The apparatus for intrinsic dissolution test recorded in United States Pharmacopeia (USP) integrating with fiber-optic drug dissolution test system (FODT) were used to real-time monitor intrinsic dissolution processes of alliin in four media which were water, solution of HCl with pH 1.2, buffer solution of acetate with pH 4.5, and buffer solution of phosphate with pH 6.8. The intrinsic dissolution rate (IDR) and the similarity factor (f2) of two intrinsic dissolution curves with two apparatuses were calculated. The IDR values of alliin with rotating disk system were 28.1.3, 33.55, 28.38 and 30.95 mg x cm(-2) x min(-1) in four media, respectively. And the IDR values of alliin with stationary disk system were 44.16, 47.07, 45.11 and 51.34 mg x cm(-2) x min(-1), respectively. The similarity factors were 56.42, 50.75, 40.30 and 40.64, respectively. The results showed that the intrinsic alliin dissolution rates were much greater than 1 mg x cm(-2) x min(-1). It inferred that alliin dissolution would not be the rate limiting step to absorption.
Cysteine ; analogs & derivatives ; chemistry ; Fiber Optic Technology ; Solubility

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.
Animals ; Cystatins/pharmacology ; Cysteine Endopeptidases/metabolism ; Cysteine Proteinase Inhibitors/chemistry/*metabolism/*pharmacology ; Helminth Proteins/*metabolism/*pharmacology ; Spirometra/*metabolism

OBJECTIVETo study effects of different chemical factors in gastrointestinal tract, i.e. pH, protein, amino acid, ionic strength, Na2S, on the dissolution of cinnabar.

METHODThe content of total mercury in various dissolution of cinnabar were analyzed by UV/VIS Spectrophotometer. The particle distributions in dissolution of cinnabar were measured by Laser Particle Size Analyzer. The constituents of dissoluble substance of cinnabar in presence of Na2S were determined using ESI-MS.

RESULTThe solubility of cinnabar could be increased significantly in the presence of Na2S/So, and strong acidic pH, respectively. While the influence of thiol amino acid on promoting dissolution remains relatively low. Cinnabar didnt dissolve mainly in the form of nanoparticle.

CONCLUSIONWe postulated that cinnabar could be dissolved in various forms of mercury complexes containing sulphur.

Cysteine ; chemistry ; Hydrogen-Ion Concentration ; Mercury Compounds ; analysis ; chemistry ; Particle Size ; Solubility ; Spectrometry, Mass, Electrospray Ionization ; Spectrophotometry, Ultraviolet ; Sulfites ; chemistry

To develop a method for the detection of surface-confined peptides containing cysteine residues or oligodeoxynucleotides (ODNs) whose 3' ends modified with thiol groups, and a thiol-specific fluorescent cross-linker, N-(9-acridinyl) maleimide (NAM) was used. The peptides studied herein include both the oxidized and reduced forms of glutathione, and a hexapeptide (FT). Peptides are first attached onto the activated 11-mercaptoundecanoic acid (MUA)-terminated alkanethiol self-assembled monolayers (SAMs) and then derivatized with NAM. The cysteine residues was determined by using electrochemical desorption and fluorescence detection. GSH concentration as low as 40 pmol x L(-1) can be measured. The fluorescence intensity in the case of FT is about 3 times as high as that for GSH, which is consistent with the molar ratio of cysteine residues in these two molecules. The analytical performance of gene analysis was also evaluated through the analyses of a complementary target and targets with varying numbers of mismatching bases. The method described here is simple, sensitive, reproducible, and does not require sophisticated analytical instrumentation and separation procedures.
Biosensing Techniques ; methods ; Cysteine ; analysis ; Electrochemistry ; methods ; Fluorescence ; Glutathione ; analysis ; chemistry ; Maleimides ; chemistry ; Oligodeoxyribonucleotides ; analysis ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVETo establish an assay system for determination of dopamine (DA) in the presence of ascorbic acid (AA) with L-cysteine modified glassy carbon electrode.

METHODSL-cysteine was modified onto glassy carbon electrode electrochemically, and with this modified electrode, dopamine was determined by linear sweep stripping voltammetry.

