脉冲电场对脂肪氧化酶及多酚氧化酶构象影响的光谱分析

以多酚氧化酶(Polyhenol oxidase, PPO)和脂肪氧化酶(Lipoxygenase, LOX)为研究材料, 利用圆二色谱(CD)和荧光光谱分析了脉冲电场(PEF)对此两种酶蛋白二级和三级构象的影响。PPO和LOX酶活在电场强度为20 kV·cm-1的同轴处理室内处理320 μs分别降低了60.3%和21.7%,并且,随着电场强度的增大, 脉冲处理时间的延长, PPO与LOX酶的活性进一步降低。通过CD光谱图发现, PEF处理后,PPO和LOX的α-螺旋含量显著降低, 而β-折叠含量则呈现增长, 表明PPO与LOX酶蛋白中的二级构象在PEF处理后发生了极大的改变, 酶活钝化和二级结构的破坏之间存在对应关系。PEF处理后LOX的荧光强度增加, 随电场强度增强荧光强度增加幅度显著增大,表明PEF破坏了LOX三级构象,酶活钝化和三级结构的破坏之间存在对应关系。     

应用FTIR研究超声对牛血清白蛋白二级结构的影响

应用傅里叶变换红外光谱(FTIR)结合荧光光谱研究了牛血清白蛋白(BSA)在超声作用下的结构变化。荧光光谱表明,超声作用使BSA溶液荧光光谱最大发射峰发生了蓝移,表明超声改变了BSA中色氨酸(Trp)残基环境;荧光强度的降低表明超声改变了蛋白质分子的构象,具有荧光猝灭效应。采用对BSA红外光谱酰胺Ⅰ带进行曲线拟合的方法,定量分析了不同超声功率、时间对BSA二级结构的影响,发现超声作用对BSA中的α-螺旋、β-折叠、β-转角及无规卷曲的相对含量有不同程度的影响;超声作用具有使BSA的二级结构由α-螺旋向β-折叠、β-转角转化的趋势,而无规则卷曲含量则基本不受超声影响而保持相对稳定。     

超声波处理对脱脂麦胚分离蛋白结构的变化研究

应用傅里叶红外光谱(FTIR)、荧光光谱研究超声处理对脱脂麦胚分离蛋白(DWGP)结构的变化,建立DWGP高效酶解与其结构变化之间的关系.研究发现,超声处理均可提高DWGP的酶解效果,特别是经超声功率600 W、时间10 min处理后,酶解液抑制活性与对照组相比提高了23.96%.通过荧光光谱发现,超声作用改变DWGP溶液荧光强度,适当超声处理可使蛋白分子伸展、生色基团外露,有助于蛋白酶解后获得活性更高的抑制肽.采用对FTIR谱酰胺I带进行曲线拟合的方法,定量分析了不同超声功率、时间对DWGP二级结构的影响,发现超声作用使β-折叠相对含量下降而β-转角含量增加,这可能是DWGP酶解后获得高效抑制肽的主要原因.     

高压脉冲电场对脂肪氧化酶二级和三级构象的影响效果

应用圆二色谱和荧光光谱研究了高压脉冲电场(PEF)对脂肪氧化酶(LOX)的二级和三级构象的影响效果。研究结果表明PEF破坏了LOX二级和三级结构。PEF处理后,LOX的CD光谱中两个特征负峰值显著降低,二级结构中α-螺旋含量显著降低(p<0.05),并且LOX中α-螺旋含量和电场强度之间存在良好线性关系。PEF处理后LOX发射光谱中特征峰(337和583 nm)的荧光强度显著增大(p<0.05),特征峰相对荧光强度和电场强度之间存在良好线性关系。结果表明PEF酶活钝化跟酶二级和三级结构的破坏有关,为PEF钝酶机理研究提供理论基础。     

