An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood

In biomedical research and clinical diagnostics, it is a major challenge to measure disease‐related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time‐consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge‐changing fluorescent peptide substrates. Charge‐changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic α‐chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both α‐chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross‐reactivity with the trypsin‐like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin‐specific), a detection limit of about 10–20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point‐of‐care diagnostics.     

草鱼胰蛋白酶GT-A的紫外光谱及结构特性

研究草鱼胰蛋白酶同工酶GT-A的结构特性,并探讨其结构特性与其性质之间的关系。用仪器分析方法,分析了草鱼肠道胰蛋白酶同工酶GT-A的紫外吸收特性以及圆二色谱(CD)。结果表明,GT-A在280nm处只有微弱的吸光度,在变性剂存在下,其紫外吸收大幅度增加,依据"双状态模型"计算得到其变性自由能为35.86KJ/mol。CD测定表明GT-A酶蛋白分子具有较少的α-螺旋、β-折叠以及β-转角等规则二级结构单元和较多的无规卷曲结构。这些信息结合起来可以合理解释GT-A的理化性质和动力学性质等。     

Protein, Oil, and Isoflavone Contents in Lipoxygenase- and Kunitz Trypsin Inhibitor-Deficient Soybean Seeds

Two traditional soybean cultivars, CAC-1 and Garimpo, two backcrossed-derived isolines lacking lipoxygenase (LOX^?KTI⁺), and two backcrossed-derived isolines lacking lipoxygenase and Kunitz trypsin inhibitor (LOX^?KTI^?) in the seeds were grown in São Gotardo and Paracatu, Brazil, and their protein, oil, and isoflavone contents were assessed. The loss of LOX or the combined loss of LOX and KTI brought about no significant changes in seed oil content. Significant decreases in seed protein content were observed in isolines of the CAC-1 genetic background, but not in isolines of the Garimpo background. Total isoflavone contents in LOX^?KTI⁺ and LOX^?KTI^? isolines were higher than those in the normal parental cultivars. Genetic eliminations also changed isoflavone composition. These results suggest that the genetic removal LOX and KTI may be an efficient approach to improve nutritional and nutraceutical values of soybean seeds, however, performances of null lines should be evaluated for undesirable modifications in seed composition.     

A novel poly(deep eutectic solvent)-based magnetic silica composite for solid-phase extraction of trypsin

Novel poly(deep eutectic solvent) grafted silica-coated magnetic microspheres (Fe₃O₄@SiO₂-MPS@PDES) were prepared by polymerization of choline chloride-itaconic acid (ChCl-IA) and γ-MPS-modified magnetic silica composites, and were characterized by vibrating sample magnetometer (VSM), Fourier transform infrared spectrometry (FT-IR), X-ray photoelectron spectra (XPS), thermal gravimetric analysis (TGA) and transmission electron microscope (TEM). Then the synthetic Fe₃O₄@SiO₂-MPS@PDES microspheres were applied for the magnetic solid-phase extraction (MSPE) of trypsin for the first time. After extraction, the concentration of trypsin in the supernatant was determined by a UV–vis spectrophotometer. Single factor experiments were carried out to investigate the effects of the extraction process, including the concentration of trypsin, the ionic strength, the pH value, the extraction time and the temperature. Experimental results showed the extraction capacity could reach up to 287.5 mg/g under optimized conditions. In comparison with Fe₃O₄@SiO₂-MPS, Fe₃O₄@SiO₂-MPS@PDES displayed higher extraction capacity and selectivity for trypsin. According to the regeneration studies, Fe₃O₄@SiO₂-MPS@PDES microspheres can be recycled six times without significant loss of its extraction capacity, and retained a high extraction capacity of 233 mg/g after eight cycles. Besides, the activity studies also demonstrated that the activity of the extracted trypsin was well retained. Furthermore, the analysis of real sample revealed that the prepared magnetic microspheres can be used to purify trypsin in crude bovine pancreas extract. These results highlight the potential of the proposed Fe₃O₄@SiO₂-MPS@PDES-MSPE method in separation of biomolecules.     

