities of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), followed by co-inactivation of nuclear respiratory factor-1(NRF1). Pharmacological interventions targeting mitochondria may be effective for developing agents against cardiotoxicity. Herein, in triptolide-treated H9C2 cardiomyocytes, we observed defective mitochondrial biogenesis and respiration, characterized by depletion of mitochondrial mass and mitochondrial DNA copy number, downregulation of mitochondrial respiratory chain complexes subunits, and disorders of mitochondrial membrane potential and mitochondrial oxidative phosphorylation. Dysregulation of mitochondria led to cardiac pathological features, such as myocardial fiber fracture, intercellular space enlargement, and elevation of serum aspartate aminotransferase, creatine kinase isoenzyme, lactate dehydrogenase, and cardiac troponin I. However, following calycosin treatment, an active compound from Astragali Radix, the mitochondrion-related disorders at both cell and tissue levels were significantly ameliorated, which was facilitated by the activation of PGC-1α via deacetylation, followed by NRF1 co-activation. Calycosin-enhanced PGC-1α deacetylation is impelled by increasing sirtuin-1 expression and NAD+/NADH ratio. PGC-1α/NRF1 signaling in calycosin-mediated mitochondrial biogenesis protection was further confirmed by NRF1 knockdown and PGC-1α inhibition with SR18292. We conclude that calycosin ameliorated triptolide-induced cardiotoxicity by protecting PGC-1α/NRF1-dependent cardiac mitochondrial biogenesis and respiration, which is the druggable pathway for cardiotoxicity mitigation.

GSK3ß-dependent lysosome biogenesis: An effective pathway to mitigate renal fibrosis with LM49.

Renal fibrosis is an incurable disorder characterised by an imbalance of the extracellular matrix (ECM) favouring excess production over degradation. The identification of actionable pathways and agents that promote ECM degradation to restore ECM homeostasis may help mitigate renal fibrosis. In this study, we identified 5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanone (LM49), a compound we previously synthesised, as a small-molecule inducer of ECM degradation. LM49 administration efficiently reduced ECM deposition in renal tissue of diabetic nephropathy rats and in transforming growth factor ß-treated renal fibroblast cells. LM49 promoted the cytosol-to-nucleus translocation of transcription factor EB (TFEB) to increase lysosome biogenesis, leading to lysosome-based degradation of the ECM. TFEB-mediated lysosome biogenesis was induced by LM49 directly inhibiting the activity of glycogen synthase kinase 3ß (GSK3ß) rather than mammalian target of rapamycin complex 1. LM49 inhibited GSK3ß kinase activity concentration-dependently via competing with ATP. Direct binding between LM49 and GSK3ß was confirmed by the bio-layer interferometry assay, cellular thermal shift assay, and drug affinity responsive target stability. A molecular docking and molecular dynamic simulation revealed that LM49 occupied the ATP pocket of GSK3ß, which was consistent with the kinase activity assay. In summary, LM49 enhances TFEB-mediated lysosome biogenesis by directly inhibiting GSK3ß, leading to the degradation of the ECM by lysosomes. The enhancement of GSK3ß-dependent lysosome biogenesis to rebalance the ECM may be a novel strategy to counteract renal fibrosis, and LM49 may be a viable clinical candidate for treating this disorder.

Improving endothelial cell junction integrity by diphenylmethanone derivatives at oxidative stress: A dual-action directly targeting caveolar caveolin-1.

Directly targeting caveolar caveolin-1 is a potential mechanism to regulate endothelial permeability, especially during oxidative stress, but little evidence on the topic limits therapeutics discoveries. In this study, we investigated the pharmacological effect of an antioxidant LM49 (5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanoe) and its five diphenylmethanone derivatives on endothelial permeability and establish two distinct mechanisms of action. Multiplex molecular assays with theoretical modeling indicate that diphenylmethanone molecules, including LM49, directly bind the caveolin-1 steric pocket of ASN53/ARG54, ILE49/ASP50, ILE18, LEU59, ASN60, GLU48 and ARG19 residues. They also indicated dynamic binding-affinity for diphenylmethanone derivatives. First, this molecular interaction at caveolin-1 pocket inhibits its phosphorylation at TYR14 residue in H2O2-injured endothelial cell. A positive correlation was established between diphenylmethanone derivative binding-affinity and caveolin-1 phosphorylation inhibition. Inhibition of caveolin-1 phosphorylation, however, was independent of the LM49-mediated variation of protein tyrosine kinase activity, suggesting a direct blockage of adenosine triphosphate substrate diffusion into cavelion-1 structure. Second, LM49 increases the expression of cellular adhesive and tight junction proteins, VE-cadherin and occludin, in H2O2-injured cell, in a dose dependent manner. A leakage assay of fluorescein isothiocyanate-labeled dextran 40 across cell monolayer suggested improvement in endothelial barrier integrity with diphenylmethanone treatments. Our results demonstrate a direct targeting effect of caveolin-1 on endothelial permeability, and should guide the diphenylmethanone therapy against oxidative stress-induced junction dysfunction, especially at caveolar membrane invagination.