超高压引发胰蛋白构象变化与酶活性间的关系

采用超高压技术处理胰蛋白酶,改变其空间结构,研究酶空间结构变化与酶活力之间的关系。采用傅立叶红外光谱(FTIR)检测超高压处理后胰蛋白酶的二级结构变化;采用荧光光谱检测处理后胰蛋白酶的三级结构;酶活力的检测采用福林酚法。结果显示,与未处理的相比,在37 ℃,不同压力(100～600 MPa)条件处理20 min,对胰蛋白酶活力影响显著(p＜0.05)。其中,300 MPa处理,胰蛋白酶活力达到最大,较未处理的酶活提高了0.386倍。FTIR检测分析显示,300 MPa处理的胰蛋白酶,α-螺旋与β-转角的峰面积比值达到最大(2.749);内源性荧光光谱检测结果显示,当激发波长为295 nm,其荧光强度达到最高值(1 353);激发波长为280 nm,其荧光强度达到最高(4 262);外源性荧光光谱结果显示,当激发波长为228 nm,疏水氨基酸残基的荧光强度达到最高(2 022); 上述荧光强度的变化较0.1 MPa处理的胰蛋白酶均有显著差异(p＜0.05)。结论：超高压处理影响胰蛋白酶的空间结构及酶活性。其中,胰蛋白酶活性与α-螺旋和β-转角的峰面积的比值、色氨酸等疏水氨基酸及酪氨酸残基暴露程度有关。     

牛α-乳白蛋白-亚油酸复合物的结构及抗肿瘤活性

利用内源性荧光光谱、荧光探针(ANS)结合荧光光谱及圆二色谱,以乳白蛋白-油酸为参照,研究了乳白蛋白结合亚油酸后,疏水性氨基酸、疏水性区域、三级结构及二级结构的变化,并利用亚甲基蓝的方法评价了该复合物的抗肿瘤活性。荧光光谱结果显示,与乳白蛋白-油酸复合物类似,乳白蛋白结合亚油酸后,其内源性荧光光谱显著红移,从331.07 nm移至337.60 nm;外源性ANS结合光谱蓝移,从516.20 nm移至508.50 nm;且荧光强度增加,表明乳白蛋白结合亚油酸后同样出现了疏水性氨基酸及疏水性区域暴露的现象。圆二色谱结果表明,与乳白蛋白-油酸复合物类似,乳白蛋白结合亚油酸后,三级结构部分丧失,二级结构中β-转角及无规卷曲的含量显著降低,β-折叠含量增加。细胞实验证实了乳白蛋白-亚油酸复合物具有良好的抗肿瘤效果。该研究从复合物结构和功能的两方面,为新型抗肿瘤乳白蛋白-亚油酸复合物的开发提供了依据。     

不同处理条件对乳铁蛋白构象的影响研究

运用荧光、圆二色谱及紫外光谱手段,研究了六种不同物理或化学条件处理后,牛乳铁蛋白(Bovine lactoferrin,bLF)三级、二级结构及二硫键的变化。荧光结果显示:6mol.L-1盐酸胍、8mol.L-1尿素和50mmol.L-1二硫苏糖醇三种处理后bLF溶液的最大发射波长从333nm红移至354nm,疏水基团大量暴露,三级结构发生明显变化;100℃加热5min、超声(450W,5s,6个脉冲)、1%巯基乙醇处理后,bLF溶液荧光强度明显减弱,最大发射波长几乎无变化。圆二色谱结果表明:经盐酸胍处理,bLF中α-螺旋结构消失,其余五种处理,二级结构的变化较小。紫外光谱数据表明:二硫苏糖醇对bLF二硫键破坏最严重,超过总二硫键含量的55%,超声次之,盐酸胍、巯基乙醇和加热破坏较少。结果对进一步明确乳铁蛋白的构效关系提供了一定的依据。     

高静压对菠菜类囊体膜叶绿素和蛋白光谱特性的影响

研究了100、250和500MPa高静压(High Hydrostatic Pressure,HHP)处理对菠菜类囊体膜叶绿素浓度、可溶性蛋白浓度、叶绿素室温吸收光谱、叶绿素发射和激发荧光光谱、蛋白荧光光谱的影响。结果表明,同热处理的菠菜相比,HHP处理的菠菜类囊体膜能保持较高的叶绿素和可溶性蛋白浓度,较高的叶绿素室温吸收光谱、叶绿素发射和激发荧光光谱、蛋白荧光光谱能力,并且在100和250MPa时效果更加显著。另外,HHP处理后的菠菜类囊体膜仍能保持光系统Ⅱ捕光和被激发的能力,同时蛋白荧光光谱的变化也反应出HHP处理后类囊体膜蛋白组分的变化。由此推断,HHP保持绿色蔬菜的颜色品质可能与类囊体膜功能特性的维持有关。     

