Simultaneous quantification of multiple amino acid adducts from sulfur mustard-modified human serum albumin in plasma at trace exposure levels by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry after propionyl derivatization.

Sulfur mustard (HD) is a highly toxic vesicant and is prohibited by the Organisation for the Prohibition of Chemical Weapons (OPCW). HD can modify human serum albumin (HSA) to generate hydroxyethylthioethyl (HETE) adducts, which could be utilized as biomarkers for verifying HD exposure in forensic analysis. Here, five amino acid adducts generated from pronase digestion of HD-exposed human serum albumin (HD-HSA) in plasma were selected as biomarkers to retrospectively detect HD exposure. HD-HSA was precipitated from plasma with acetone, digested by pronase, derivatized with propionic anhydride (PA), and analysed with ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-TQ MS). The limits of detection (LODs) and limits of quantification (LOQs) of the HD exposure concentrations were evaluated as 1.00 ng/mL at S/N≥3 and 3.00 ng/mL at S/N≥10, respectively, which are approximately 60 times lower than those of the reported method. The approach shows good linearity (R2≥0.997) from 3.00 ng/mL to 10.0 µg/mL of HD-exposed human plasma with satisfactory precision and accuracy. The developed approach was applied to analysing samples from the 6th OPCW Biomedical Proficiency Test (BioPT). The study showed that the developed approach was also suitable for analysing human plasma samples that were exposed to six of HD analogues, which were common impurities in sulfur mustard mixtures. Moreover, the method was successfully applied to plasma from other species, including rabbits, rats and cattle. This study provides a reliable and sensitive tool for the retrospective detection of vesicants exposure based on multiple biomarkers.

Structural mapping techniques distinguish the surfaces of fibrillar 1N3R and 1N4R human tau.

The rigid core of intracellular tau filaments from Alzheimer's disease (AD), Pick's disease (PiD), and Corticobasal disease (CBD) brains has been shown to differ in their cryo-EM atomic structure. Despite providing critical information on the intimate arrangement of a fraction of htau molecule within the fibrillar scaffold, the cryo-EM studies neither yield a complete picture of tau fibrillar assemblies structure nor contribute insights into the surfaces that define their interactions with numerous cellular components. Here, using proteomic approaches such as proteolysis and molecular covalent painting, we mapped the exposed amino acid stretches at the surface and those constituting the fibrillar core of in vitro-assembled fibrils of human htau containing one N-terminal domain and three (1N3R) or four (1N4R) C-terminal microtubule-binding repeat domains as a result of alternative splicing. Using limited proteolysis, we identified the proteolytic fragments composing the molecular "bar-code" for each type of fibril. Our results are in agreement with structural data reported for filamentous tau from AD, PiD, and CBD cases predigested with the protease pronase. Finally, we report two amino acid stretches, exposed to the solvent in 1N4R not in 1N3R htau, which distinguish the surfaces of these two kinds of fibrils. Our findings open new perspectives for the design of highly specific ligands with diagnostic and therapeutic potential.

Fabrication of protease XIV-loaded microspheres for cell spreading in silk fibroin hydrogels.

Due to their excellent mechanical strength and biocompatibility, silk fibroin(SF) hydrogels can serve as ideal scaffolds. However, their slow rate of natural degradation limits the space available for cell proliferation, which hinders their application. In this study, litchi-like calcium carbonate@hydroxyapatite (CaCO3@HA) porous microspheres loaded with proteases from Streptomyces griseus (XIV) were used as drug carriers to regulate the biodegradation rate of SF hydrogels. The results showed that litchi-like CaCO3@HA microspheres with different phase compositions could be prepared by changing the hydrothermal reaction time. The CaCO3@HA microspheres controlled the release of Ca ions, which was beneficial for the osteogenic differentiation of mesenchymal stem cells (MSCs). The adsorption and release of protease XIV from the CaCO3@HA microcarriers indicated that the loading and release amount can be controlled with the initial drug concentration. The weight loss test and SEM observation showed that the degradation of the fibroin hydrogel could be controlled by altering the amount of protease XIV-loaded CaCO3@HA microspheres. A three-dimensional (3D) cell encapsulation experiment proved that incorporation of the SF hydrogel with protease XIV-loaded microspheres promoted cell dispersal and spreading, suggesting that the controlled release of protease XIV can regulate hydrogel degradation. SF hydrogels incorporated with protease XIV-loaded microspheres are suitable for cell growth and proliferation and are expected to serve as excellent bone tissue engineering scaffolds.