Monitoring of morphology and physical properties of cultured cells using a micro camera and a quartz crystal with transparent indium tin oxide electrodes after injections of glutaraldehyde and trypsin

For investigating the effects of chemical stimulation to cultured cells, we have developed a quartz crystal sensor system with a micro charge-coupled device (CCD) camera that enables microphotograph imaging simultaneously with quartz crystal measurement. Human hepatoma cell line (HepG2) cells were cultured on the quartz crystal through a collagen film. The electrode of the quartz crystal was made of indium tin oxide (ITO) transparent electrodes that enable to obtain a transparent mode photograph. Glutaraldehyde and trypsin were injected to the chamber of the cells, respectively. The response of the quartz crystal was monitored and microphotographs were recorded, and the resonance frequency and resonance resistance were analyzed with an F-R diagram that plotted the resonance frequency and resonance resistance. In the case of the glutaraldehyde injection, the cells responded in two steps that included the fast response of the cross-linking reaction and the successive internal change in the cells. In the case of the trypsin injection, the responses included two processes. In the first step, cell adhesion factors were cleaved and the cell structure became round, and in the next step, the cells were deposited on the quartz crystal surface and the surface of the cells was directly in contact with the quartz crystal surface.     

Diffusion tensor imaging of native and degenerated human articular cartilage

Diffusion tensor imaging (DTI) is potentially sensitive to collagen degeneration in cartilage. In this study, DTI was measured on human cartilage samples with interventions of trypsin and collagenase. The measured preferred diffusion direction was consistent with the zonal structure of collagen network. The glycosaminoglycan concentration decreased and apparent diffusion coefficient increased with both interventions. The fractional anisotropy (FA) was not affected by trypsin and showed a slight increase with combined trypsin and collagenase intervention. DTI in cartilage is technically challenging due to the low FA and the almost undetectable change with collagen disruption seen here.     

Supramolecular-mediated Immobilization of Trypsin on Cyclodextrin-modified Gold Nanospheres

Bovine pancreatic trypsin was immobilized on β- and γ-cyclodextrin coated gold nanospheres via supramolecular associations. The enzyme retained 100%–120% of its catalytic activity and its thermal stability at 50°C was 2–2.5 fold increased in the presence of the β- and γ-cyclodextrin modified metal nanoparticles, respectively. The influence of these immobilization processes on the conformational properties of the enzyme was studied by fluorescence spectroscopy. These results open a new perspective to the possible application of cyclodextrin-modified gold nanospheres as water-soluble carriers for enzyme immobilization.     

AgInS_2∶Mn@ZnS量子点在测定胰蛋白酶中的应用

基于胰蛋白酶能够选择性地猝灭AgInS_2∶Mn@ZnS量子点(QDs)的荧光和磷光,建立了一种检测胰蛋白酶的新方法。实验考察了AgInS_2∶Mn@ZnS QDs对常见蛋白质的选择性以及酸度的影响,优化了测定胰蛋白酶的条件。结果表明,在pH=8.0的磷酸盐缓冲溶液中,荧光猝灭法测定胰蛋白酶的线性范围为5.0×10~(-7)~4.0×10~(-6)mol/L(R=0.9975),检出限为5.0×10~(-8) mol/L;磷光猝灭法测定胰蛋白酶的线性范围为5.0×10~(-7)~3.5×10~(-6) mol/L(R=0.9940),检出限为4.7×10~(-8) mol/L。不同加标水平下的加标回收率在94.1%~107.2%之间,相对标准偏差小于3%。该方法成功地应用于尿样中胰蛋白酶的测定。     

An ultra-fast and highly efficient multiple proteases digestion strategy using graphene-oxide-based immobilized protease reagents

Highly efficient and rapid proteolytic digestion of proteins into peptides is a crucial step in shotgun-based proteome-analysis strategy.Tandem digestion by two or more proteases is demonstrated to be helpful for increasing digestion efficiency and decreasing missed cleavages,which results in more peptides that are compatible with mass-spectrometry analysis.Compared to conventional solution digestion,immobilized protease digestion has the obvious advantages of short digestion time,no self-proteolysis,and reusability.We proposed a multiple-immobilized proteases-digestion strategy that combines the advantages of the two digestion strategies mentioned above.Graphene-oxide(GO)-based immobilized trypsin and endoproteinase Glu-C were prepared by covalently attaching them onto the GO surface.The prepared GO-trypsin and GO-Glu-C were successfully applied in standard protein digestion and multiple immobilized proteases digestion of total proteins of Thermoanaerobacter tengcongensis.Compared to 12-hour solution digestion using trypsin or Glu-C,14%and 7%improvement were obtained,respectively,in the sequence coverage of BSA by one-minute digestion using GO-trypsin and GO-Glu-C.Multiple immobilized-proteases digestion of the total proteins of Thermoanaerobacter tengcongensis showed 24.3%and 48.7%enhancement in the numbers of identified proteins than was obtained using GO-trypsin or GO-Glu-C alone.The ultra-fast and highly efficient digestion can be contributed to the high loading capacity of protease on GO,which leads to fewer missed cleavages and more complete digestion.As a result,improved protein identification and sequence coverage can be expected.