The protective effects of Mai-Luo-Ning injection against LPS-induced acute lung injury via the TLR4/NF-κB signalling pathway.

BACKGROUND: Acute lung injury (ALI) is a severe inflammatory disorder associated with high morbidity and mortality rates. Various therapeutic strategies for ALI have been proposed over the last few decades; however, the treatment options remain limited. Mai-Luo-Ning injection (MLN), a traditional Chinese medical formulation, has been extensively used for the treatment of respiratory diseases. Nevertheless, the effects of MLN on ALI remain unclear. PURPOSE: This study aimed to investigate the protective and therapeutic effects of MLN on lipopolysaccharide-induced ALI mouse models and RAW 264.7 cells, and further explore the underlying mechanism of these effects. METHODS: The therapeutic activity of MLN was evaluated using an in vivo ALI model and an in vitro model of RAW 264.7 macrophages. UHPLC-ESI-Q-TOF-MS/MS was used to investigate the chemical constituents of the MLN. The material basis and potential protective mechanism of MLN were analyzed using network pharmacology. The roles of MLN in inhibiting the Toll-like receptor 4 (TLR4)/ nuclear factor kappa B (NF-κB) signalling pathway were investigated via western blotting, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunofluorescence staining. RESULTS: In vivo experiments demonstrated that MLN ameliorated LPS-induced histological changes in lung tissues and reduced lung wet/dry weight ratio, total protein concentration in the bronchoalveolar lavage fluid and myeloperoxidase activity. Furthermore, MLN downregulated the in vivo and in vitro expression of pro-inflammatory cytokines such as tumour necrosis factor-alpha, interleukin-6, and interleukin-1ß. Network pharmacology analysis revealed that MLN could act synergistically through multiple targets and pathways and exert a protective effect, possibly through inhibiting TLR4/ NF-κB signalling pathways. Western blotting and immunofluorescence experiments further confirmed that MLN could regulate the expression of TLR4, MyD88, phospho-IκB-α, and phospho-NF-κB p65 in the TLR4/NF-κB signalling pathway and decrease the translocation of phospho-NF-κB p65 into the nucleus. CONCLUSION: This study suggests that MLN has a potential protective effect against LPS-induced ALI, which might be associated with the inhibition of the TLR4/NF-κB signalling pathway. Therefore, MLN is worthy of further investigation as a potential candidate for the treatment of ALI in the future.

The Chemical Constituents of Diaphragma Juglandis Fructus and Their Inhibitory Effect on α-Glucosidase Activity.

In our current investigation, 37 constituents (1-37), including 11 megastigmanes (1-11), 17 flavonoids (12-28) and 9 phenylpropanoids (29-37), were isolated from a 70%-EtOH extract of Diaphragma juglandis Fructus. Among them, compounds 1-3, 12 and 29 were new compounds and their structures were elucidated on the basis of physicochemical evidence and meticulous spectroscopic analysis (NMR, HRESIMS and CD). Compounds 13, 16, 21 and 28 showed moderate inhibitory effect on α-glycosidase inhibitory activities, with IC50 values being in the range of 29.47-54.82 µM and stronger than the positive control (acarbose, 60.01 ± 4.82 µM).

The Role of Chronic Inflammation in Various Diseases and Anti-inflammatory Therapies Containing Natural Products.

