Self-Assembly of a Bile Acid Dimer in Aqueous Solutions: From Nanofibers to Nematic Hydrogels.

A mixture of a cholic acid dimer with a secondary amine group and formic acid at a molar ratio of 1/1 is regarded as an organic salt, and it self-assembles in aqueous solutions to form monodisperse nanofibers. The nanofibers are separated at low concentrations of the mixture but entangle with each other at high concentrations to form well-dispersed and randomly arranged 3D fibrous networks. Above the minimum gelation concentration of the dimer, the fibrous network is strong enough to gelate the aqueous solutions to form a hydrogel. Hydrogels obtained from the dimer salt at a lower concentration are isotropic and show extinction between crossed polarizers in the polarizing microscope, whereas they become anisotropic (i.e., nematic hydrogels) upon increasing the dimer salt concentration or under physical stirring. The parallel arrangement of nanofibers from randomly directed fibrous networks may be responsible for the formation of such nematic hydrogels.

Supramolecular hydrogelation with bile acid derivatives: structures, properties and applications.

Hydrogelation of small molecules in aqueous solutions results from a balance between solubilization and precipitation (or crystallization). The hydrophobic moieties of amphiphiles tend to aggregate and the hydrophilic units may stabilize the aggregates in aqueous solutions. Morphologies vary according to the chemical structure of the amphiphiles. The formation of nanofibers or worm-like micelles is a prerequisite for hydrogels. Molecular hydrogels often show better degradability and functional diversity than polymeric hydrogels and may be useful in biomedical applications. Bile acids have attracted increasing attention for designing various biomaterials, including molecular hydrogels. They are naturally occurring amphiphilic compounds that exist in our body and help with the dissolution and digestion of fat by the formation of micelles. This review highlights the recent progress in the field of molecular hydrogelators based on bile acids, including bile salts, anionic, cationic and neutral bile acid derivatives, two-component hydrogelation systems, and polymeric supramolecular hydrogels, along with their potential applications.

A rapid, quantitative liquid chromatography-mass spectrometry screening method for 71 active and 11 natural erectile dysfunction ingredients present in potentially adulterated or counterfeit products.

A rapid LC-MS/MS method has been developed to simultaneously separate 71 erectile dysfunction (ED) drugs and 11 natural ingredients that are sometimes found alongside ED drugs, present in suspected adulterated or counterfeit samples. The separation was achieved in 10min using 2.6µm fused-core C18 particles in a 100×2.1mm column coupled to an LTQ Orbitrap XL mass spectrometer operated in positive electrospray mode. Using a straightforward methanolic extraction procedure, recovery from real samples (tablets, capsules, oral liquids and herbal products) was 92-111% and the lower and upper limits of detection and quantification were in the sub ng/mL and the sub µg/mL ranges, respectively. The intra- and inter-assay precision were ≤3.2% and 10.4% respectively across three concentrations of standards (50, 250 and 1000ng/mL) measured for 4 representative drugs spiked into a tablet-based matrix. This behavior was consistently observed for all the other compounds. The mass accuracy was less than 3ppm. Moreover, an advantage of this method is that the full scan event in the acquisition method associated with the high resolution of the Orbitrap XL allows post-analysis identification, in an untargeted approach, of additional species in the complex matrices. Our LC-MS/MS method for ED drugs was successfully applied to 32 samples and the drug identifications were in 100% agreement with those obtained by the conventional methods HPLC-UV and GC-MS. Following the complete validation of the ED method, it has been introduced in the current counterfeit identification procedures at Health Canada.

Development of glutaraldehyde-crosslinked chymotrypsin and an in situ immobilized enzyme microreactor with peptide mapping by capillary electrophoresis.

Immobilized proteolytic enzymes present several advantages over their soluble form, not the least of which is suppression of autoproteolysis peaks even at high enzyme-to-substrate ratios. We have made immobilized chymotrypsin by directly crosslinking it with glutaraldehyde to produce polymeric particles. Digestion of two model substrates using the particles was followed by CE peptide mapping with detection by UV absorbance or LIF. Results showed that autoproteolysis was highly suppressed and that different storage conditions of the particles in the short term (24 h) did not affect digestion of denatured BSA. As well, the chymotrypsin particles were indifferent to the presence of fluorescein groups on a casein substrate. Glutaraldehyde crosslinking of chymotrypsin inside a fused silica capillary column to make an immobilized enzyme reactor (IMER) was achieved in a series of reagent addition and washing steps, entirely automated using a commercial CE instrument. Digestion of myoglobin in the IMER for 30 min at 37°C followed by peptide mapping by CE-MS of the collected digest allowed identification of 17 chymotryptic peptides of myoglobin, or 83% primary sequence coverage.

