基于真空LHPEF结合胰蛋白酶降解猪血红蛋白

在真空及低压高频脉冲（LHPEF）条件下,结合胰蛋白酶降解猪Hb,利用高效凝胶色谱、Native-PAGE蛋白电泳及紫外扫描来测定其降解效果。结果：在37℃温度下,1.88 V、200 kHz的LHPEF处理猪Hb,电导率为3.45326 ×10^5 μs/cm（对照组为0.645×10^4 μs/cm）。紫外扫描曲线图中的真空LHPEF胰蛋白酶降物中有8个特征吸收峰,而对照组中为6个吸收峰,Native- PAGE蛋白质电泳图中有13个明显色带,而对照组中只有6个色带,高效凝胶色谱图中的特征吸收峰为48个,对照组中7个特征吸收峰。     

棉铃虫中肠类胰蛋白酶的部分纯化和性质测定及其对苏云金杆菌δ—内毒素的降解作用

将棉铃虫Helicoverpa armingera中肠液经SephadexG-75、DEAE-SephadexA-50阴离子交换和CM-SephadexC-50阳离子交换柱层析进行分离纯化,得到部分纯化的24.5kD蛋白,该蛋白可以水解胰蛋白酶的专性底物BApNA和TAME,不能水解胰凝乳蛋白酶的专性底物BTEE,蛋白酶抑制剂的抑制试验显示,TLCK、PMSF、STI及SBBI均可显著抑制该蛋白对BApNA的水解作用,而TPCK无抑制作用,由此推断,24.5kD蛋白是棉铃虫的类胰蛋白酶。以BApNA为底物,类胰蛋白酶的最适pH为10.5-11.0。SephadexG-75分离得到了4个洗脱峰,电泳结果显示,各洗脱峰均能使苏云金杆菌库斯塔克变种Bacillus thuringiensis var.kurstakiHD-1原毒素降解为60.5kD的活力片段,峰Ⅳ虽然不能水解BApNA和TAME,但仍可对原毒素进行降解,而类胰蛋白酶活力最高的峰Ⅱ已将60.5kD蛋白完全降解,当减少用量至1/3时,才出现60.5kD蛋白。因此,苏云金杆菌δ-内毒素在棉铃虫中肠内的降解有多种蛋白酶参与,类胰蛋白酶在其中起重要作用,CM-SephadexC-50进一步分离得到的类胰蛋白酶可对原毒进行降解,其降解作用受到TLCK、PMSF、STI及SBBI的显著抑制,不受TPCK的影响。     

从半夏中提取的胰蛋白酶抑制剂及其特征

应用硫酸铵分级、无离子水沉淀、2.5%TCA 处理、DEAE-Sephadex A-50离子交换层析以及胰蛋白酶-琼脂糖凝胶亲和层析,从植物半夏新鲜块茎中分离纯化到一种胰蛋白酶抑制剂。半夏抑制剂只抑制胰蛋白酶对酰胺、酯、血红蛋白和酪蛋白的水解,不能抑制胰凝乳蛋白酶、舒缓激肽释放酶、枯草杆菌蛋白酶和木瓜蛋白酶对各自底物的水解。抑制剂对猪胰蛋白酶水解酰胺、酯、血红蛋白和酪蛋白的重量抑制比值分别为1∶0.71、1∶0.88、1∶0.71和1∶0.71。从分子大小的范围看,半夏胰蛋白酶抑制剂应属大分子抑制剂。     

从半夏中提取的胰蛋白酶抑制剂及其特征

应用硫酸铵分级、无离子水沉淀、2.5%TCA处理、DEAE-Sephadex A-50离子交换层析以及胰蛋白酶-琼脂糖凝胶亲和层析,从植物半夏新鲜块茎中分离纯化到一种胰蛋白酶抑制剂。半夏抑制剂只抑制胰蛋白酶对酰胺、酯、血红蛋白和酪蛋白的水解,不能抑制胰凝乳蛋白酶、舒缓激肽释放酶、枯草杆菌蛋白酶和木瓜蛋白酶对各自底物的水解。抑制剂对猪胰蛋白酶水解酰胺、酯、血红蛋白和酪蛋白的重量抑制比值分别为1∶0.71、1∶0.88、1∶0.71和1∶0.71。从分子大小的范围看,半夏胰蛋白酶抑制剂应属大分子抑制剂。     

芦丁与胰蛋白酶相互作用的光谱性质研究

利用紫外光谱和荧光光谱研究了芦丁和胰蛋白酶的相互作用机制。结果表明,生理pH 7.40条件下芦丁使胰蛋白酶的紫外吸收峰增强,特征荧光峰淬灭。并利用荧光淬灭反应测得芦丁和胰蛋白酶之间结合常数KA=6.8786×104(mol/L)-1,结合位点数n=1.0173。     

