Analytical Methods
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2014
·Vol. 7
·期 2
·638-642
·
IF 1.938
Wei Wang,Lei Zhan,Yu Qing Du,Fei Leng,Cheng Zhi Huang
A novel and selective spectrofluorometric method for pyrophosphate (PPi) assay in aqueous solution was developed in this contribution on the basis of the enhanced fluorescence intensity of trypsin-stabilized copper clusters (CuNCs). Mechanism investigation showed that PPi can make the CuNCs dispersed, concomitantly resulting in the improved quantum yield and the enhanced fluorescence emission of the CuNCs. A good linear relationship in the range of 78 μM–20 mM was achieved between the enhanced fluorescence intensity of the CuNCs at 455 nm and the logarithm of PPi concentration with the detection limit (3σ) of 101 nM. By this approach, we can achieve simple, fast and inexpensive detection of PPi.
研究领域:
分析化学
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光谱学
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纳米材料
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2018
·
IF 3.909
Nan Wu,Siming Wang,Ye Yang,Jiayi Song,Ping Su,Yi Yang
A novel type of trypsin capillary microreactor was developed based on a DNA-directed immobilization (DDI) technique applied to a fused-silica capillary modified with polyamidoamine (PAMAM) dendrimers. Trypsin binding to the inner wall of the capillary was confirmed by confocal laser scanning microscopy. The properties of the trypsin–DNA conjugated, PAMAM-modified capillary microreactor were investigated by monitoring hydrolysis of N α-benzoyl- L -arginine ethyl ester. Through the hybridization and dehybridization of the DNA, the inner wall of the capillary functionalized with trypsin can be regenerated, thus indicating the renewability of this enzyme microreactor. In addition, these results demonstrated that introduction of PAMAM enabled higher amounts of trypsin to be immobilized, markedly improving the enzymolysis efficiency, compared with traditional modified capillaries. The digestion performance of the trypsin capillary microreactor was further evaluated by digesting cytochrome C, and a peptide numbers of 8, and a sequence coverage of 59% were obtained. This renewable and efficient immobilized trypsin capillary microreactor combines advantages of both DDI technology and PAMAM, and is potentially adaptable to high-throughput enzyme assays in biochemical and clinical research.
研究领域:
生物化学
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酶学
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分析化学
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生物医学工程
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SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
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2023
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IF 4.4
Qi Zhou,Deyuan Yang,Xiujuan Huang,Yuanyuan Chen,Yifeng Tu,Jilin Yan
Trypsin is a serine protease playing a key role in regulating pancreatic exocrine function and can be applied as a marker for the diagnosis of pancreatitis. In this work, a convenient and sensitive fluorescent assay was developed toward trypsin. Hydrogen peroxide slowly oxidized a non-fluorescent o-phenylenediamine (OPD) into a fluorescent product 2,3-diaminophenothiazine (DAP) under the catalytic from copper ions. After the introduction of bovine serum albumin (BSA), the combination of BSA with copper ions formed a peroxidase mimic and significantly accelerated the reaction rate. As an efficient protease, trypsin cleaved the lysine and arginine residues in BSA. This destroyed the binding between Cu 2+ and BSA, and brought in a reduction of the catalytic effect. The accompanying decrease in fluorescence provided a response to trypsin in the range of 0.01–600 ng/mL, with a detection limit of 0.007 ng/mL. The scheme had a good selectivity and was successfully applied to the detection of real samples.
研究领域:
生物化学
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分析化学
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医学诊断
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Min Hu,Jia-Xu Song,Shi-Tao Miao,Cheng-Kai Wu,Xing-Wen Gong,Hong-Ju Sun
Acetaldehyde dehydrogenase 2 (ALDH2) is a crucial enzyme in alcohol metabolism, and oral administration of ALDH2 is a promising method for alcohol detoxification. However, recombinant ALDH2 is susceptible to hydrolysis by digestive enzymes in the gastrointestinal tract and is expressed as inactive inclusion bodies in E. coli . In this study, we performed three rounds of rational design to address these issues. Specifically, the surface digestive sites of pepsin and trypsin were replaced with other polar amino acids, while hydrophobic amino acids were incorporated to reshape the catalytic cavity of ALDH2. The resulting mutant DE2–852 exhibited a 45-fold increase in soluble expression levels, while its stability against trypsin and pepsin increased by eightfold and twofold, respectively. Its catalytic efficiency ( k cat /K m ) at pH 7.2 and 3.2 improved by more than four and five times, respectively, with increased V max and decreased K m values. The enhanced properties of DE2–852 were attributed to the D457Y mutation, which created a more compact protein structure and facilitated a faster collision between the substrate and catalytic residues. These results laid the foundation for the oral administration and mass preparation of highly active ALDH2 and offered insights into the oral application of other proteins.