Chemical profiling of the deacetylase activity of acetyl xylan esterase A (AxeA) variants on chitooligosaccharides using hydrophilic interaction chromatography-mass spectrometry.

Chitosan oligosaccharides (oligomers of (GlcNAc)(x)(GlcN)(y)) are used in the pharmaceutical, cosmetic and food industries and are reported to have therapeutic benefits. However, it is unknown whether their biological activity depends on the degree of deacetylation or the sequence of residues within the oligomer. We report here the development of a random mutagenesis method for directed evolution of Streptomyces lividans acetyl xylan esterase (AxeA), which we previously showed is able to deacetylate chitinous substrate, in order to obtain chitooligosaccharides with well-defined structural properties. A colorimetric assay was used to pre-screen libraries for p-nitrophenol acetate hydrolysis activity and an HPLC-UV absorbance assay was optimized to subsequently screen for deacetylase activity toward hexa-N-acetyl-glucosamine substrate (GlcNAc)(6). Native AxeA and two variants displaying>50% deacetylation of the oligohexamer substrate after reaction at 50°C for 24h in diluted culture supernatant were then selected for detailed analysis of the enzymatic products. A HILIC (hydrophilic interaction chromatography)-mode LC method was developed for profiling the deacetylated chitooligosaccharide products and HILIC-MS/MS sequencing revealed that ca. 30 different deacetylation products ranging from (GlcNAc)(5)(GlcN)(1) to (GlcNAc)(1)(GlcN)(5) and isomers thereof were produced. The AxeA variants produced, on average, 26% more unique products than the native enzyme; however, none were able to fully deacetylate the substrate to make (GlcN)(6). The long term goal of this multidisciplinary approach is to improve the activity of chitosan oligosaccharides to an industrially applicable level.

Headspace stir bar sorptive extraction-gas chromatography/mass spectrometry characterization of the diluted vapor phase of cigarette smoke delivered to an in vitro cell exposure chamber.

Advanced smoke generation systems, such as the Borgwaldt RM20S(®) smoking machine used in combination with the BAT exposure chamber, allow for the generation, dilution and delivery of fresh cigarette smoke to cell or tissue cultures for in vitro cell culture analyses. Recently, our group confirmed that the Borgwaldt RM20S(®) is a reliable tool to generate and deliver repeatable and reproducible exposure concentrations of whole smoke to in vitro cultures. However, the relationship between dose and diluted smoke components found within the exposure chamber has not been characterized. The current study focused on the development of a headspace stir bar sorptive extraction (HSSE) method to chemically characterize some of the vapor phase components of cigarette smoke generated by the Borgwaldt RM20S(®) and collected within a cell culture exposure chamber. The method was based on passive sampling within the chamber by HSSE using a Twister™ stir bar. Following exposure, sorbed analytes were recovered using a thermal desorption unit and a cooled injection system coupled to gas chromatograph/mass spectrometry for identification and quantification. Using the HSSE method, sixteen compounds were identified. The desorption parameters were assessed using ten reference compounds and the following conditions led to the maximal response: desorption temperature of 200°C for 2 min with cryofocussing temperature of -75°C. During transfer of the stir bars to the thermal desorption system, significant losses of analytes were observed as a function of time; therefore, the exposure-to-desorption time interval was kept at the minimum of 10±0.5 min. Repeatability of the HSSE method was assessed by monitoring five reference compounds present in the vapor phase (10.1-12.9% RSD) and n-butyl acetate, the internal standard (18.5% RSD). The smoke dilution precision was found to be 17.2, 6.2 and 11.7% RSD for exposure concentrations of 1, 2 and 5% (v/v) cigarette vapor phase in air, respectively. A linear response of analyte abundance was observed as a function of dilution. Extrapolation to 100% (v/v) cigarette vapor phase, i.e., undiluted smoke, gave yields for the five compounds ranging from 6 to 450 ng for 10 min exposure.

Evaluation of precision and accuracy of the Borgwaldt RM20S(®) smoking machine designed for in vitro exposure.

