Development of a liquid chromatography-based screening methodology for proteolytic enzyme activity

A new methodology for the detection and isolation of serine proteases in complex mixtures has been developed. It combines the characterization of crude samples by electrospray tandem mass spectrometry (ESI-MS/MS) in a multi-substrate assay and the differentiated sensitive detection of the responsible enzymes by means of liquid chromatography hyphenated online to biochemical detection (BCD). First, active samples are identified in the multi-substrate assay monitoring the conversion of eight substrates in multiple reaction monitoring in parallel within 60 s. Hereby, the product patterns are investigated and the suitable peptide as substrate for BCD analysis is selected. Subsequently, the active proteases are identified online in the continuous-flow reactor serving as BCD after non-denaturing separation by size-exclusion chromatography and ion-exchange chromatography. For BCD, the selected para-nitroaniline (pNA) labeled peptide is added post-column and is cleaved by eluting proteases under release of the coloured pNA in a reaction coil (reaction time 5 min). The method was optimized and the figures of merit were characterized with trypsin and chymotrypsin serving as the model proteases. For trypsin, a limit of detection in LC–BCD of 0.1 U/mL corresponding to an injected amount of 0.4 ng protein (∼18 fmol) was observed. The BCD signal remained linear for an injected enzyme concentration of 0.3–10 U/mL (1.3–42 ng enzyme). The method was applied to the characterization of the crude venom of the pit viper Bothrops moojeni and the extracellular protease of the pathogenic amoeba Acanthamoeba castellanii. In the two samples, fractions with proteolytic activity potentially interfering with the blood coagulation cascade were identified. The described methodology represents a tool for serine protease screening in complex mixtures by a fast ESI-MS/MS identification of active samples followed by the separation and isolation of active sample constituents in LC–BCD.     

Propagation rate coefficients of isobornyl acrylate,tert‐butyl acrylate and 1‐ethoxyethyl acrylate: A high frequency PLP‐SEC study

Pulsed laser polymerization (PLP) coupled to size exclusion chromatography (SEC) is considered to be the most accurate and reliable technique for the determination of absolute propagation rate coefficients, k_p. Herein, k_p data as a function of temperature were determined via PLP‐SEC for three acrylate monomers that are of particular synthetic interest (e.g., for the generation of amphiphilic block copolymers). The high‐T_g monomer isobornyl acrylate (iBoA) as well as the precursor monomers for the synthesis of hydrophilic poly(acrylic acid), tert‐butyl acrylate (tBuA), and 1‐ethoxyethyl acrylate (EEA) were investigated with respect to their propagation rate coefficient in a wide temperature range. By application of a 500 Hz laser repetition rate, data could be obtained up to a temperature of 80 °C. To arrive at absolute values for k_p, the Mark‐Houwink parameters of the polymers have been determined via on‐line light scattering and viscosimetry measurements. These read: K = 5.00 × 10⁵ dL g⁻¹, a = 0.75 (piBoA), K = 19.7 × 10⁵ dL g⁻¹, a = 0.66 (ptBA) and K = 1.53 × 10⁵ dL g⁻¹, a = 0.85 (pEEA). The bulky iBoA monomer shows the lowest propagation rate coefficient among the three monomers, while EEA is the fastest. The activation energies and Arrhenius factors read: (iBoA): log(A/L mol⁻¹ s⁻¹) = 7.05 and E_A = 17.0 kJ mol⁻¹; (tBuA): log(A/L mol⁻¹ s⁻¹) = 7.28 and E_A = 17.5 kJ mol⁻¹ and (EEA): log(A/L mol⁻¹ s⁻¹) = 6.80 and E_A = 13.8 kJ mol⁻¹. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 6641–6654, 2009     

Photooxidation of poly(N-vinylpyrrolidone) (PVP) in the solid state and in aqueous solution

The photooxidation of poly(N-vinylpyrrolidone) (PVP) in the solid state and in an aqueous solution has been studied under irradiation at long wavelengths (at λ ≥ 300 nm) and in the presence of oxygen, as these conditions of irradiation are those of natural outdoor aging. Infrared spectroscopy was used to follow the photochemical evolution of the polymer either for solid films of PVP or for PVP irradiated in an aqueous solution. Chemical treatments (NH₃, SF₄) were carried out on the photooxidized samples, and aqueous solutions of PVP were characterized by viscometry and size exclusion chromatography (SEC). Based on photooxidation results obtained in both the solid state and in an aqueous solution, a general mechanism that accounts for the main routes of oxidation is proposed. It was shown that the rates of oxidation and the stoichiometries of the reactions were strongly influenced by the physical state of the polymer (solid state or aqueous solution).     