Chronic inflammation represents a long-term reaction of the body's immune system to noxious stimuli. Such a sustained inflammatory response sometimes results in lasting damage to healthy tissues and organs. In fact, chronic inflammation is implicated in the development and progression of various diseases, including cardiovascular diseases, respiratory diseases, metabolic diseases, neurodegenerative diseases, and even cancers. Targeting nonresolving inflammation thus provides new opportunities for treating relevant diseases. In this review, we will go over several chronic inflammation-associated diseases first with emphasis on the role of inflammation in their pathogenesis. Then, we will summarize a number of natural products that exhibit therapeutic effects against those diseases by acting on different markers in the inflammatory response. We envision that natural products will remain a rich resource for the discovery of new drugs treating diseases associated with chronic inflammation.

Dataset on preparation of the phosphorylated counterparts of a Momordica charantia protein for studying antifungal activities against susceptible dose-dependent C. albicans to antimycotics.

The data presented here are related to a research article entitled "Development of a phosphorylated Momordica charantia protein system for inhibiting susceptible dose-dependent C. albicans to available antimycotics: An allosteric regulation of protein" (Qiao et al., 2017) [1]. The data set includes three portions: (1) a relationship between reaction velocities of protein phosphorylation as a function of the substrate concentrations, determined in enzymatic reactions in aid of protein kinases; (2) a result of antifungal susceptibility testing of C. albicans after it is selected in antimycotics; and (3) a comparison of protein expression in the susceptible dose-dependent fungus relative to the wild C. albicans. In the first portion, the relationship of reaction velocities and substrate concentrations is expressed as an output from the inverse variation model. All data and analyses are made publicly available and citied in the research article using a style for the Data in Brief.

Development of a phosphorylated Momordica charantia protein system for inhibiting susceptible dose-dependent C. albicans to available antimycotics: An allosteric regulation of protein.

A regulatory Momordica charantia protein system was constructed allosterically by in vitro protein phosphorylation, in an attempt to evaluate antimycological pluripotency against dose-dependent susceptibilities in C. albicans. Fungal strain lineages susceptible to ketoconazole, econazole, miconazole, 5-flucytosine, nystatin and amphotericin B were prepared in laboratory, followed by identification via antifungal susceptibility testing. Protein phosphorylation was carried out in reactions with 5'-adenylic, guanidylic, cytidylic and uridylic acids and cyclic adenosine triphosphate, through catalysis of cyclin-dependent kinase 1, protein kinase A and protein kinase C respectively. Biochemical analysis of enzymatic reactions indicated the apparent Michaelis-Menten constants and maximal velocity values of 16.57-91.97mM and 55.56-208.33µM·min-1, together with an approximate 1:1 reactant stoichiometric ratio. Three major protein phosphorylation sites were theoretically predicted at Thr255, Thr102 and Thr24 by a KinasePhos tool. Additionally, circular dichroism spectroscopy demonstrated that upon phosphorylation, protein folding structures were decreased in random coil, ß6-sheet and α1-helix partial regions. McFarland equivalence standard testing yielded the concentration-dependent inhibition patterns, while fungus was grown in Sabouraud's dextrose agar. The minimal inhibitory concentrations of 0.16-0.51µM (at 50% response) were obtained for free protein and phosphorylated counterparts. With respect to the 3-cycling susceptibility testing regimen, individuals of total protein forms were administrated in-turn at 0.14µM/cycle. Relative inhibition ratios were retained to 66.13-81.04% of initial ones regarding the ketoconazole-susceptible C. albicans growth. An inhibitory protein system, with an advantage of decreasing antifungal susceptibilities to diverse antimycotics, was proposed because of regulatory pluripotency whereas little contribution to susceptibility in itself.

A review on architecture of the gag-pol ribosomal frameshifting RNA in human immunodeficiency virus: a variability survey of virus genotypes.

Programmed '-1' ribosomal frameshifting is necessary for expressing the pol gene overlapped from a gag of human immunodeficiency virus. A viral RNA structure that requires base pairing across the overlapping sequence region suggests a mechanism of regulating ribosome and helicase traffic during expression. To get precise roles of an element around the frameshift site, a review on architecture of the frameshifting RNA is performed in combination of reported information with augments of a representative set of 19 viral samples. In spite of a different length for the viral RNAs, a canonical comparison on the element sequence allocation is performed for viewing variability associations between virus genotypes. Additionally, recent and historical insights recognized in frameshifting regulation are looked back as for indel and single nucleotide polymorphism of RNA. As specially noted, structural changes at a frameshift site, the spacer sequence, and a three-helix junction element, as well as two Watson-Crick base pairs near a bulge and a C-G pair close a loop, are the most vital strategies for the virus frameshifting regulations. All of structural changes, which are dependent upon specific sequence variations, facilitate an elucidation about the RNA element conformation-dependent mechanism for frameshifting. These facts on disrupting base pair interactions also allow solving the problem of competition between ribosome and helicase on a same RNA template, common to single-stranded RNA viruses. In a broad perspective, each new insight of frameshifting regulation in the competition systems introduced by the RNA element construct changes will offer a compelling target for antiviral therapy.