Cyclic Tripeptide-based Potent and Selective Human SIRT5 Inhibitors.

BACKGROUND: SIRT5 is one of the seven members (SIRT1-7) of the mammalian sirtuin family of protein acyl-lysine deacylase enzymes. In recent years, important regulatory roles of SIRT5 in (patho)physiological conditions (e.g. metabolism and cancer) have been increasingly demonstrated. For a better biological understanding and therapeutic exploitation of the SIRT5- catalyzed deacylation reaction, more effort on identifying potent and selective SIRT5 inhibitors beyond those currently known would be rewarding. OBJECTIVE: In the current study, we would like to see if it would be possible to develop potent and selective SIRT5 inhibitory lead compounds with a novel structural scaffold than those of the currently known potent and selective SIRT5 inhibitors. METHODS: In the current study, six N-terminus-to-side chain cyclic tripeptides (i.e. 8-13) each harboring the thiourea-type catalytic mechanism-based SIRT5 inhibitory warhead Nε-carboxyethylthiocarbamoyl- lysine as the central residue were designed, synthesized by the Nα-9- fluorenylmethoxycarbonyl (Fmoc) chemistry-based solid phase peptide synthesis (SPPS) on the Rink amide 4-methylbenzhydrylamine (MBHA) resin, purified by the semi-preparative reversedphase high performance liquid chromatography (RP-HPLC), characterized by the high-resolution mass spectrometry (HRMS); and were evaluated by the in vitro sirtuin inhibition assay and the in vitro proteolysis assay. RESULTS: Among the cyclic tripeptides 8-13, we found that 10 exhibited a potent (IC50 ~2.2 µM) and selective (≥60-fold over the SIRT1/2/3/6-catalyzed deacylation reactions) inhibition against the SIRT5-catalyzed desuccinylation reaction. Moreover, 10 was found to exhibit a ~42.3-fold stronger SIRT5 inhibition and a greater proteolytic stability than its linear counterpart 14. CONCLUSION: With a novel and modular structural scaffold as compared with those of all the currently reported potent and selective SIRT5 inhibitors, 10 could be also a useful and feasible lead compound for the quest for superior SIRT5 inhibitors as potential chemical/pharmacological probes of SIRT5 and therapeutics for human diseases in which SIRT5 desuccinylase activity is upregulated.

Autonomous Self-Healing Silk Fibroin Injectable Hydrogels Formed via Surfactant-Free Hydrophobic Association.

Many natural materials, such as silk, animal bone, nacre, and plant fibers, achieve outstanding strength and toughness through the rupture of sacrificial bonds between chain segments in the organic phase. In this work, we present a bioinspired strategy to fabricate silk fibroin-based hydrophobic-association (HA) hydrogels by incorporating the hydrophobic interaction as a sacrificial bond into the alginate ionic network, which not only enhanced the mechanical extensibility, strength, and toughness of the hydrogels but also enabled self-recovery and self-healing properties via reversible hydrophobic interactions without external stimuli at room temperature. The hydrophobic interaction system consisted of the hydrophobic monomer stearyl methacrylate (C18M) and an amphiphilic regenerated silk fibroin (RSF) solution. The mechanical tests and rheometry indicated that the hydrophobic interaction served as the sacrificial bond that preferentially ruptures prior to the alginate ionic network under an external load, which dissipated enormous amounts of energy and conferred an improved mechanical performance. Moreover, the structure of HA gels could be quickly recovered after injection due to the existence of hydrophobic interactions. In addition, the degradability of the HA gels in a protease XIV solution was strongly dependent upon the C18M component, which significantly promoted the degradation rate of HA gels. The biomimetic mineralization process of HA gels within a simulated body fluid (SBF), mimicking the inorganic composition of human blood plasma, was performed and the calcium phosphate nanoparticles on the hydrogel were observed. Importantly, in vivo experiments illustrated that the HA gels exhibited satisfactory biocompatibility, and the mouse osteoblasts (MC3T3-E1) could attach and spread on the hydrogels. Overall, the self-healing, biocompatibility, and high mechanical properties of the HA gels render them potentially suitable for load-bearing applications in drug delivery or other soft tissue-engineering applications.

HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis, Multilaboratory Testing Study: Final Action 2017.03.

Background: A multi-laboratory study was completed with AOAC First Action Method 2017.03, HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis. Objective: Ten laboratories from seven countries participated in the multi-laboratory study. Each laboratory analyzed 14 infant, pediatric, and adult nutritionals in duplicate. Product matrices analyzed included milk, soy, partially hydrolyzed milk, partially hydrolyzed soy, and elemental-based infant formula powders, milk-based infant formula ready-to-feed (RTF) liquids, adult low-fat powders, and adult high-fat and high-protein ready-to-drink nutritionals. Methods: Tryptophan was released from the intact protein in product matrices with a combination of proteolytic enzymes found in pronase. Following proteolysis, tryptophan was quantitated by reverse-phase isocratic HPLC and fluorescence detection. Prepared samples were injected onto a C8 HPLC column with a methanol/0.05 M phosphate buffer mobile phase. Results: Overall for tryptophan, repeatability averaged 2.1% relative SD (RSD) with a range of 0.9-3.6% RSD, and reproducibility averaged 4.2% RSD with a range of 3.0-9.9% RSD. Conclusions: Repeatability Standard Method Performance Requirements (SMPRs®) were met for 13 of the 14 matrices and reproducibility SMPRs were met for 11 of the 14 product matrices analyzed.

A sensitive quantification approach for detection of HETE-CP adduct after benzyl chloroformate derivatization using ultra-high-pressure liquid chromatography tandem mass spectrometry.

Sulfur mustard (HD) reacts with human serum albumin (HSA) at Cys34 and produces a long-term biomarker of HD exposure. Here, we present a novel, sensitive, and convenient method for quantification of HD exposure by detection of HD-HSA adducts using pronase digestion, benzyl chloroformate (Cbz-Cl) derivatization, and ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). The HSA in HD-exposed plasma in vitro was precipitated with acetone and digested (2 h, 50 °C) with pronase to form the alkylated dipeptide, S-hydroxyethylthioethyl-CysPro (HETE-CP). The HETE-CP adduct was derivatized with Cbz-Cl to generate N-carbobenzoxy HETE-CP (HETE-C(Cbz)P). The derivatized product was analyzed by UHPLC-MS/MS. HD surrogate, 2-chloroethyl ethyl sulfide (2-CEES), was introduced as a non-isotope internal standard (ISTD) instead of traditional d8-HD for quantification. The method was found to be linear between 1.00 and 200 ng/mL HD exposure (R2 > 0.998) with precision of ≤ 9.0% relative standard deviation (RSD) and accuracy ranged between 97.1 and 111%. The limit of detection (LOD) is 0.500 ng/mL (S/N~5), over 15 times lower than that of the previous method (7.95 ng/mL). Time-consuming affinity purification or solid phase extraction (SPE) is not needed in the experiment and the operation takes less than 5 h. This study provides a new strategy and useful tool for retrospective analysis of HD exposure by HETE-CP biomarker detection. Graphical abstract Flow diagram for quantification of sulfur mustard exposure by detection of HETE-CP dipeptide adduct after benzyl chloroformate derivatization using ultra-high-pressure liquid chromatography tandem mass spectrometry.

Improved flow cytometry crossmatching in kidney transplantation.