The Borgwaldt RM20S(®) smoking machine enables the generation, dilution, and transfer of fresh cigarette smoke to cell exposure chambers, for in vitro analyses. We present a study confirming the precision (repeatability r, reproducibility R) and accuracy of smoke dose generated by the Borgwaldt RM20S(®) system and delivery to exposure chambers. Due to the aerosol nature of cigarette smoke, the repeatability of the dilution of the vapor phase in air was assessed by quantifying two reference standard gases: methane (CH(4), r between 29.0 and 37.0 and RSD between 2.2% and 4.5%) and carbon monoxide (CO, r between 166.8 and 235.8 and RSD between 0.7% and 3.7%). The accuracy of dilution (percent error) for CH(4) and CO was between 6.4% and 19.5% and between 5.8% and 6.4%, respectively, over a 10-1000-fold dilution range. To corroborate our findings, a small inter-laboratory study was carried out for CH(4) measurements. The combined dilution repeatability had an r between 21.3 and 46.4, R between 52.9 and 88.4, RSD between 6.3% and 17.3%, and error between 4.3% and 13.1%. Based on the particulate component of cigarette smoke (3R4F), the repeatability (RSD = 12%) of the undiluted smoke generated by the Borgwaldt RM20S(®) was assessed by quantifying solanesol using high-performance liquid chromatography with ultraviolet detection (HPLC/UV). Finally, the repeatability (r between 0.98 and 4.53 and RSD between 8.8% and 12%) of the dilution of generated smoke particulate phase was assessed by quantifying solanesol following various dilutions of cigarette smoke. The findings in this study suggest the Borgwaldt RM20S(®) smoking machine is a reliable tool to generate and deliver repeatable and reproducible doses of whole smoke to in vitro cultures.

Immobilization of trypsin onto 1,4-diisothiocyanatobenzene-activated porous glass for microreactor-based peptide mapping by capillary electrophoresis: effect of calcium ions on the immobilization procedure.

The immobilization conditions and kinetic behaviour of trypsin, covalently immobilized via the 1,4-diisothiocyanatobenzene (DITC) linker onto aminopropylated controlled pore glass (CPG) particles, have been evaluated to establish a rapid and efficient protocol for fabrication of an immobilized enzyme microreactor (IMER) for protein hydrolysis and subsequent peptide mapping. Addition of calcium ions to either the immobilization reaction solution or hydrolysis assay was studied for a synthetic substrate. Activity was slightly higher when immobilization was carried out in the presence of Ca(2+) whereas more enzyme could be immobilized in its absence. A protocol requiring less than 3 h was devised to obtain maximal enzymatic activity with the lowest ratio of soluble trypsin to DITC-CPG particles. The resulting immobilized enzyme was found to retain an acceptable percentage (ca. 35%) of its activity after immobilization. The particles were dry-packed into a capillary to make a microscale IMER. Repeatability, reusability and digestion efficiency of the microIMER were investigated for the substrate beta-casein using capillary electrophoretic-based peptide mapping. In initial tests, a single device showed reproducible peptide maps for 21 digestions lasting 2 h each, carried out over a period of 2 months. Complete digestion of beta-casein could be achieved in a few minutes (86 s residence time in the microIMER followed by a wash step).

Development of an enzymatic microreactor based on microencapsulated laccase with off-line capillary electrophoresis for measurement of oxidation reactions.

Microencapsulation is used here as a new technique to immobilize enzymes in a microreactor coupled off-line to capillary electrophoresis (CE), allowing the determination of enzymatic reaction products. The redox enzyme laccase was encapsulated using the method of interfacial cross-linking of poly(ethyleneimine) (PEI). The 50 microm diameter capsules were slurry packed from a suspension into a capillary-sized reactor made easily and quickly from a short length of 530 microm diameter fused-silica tubing. The volume of the bed of laccase microcapsules in the microreactor was in the order of 1.1 microL through which 50 microL of the substrate o-phenylenediamine (OPD) was flowed. The oxidation product 2,3-diaminophenazine (DAP) and the remaining OPD were quantified by CE in a pH 2.5 phosphate buffer. Peak migration time reproducibility was in the order of 0.4% RSD and peak area reproducibility was less than 1.7% RSD within the same day. Using the OPD peak area calibration curve, a conversion efficiency of 48% was achieved for a 2-min oxidation reaction in the microreactor.

Sequential fractionation with concurrent chemical and toxicological characterization of the combustion products of chlorogenic acid.