Twenty-six circulating antigens and two novel diagnostic candidate molecules identified in the serum of canines with experimental acute toxoplasmosis.

BACKGROUND: The protozoan Toxoplasma gondii is a pathogen that causes severe opportunistic disease in a wide range of hosts. Efficient methods to diagnose acute T. gondii infection are essential for the administration of appropriate treatments and to reduce economic losses. In animals with acute infections, circulating antigens (CAgs) were detected as early as two days post-infection; these CAgs were reliable diagnostic indicators of acute infection. However, only a limited number of CAgs have been identified to date. The objective of this study was to identify a broader spectrum of CAgs and to explore novel diagnostic candidates in serum. METHODS: A canine model of acute toxoplasmiosis was established. For this purpose, six dogs were infected by intraperitoneal inoculation of tachyzoites. The CAgs spectrum in the serum was identified with the immunoprecipitation-shotgun approach. Two CAgs with low homology to other species, coronin protein (TgCOR) and ELMO protein (TgELMO), were heterologously expressed in Escherichia coli. Polyclonal antibodies against these two proteins were prepared, and the presence of these proteins in the serum was verified by Western blotting. The two CAgs were detected and evaluated by indirect ELISA methods. RESULTS: The CAgs levels peaked between two and five days after inoculation, and twenty-six CAgs were identified. Western blotting showed the presence of the two proteins in the serum during acute infection. Based on ELISA tests, the two CAgs were detected during acute infection. CONCLUSIONS: We identified twenty-six CAgs in the serum of canines with experimental acute toxoplasmosis and discovered two novel diagnostic candidates. We also provide new insights into the diagnosis of acute toxoplasmosis.

5-(4-Hy-droxy-phen-yl)imidazolidine-2,4-dione.

The title compound, C9H8N2O3, was prepared by reaction of phenol, glyoxylic acid and urea in water. The imidazolidine ring adopts an almost planar conformation (r.m.s. deviation = 0.012 Å) and is twisted by 89.3 (1)° relative to the benzene ring. In the crystal, mol-ecules are linked by N-H⋯O and O-H⋯O hydrogen bonds into a three-dimensional framework.

Crystal structure of bis-(1-benzyl-1H-1,2,4-triazole) perchloric acid monosolvate.

The title compound, 2C9H9N3·HClO4, was prepared by reaction of 1-benzyl-1H-1,2,4-triazole and HClO4 in ethanol at room temperature. The asymmetric unit consists of two mol-ecules of 1-benzyl-1H-1,2,4-triazole and one of HClO4 mol-ecule. The benzene and triazole rings make dihedral angles of 85.45 (8) and 84.76 (8)° in the two mol-ecules. The H-atom position of the perchloric acid mol-ecule is split over two O atoms (real peaks on difference map), with site-occupation factors of 0.5. These H atoms form two classical hydrogen bonds [2.546 (5) and 2.620 (4) Å] with the same N atoms in both mol-ecules. Five inter-molecular non-classical C-H⋯O inter-actions, with C⋯O distances in the range 3.147 (5)-3.483 (5) Å, are found in the crystal structure.

A novel chemiluminescent ELISA for detecting furaltadone metabolite, 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) in fish, egg, honey and shrimp samples.