Flow cytometry crossmatching (FC-XM) assay is the most sensitive cell-based method for detecting donor-specific antibodies (DSAs). However, the use of FC-XM remains limited by methodological and clinical variations. This basic assay cannot discriminate between complement-fixing and noncomplement-fixing antibodies. FC-XM also detects patient all antibodies bound to donor cells and not only DSAs against to HLA molecules. Pretest factors associated with a donor's medical care can affect test results by changing the number, viability and target on lymphocytes (such as rituximab on CD20+ B-cells). Assay adjustment can be performed to improve the sensitivity and specificity of FC-XM. Pronase treatment (0.5-1 mg/mL) prevents false-positive B-cell FC-XM due to nonspecific immunoglobulin binding by Fc receptors and binding of surface immunoglobulins onto the surface of B-cells. Pronase treatment (2 mg/mL) or a serum incubation step with an anti-rituximab monoclonal antibody (Ab) prevents the interference induced by rituximab therapy. The use of 7 aminoactinomycin-D (7-AAD) or fluorochrome-conjugated C4d Ab, after complement incubation, allows complement-fixing antibodies to be distinguished from noncomplement-fixing antibodies. The use of donor endothelial precursor cells as target cells allows the detection of nonmajor histocompatibility complex Ab-binding endothelial cells. However, lymphocyte crossmatches still had some limits in specificity and sensitivity. This implies that this assay must be interpreted with the virtual crossmatch.

HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis: Single-Laboratory Validation, First Action 2017.03.

A method for tryptophan (Trp) analysis designed to comply with AOAC Standard Method Performance Requirement 2014.013 is described. Unlike AOAC 988.15, which uses alkaline hydrolysis, this method uses enzymatic hydrolysis to release the Trp from the intact protein. The method achieves an LOQ of 0.18 mg/100 g Trp on a ready-to-feed basis with mean recoveries ranging from 93.8 to 104.9%. Repeatability ranged from 0.2 to 5.0%. Intermediate precision ranged from 1.0 to 6.9%. The analytical range was determined to be 0.18-300 mg/100 g, with linearity over eight calibration standard levels giving an average deviation from theoretical levels of 0.3%. No single calibration point had a deviation of >5.0%. Two standard reference materials (SRMs 1849a and 927e) were analyzed, and the average deviation from the certified value was 98.5% for SRM 1849a and 101.2% for SRM 927e. Sample preparation is very similar to existing methods in terms of time and complexity. The use of an internal standard reduces laboratory error and allows for reproducible results.

Measuring protein structural changes on a proteome-wide scale using limited proteolysis-coupled mass spectrometry.

Protein structural changes induced by external perturbations or internal cues can profoundly influence protein activity and thus modulate cellular physiology. A number of biophysical approaches are available to probe protein structural changes, but these are not applicable to a whole proteome in a biological extract. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a recently developed proteomics approach that enables the identification of protein structural changes directly in their complex biological context on a proteome-wide scale. After perturbations of interest, proteome extracts are subjected to a double-protease digestion step with a nonspecific protease applied under native conditions, followed by complete digestion with the sequence-specific protease trypsin under denaturing conditions. This sequential treatment generates structure-specific peptides amenable to bottom-up MS analysis. Next, a proteomics workflow involving shotgun or targeted MS and label-free quantification is applied to measure structure-dependent proteolytic patterns directly in the proteome extract. Possible applications of LiP-MS include discovery of perturbation-induced protein structural alterations, identification of drug targets, detection of disease-associated protein structural states, and analysis of protein aggregates directly in biological samples. The approach also enables identification of the specific protein regions involved in the structural transition or affected by the binding event. Sample preparation takes approximately 2 d, followed by one to several days of MS and data analysis time, depending on the number of samples analyzed. Scientists with basic biochemistry training can implement the sample preparation steps. MS measurement and data analysis require a background in proteomics.

Pronase treatment improves flow cytometry crossmatching results.