几种蛋白酶对文蛤肉的酶解工艺条件研究

以文蛤为原料,水解度为指标,从胰蛋白酶,木瓜蛋白酶,胃蛋白酶和风味蛋白酶中选出水解效果较好的胰蛋白酶和木瓜蛋白酶.并通过实验确定了胰蛋白酶,木瓜蛋白酶单酶水解及两者组合复合酶解文蛤肉的最佳工艺.结果表明:复合水解效果最佳.最佳工艺:先添加胰酶6000 u/g(原料),水解温度50℃,固液比1:3(V/W),pH 8.0,在此条件下水解6 h;然后改变条件,温度55℃,pH 5.5,底物浓度1:3(V/W),添加木瓜蛋白酶2000 u/g(原料),在此条件下水解2 h.通过实验验证,胰蛋白酶和木瓜蛋白酶组合在该条件下对文蛤肉蛋白具有较好的水解效果,其水解度为28.15%.     

基于串联质谱的鱼皮明胶鉴别研究

在胶原蛋白序列比对基础上,以虹鳟鱼明胶、猪明胶和牛明胶为模型,利用高效液相色谱－串联质谱技术（HPLC-MS/MS）研究了3种明胶降解多肽组成的差异。使用胰蛋白酶将鱼皮明胶进行了酶解处理,使用HPLC-MS/MS对酶解产物中的多肽组成进行了分析,并与猪和牛明胶酶解产物中的多肽进行了比较。结果表明鱼明胶酶解产物中存在特征多肽,通过特征多肽的种类可区别鱼明胶与猪和牛明胶,研究了明胶多肽中脯氨酸羟基化修饰、明胶分子量范围和脱酰胺化对特征多肽识别的影响。研究表明利用HPLC-MS/MS技术通过识别明胶酶解产物中的特征多肽进行鱼皮明胶鉴别具有可行性。     

猪血红蛋白的酶解及氨基酸含量的研究

猪血是一种营养价值高而价格低廉的蛋白质资源.猪血中含有丰富的蛋白质,其中血红蛋白占主要部分.因此,对血红蛋白及其水解产物的研究受到了国内外众多学者的关注.为选出适合于水解猪血红蛋白的蛋白酶,笔者采用碱性蛋白酶、木瓜蛋白酶、中性蛋白酶对猪血进行单酶和复合酶水解试验,确定碱性蛋白酶为水解猪血的适用酶.通过质谱仪测定,碱性蛋白酶酶解产物分子量集中在800～2400道尔顿,而800～1260道尔顿区间占主要部分;经全自动氨基酸分析仪测出其产物含有17种氨基酸,其中含8种必需氨基酸,必需氨基酸占总氨基酸量的50.93%,说明碱性蛋白酶酶解产物是制作食品和饲料添加剂的理想资源.     

花蚬酶解物清除羟自由基作用研究

探讨了不同蛋白酶酶解花蚬蛋白所得酶解物对Fenton体系产生的羟自由基(.OH)的清除效果,然后进行Sephadex G-25凝胶柱分离酶解产物中的抗氧化活性肽,并测定活性肽相对分子质量分布。结果表明:木瓜蛋白酶在50℃、酶解30 min、pH=7.5、酶质量分数0.15%、m(底物)∶m(水)=1∶2的水解条件下,酶解物对羟自由基的清除效果最佳,清除率为86.9%;胰蛋白酶在温度55℃、酶解时间85 min、pH=8.0、酶质量分数0.30%、m(底物)∶m(水)=1∶2的水解条件下,酶解物对羟自由基清除效果最佳,清除率为89.5%。木瓜蛋白酶酶解物在最大洗脱峰时,清除率为84.73%,在最大峰处酶解物中活性肽的相对分子质量为5.68×103;胰蛋白酶酶解物有两个洗脱峰,在最大洗脱峰处分离组分对羟自由基的清除率很低,在较小洗脱峰处,其清除率为88.49%,该峰处活性肽的相对分子质量为1.165×104。     

高功率微波辐照对视细胞脂质结构的影响

目的:研究高功率微波( HPM)照射后视网膜感光细胞中膜脂质的分子结构改变,探讨HPM的损伤机制.方法:通过显微FT-IR技术观察HPM照射前后大鼠视网膜冰冻切片中感光细胞外节的膜脂质分子特征吸收峰的位移与吸收值差异,分析脂质分子结构的改变情况.结果:HPM照后外节发生了膜脂质分子特征吸收峰的位移,并出现=C-H基团和...     