Polyamide‐6,6‐based blocky copolyamides obtained by solid‐state modification

Copolyamides based on polyamide‐6,6 (PA‐6,6) were prepared by solid‐state modification (SSM). Para‐ and meta‐xylylenediamine were successfully incorporated into the aliphatic PA‐6,6 backbone at 200 and 230 °C under an inert gas flow. In the initial stage of the SSM below the melting temperature of PA‐6,6, a decrease of the molecular weight was observed due to chain scission, followed by a built up of the molecular weight and incorporation of the comonomer by postcondensation during the next stage. When the solid‐state copolymerization was continued for a sufficiently long time, the starting PA‐6,6 molecular weight was regained. The incorporation of the comonomer into the PA‐6,6 main chain was confirmed by size exclusion chromatography (SEC) with ultraviolet detection, which showed the presence of aromatic moieties in the final high‐molecular weight SSM product. The occurrence of the transamidation reaction was also proven by ¹H nuclear magnetic resonance (NMR) spectroscopy. As the transamidation was limited to the amorphous phase, this SSM resulted in a nonrandom overall structure of the PA copolymer as shown by the degree of randomness determined using ¹³C NMR spectroscopy. The thermal properties of the SSM products were compared with melt‐synthesized copolyamides of similar chemical composition. The higher melting and higher crystallization temperatures of the solid state‐modified copolyamides confirmed their nonrandom, block‐like chemical microstructure, whereas the melt‐synthesized copolyamides were random. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013 , 51, 5118–5129     

Effect of Fusarium oxysporum f. sp. lycopersici on the degradation of humic acid associated with Cu, Pb, and Ni: an in vitro study

The intent of this work was to gain further insight on the fungus-assisted degradation/solubilization of humic acid and the related changes in metal-binding profiles. In the experimental design, Aldrich reagent humic acid (HA) or HA enriched with Cu, Pb, and Ni (HA(Me)) was added to Fusarium oxysporum f. sp. lycopersici cultures in vitro. The cultures were supplied by different carbon- and nitrogen-containing nutrients (glucose, Glc, or glutamate, Glu and ammonium, NH₄⁺, or nitrate, NO₃⁻, ions, respectively) in order to examine their possible effect on HA and HA(Me) decomposition. During the first 48 h of fungus growth, gradual acidification to pH 2 was observed in medium containing Glc + NH₄⁺, while for other cultures, alkalinization to pH 9 occurred and then, the above conditions were stable up to at least 200 h. Size exclusion chromatography (SEC) with UV/Vis detection showed progressive degradation and solubilization of both HA and HA(Me) with the increasing time of fungus growth. However, the molecular mass distributions of HA-related soluble species were different in the presence of metals (HA(Me)) as referred to HA and were also influenced by the composition of growth medium. The solubilization of Pb, Cu, and Ni and their association with HA molecular mass fractions were studied using inductively coupled plasma mass spectrometry (ICP-MS) detection. Under acidic conditions, relatively high concentrations of low-molecular-mass metallic species were found in culture supernatants, while in alkaline media, metal solubilization was generally poorer. In contrast to low pH culture, SEC-ICP-MS results obtained in alkaline supernatants indicated metal binding to degradation products of humic substances of MM > 5 kDa. In summary, the results of this study suggest that fungus-assisted degradation of HA and HA(Me) might be controlled using appropriate N- and C- sources required for fungus growth, which in turn would affect molecular mass distribution of soluble metallic species thus potentially influencing their actual bioaccessibility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Poly(vinyl phosphonic acid): Hydrodynamic Properties and SEC‐Calibration in Aqueous Solution

Poly(vinyl phosphonic acid) (PVPA) as obtained by free radical polymerization of aqueous vinyl phosphonic acid was studied by light scattering (SLS, DLS) and size exclusion chromatography (SEC) in dilute aqueous solutions containing sufficient salt in order to screen long range electrostatic interactions. Samples of 37< < 110 × 10³ were studied. The polymers showed positive A₂‐values in aqueous NaH₂PO₄ solution (0.04 M ), and self‐diffusion behavior and R_H/R_G—ratios indicative of the structure of random coiled chains. A comparison of the SEC‐elugrams of the PVPA‐samples with those of commercially available standards of poly(acrylic acid) sodium salt gave a fit to the same calibration curve described by log P_n(PVPA) = −0.21ν_e + 7.0(+0.1) which correlates the number average degree of polymerization (P_n) with the elution volume ν_e. This indicates that PVPA and PAA have the same hydrodynamic structure under given solution conditions.