研究领域:
生物化学
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蛋白质工程
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药物开发
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BIOSENSORS & BIOELECTRONICS
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2014
·
IF 6.451
Guang-Li Wang,Lu-Yi Jin,Yu-Ming Dong,Xiu-Ming Wu,Zai-Jun Li
In this research, a novel enzyme mimetics based on the photochemical property of gold nanoclusters was demonstrated. It was found that the bovine serum albumin (BSA) stabilized red or blue emitting gold nanoclusters (Au NCs) exhibited enzyme-like activity under visible light irradiation . The BSA–Au NCs had better stability against stringent conditions compared to natural enzyme. In addition, the photostimulated enzyme mimetics of BSA–Au NCs showed several unprecedented advantages over natural peroxidase or other existing alternatives based on nanomaterials , such as the independence of hydrogen peroxide on activity and the easily regulated activity by light irradiation. The mechanism of the photoresponsive enzyme-like activity of BSA–Au NCs was investigated. The photoactivated BSA–Au NCs was designed to develop a facile, cheap, and rapid colorimetric assay to detect trypsin through trypsin digestion of the protein template of BSA-stabilized Au NCs. The limit of detection for trypsin was 0.6 μg/mL, which was much lower than the average level of trypsin in patient′s urine or serum.
研究领域:
生物化学
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纳米医学
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分析化学
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JOURNAL OF FOOD COMPOSITION AND ANALYSIS
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2024
·
IF 4
Yuyin Wu,Pengfei Yu,Mati Ullah Khan,Ziwei Han,Jun Yang,Jiaqi Cui,Anqi Liu,Zhiwei Chen,Ling Kong
Trypsin, a globular protein hydrolase, catalyzes the hydrolysis of peptide bonds at carboxyl groups of arginine and lysine residues, its detection holds paramount significance in the dairy industry, aiding in the diagnosis, management of animal diseases, and preservation of dairy products. This study introduces a highly sensitive fluorometric detection platform utilizing bimetallic gold/silver nanoclusters (Au/Ag NCs) and gold nanoparticles (AuNPs) in an "off-on-off" configuration for precise quantification of dairy trypsin and trypsin inhibitors. This fluorescence variation is leveraged to determine the concentration of trypsin and its inhibitors. Under optimal experimental conditions, the detection of trypsin demonstrates a linear range of 5 ∼ 2000 ng·mL −1 , a lowest detection limit of 1.18 ng·mL −1 , and an observed IC 50 of 5.94 μg·mL −1 for the soybean trypsin inhibitor (STI). Therefore, it could be inferred that the development of a fluorescence sensing platform presents an important basis for the detection of dairy trypsin.
研究领域:
分析化学
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食品科学
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纳米技术
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ACS Applied Materials & Interfaces
·
2024
·
IF 8.3
Hanyue Song,Wenkang Zhang,Shuyi Zhang,Ying Liu,Ping Su,Jiayi Song,Yi Yang
Low-molecular weight proteins (LWPs) are important sources of biological information in biomarkers, signaling molecules, and pathology. However, the separation and analysis of LWPs in complex biological samples are challenging, mainly due to their low abundance and the complex sample pretreatment procedure. Herein, trypsin modified by poly(acrylic acid) (PAA) was encapsulated by a zeolitic imidazolate framework (ZIF-L). Mesopores were formed on the ZIF-L with the introduction of PAA. An alternative strategy for separation and pretreatment of LWPs was developed based on the prepared ZIF-L-encapsulated trypsin with adjustable pore size. The mesoporous structure of the prepared materials selectively excluded high-molecular weight proteins from the reaction system, allowing LWPs to enter the pores and react with the internal trypsin, resulting in an improved separation efficiency. The hydrophobicity of the ZIF-L simplified the digestion process by inducing significant structural changes in substrate proteins. In addition, the enzymatic activity was significantly enhanced by the developed encapsulation method that maintained the enzyme conformation, allowed low mass transfer resistance, and possessed a high enzyme-to-substrate ratio. As a result, the ZIF-L-encapsulated trypsin can achieve highly selective separation, valid denaturation, and efficient digestion of LWPs in a short time by simply mixing with substrate proteins, greatly simplifying the separation and pretreatment process of the traditional hydrolysis method. The prepared materials and the developed strategy demonstrated an excellent size-selective assay performance in model protein mixtures, showing great potential in the application of proteomics analysis.
