圆二色光谱揭示光系统Ⅱ在热处理过程中叶绿素荧光动力学变化的原因

研究了光系统Ⅱ(PSⅡ)在热处理过程中的叶绿素a荧光和圆二色(CD)光谱.在30℃～40℃热处理过程中,PSⅡ的叶绿素荧光参数Fo′保持稳定不变;当温度大于40℃时,Fo'逐渐升高并在55℃达到最大值.在PSⅡ颗粒和富含捕光色素(LHCⅡ)的复合物的热处理过程中,具有超大振幅的CD异常信号出现,并且在40℃时,677 nm的异常CD峰强度达到最大.这些结果暗示在PS Ⅱ颗粒热处理过程中,PSⅡ颗粒中的LHCⅡ的聚集状态和异常CD信号相关,并且也可能是影响Fo′的一个重要因素.     

克山病心肌线粒体氧化损伤的实验研究

用克山病区粮喂养豚鼠证明其红细胞、肝等组织及其线粒体、上清中的硒及谷胱甘肽过氧化物酶活性明显降低,心肌线粒体脂质氢过氧化物、荧光色脂等增加、同时膜脂组成异常,内膜界面脂的心磷脂(CL)减少,细胞色素C氧化酶(CCO)活性降低;园二色性(CD)异常,近208nm及222nm区的峰值降低,病区粮加喂青菜的动物,随其线粒体硒及CL水平的增加。CCO活性和CD谱均明显恢复;通过外源性磷脂与纯化CCO的重组复性实验,只有CL可使病区粮组酶活性完全达到正常对照水平。表明心肌线粒体膜结合酶CCO活性的降低是CL含量降低引起酶的二级结构(构象)改变的结果。     

非酶糖基化对α-synuclein分子构象的影响

将纯化后的α－synuclein分别与果糖和葡萄糖孵育,通过内源荧光、非酶糖基化衍生物特征荧光、圆二色光谱以及电子显微镜等技术进行检测发现：α—synuclein与还原糖共同孵育后,308nm内源荧光强度明显降低,同时在447nm产生一个非酶糖基化衍生物特征荧光．与果糖孵育的蛋白质样品其非酶糖基化特征荧光的出现速度快于葡萄糖孵育样品．内源荧光与非酶糖基化特征荧光之间存在能量传递现象,提示Tyr残基与非酶糖基化特征荧光发色团在空间距离上彼此接近．圆二色光谱测定结果显示,α－synuclein与果糖孵育后,其α－螺旋含量增加．非酶糖基化的α－synuclein在电子显微镜下表现为短纤维状．非酶糖基化可以诱导α－synuclein蛋白分子聚集,且果糖较葡萄糖更容易使α－synuclein发生非酶糖基化．以上结果提示,非酶糖基化似乎可以导致α－synuclein在细胞内的错误折叠和分子聚集．     

圆二色光谱揭示光系统Ⅱ在热处理过程中叶绿素荧光动力学变化的原因

研究了光系统Ⅱ(PSⅡ)在热处理过程中的叶绿素a荧光和圆二色(CD)光谱。在30 ℃～40 ℃热处理过程中,PSⅡ的叶绿素荧光参数Fo'保持稳定不变;当温度大于40 ℃时,Fo' 逐渐升高并在55 ℃达到最大值。在PSⅡ颗粒和富含捕光色素(LHCⅡ)的复合物的热处理过程中,具有超大振幅的CD异常信号出现,并且在40 ℃时, 677 nm的异常CD峰强度达到最大。这些结果暗示在PSⅡ颗粒热处理过程中,PSⅡ颗粒中的LHCⅡ的聚集状态和异常CD信号相关,并且也可能是影响Fo'的一个重要因素。     

热激诱导的玉米幼苗耐热性及其与脯氨酸的关系

研究了热激对玉米幼苗耐热性的效应及其与脯氨酸的关系。结果表明,培养2.5 d的玉米幼苗经过42℃热激4 h并于26.5℃下恢复4 h后,提高了玉米幼苗在48℃下的存活率,并且热激及其后的恢复过程中都表现出脯氨酸的积累。不同浓度的外源脯氨酸预处理也可提高玉米幼苗内源脯氨酸的水平和抗氧化酶抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽还原酶(GR)、过氧化物酶(GPX)的活性,从而提高玉米幼苗在高温胁迫下的存活率。这些结果暗示热激过程中脯氨酸的积累所诱发的抗氧化酶活性的增强可能是热激诱导的玉米幼苗耐热性形成的生理基础之一。     