Hemoglobins, XXI: sequence analysis of porcine hemoglobin (author's transl)

The hemoglobin of a bavarian domestic pig (Suidae) was isolated. The chains were separated by countercurrent distribution, then cleaved with trypsin. The isolated peptides were sequenced with hydrophilic phenylisothiocyanate I and IV, or with a dimethylaminopropyne program in the sequenator. The sequences of the chains are given. Some methodical aspects of automatic sequencing are discussed and the sequences of human porcine hemoglobin are compared. The role of adult pig haemoglobin as foetal hemoglobin is discussed.     

Comparison of membrane electroporation and protein denature in response to pulsed electric field with different durations

In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field‐induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity. Bioelectromagnetics 34:253–263, 2013. © 2012 Wiley Periodicals, Inc.     

Improvement of antioxidant activity of peptides with molecular weights ranging from 1 to 10 kDa by PEF technology

Egg white protein powder was hydrolyzed with Alcalase to produce antioxidant peptides. Then, the peptides were fractionated with ultrafiltration membranes. The peptides (1-10 kDa) were further treated by pulsed electric field (PEF) to investigate its effect on the antioxidant activity of the peptides. Antioxidant activity was evaluated using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical inhibition assay. The results indicated that optimal electric field intensity and standing times of PEF can enhance the antioxidant activity of the peptides. Therefore, a Box-Behnken design (BBD) with three independent variables including concentration, electric field intensity and pulse frequency was used to establish the regression equation of second-order response surface. The optimal conditions were as follows: concentration 8 mg/ml, electric field intensity 10 kV/cm and pulse frequency 2400 Hz. Under these conditions, the peptides antioxidant activity was 62.64% ± 0.98%. The present study demonstrated that the antioxidant activity of peptides (1-10 kDa) could be improved using PEF.     

Effects of Electromagnetic Fields on the Bioactivities of an Osteoblast-Like Cell Line (UMR-106)

In the present study, we first investigated the effects of various types of low-energy, low-frequency electromagnetic fields (EMFs) on DNA synthesis in UMR-106 osteoblast-like cells. The experimental groups were exposed to EMFs for 2 days (twice/day, 30 min/time), and DNA synthesis was measured. The results showed that the cells responded most sensitively to EMFs of some specific combinations of the parameters by an increase in DNA synthesis, implying that EMFs with a specific waveform rather than a complex one can be used in clinical electrotherapy. The parameters were as follows: pulsed electric field (PEF) with pulse width 0.2 ms, field strength 10 V/cm, frequency 125 Hz; sinusoidal electric field (SEF) with field strength 1 V/cm, frequency 10 Hz; and alternating magnetic field (AMF) with field density 0.5 mT, frequency 5 Hz. In addition to frequency, the field strength or field density within a suitable intensity scale played a dominant role in causing the DNA synthesis response. We then compared the effects of two kinds of fields, PEF and AMF, with the optimum parameters identified by the experiments, on alkaline phosphatase (ALP) activity, protein and collagen synthesis, and intracellular levels of cyclic adenosine monophosphate (cAMP). The results indicated that both fields could not only affect UMR-106 cells proliferation but could particularly affect a series of characteristic bioactivities of UMR-106 such as ALP activity and collagen synthesis. The intracellular cAMP levels were increased rapidly and greatly with exposure to both PEF and AMF, implying that the action of low-frequency EMFs proceeds via second messenger-dependent processes originating from signals at the cell membrane. The difference in action between PEF and AMF suggests that they may couple to the cell membrane in a partially different way.     

Influence of a pulsed electric field on the spores and oxygen consumption of Aspergillus niger and its citric acid production

Spores of Aspergillus niger were exposed to a pulsed electric field. After treatment by the electric field, the viability of the conidia of A. niger varied depending on the field strength, pulse width and frequency. In all cases, these parameters reduced the viability rate of the conidia from 2.0 × 10⁷ to a range from 6.2 × 10⁶ to 8.5 × 10⁶ spores/ml (3.1 to 42.6%). After pulse treatment, the conidia were used as the inoculum for citric acid fermentation in shake flasks. The highest increase in citric acid yield (about 1.4-fold) was reached at a field strength of 2.85 kV/cm, a frequency of 1 Hz and a pulse width of 1 ms. When the parameters of the electric field increased there were important changes in the respiration rate of the Aspergillus niger mycelium (48-h-old) after electric shock treatment. The highest consumption of dissolved oxygen (22.9%) in the medium by Aspergillus niger mycelium was observed at an electric field strength of 2.85 kV/cm, a 1 Hz frequency, a pulse width of 1 ms and a 1-min exposure period. It seems that an electric-field stimulation of the conidia prior to inoculation may offer an important method of improving the efficiency of citric acid. The treatment of the conidia is both simple from the technical point of view and extremely rapid.     