研究领域:
生物化学
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蛋白质组学
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分析化学
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材料科学
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PubMed
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Analytical Methods
·
2025
·
IF 2.6
Jing Chen,Yuzhi Wang,Xiyan He
Three novel polymethacrylic acid-based deep eutectic solvents (DESs)-functionalized magnetic graphene oxide composites were designed and successfully synthesized via the radical polymerization method. The aim was to achieve the selective solid - phase extraction of trypsin (Tryp). Among them, the magnetic extractant (MGO@PDES2) functionalized with a poly-deep eutectic solvent composed of tetraethylammonium chloride and methylpropionic acid at a molar ratio of 1:2 exhibited the highest extraction efficiency for Tryp. Owing to the high specific surface area of MGO and the abundant carboxyl functional groups in PDES2, the prepared MGO@PDES2 exhibited excellent selectivity and stability for Tryp extraction. Under optimal conditions, the extraction capacity of Tryp by MGO@PDES2 reached 708.85 mg·g-1. The extraction driving forces between MGO@PDES2 and Tryp were hydrogen-bonding interactions and electrostatic interactions. Among the seven biomacromolecules, MGO@PDES2 displayed outstanding selectivity for Tryp. In addition, the results of cycling experiments indicated that MGO@PDES2 could be reused many times without a significant change in extraction capacity. Moreover, the proposed method was successfully applied to extract Tryp from the crude extract of bovine pancreas, yielding satisfactory results. All the results suggest that MGO@PDES2 is a promising magnetic extractant, which is expected to provide new ideas for the extraction and separation of proteins.
研究领域:
生物化学
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分析化学
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PubMed
高影响因子
PLoS One
·
2026
·Vol. 21
·期 2
·e0342746
·
IF 2.8
Dandan Feng,Yakun Song,Zuqi Wu,Wuruo Liu,Yuting Pu,Yangcan Gao,Yuying Sun,Jiquan Zhang
Trypsin, a canonical serine protease in crustaceans, plays a crucial role in ontogeny and antibacterial defense. Whether these biological functions correlate with its catalytic characteristics remains unresolved in the freshwater shrimp Neocaridina denticulata sinensis . To address this gap, we characterized a trypsin gene from N. denticulata sinensis ( NdTryp ) and assessed both its biological roles and its prospective utility. NdTryp was predominantly expressed in the hepatopancreas, where it localized to storage cells (R-cells) and tubule-lining epithelial cells (ECTs). Across development, NdTryp transcripts were essentially absent during early embryogenesis but rose sharply at late stages, temporally coincident with hatching and the onset of feeding. After a challenge with Vibrio parahaemolyticus , the expression of NdTryp was induced, with the expression level significantly increased relative to the baseline expression level. RNA interference-mediated knockdown rendered shrimp more susceptible to infection and was accompanied by extensive hepatopancreatic injury, including epithelial detachment and disruption of the basement membrane. Biochemically, recombinant NdTryp (rNdTryp) displayed proteolytic activity over a broad temperature and pH span. Activity was differentially tuned by metal ions, with several divalent cations producing marked enhancement, whereas ferric iron exerted strong inhibition. Overall, our results showed that NdTryp functions as a multifunctional protease involved in both late embryonic development and innate antimicrobial defense. Furthermore, the robust stability of rNdTryp underscores its potential as an aquafeed additive and candidate for enzymatic biotransformation.