In this study, an indirect competitive enzyme-linked immunosorbent assay with chemiluminescent (CLELISA) detection for 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) was developed. A monoclonal antibody (MAb) against AMOZ was prepared through immunizing BALB/c mice with 4-carboxybenzaldehyle derivatized AMOZ (CPAMOZ), conjugated with bovine serum albumin (BSA) as antigen. The effects of the substrates luminol, p-iodophenol and urea peroxide on the performance of the assay were studied and optimized. In addition, the specificity of the MAb, estimated as the cross-reactivity values with 4-nitrobenzaldehyde derivatized AMOZ (NPAMOZ), CPAMOZ and AMOZ, was 100%, 27.45% and 0.18%, respectively. The sensitivity of the developed CLELISA was estimated as 50% inhibitory concentration (IC50) value (0.14µg/l) with a linear working range between 0.03 and 64µg/l, and a limit of detection of 0.01µg/l. The CLELISA described in this study was 5-fold more sensitive than the indirect competitive ELISA previously developed in our laboratory. Finally, this new CLELISA was compared with a commercial kit to detect NPAMOZ in spiked fish, shrimp, honey and egg samples. The recovery values from four spiked fish, shrimp, honey and egg samples with different concentrations of NPAMOZ in CLELISA were 92.1-107.7%. Thus, the immunoassay method described here has a broad detection range and high sensitivity and is a valid and cost-effective means for high throughput monitoring of residual AMOZ levels in fish, shrimps, honey and eggs with potential applications in other animal tissues.

Development and validation of a rapid capillary zone electrophoresis method for determining charge variants of mAb.

This work aimed to develop a rapid capillary zone electrophoresis (CZE) method to provide abundant purity and identity information of monoclonal antibodies. The CZE running buffer system was optimized to be 20mM acetate-acetic acid (pH 6.0) together with the co-addition of 0.3% polyethylene oxide (PEO) and 2mM triethylenetetramine (TETA), which was further tested with advantages on the peak resolution improvements. The conditioning period was scheduled to 1 min for both 0.1M HCl and CZE running buffer to reduce total separation time. Additionally, the applied voltage and effective separation length were optimized at 30 kV and 20 cm separately. Compared with the method reported by Yan [1], this newly developed method showed a higher resolution in separating the two unknown basic peaks by testing monoclonal antibody sample (mAb1). The further validation results showed that for all five of charge isoform peaks of test mAb1, repeatability, intraday and interday precision had a RSD less than 0.58% for migration time and less than 3.18% for corrected area percent. The correlation coefficients of more than 0.98 for all peaks also demonstrated the good linearity for the method. In addition to the application of distinguishing intact antibody from C-terminal Lys variants, the method also has advantage in separating the Fab, Fc and intact antibody-relevant substances quickly, which facilitated the rough evaluation of papain induced digestion.

Bioconjugation of zirconium uridine monophosphate: application to myoglobin direct electrochemistry.

Porous nano-granule of zirconium uridine monophosphate, Zr(UMP)2.H2O is, for the first time, synthesized under mild experimental conditions and applied to the bioconjugation of myoglobin (Mb) to realize its direct electron transfer. UV-vis and resonance Raman spectroscopies prove that Mb in the Zr(UMP)2.H2O film maintains its secondary structure similar to the native state. The conjugation film of the Mb-Zr(UMP)2.H2O on the glassy carbon (GC) electrode gives a well-defined and quasi-reversible cyclic voltammogram, which reflects the direct electron transfer of the heme Fe III/Fe II couple of Mb. On the basis of the satisfying bioelectrocatalysis of the nano-conjugation of Mb and genetic substrate, a kind of mediator-free biosensor for H2O2 is developed. The linear range for H2O2 detection is estimated to be 3.92-180.14 microM. The apparent Michaelis-Menten constant (Km) and the detection limit based on the signal-to-noise ratio of 3 are found to be 196.1 microM and 1.52 microM, respectively. Both the apparent Michaelis-Menten constant and the detection limit herein are much lower than currently reported values from other Mb films. This kind of sensor possesses excellent stability, long-term life (more than 20 days) and good reproducibility.

Composite films of lecithin and heme proteins with electrochemical and electrocatalytic activities.

Functional composite films made from lecithin micelles and the two heme proteins of met-myoglobin (Mb) and met-hemoglobin (Hb) are reported in this paper. Proteins in functional composite films have much higher rates of electron transfer than proteins in solutions on carbon paste (CP) electrodes. Cyclic voltammograms (CVs) all give a pair of well-defined and quasi-reversible peaks, corresponding to the heme FeIII/FeII redox couple of proteins. Differential pulse voltammograms (DPVs) also show the same formal potential (E0') values of proteins under identical conditions. Electronic and vibrational spectra indicate that proteins in these films are not denatured, but their conformational differences from native states may exist. The E0' value for Mb in the lecithin film is found to be pH dependent. The Mb lecithin film can catalytically reduce O2 and H2O2, and its analytical application to H2O2 determination is established.