硒、谷胱甘肽过氧化物酶和不饱和脂肪酸在鼠脑亚细胞中的分布

采用梯度离心和放射性同位素等方法从鼠脑中分离得到髓磷脂、突触囊、轻突触体、重突触体、线粒体６个亚细胞组分。分别测定了各亚细胞中硒－７５、谷胱甘肽过氧化物酶和不饱和脂肪酸的含量,结果表明这些成分在鼠脑亚细胞中的分布呈现明显的相关性,同时首次在突触囊、线粒体和微粒体中检测到三种不同的谷胱甘肽过氧化物酶的活性峰,其中之一可能是红细胞谷胱甘肽过氧化物酶（ＥＣ１．１１．１．９）．还就机体的自我保护机制和硒在脑组织中的重要作用进行了讨论。     

褐飞虱高温条件下应激反应及体内保护酶系活性的研究

利用34℃、36℃、38℃3个温度处理褐飞虱的若虫和不同型成虫,研究其在热休克过程中体内保护酶系活性及脂质过氧化物含量的变化。结果表明,热休克对4龄若虫过氧化氢酶（CAT）活性影响大于3龄、5龄,而其成虫期CAT活性则随日龄增加而增加,36℃为CAT清除H2O2最适温度。高龄若虫全内谷胱甘肽过氧化物本(GSH-px）活性高于低龄若虫,老熟成虫体内GSH－px清除H2O2能力强于初羽化成虫,超氧化物歧化酶（SOD）活性与处理温度呈正相关,而同一处理温度下SOD活性随成虫龄期和成虫日龄增加而降低,长型成虫体内CAT、GSH－px、SOD活性均高于短翅型,而雌、雄成虫间CAT、GSH－px、SOD活性无明显差异（α＞0.05）。褐飞虱体内脂质过氧化物（LPO）含量随处理温度升高而上升,同一处理温度下随若虫龄期和成虫日龄的增加而增加,短型成虫体内LPO含量高于长翅型。     

荧光标记脂肪酶的固定化及其稳定性

将标记有荧光探针FITC(异硫氰基荧光素)的脂肪酶固定化,通过测定活性和荧光光谱,探究各种因素对固定化后荧光标记脂肪酶性质的影响,并分析活性、构象和荧光光谱三者之间的联系。研究结果表明:在固定化脂肪酶过程中,聚乙二醇400二丙烯酸酯能形成合理的网格结构,使酶活较高;配体诱导酶的催化构象,使酶活性提高到未诱导酶的2倍以上;配体抽提能使脂肪酶活性中心得到释放从而提高催化活力。固定化脂肪酶的稳定性大大提高,在90℃、强酸强碱下固定化酶仍保有原酶70%、60%以上的活性;用盐酸胍、脲等溶解变性剂浸泡15d后,酶活性仍然可以保持初始活性的70%以上。荧光光谱能较好地反映脂肪酶的活性和构象变化,最适pH和温度下脂肪酶的荧光强度最低,在溶解变性剂中,荧光强度随时间延长而逐渐降低,这表明不同条件下脂肪酶构象经历的去折叠过程不同。     

嗜热脂肪芽孢杆菌HY-69耐热金属蛋白酶基因表达产物的纯化及性质研究

利用CM纤维素离子交换层析法,从宿主细胞枯草芽孢杆菌MI113中纯化了嗜热脂肪芽孢杆菌HY69的耐热金属蛋白酶基因的表达产物,达电泳纯。该酶的最适反应温度为70℃,有着较好的热稳定性和极高的盐酸胍抗性。70℃的半寿期为45min。在3mol/L的盐酸胍中变性20min,仍残余近40%的酶活。利用凝胶过滤和SDSPAGE,测定其分子量均为27 000±1 000。通过CD光谱得知,该酶含有66%的α-螺旋,28%的β-转角,6%的无规则卷曲,无β-折叠。利用CD光谱和荧光光谱研究了该酶在盐酸胍变性过程中的构象变化,推测其主要是通过增加包装效率,减少无规则卷曲来提高酶的稳定性。     

硒,谷胱甘肽过氧化物酶和不饱和脂肪酸在鼠亚细胞中的分布

采和梯度离心和放射性同位素等方法从鼠脑中分离得到髓磷脂、突触囊、轻突触体、重突触体、线粒体６个亚细胞组分,分别测定了各亚细胞中硒－７５、谷胱甘肽过氧化物酶和不饱和脂肪酸的含量,结果表明这上结成分在鼠脑亚细胞中的分布呈明显的相关性,同时首次在突触囊、线粒体和微粒体中检测到三咱不同的谷胱甘肽过氧化物酶的活性峰,其中之一可能是红细胞谷胱甘过氧化物酶（ＥＣ１．１１．１．９）。还就机体的自我保护机制和硒在脑     

Association of heat-induced conformational change with activity loss of Rubisco.

Circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) were used to investigate the thermal conformational change associated with the activity loss of spinach Rubisco. CD and intrinsic fluorescence demonstrated a three stage thermal unfolding of Rubisco. At 25-45 degrees C, the secondary structure did not change but the tertiary and/or quaternary structure changed obviously with increased temperature. In 45-60 degrees C, the secondary structure showed much change with increased temperature and the tertiary and/or quaternary structure changed much faster. Over 60 degrees C, whole conformation changed abruptly with increased temperature and finally unfolded completely. DSC, CD and activity assays after annealing showed that the conformational change and the activity loss of Rubisco were completely reversible if the heating temperature was below 45 degrees C, partly reversible between 45 and 60 degrees C, and irreversible beyond 60 degrees C.     

Purification and physicochemical characterization of a recombinant phospholipid hydroperoxide glutathione peroxidase from Oryza sativa

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an unique antioxidant enzyme that directly reduces lipid hydroperoxides in biomembranes. In the present work, the entire encoding region for Oryza sativa PHGPx was expressed in Escherichia coli M15, and the purified fusion protein showed a single band with 21.0 kD and pI = 8.5 on SDS- and IFE-PAGE, respectively. Judging from CD and fluorescence spectroscopy, this protein is considered to have a well-ordered structure with 12.2% alpha-helix, 30.7% beta-sheet, 18.5% gamma-turn, and 38.5% random coil. The optimum pH and temperature of the enzyme activity were pH 9.3 and 27 degrees C. The enzyme exhibited the highest affinity and catalytical efficiency to phospholipid hydroperoxide employing GSH or Trx as electron donor. Moreover, the protein displayed higher GSH-dependent activity towards t-Butyl-OOH and H(2)O(2). These results show that OsPHGPx is an enzyme with broad specificity for hydroperoxide substrates and yielded significant insight into the physicochemical properties and the dynamics of OsPHGPx.     

Structural stability of the PsbQ protein of higher plant photosystem II

We have characterized the stability and folding behavior of the isolated extrinsic PsbQ protein of photosystem II (PSII) from a higher plant, Spinacia oleracea, using intrinsic protein fluorescence emission and near- and far-UV circular dichroism (CD) spectroscopy in combination with differential scanning calorimetry (DSC). Experimental results reveal that both chemical denaturation using guanidine hydrochloride (GdnHCl) and thermal unfolding of PsbQ proceed as a two-state reversible process. The denaturation free-energy changes (DeltaG(D)) at 20 degrees C extrapolated from GdnHCl (4.0 +/- 0.6 kcal mol(-1)) or thermal unfolding (4.4 +/- 0.8 kcal mol(-1)) are very close. Moreover, the far-UV CD spectra of the denatured PsbQ registered at 90 degrees C in the absence and presence of 6.0 M GdnHCl superimpose, leading us to conclude that both denatured states of PsbQ are structurally and energetically similar. The thermal unfolding of PsbQ has been also characterized by CD and DSC over a wide pH range. The stability of PsbQ is at its maximum at pH comprised between 5 and 8, being wider than the optimal pH for oxygen evolution in the lumen of thylakoid membranes. In addition, no significant structural changes were detected in PsbQ between 50 and 55 degrees C in the pH range of 3-8, suggesting that PsbQ behaves as a soluble and stable particle in the lumen when it detaches from PSII under physiological stress conditions such as high temperature (45-50 degrees C) or low pH (<5.0). Sedimentation experiments showed that, in solution at 20 degrees C, the PsbQ protein is a monomer with an elongated shape.     