RESULTSL-cysteine polymer-modified electrode had strong catalytic effect towards the electrochemical oxidation of DA. The modified electrode showed good properties in determination of DA with coexisting AA. Under selected conditions, the linearity of DA was in the range of 2.0 x 10(-7) - 1.0 x 10(-4) mol/L with the detection limit of 2.0 x 10(-8) mol/L. The stability, reliability and recovery of this L-cysteine-modified electrode based on electrochemical method were also satisfactory.

CONCLUSIONL-cysteine-modified electrode can avoid the interference by AA for determination of DA.

Ascorbic Acid ; chemistry ; pharmacology ; Carbon ; chemistry ; Cysteine ; chemistry ; Dopamine ; analysis ; chemistry ; Electrochemistry ; instrumentation ; methods ; Electrodes ; Oxidation-Reduction ; drug effects ; Reproducibility of Results

Several studies have reported that the citrus red mites Panonychus citri were an important allergen of citrus-cultivating farmers in Jeju Island. The aim of the present study was to purify and assess properties of a cysteine protease from the mites acting as a potentially pathogenic factor to citrus-cultivating farmers. A cysteine protease was purified using column chromatography of Mono Q anion exchanger and Superdex 200 HR gel filtration. It was estimated to be 46 kDa by gel filtration column chromatography and consisted of 2 polypeptides, at least. Cysteine protease inhibitors, such as trans poxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) and iodoacetic acid (IAA) totally inhibited the enzyme activities, whereas serine or metalloprotease inhibitors did not affect the activities. In addition, the purified enzyme degraded human IgG, collagen, and fibronectin, but not egg albumin. From these results, the cysteine protease of the mites might be involved in the pathogenesis such as tissue destruction and penetration instead of nutrient digestion.
Animals ; Chromatography, Gel ; Chromatography, Ion Exchange ; Collagen/metabolism ; Cysteine Proteases/chemistry/*isolation & purification ; Cysteine Proteinase Inhibitors/metabolism ; Fibronectins/metabolism ; Humans ; Immunoglobulin G/metabolism ; Molecular Weight ; Protein Subunits/chemistry/isolation & purification ; Proteolysis ; Substrate Specificity ; Tetranychidae/*enzymology

OBJECTIVE: To perform the proteome analysis of conditioned medium prepared from mouse embryonic fibroblast feeder layers by 2-dimensional (2D) electrophoresis and mass spectrometry and to find out the possible differentiation-inhibitory factor in conditioned medium. METHODS: Feeder layers were prepared by 60Co gamma-irradiation on mouse embryonic fibroblast. Insulin-transferrin-sodium selenite supplemented medium was used to culture the feeder layers for 24 hours. The condioned medium prepared from mouse embryonic fibroblast feeder layers were made into powder by lyophilization, the redissolved solution was applied to Sephadex G-50 gel filtration chromatography, and then cold acetone was used to precipitate the proteins in the eluted solution. The protein samples were applied to 2D electrophoresis. The 2D images were analyzed by 2D image analysis software. Selected protein spots were digested by trypsin, analyzed by mass spectrometry, and then searched against the NCBInr batabase using Mascot MS/MS Ions Search. RESULTS: The protein samples extracted from mouse embryonic fibroblast feeder layers conditioned medium could be used for 2D electrophoresis. On 2D images, there were (221+/-67) spots. Most of the proteins were located in the region of MW 20 approximately 70 kD, pI 4 approximately 8. Using mass spectrometry, we preliminarily identified 13 spots: 3 keratins, 3 transferrins, 1 trypsin precursor, 2 unknown proteins (3 spots), 1 connexin 46, 1 beta-galactoside binding protein, and 1 secreted protein, acidic and rich in cysteine. CONCLUSION Conditioned medium prepared from mouse embryonic fibroblast feeder layers contain beta-galactoside binding protein and secreted protein, acidic and rich in cysteine.
Animals ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; Cysteine ; chemistry ; Electrophoresis, Gel, Two-Dimensional ; Embryo, Mammalian ; Fibroblasts ; cytology ; Galactosides ; chemistry ; Mass Spectrometry ; Mice ; Proteome