Chlorogenic acid is the most abundant polyphenol found in the tobacco plant. The biological effects of its combustion products remain largely unknown. In this study, chlorogenic acid was burned at 640 degrees C for 2 min and the particulate matter of the smoke was collected onto Cambridge filter pads followed by selective extraction in five different solvents. Various fractions of the chlorogenic acid combustion products were tested for induction of micronuclei in V79 Chinese hamster fibroblast cells. Over 40 compounds were identified in the dimethyl sulfoxide (DMSO) extract by high-performance liquid chromatography coupled to electrospray time-of-flight mass spectrometry (HPLC/TOF-MS). The DMSO extract was then fractionated into three major fractions by preparative LC. The fraction inducing the highest degree of toxicity was further separated into four sub-fractions. The sub-fraction responsible for the most toxic response was determined to contain catechol as its major component. The overall reproducibility of the combustion, the extraction procedure and the chemical characterization of the compounds responsible for the toxicity in the chlorogenic acid smoke were evaluated by LC/TOF-MS.

Synthesis and enzymatic stability of PEGylated oligonucleotide duplexes and their self-assemblies with polyamidoamine dendrimers.

The objectives of the current study were to design and characterize poly(ethylene glycol) (PEG)-based carriers for antisense oligonucleotide (AON) delivery that would gradually release the AON upon the enzymatic degradation of a complementary nuclease-sensitive sequence (SON). A phosphodiester SON was conjugated to one extremity or to the central part of PEG (molecular weight 10 or 20 K). The PEG-SON was hybridized to a nuclease-resistant phosphorothioate AON analog. Compared to the non-PEGylated duplex, the PEG-SON/AON derivative had a modest impact on the degradation kinetics of SON as monitored by a fluorescence dequenching assay performed in the presence of DNase 1. The reaction rate depended on the grafting position of SON and on the PEG's molecular weight. To further control the release rate, PEG-SON/AON conjugates were complexed to poly(amidoamine) (PAMAM) dendrimers of different generations (G). Interaction with PAMAMs of G3 and G5 yielded monodisperse polyion complex micelles (PICMs) with average mean sizes ranging from 70 to 100 nm. The PICMs were found to decrease the catalytic reaction rate by 20 to 100 fold; the slowest release kinetics being achieved with PEG10K-SON/AON/G5 PAMAM. The PEGylated conjugates reported in this manuscript as well as their self-assemblies with PAMAMs, could prove potentially useful to confer prolonged circulating properties to nucleic acid drugs and release them in a sustained manner.

Identification of unusual bacterial glycosylation by tandem mass spectrometry analyses of intact proteins.

The characterization of protein glycosylation can be a complex and time-consuming procedure, especially for prokaryote O-linked glycoproteins, which often comprise unusual oligosaccharide structures with no known glycosylation motif. In this report, we describe a "top-down" approach that provides information on the extent of glycosylation, the molecular masses, and the structure of oligosaccharide residues on bacterial flagella, important structural proteins involved in the motility of pathogenic bacteria. Flagella from four bacterial pathogens, namely, Campylobacter jejuni, Helicobacter pylori, Aeromonas caviae, and Listeria monocytogenes, were analyzed by this top-down mass spectrometry approach. The approach needs minimal sample preparation and can be performed within a few minutes compared to the tedious and often time-consuming "bottom-up" approach involving proteolytic digestion and LC-MS-MS analyses of the suspected glycopeptides. Multiply protonated protein precursor ions subjected to low-energy collisional activation in a quadrupole time-of-flight instrument showed extensive and specific gas-phase deglycosylation resulting in the formation of abundant oxonium ions with very few fragment ions from peptidic bond cleavages. Structural information on individual carbohydrate residues is obtained using a second-generation product ion scan of oxonium ions formed by collisional activation of the intact protein ions in the source region. The four bacterial flagella examined differed not only by the extent of glycosylation but also by the nature of carbohydrate substituents. For example, the flagellin from the Gram-positive bacterium, L. monocytogenes showed O-linked GlcNAc residues at up to 6 sites/protein monomer. In contrast, the three Gram-negative bacterial pathogens C. jejuni, H. pylori and A. caviae displayed up to 19 Ser/Thr O-linked sites modified with residues structurally related to N-acetylpseudaminic acid (Pse5Ac7Ac) and in the case of Campylobacter include a novel N-acetylglutamine substituent on Pse5Am7Ac.

Capillary electrophoresis separation of a mixture of chitin and chitosan oligosaccharides derivatized using a modified fluorophore conjugation procedure.