Double thermal transitions of type I collagen in acidic solution

Contributed equally to this work. To further understand the origin of the double thermal transitions of collagen in acidic solution induced by heating, the denaturation of acidic soluble collagen was investigated by micro-differential scanning calorimeter (micro-DSC), circular dichroism (CD), dynamic laser light scattering (DLLS), transmission electron microscopy (TEM), and two-dimensional (2D) synchronous fluorescence spectrum. Micro-DSC experiments revealed that the collagen exhibited double thermal transitions, which were located within 31–37?°C (minor thermal transition, T _s?～?33?°C) and 37–55?°C (major thermal transition, T _m?～?40?°C), respectively. The CD spectra suggested that the thermal denaturation of collagen resulted in transition from polyproline II type structure to unordered structure. The DLLS results showed that there were mainly two kinds of collagen fibrillar aggregates with different sizes in acidic solution and the larger fibrillar aggregates (T _p2?=?40?°C) had better heat resistance than the smaller one (T _p1?=?33?°C). TEM revealed that the depolymerization of collagen fibrils occurred and the periodic cross-striations of collagen gradually disappeared with increasing temperature. The 2D fluorescence correlation spectra were also applied to investigate the thermal responses of tyrosine and phenylalanine residues at the molecular level. Finally, we could draw the conclusion that (1) the minor thermal transition was mainly due to the defibrillation of the smaller collagen fibrillar aggregates and the unfolding of a little part of triple helices; (2) the major thermal transition primarily arose from the defibrillation of the larger collagen fibrillar aggregates and the complete denaturation of the majority part of triple helices.     

Enhanced Unfolding of Bovine β-Lactoglobulin Structure Using Microwave Treatment: a Multi-Spectroscopic Study

Commercial whey protein hydrolysates containing bovine β-lactoglobulin (β-Lg) may have residual allergenicity due to the inaccessibility of some sequential epitopes to proteases. Microwave may enhance unfolding pathways in protein structure due to its non-thermal effects. This research compared the effects of microwave heating (MW) and conventional heating (CH) on the unfolding in the secondary and tertiary structures of β-Lg over a temperature range of 40-90 °C using circular dichroism (CD), fluorescence spectroscopy, and two dimensional (2D) ¹H nuclear magnetic resonance (NMR) spectroscopy. Above 50 °C, β-sheet and α-helical secondary structures decreased during MW and CH, with a higher decrease being observed during MW. The near-UV spectra of MW β-Lg showed lower intensity suggesting higher tertiary structure loss than in CH β-Lg at all temperatures. The fluorescence spectra of MW β-Lg showed increased exposure of tryptophan residues to solvent as compared to CH β-Lg and suggested greater unfolding in tertiary structure in MW β-Lg at 60 °C than in CH β-Lg at 70 °C. 2D ¹H NMR spectra confirmed more extensive H-D exchange in MW β-Lg explained by the exposure of β-sheets (C, G, and H) at 50 °C under microwave treatment, which are thermally resistant to H-D exchange up to 75 °C during conventional heating. These results revealed a substantial enhancing effect of microwave treatment on the thermal unfolding and exposure of buried amide groups in β-Lg compared to conventional heating. Microwave processing could be a promising alternative to produce hydrolysates with lower allergenicity and improved bioactivity through structure modification.     

Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: a calorimetric and spectroscopic study

The effect of methylurea, N,N'-dimethylurea, ethylurea, and butylurea as well as guanidine hydrochloride (GuHCl), urea and pH on the thermal stability, structural properties, and preferential solvation changes accompanying the thermal unfolding of ribonuclease A (RNase A) has been investigated by differential scanning calorimetry (DSC), UV, and circular dichroism (CD) spectroscopy. The results show that the thermal stability of RNase A decreases with increasing concentration of denaturants and the size of the hydrophobic group substituted on the urea molecule. From CD measurements in the near- and far-UV range, it has been observed that the tertiary structure of RNase A melts at about 3 degrees C lower temperature than its secondary structure, which means that the hierarchy in structural building blocks exists for RNase A even at conditions at which according to DSC and UV measurements the RNase A unfolding can be interpreted in terms of a two-state approximation. The far-UV CD spectra also show that the final denatured states of RNase A at high temperatures in the presence of different denaturants including 4.5 M GuHCl are similar to each other but different from the one obtained in 4.5 M GuHCl at 25 degrees C. The concentration dependence of the preferential solvation change delta r23, expressed as the number of cosolvent molecules entering or leaving the solvation shell of the protein upon denaturation and calculated from DSC data, shows the same relative denaturation efficiency of alkylureas as other methods.     