纳米发光材料LnVO4:Eu(Ln=La,Gd,Y)的光谱研究

采用络合溶胶 凝胶法制备了系列纳米发光材料LnVO4:Eu(Ln =La ,Gd ,Y) ,并采用X射线衍射(XRD)、红外光谱 (FTIR)、荧光光谱以及紫外 可见光谱 (UV Vis)对三种发光材料的结构以及发光性质进行研究。结果表明 :YVO4与GdVO4具有相同的结构 ,均属于四方晶系 ,二者具有相似的光谱性质 ;而LaVO4属于单斜晶系 ,受对称性的影响其光谱也相应的发生了变化 ,红外光谱和发射光谱的特征峰发生明显的宽化 ,紫外光谱峰的数目增多     

丹参酚酸A和丹参酚酸B与胰岛素相互作用的分子光谱学研究以及葡萄糖的影响

采用紫外-可见光谱、荧光光谱和傅里叶变换衰减全反射红外光谱等技术,研究在模拟人体生理Ph值条件下,丹参酚酸A(或丹参酚酸B)与胰岛素分子之间的结合作用,以及丹参酚酸A(或丹参酚酸B)对胰岛素二级结构的影响,并考察葡萄糖对它们的影响。实验结果表明,丹参酚酸A和丹参酚酸B均能导致胰岛素内源性荧光静态猝灭;同步荧光和三维荧光谱图表明胰岛素与丹参酚酸A(或丹参酚酸B)结合后,构象没有发生明显变化。红外光谱研究表明,胰岛素与丹参酚酸A(或丹参酚酸B)结合后二级结构发生了一些改变,β-转角和无规则卷曲的相对含量略有增加,α-螺旋和β-折叠没有发生明显改变。葡萄糖的加入会改变丹参酚酸A(或丹参酚酸B)与胰岛素的结合程度,并加剧胰岛素构象变化以及二级结构中α-螺旋相对含量改变,从而影响丹参酚酸A(或丹参酚酸B)-胰岛素体系中胰岛素的生物活性。     

Effects of flat sweep frequency and pulsed ultrasound on the activity,conformation and microstructure of mushroom polyphenol oxidase

The effects of thermal processing (TP) and flat sweep frequency and pulsed ultrasound (FSFPU) treatment with different frequency modes on the activity, conformation and physicochemical properties of mushroom polyphenol oxidase (PPO) were investigated. The results showed that the relative enzymatic activity of PPO gradually decreased with increasing temperature and duration, and thermosonication decreased the PPO activity to a greater extent compared with thermal processing. FSFPU treatment with dual-frequency of 22/40 kHz mode showed the most significant effect. Circular dichroism (CD) showed that the content of α-helix and β-turn dropped, while that of β-sheet and random coil raised after FSFPU treatment. The intensity of endogenous fluorescence decreased, indicating that PPO protein unfolded and the tertiary structure was destroyed. The amount of free sulfhydryl, protein aggregation index, and turbidity all rose. Moreover, FSFPU treatment led to the aggregation of protein from the analysis of atomic force microscope (AFM). Conclusively, FSFPU can be used as an effective method to inhibit the activity of endogenous enzymes in food.     

动态超高压微射流对卵清蛋白微观结构的影响

采用圆二色谱(CD)、X射线衍射(XRD)、ANS荧光探针和紫外(UV)光谱研究了动态超高压微射流对卵清蛋白微观结构的影响。结果表明：卵清蛋白的微观结构的变化与处理压力有关,CD显示不同压力处理的卵清蛋白二级结构中的α-螺旋,β-折叠,β-转角和无规卷曲之间发生相互转化,二级结构的有序性提高;X射线衍射图谱直观显示不同压力处理的卵清蛋白晶体结构增加,160 MPa处理下结晶区最大,说明蛋白结构的有序性提高,与CD分析结果相似;ANS荧光探针光谱显示卵清蛋白的表面疏水性随着处理压力的增大而提高,120 MPa处理下达到最大;紫外光谱显示随着处理压力的增大,卵清蛋白最大紫外吸光值下降,卵清蛋白分子表面的具有紫外吸收的芳香氨基酸残基被包埋于分子内部,卵清蛋白的三维结构发生改变。     

Synchrotron radiation circular dichroism spectroscopy: vacuum ultraviolet irradiation does not damage protein integrity

Synchrotron radiation circular dichroism (SRCD) spectroscopy is an emerging technique for sensitive determination of protein secondary structures and for monitoring of conformational changes. An important issue for its adoption as a useful technique is whether the high-intensity low-wavelength vacuum ultraviolet radiation in the SRCD chemically damages proteins. In this paper, using horse myoglobin as a test sample, it is shown that extensive irradiation in the SRCD does not produce any change in the chemical nature of the protein as detected by either SDS gel electrophoresis or mass spectrometry. In addition, no changes in the protein secondary structure are detectable from the SRCD spectra after extensive exposure to the SRCD beam.     