Proteolytic processing of viral polyproteins is indispensible for the lifecycle of coronaviruses. The main protease (M(pro)) of SARS-CoV is an attractive target for anti-SARS drug development as it is essential for the polyprotein processing. M(pro) is initially produced as part of viral polyproteins and it is matured by autocleavage. Here, we report that, with the addition of an N-terminal extension peptide, M(pro) can form a domain-swapped dimer. After complete removal of the extension peptide from the dimer, the mature M(pro) self-assembles into a novel super-active octamer (AO-M(pro)). The crystal structure of AO-M(pro) adopts a novel fold with four domain-swapped dimers packing into four active units with nearly identical conformation to that of the previously reported M(pro) active dimer, and 3D domain swapping serves as a mechanism to lock the active conformation due to entanglement of polypeptide chains. Compared with the previously well characterized form of M(pro), in equilibrium between inactive monomer and active dimer, the stable AO-M(pro) exhibits much higher proteolytic activity at low concentration. As all eight active sites are bound with inhibitors, the polyvalent nature of the interaction between AO-M(pro) and its polyprotein substrates with multiple cleavage sites, would make AO-M(pro) functionally much more superior than the M(pro) active dimer for polyprotein processing. Thus, during the initial period of SARS-CoV infection, this novel active form AOM(pro) should play a major role in cleaving polyproteins as the protein level is extremely low. The discovery of AOM(pro) provides new insights about the functional mechanism of M(pro) and its maturation process.
Coronavirus ; metabolism ; Cysteine Endopeptidases ; Endopeptidases ; metabolism ; Humans ; Peptides ; chemistry ; metabolism ; Polyproteins ; chemistry ; metabolism ; Protein Binding ; SARS Virus ; chemistry ; metabolism ; Viral Proteins

The recombinant plasmid carrying the gene encoding 3C protease of Enterovirus 71 (EV71) was constructed, the recombinant protein was then expressed and purified, the functional activity was also measured. Firstly, the 3C protease gene was inserted into pET28a vector, the constructed recombinant plasmid was transformed into E. coli BL21 (DE3) for expression under the induction of IPTG. The expressed protein was purified by affinity chromatography (Ni-NTA) and the N-terminus His-tag was cleaved by enterokinase from 3C protease. The activity of 3C protease was evaluated with fluorescent peptide substrates. It was verified by restriction analysis and sequencing that recombinant plasmid pET28a-3C was constructed correctly and functionally expressed in E. coli BL21 (DE3) resulting in the production of recombinant 3C protease with a size of 22kD. Both His-tag and non-His-tag (cleaved by enterokinase) 3C protease exhibited similar enzyme activity to 3B-3C fluorescent peptide with Km, Vmax and Kcat values of 22 microM, 434nM. Min(-1) and 0.0669 Min(-1), respectively. The optimial pH and temperature were 7.0 and 30-37 degrees C, respectively. The acquirement of recombinant purified 3C protease with high activity has paved the way of further studies on anti-viral inhibitors, structural protein assembly, vaccine development and detection methods of EV71.
Cloning, Molecular ; Cysteine Endopeptidases ; chemistry ; genetics ; metabolism ; Enterovirus A, Human ; enzymology ; genetics ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Kinetics ; Viral Proteins ; chemistry ; genetics ; metabolism

Redox signal transduction, especially the oxidative modification of proein thiols, correlates with many diseases and becomes an expanding research area. However, there was rare method for quick and specific detection of protein thiols and their oxidative modification in living cells. In this article, we review the current chemical strategies for the detection and quantification of protein thiols and related cysteine oxidation. We also look into the future of the development of fluorescent probes for protein thiols and their potential application in the research of reactive cysteine proteomes and early detection of redox-related diseases.
Animals ; Cysteine ; metabolism ; Fluorescent Dyes ; Humans ; Nitrosation ; Oxidation-Reduction ; Proteins ; chemistry ; metabolism ; Reactive Nitrogen Species ; metabolism ; Reactive Oxygen Species ; metabolism ; Sulfenic Acids ; analysis ; Sulfhydryl Compounds ; analysis ; chemistry ; metabolism