Effect of pulsed electric fields on the activity of neutral trehalase from beer yeast and RSM analysis

The trehalase activity plays an important role in extraction of trehalose from beer yeast. In this study, the effect of pulsed electric field processing on neutral trehalase activity in beer yeast was investigated. In order to develop and optimize a pulsed electric field (PEF) mathematical model for activating the neutral trehalase, we have investigated three variables, including electric field intensity (10-50 kV/cm), pulse duration (2-10 μs) and liquid-solid ratio (20-50 ml/g) and subsequently optimized them by response surface methodology (RSM). The experimental data were fitted to a second-order polynomial equation and profiled into the corresponding contour plots. Optimal condition obtained by RSM is as follows: electric field intensity 42.13 kV/cm, liquid-solid ratio 30.12 ml/g and pulse duration 5.46 μs. Under these conditions, with the trehalose decreased 8.879 mg/L, the PEF treatment had great effect on activating neutral trehalase in beer yeast cells.     

In vitro increase of the fluid-phase endocytosis induced by pulsed radiofrequency electromagnetic fields: importance of the electric field component

Nowadays, due to the wide use of mobile phones, the possible biological effects of electromagnetic fields (EMF) become a public health general concern. Despite intensive research, there are no widely accepted theories about the interactions between EMFs and living cells, and the experimental data are often controversial. We examined the effects of mobile phones EMF (envelope frequency of 217 Hz, carrier frequency of 900 MHz and pulse duration of 580 micros) or its pure, low-frequency pulsed electric field component on fluid-phase endocytosis. In both cases, with exposures exceeding 10 min, an increase of the fluid-phase endocytosis rate was observed ( approximately 1.5-fold), on three different cell types. This increase is an all-or-nothing type of response that is occurring for threshold values comprised between 1.3 and 2.6 W/kg for the delivered EMF powers and between 1.1 and 1.5 V/cm for the electric fields intensities depending upon the cell type. The electric component of these EMFs is shown to be responsible for the observed increase. Variations of frequency or pulse duration of the electric pulses are shown to be without effect. Thus, EMF, via their electrical component, can perturb one of the most fundamental physiological functions of the cells-endocytosis.     

The Effect of Electrical Deformation Forces on the Electropermeabilization of Erythrocyte Membranes in Low- and High-Conductivity Media

Electrical breakdown of erythrocytes induces hemoglobin release which increases markedly with decreasing conductivity of the pulse medium. This effect presumably results from the transient, conductivity-dependent deformation forces (elongation or compression) on the cell caused by Maxwell stress. The deformation force is exerted on the plasma membrane of the cell, which can be viewed as a transient dipole induced by an applied DC electric field pulse. The induced dipole arises from the free charges that accumulate at the cell interfaces via the Maxwell-Wagner polarization mechanism. The polarization response of erythrocytes to a DC field pulse was estimated from the experimental data obtained by using two complementary frequency-domain techniques. The response is very rapid, due to the highly conductive cytosol. Measurements of the electrorotation and electrodeformation spectra over a wide conductivity range yielded the information and data required for the calculation of the deformation force as a function of frequency and external conductivity and for the calculation of the transient development of the deformation forces during the application of a DC-field pulse. These calculations showed that (i) electric force precedes and accompanies membrane charging (up to the breakdown voltage) and (ii) that under low-conductivity conditions, the electric stretching force contributes significantly to the enlargement of ``electroleaks' in the plasma membrane generated by electric breakdown. Received: 12 December 1997/Revised: 13 March 1998     

In vitro increase of the fluid-phase endocytosis induced by pulsed radiofrequency electromagnetic fields: importance of the electric field component

Nowadays, due to the wide use of mobile phones, the possible biological effects of electromagnetic fields (EMF) become a public health general concern. Despite intensive research, there are no widely accepted theories about the interactions between EMFs and living cells, and the experimental data are often controversial. We examined the effects of mobile phones EMF (envelope frequency of 217 Hz, carrier frequency of 900 MHz and pulse duration of 580 μs) or its pure, low-frequency pulsed electric field component on fluid-phase endocytosis. In both cases, with exposures exceeding 10 min, an increase of the fluid-phase endocytosis rate was observed (≈1.5-fold), on three different cell types. This increase is an all-or-nothing type of response that is occurring for threshold values comprised between 1.3 and 2.6 W/kg for the delivered EMF powers and between 1.1 and 1.5 V/cm for the electric fields intensities depending upon the cell type. The electric component of these EMFs is shown to be responsible for the observed increase. Variations of frequency or pulse duration of the electric pulses are shown to be without effect. Thus, EMF, via their electrical component, can perturb one of the most fundamental physiological functions of the cells—endocytosis.