Flow cytometry crossmatching (FC-XM) is the most sensitive cell-based method for detecting donor-specific antibodies in clinical organ transplantation. Unfortunately, background FC-XM reactivity is elevated in assays with B lymphocytes-partly because of nonspecific immunoglobulin binding by Fc receptors and B-cell surface immunoglobulins. To reduce the background reactivity in a B-cell FC-XM assay, we treated lymphocytes with pronase (1 mg/mL for 30 minutes). This treatment drastically reduced the presence of kappa light chains and Fc receptors (CD32b), while the concomitant decrease in CD19, CD20 and major histocompatibility complex (MHC) I and II expression on B-cells was acceptable. Higher pronase concentrations (>2 mg/mL) started to significantly affect CD19, CD20, MHC-I and -II expression on B-cells. In subsequent prospective experiments (on 42 donor cells tested with 102 sera), we found that pronase treatment was associated with a relative increase of the sensitivity and specificity in our B-cell FC-XM assay.

Facile method to prepare silk fibroin/hyaluronic acid films for vascular endothelial growth factor release.

A facile approach was proposed to prepare silk fibroin (SF) and hyaluronic acid (HA) composite films from aqueous solution without crosslinking or any post treatment. Only by controlling the HA content and film formation temperature during the film casting, the HA/SF films with different composition were prepared. The films were then characterized by structural characteristics, thermal stability, morphology, water stability, water absorption, mechanical properties. After immersing in water for 24h, all of the films showed good structural integrity. The degradation rate of the HA/SF films in protease XIV can be controlled by changing the film formation temperature and HA content. Decreasing the temperature and adding HA resulted in the rapid release of VEGF (vascular endothelial growth factor) from the HA/SF films. Overall, the 5% HA/SF films formed at 37°C with more rapid VEGF release exhibited great potential in drug delivery, especially when the rapid vascularization was needed.

Preparation of asparagine-linked monoglucosylated high-mannose-type oligosaccharide from egg yolk.

Monoglucosylated high-mannose-type glycan (Glc1Man9GlcNAc2: G1M9) is well-known as a key glycoform in the glycoprotein folding process, which is specifically recognized by lectin chaperones calnexin (CNX) and calreticulin (CRT) in the endoplasmic reticulum (ER). In this work, we developed an efficient method for the preparation of G1M9-Asn. The G1M9-Asn was obtained from the IgY-rich fraction derived from hen egg yolk by the digestion with pronase. The α-amino group of asparagine in G1M9-Asn was protected with the 9-fluorenylmethyloxycarbonyl (Fmoc) group and the labeled glycans were subsequently purified using high performance liquid chromatography (HPLC). This method will provide useful substrates for analysis of the glycoprotein folding cycle in the ER.

Evidence of Sulfur Mustard Exposure in Human Plasma by LC-ESI-MS-MS Detection of the Albumin-Derived Alkylated HETE-CP Dipeptide and Chromatographic Investigation of Its Cis/Trans Isomerism.

Sulfur mustard (SM) is a chemical warfare agent that causes painful blisters and chemically modifies endogenous biomacromolecules by alkylation to hydroxyethylthioethyl (HETE) adducts representing valuable long-term markers for post-exposure analysis. The albumin adduct formed in human plasma in vitro (HETE bound to the side chain of cysteine 34) was isolated and cleaved by current lots of pronase primarily generating the internal modified dipeptide (HETE-cysteine-proline, HETE-CP) instead of the formerly reported HETE-CPF tripeptide. The analyte was detected by liquid chromatography-electrospray ionization tandem-mass spectrometry (LC-ESI-MS-MS). In principle, HETE-CP undergoes a dynamic on-column equilibrium of cis-trans isomerism thus requiring separation at 50°C to obtain one narrow peak. Accordingly, we developed both a novel longer lasting but more sensitive microbore (1 mm i.d., flow 30 µL/min, cycle time 60 min, LOD 50 nM) and a faster, less sensitive narrowbore (2.1 mm i.d., 200 µL/min, cycle time 16 min, LOD 100 nM, both on Atlantis T3 material at 50°C) LC-ESI-MS-MS method suitable for verification analysis. The corresponding tri- and tetrapeptide, Q(HETE)-CPF were monitored simultaneously. HETE-CP peak areas were directly proportional to SM concentrations added to plasma in vitro (0.05-100 µM). Albumin adducts formed by deuterated SM (d8-SM) served as internal standard.