A capillary electrophoresis (CE) method was developed for the simultaneous analysis of small chitin and chitosan oligosaccharides. For detection purposes, the oligomers were derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS), a well known fluorophore for oligosaccharides analysis. The detection was performed by laser-induced fluorescence (LIF) with an argon ion laser having an excitation wavelength of 488 nm and with emission monitored at 520 nm. Derivatization parameters such as reaction time and conditions were examined. Separation conditions were also varied by testing a range of buffer pHs and concentrations. The best conditions were found using an 80 mM borate buffer at pH 8.4. This CE-LIF optimized method was used for the analysis of an enzymatically produced oligo-chitosan sample composed of a complex mixture and having an average degree of polymerization of 3.7 monomer units and 80% deacetylation. The oligo-chitosan sample was treated with a chitin deacetylase-like enzyme, the products were derivatized with APTS, and then analyzed without purification. The goal was to determine whether the deacetylase-like enzyme could increase the extent of deacetylation of the oligo-chitosan sample.

Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking.

Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.

Comparison of two glutaraldehyde immobilization techniques for solid-phase tryptic peptide mapping of human hemoglobin by capillary zone electrophoresis and mass spectrometry.

Stabilization of proteolytic enzymes, especially by immobilization, is of considerable interest because of their potential applications in medicine and the chemical and pharmaceutical industries. We report here a detailed comparison of two procedures for trypsin immobilization using the same homobifunctional agent, glutaraldehyde, for the purpose of peptide mapping. These methods include covalent coupling either to controlled pore glass (solid support) or via a cross-linking reaction (without any solid support). The immobilized trypsin preparations were characterized by the determination of immobilization efficiency, which ranged from 68 to > 95%, and measurement of apparent kinetic parameters toward a synthetic peptide-like substrate. Batch digestions of whole denaturated human normal adult hemoglobin (HbA) were performed to obtain peptide maps by capillary zone electrophoresis (CZE). Migration time reproducibility of the CZE maps was excellent, with a mean relative standard deviation of 1.5%. Moreover, the two immobilized enzyme preparations showed excellent reproducibility for repeated digestions. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry was also used for peptide mass mapping of denaturated HbA digested using the two immobilized trypsin preparations. Even though the two immobilized trypsin preparations do not behave identically, similar sequence coverages of 57% and 61% (for the two HbA chains merged) were achieved for the support-based and cross-linked trypsin preparations, respectively.

Oxidative aggregation of ceruloplasmin induced by hydrogen peroxide is prevented by pyruvate.

Ceruloplasmin (CP) is a blue copper glycoprotein with multiple physiological functions including ferroxidase and oxidase activities. CP is also an important serum oxygen free radical (OFR) scavenger and antioxidant, exerting cardioprotective and antifibrillatory actions. Although it has been reported that CP activities can be inhibited by OFR, the intimate mechanism of this inactivation is still not clear. Exposure of bovine CP to H2O2 induced inactivation of the protein as well as structural alterations as indicated by loss of protein bands by SDS-PAGE. Both phenomena were H2O2 concentration and time dependent. HPLC gel filtration and capillary electrophoresis analysis of CP treated with H2O2 revealed an aggregation of the protein. Quantification of dityrosine formation by fluorescence indicated the involvement of dityrosine bridging, which could be responsible for aggregation of CP under oxidative attack. Oxidative damage to CP under H2O2 treatment was completely prevented by pyruvate, suggesting that the association of CP with antioxidants could extend the range of the protective action of this protein.

A comparison between electrokinetic capillary chromatography and absorption spectroscopy for the analysis of peptide-micelle association by weak hydrophobic interactions.

Micellar electrokinetic capillary chromatography (MEKC) was compared to absorption spectroscopy to estimate equilibrium association constans (K(as)) for peptide-micelle systems involving three peptides (leucine-enkephalin, methionine-enkephalin and leucine-phenylalanine (LF)) and two surfactant micelles (sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB)). Buffer pH was chosen to minimize purely electrostatic interactions between peptides and micelles that could not be interrogated by absorption spectroscopy. Viscosity-corrected MEKC mobilities gave reasonably similar estimates of K(as) between the two methods for all three peptide-SDS micelle systems, with K(as) values ranging from 13.7 +/- 0.3 to 49 +/- 1 M(-1). For CTAB, estimates of K(as) for LF-CTAB micelle association were of the same order of magnitude as the SDS micelle by the two methods of estimation. On the other hand, enkephalin-CTAB micelle binding was about 10 times stronger (K(as) = 122 +/- 3 M(-1) to 311 +/- 9 M(-1)) than the enkephalin-SDS micelle binding. In addition, MEKC underestimated the K(as) values relative to spectroscopy by a factor of 2-3 for the enkephalin-CTAB system.