Three steps in the thermal unfolding of F-actin: an experimental evidence

The development of differential scanning calorimetry has resulted in an increased interest in studies of the unfolding process in proteins with the aim of identifying domains and interactions with ligands or other proteins. Several of these studies were done with actin and showed that the thermal unfolding of F-actin occurs in at least three steps; this was interpreted as the denaturation of independent domains. In the present work, we have followed the thermal unfolding of F-actin using differential scanning calorimetry (DSC), CD spectroscopy, and probe fluorescence. We found that the three steps revealed through DSC are not the denaturation of independent domains. These three steps are a change in the environment of cys 374 at 49.5 degrees C; a modification at the nucleotide-binding site at 55 degrees C; and the unfolding of the peptide chain at 64 degrees C. Previous interpretations of the thermograms of F-actin were thus erroneous. Since DSC is now widely used to study proteins, our experimental approach and conclusions may also be relevant in denaturation studies of proteins in general.     

Spectroscopic and differential scanning calorimetric studies on the unfolding of <Emphasis Type="Italic">Trichosanthes dioica</Emphasis> seed lectin. Similar modes of thermal and chemical denaturation

Physico-chemical and unfolding studies have been carried out on Trichosanthes dioica seed lectin (TDSL). The lectin exhibited maximum activity between pH 7.0 and 10.0, which decreased steeply at lower pH. The hemagglutination activity of TDSL was unaffected in the temperature range 4–50°C, but decreased rapidly at higher temperatures. Differential scanning calorimetric studies indicate that thermal unfolding of TDSL is an irreversible process, which could be described by a three-state model. The calorimetric scan recorded at pH 7.0 consists of two transitions, occurring at around 338.6 K, and 342.8 K. In the presence of carbohydrate ligands both these transitions shifted to higher temperatures, suggesting that ligand binding stabilizes the native conformation of the protein. The unfolding temperature was highest at pH 5.0 indicating that TDSL is more stable at acidic pH. Gdn.HCl induced unfolding, monitored by following changes in the intrinsic fluorescence properties of the protein, was also observed to be a three-state process involving an intermediate. CD spectroscopy indicates that the secondary and tertiary structures of TDSL are rather similar at different pH values, indicating that the lectin structure remains essentially unchanged over a wide range of pH.     

Functional stability and structural transitions of Kallikrein: spectroscopic and molecular dynamics studies

Kallikrein, a physiologically vital serine protease, was investigated for its functional and conformational transitions during chemical (organic solvents, Gdn-HCl), thermal, and pH induced denaturation using biochemical and biophysical techniques and molecular dynamics (MD) simulations approach. The enzyme was exceptionally stable in isopropanol and ethanol showing 110% and 75% activity, respectively, after 96 h, showed moderate tolerance in acetonitrile (45% activity after 72 h) and much lower stability in methanol (40% activity after 24 h) (all the solvents [90% v/v]). Far UV CD and fluorescence spectra indicated apparent reduction in compactness of KLKp structure in isopropanol system. MD simulation studies of the enzyme in isopropanol revealed (1) minimal deviation of the structure from native state (2) marginal increase in radius of gyration and solvent accessible surface area (SASA) of the protein and the active site, and (3) loss of density barrier at the active site possibly leading to increased accessibility of substrate to catalytic triad as compared to methanol and acetonitrile. Although kallikrein was structurally stable up to 90 °C as indicated by secondary structure monitoring, it was functionally stable only up to 45 °C, implicating thermolabile active site geometry. In GdnHCl [1.0 M], 75% of the activity of KLKp was retained after incubation for 4 h, indicating its denaturant tolerance. A molten globule-like structure of KLKp formed at pH 1.0 was more thermostable and exhibited interesting structural transitions in organic solvents. The above results provide deeper understanding of functional and structural stability of the serine proteases at molecular level.     

A temperature‐dependent conformational change of NADH oxidase from Thermus thermophilus HB8

Using molecular dynamics simulations and steady‐state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). The enzyme's far‐UV circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75°C. However, the fluorescence of the tightly bound FAD cofactor increased approximately fourfold over this temperature range. This effect appears not to be due to aggregation, unfolding, cofactor dissociation, or changes in quaternary structure. We therefore attribute the change in flavin fluorescence to a temperature‐dependent conformational change involving the NOX active site. Molecular dynamics simulations and the effects of mutating aromatic residues near the flavin suggest that the change in fluorescence results from a decrease in quenching by electron transfer from tyrosine 137 to the flavin. Proteins 2012. © 2011 Wiley Periodicals, Inc.