Impact of sterilization on the enzymatic degradation and mechanical properties of silk biomaterials.

The effect of some sterilization methods (autoclaving and ethanol treatments) on the degradation rate and mechanical properties of two types of porous silk scaffolds (aqueous- and hexafluoroisopropanol-derived) is evaluated. Changes in secondary structure, crystal size, and supramolecular features of silk fibroin, resulting from sterilization, are tracked to elucidate molecular level effects on protease XIV enzymatic degradation and compressive mechanical properties. The structural features and pore sizes of the silk scaffolds remain intact after both sterilization processes. Autoclave sterilization dramatically reduce the degradation rate of the silk scaffolds in response to protease XIV and significantly increase mechanical properties, in contrast to scaffolds sterilized with 70% ethanol. Higher ß-sheet content and larger crystal size are observed after autoclaving, unlike in response to 70% ethanol sterilization, based on examination of Fourier transform (FT) IR spectroscopy and wide-angle X-ray scattering (WAXS). In addition, thermal analysis finds supramolecular features within silk fibroin amorphous regions, including the glass transition temperature (Tg ), heat capacity of glass transition (ΔCp-Tg ), and thermal gravimetric degradability. Such supramolecular level changes are related to the shift in enzymatic degradation and mechanical properties due to autoclaving versus treatment with 70% EtOH. The changes in supramolecular organization in amorphous regions can retard enzyme diffusion through the glassy regions of the silk matrix or/and hinder binding of enzymes, while also stiffening these matrices.

Conditions for sample preparation and quantitative HPLC/MS-MS analysis of bulky adducts to serum albumin with diolepoxides of polycyclic aromatic hydrocarbons as models.

Stable adducts to serum albumin (SA) from electrophilic and genotoxic compounds/metabolites can be used as biomarkers for quantification of the corresponding in vivo dose. In the present study, conditions for specific analysis of stable adducts to SA formed from carcinogenic polycyclic aromatic hydrocarbons (PAH) were evaluated in order to achieve a sensitive and reproducible quantitative method. Bulky adducts from diolepoxides (DE) of PAH, primarily DE of benzo[a]pyrene (BPDE) and also DE of dibenzo[a,l]pyrene (DBPDE) and dibenzo[a,h]anthracene (DBADE), were used as model compounds. The alkylated peptides obtained after enzymatic hydrolysis of human SA modified with the different PAHDE were principally PAHDE-His-Pro, PAHDE-His-Pro-Tyr and PAHDE-Lys. Alkaline hydrolysis under optimised conditions gave the BPDE-His as the single analyte of alkylated His, but also indicated degradation of this adduct. It was not possible to obtain the BPDE-His as one analyte from BPDE-alkylated SA through modifications of the enzymatic hydrolysis. The BPDE-His adduct was shown to be stable during the weak acidic conditions used in the isolation of SA. Enrichment by HPLC or SPE, but not butanol extraction, gave good recovery, using Protein LoBind tubes. A simple internal standard (IS) approach using SA modified with other PAHDE as IS was shown to be applicable. A robust analytical procedure based on digestion with pronase, enrichment by HPLC or SPE, and analysis with HPLC/MS-MS electrospray ionisation was achieved. A good reproducibility (coefficient of variation (CV) 11 %) was obtained, and the achieved limit of detection for the studied PAHDE, using standard instrumentation, was approximately 1 fmol adduct/mg SA analysing extract from 5 mg SA.

Identification of serum component involved in generation of neo-lectin with agglutinating and phenoloxidase activities in human serum.