     

Design of Experiment (DoE) as a Tool for the Optimization of Source Conditions in SEC‐ESI‐MS of Functional Synthetic Polymers Synthesized via ATRP

Design of experiment (DoE) is applied to establish the optimum ionization conditions for analyzing synthetic polymers via coupled size exclusion chromatography electrospray ionization mass spectrometry (SEC‐ESI‐MS) yielding maximum ionization efficiency. The ion source conditions were optimized with regard to the ionization efficiency, the amount of fragmentation, as well as the formation of salt adducts. A D ‐optimal experimental design was employed for this purpose and the recorded data were evaluated by a quadratic response surface model, accounting for possible interactions between the individual source settings. It was established that the ionization efficiency can be improved by up to one order of magnitude without compromising the softness of the ionization process and that optimal ionization conditions are found at similar source settings regardless of the charge state. The present optimization exercise therefore provides a hands‐on guide for the use of experimental design to determine optimum ionization conditions during the SEC‐ESI‐MS of functional polymers.

     

Hg2+在大鼠组织器官中的分布及其存在形态研究

汞是环境中重要的污染物之一,对人类的健康构成了巨大的威胁。文章通过建立电感耦合等离子体质谱及凝胶排阻色谱分离法与紫外和电感耦合等离子体质谱在线检测的联用技术,研究大鼠灌喂HgCl2后Hg在大鼠中的分布累积规律和及其与蛋白的结合,实验结果表明肝脏和肾脏中在Hg刺激下诱导产生大量硫蛋白,大量产生的金属硫蛋白可能因此而优先与通过胃肠吸收摄入的Hg结合,减少了Hg对其它蛋白的正常作用的干扰,从而大大降低了Hg^2＋的毒性。     

Determination of the Molar Mass and the Radius of Gyration, Together with their Distributions for Methylhydroxyethylcelluloses

Three molar mass series were produced by different methods of degradation (namely ultrasonic (seven samples), oxidation (seven samples) and autoclaving (eight samples)) from a methylhydroxyethylcellulose (MHEC) sample with an average degree of substitution (DS) of 1.3, a molar degree of substitution (MS) of 0.46, a radius of gyration of 67 nm and a weight-average molar mass, M _w, of 318,000 g/mol. The degraded samples were characterized in terms of their molar mass and particle size together with their respective distributions with a hyphenated apparatus consisting of size exclusion chromatography and multi-angle laser light scattering and concentration detector (SEC/MALLS/DRI) at 25 °C in 0.1 M NaNO₃ solution (with 200 ppm NaN₃ as antibactericide). The refractive index increment was determined as dn/dc = 0.135 cm³/g. It was possible to reduce the weight-average molar mass down to approximately 10% of the initial molar mass using all the methods. In a comparison of the three degradation methods it was shown that only ultrasonic degradation retains the monomodal distribution, whereas the other two degradation methods yield a bimodal molar mass distribution. Consequently, only ultrasonic degradation represents a suitable method for producing homologous molar mass series. An R _G–M relationship of R _G = 0.0511 × M ^0.56 was established for the sample used in this case, and from this it was possible to calculate an []–M relationship of [] = 0.3587 × M ^0.68.     

Towards a less biased dissolution of chitosan

The dissolution of polysaccharides is notoriously challenging, especially when one needs a “true” solution. Factors influencing chitosan's solubility include composition, also known as degree of acetylation (DA). The dissolution of chitosan was investigated by visual observation, size-exclusion chromatography (SEC), pressure mobilization (PM), free-solution capillary electrophoresis (CE) and real-time solution-state NMR spectroscopy. Aqueous HCl dissolves around 15% more chitosan than the commonly used aqueous acetic acid (AcOH), however aggregates were detected in SEC suggesting incomplete dissolution. Significant deacetylation of chitosan over the period needed for dissolution at high temperature was observed by NMR spectroscopy in DCl by about 20% of the initial DA value. Accurate DA determination by NMR spectroscopy may thus be possible only in the solid state (with a precision within 1% on the DA % scale above a DA of 10%). Overall a compromise between maximum solubilization and minimum degradation is required in attempting to obtain a “true” solution of chitosan. The completeness of the dissolution may be more influenced by the average DA than by molar mass.