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COLLOIDS AND SURFACES A-PHYSICOCHEMICAL AND ENGINEERING ASPECTS
·
2022
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IF 5.518
Xian-song Wang,Shen Zhang
Herein, a platform for sensitive fluorescence sensing of morin was developed based on the copper nanoclusters , which was carried out by using a one-pot method. The copper nanoclusters (Trypsin@Cu NCs) were fabricated through CuCl 2 as a metal precursor, trypsin as a protective agent and hydrazine hydrate as a reducing agent. The Trypsin@Cu NCs exhibited blue fluorescence under the excitation wavelength of 378 nm. Using Trypsin@Cu NCs as the patterned nanosensors , the fluorescent “turn off” phenomenon was developed for the measurement of morin with the linear decline of emissive intensity at 459 nm. The fluorescence quenching reason was subjected to the static quenching and inner filter effect . Under the optimal conditions, an excellent linear response between ln(F 0 /F) of Trypsin@Cu NCs and morin amounts in the light of the range of 0.5–150 μM, and the limit of detection was 0.083 μM. Moreover, this proposed nanosensor could be applied for the sensing of temperature. This sensing method was displayed to be a practical determination system for the measurement of morin in real samples, which could open new horizons to develop novel detection models for morin.
研究领域:
分析化学
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生物传感
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材料科学
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DOI
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FOOD CHEMISTRY
·
2019
·
IF 5.399
Hao Zhang,Zhao-Yu Wu,Yin-Zhen Wang,Dong-Dong Zhou,Feng-Qing Yang,De-Qiang Li
Phenolic acids, which are important aromatic secondary metabolites, are widely distributed in plant foods. In this study, a simple, economical and fast on-line immobilized trypsin microreactor was developed for evaluating the inhibitory activity of phenolic acids by capillary electrophoresis. The Michaelis-Menten constant ( K m ) of immobilized trypsin was determined as 0.99 mM, and the half-maximal inhibitory concentration (IC 50 ) and inhibition constant ( K i ) of benzamidine were measured as 3.39 and 1.68 mM, respectively. Then, the developed strategy was applied to investigate the inhibitory activity of six phenolic acids on trypsin. The results showed that gallic acid, caffeic acid and ferulic acid had high inhibitory activity at concentration of 150 μM. Molecular docking results illustrated that gallic acid, caffeic acid and ferulic acid can interact indirectly with the catalytic and substrate-binding sites of trypsin. The developed strategy is an effective tool for evaluating inhibitory activity of phenolic acids on trypsin.
研究领域:
分析化学
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生物化学
·
药学
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PubMed
高影响因子
Food & Function
·
2019
·Vol. 10
·期 12
·8182-8194
·
IF 3.241
Min Liu,Tingting Liu,Yabo Shi,Yanna Zhao,Hui Yan,Bin Sun,Qingpeng Wang,Zhengping Wang,Jun Han
Natural polyphenols showing a variety of beneficial effects will interact with multiple proteases after administration. The interactions of oxyresveratrol and piceatannol with trypsin and lysozyme were investigated using fluorescence spectroscopy, UV-vis absorption spectroscopy, circular dichroism spectroscopy, differential scanning calorimetry and molecular docking. Fluorescence quenching results and UV-vis absorption difference spectra revealed that the quenching process was a static mode initiated by ground-state complex formation. The different binding ability of oxyresveratrol and piceatannol with trypsin and lysozyme was discussed based on their different molecular structures. Moreover, the major driving force for the binding process was elucidated as hydrogen bonding and van der Waals forces by the negative enthalpy and entropy changes. Synchronous fluorescence, three-dimensional fluorescence and circular dichroism spectral analysis suggested that the binding of oxyresveratrol and piceatannol to trypsin and lysozyme induced some microenvironmental and conformational changes of the two enzymes. The thermal stability of the enzymes in the presence of polyphenols was studied based on the change in melting temperature by differential scanning calorimetry. The above experimental results were validated by the protein–ligand docking studies which showed the location of the two ligands in the enzymes and the surrounding amino acid residues. Furthermore, enzyme activity assays indicated that the enzymatic activity of trypsin and lysozyme was inhibited by oxyresveratrol and piceatannol. The effect of trypsin and lysozyme on the antioxidant activity and stability of oxyresveratrol and piceatannol was also investigated. In conclusion, the comparative study on the interaction of oxyresveratrol and piceatannol with trypsin and lysozyme showed that the positions of hydroxyl groups of the polyphenols had an important influence on their interaction with enzymes and their antioxidant activity and stability as well as the enzyme activities. The obtained results are expected to provide a theoretical basis for the application of polyphenols in functional foods and pharmaceuticals.
研究领域:
生物化学
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药理学
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食品科学
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PubMed
高影响因子
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