Human serum albumin (HSA) was identified as the component involved in generation of neo-lectin molecules with both lectin and phenoloxidase activities. Pronase treated HSA was able to agglutinate hen RBC and oxidize hydroquinone. Sodium dodecyl sulphate (SDS) treated HSA agglutinated both hen and sheep RBC as well as oxidized dopamine. The hemagglutinating activities of pronase/SDS treated HSA observed against hen RBC were dosimetric. The oxidation of pronase/SDS treated HSA with hydroquinone/dopamine, respectively, was inhibitable by inhibitors of phenoloxidase, namely, phenylthiourea and tropolone. Very low concentrations of HSA could generate these humoral neo-lectin molecules.

Role of glycosylation in secretion and stability of micromycetes α-galactosidase.

The effect of the glycosylation inhibitors (tunicamycin and 2-deoxy-D-glucose) on the activity, stability and production of fungal glycosidases has been studied. It was shown that inhibition of N-glycosylation sites did not affect the secretion of Aspergillus niger α-galactosidase, however reduced yield of Cladosporium cladosporioides and Penicillium canescens α-galactosidases. Changes in the level of O-glycosylation resulted in a significant reduction in the activity and stability of α-galactosidases of all three producers tested. Activity of the modified enzymes was significantly lower than that of the native ones, and was 2.6 and 0.33 U/mg for A. niger α-galactosidase, 3.3 and 32.5 U/mg for C. cladosporioides α-galactosidase, 11.66 and 31.1 U/mg for P. canescens α-galactosidase, respectively. A. niger α-galactosidase completely lost activity during purification and storage. The decrease of thermal stability at 55 °C by 20% was shown for C. cladosporioides and P. canescens α-galactosidases. It was also noted that O-deglycosylation led to a decrease in resistance of these enzymes to the action of proteases.

Determination of selenomethionine and seleno-methyl-selenocysteine in biota by ultrasonic-assisted enzymatic digestion and multi-shot stir bar sorptive extraction-thermal desorption-gas chromatography-mass spectrometry.

A method based on stir bar sorptive extraction (SBSE) and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS) has been optimized for the determination of seleno-methyl-selenocysteine (SeMetSeCys) and selenomethionine (SeMet) in biota samples. Aliquots of freeze-dried tissue, a mixture of protease XIV-lipase and water were sonicated for 2min. After extraction, the extract was separated by centrifugation and subjected to derivatization and SBSE-TD-GC-MS. The parameters affecting derivatization, absorption and desorption steps were investigated. The optimized conditions consist of a derivatization with 40µL of ethyl chloroformate (ECF) in 400µL of a water:ethanol:pyridine (60:32:8) mixture, followed by dilution to 1.5mL of 70g NaClL(-1) in water at neutral pH and an extraction step using 10mm×1mm PDMS stir bar, stirring at 800rpm for 20min at room temperature (23±1°C). Three stir bars were used for the extraction of three different aliquots of the same sample and then placed in a single glass desorption liner and simultaneously desorbed for GC-MS analysis. The desorption step required the following conditions: 300°C (desorption temperature), 6min (desorption time), 50mLmin(-1) (vent flow) and -5°C (cryotrapping temperature). The method provided precise (8.1%) and accurate results in the mgSekg(-1) range (using the selected-ion monitoring-SIM mode) against certified reference material SELM-1 yeast, with recoveries higher than 80% for spiked algae and clams samples.

Controlled enzyme-catalyzed degradation of polymeric capsules templated on CaCO3: influence of the number of LbL layers, conditions of degradation, and disassembly of multicompartments.

Enzyme-catalyzed degradation of CaCO3-templated capsules is presented. We investigate a) biodegradable, b) mixed biodegradable/synthetic, and c) multicompartment polyelectrolyte multilayer capsules with different numbers of polymer layers. Using confocal laser scanning microscopy we observed the kinetics of the non-specific protease Pronase-induced degradation of capsules is slowed down on the order of hours by either increasing the number of layers in the wall of biodegradable capsules, or by inserting synthetic polyelectrolyte multilayers into the shell comprised of biodegradable polymers. The degradation rate increases with the concentration of Pronase. Controlled detachment of subcompartments of multicompartment capsules, with potential for intracellular delivery or in-vivo applications, is also shown.