热处理对蛋清卵类粘蛋白过敏原性及构象的影响

采用酶联免疫吸附法(ELISA)、圆二色谱(CD)、ANS荧光探针和紫外光谱(UV)系统研究了热处理对蛋清卵类粘蛋白过敏原性及构象的影响。结果显示,加热处理卵类粘蛋白的过敏原性降低,且随加热温度升高和加热时间延长,不断降低。经不同温度热处理后的卵类粘蛋白二级结构的α-螺旋,β-折叠,β-转角和无规卷曲之间相互转化,分子有序性降低;卵类粘蛋白的表面疏水性随加热温度的升高而降低;随加热的温度的升高,具有紫外吸收的氨基酸残基逐渐暴露,最大吸光度逐渐增大。由此可以推断,卵类粘蛋白的构象改变导致其过敏原性变化。     

Study of the interaction of gemini surfactant NAE12-4-12 with bovine serum albumin

The interaction of 1,4-bis(3-(dodecyloxylacyl)pyridinium)butane dibromide (designated as NAE12-4-12) and bovine serum albumin (BSA) was investigated by UV–vis absorption, FTIR and fluorescence spectroscopies. The results showed that NAE12-4-12 had strong ability to quench the intrinsic fluorescence of BSA and caused the emission peak blueshift through a static quenching process. The binding constant of NAE12-4-12 with BSA decreased with increasing temperature. The binding process was exothermic, spontaneous and enthalpy driven. The distance between BSA and NAE12-4-12 decreased with incremental concentration of NAE12-4-12. Furthermore, FTIR spectra of BSA–NAE12-4-12 reflected that the secondary structure of BSA changed in the presence of NAE12-4-12, and the curve fitting of IR spectra revealed that the content of α-helix decreased while those of β-sheet, β-turn and random coil rose.     

Effect of high intensity ultrasound on the structure and solubility of soy protein isolate-pectin complex

In this study, a soy protein isolate (SPI)-pectin (PC) complex was prepared, and the effects of different high intensity ultrasound (HIU) powers on the structure and solubility of the complex were studied. Fourier transform infrared (FTIR) spectroscopy analysis exhibited that with increasing HIU power, the α-helix content of the SPI in the complex was significantly reduced, and the random coil content increased; however, an opposite trend appeared after higher power treatments. Fluorescence spectra showed that HIU treatment increased the fluorescence intensity of the complex, and the surface hydrophobicity was increased. The trend of the protein structure studied by Raman spectroscopy was similar to that of FTIR and fluorescence spectroscopy. When the HIU treatment was performed for 15 min and at 450 W power, the particle size of the complex was 451.85 ± 2.17 nm, and the solubility was 89.04 ± 0.19 %, indicating that the HIU treatment caused the spatial conformation of the protein to loosen and improved the functional properties of the complex. Confocal laser scanning microscopy (CLSM) revealed that the complex after HIU treatment exhibited improved dispersibility in water and smaller particle size. Gel electrophoresis results indicated that HIU treatment did not affect the protein subunits of the complex. Therefore, the selection of a suitable HIU treatment power can effectively improve the structural properties and solubility of SPI in the complex, and promote the application of the SPI-PC complex in food processing and industries.     

S-构型转化对卵白蛋白微观结构的影响

采用圆二色谱(CD)、X射线衍射(XRD)、ANS荧光探针和紫外光谱(UV)研究了S-构型转化对卵白蛋白微观结构的影响.结果显示,不同诱导时间处理的卵白蛋白二级结构的α-螺旋,β-折叠,β-转角和无规卷曲之间相互转化,α-螺旋略有减少,β-折叠相应增加,分子有序性提高;S-构型转化后卵白蛋白晶体结构增加,72 h处理后...