pH值对紫色光合细菌Rhodopseudomonas palustris外周捕光天线的影响

采用稳态吸收光谱、圆二色谱、亚微秒时间分辨光谱手段检测了不同pH值对Rhodopseudomonas palustris的外周捕光天线LH2复合物的结构及色素功能的影响, 发现: (i) 在强酸性pH诱导下, B800细菌叶绿素分子在分钟时间尺度上逐渐转化成游离色素, 而类胡萝卜分子(Car)的光谱变化与B850的变化基本同步. 伴随着B800的缺失, 其Qy带对应的圆二色信号消失; (ii) 在强碱性条件下, B800分子比较稳定, B850分子的Qy带从852 nm蓝移至837 nm左右, 其近红外区域的CD 信号也发生了相应蓝移; 不同pH值条件下可见光区域Car分子均呈现出特征的CD信号; (iii) 在生理及碱性情况下, 采用532 nm激光脉冲直接激发Car分子, 在亚微秒时间尺度上观察到类胡萝卜素的TnT1吸收; 而在强酸性pH下仅观察到无特征的弱吸收带. 以上实验结果表明Rps. palustris的LH2复合物在酸性条件下B800发生缺失, Car的光保护功能受到影响; 而在强碱性条件下环状聚集体结构仍然保持, Car能正常发挥其光保护功能.     

共振瑞利散射测定痕量甲胎蛋白含量研究

用共振瑞利散射光谱研究磷钼杂多酸与甲胎蛋白的相互作用的过程中,发现其结合会引起共振瑞利散射(RRS),最大RRS峰均位于480 nm。在一定浓度范围内,AFP浓度与散射强度成正比,这样就产生了一种新的利用共振光散射强度定量测定甲胎蛋白的方法。本文对该反应体系的适宜反应条件、主要影响因素、散射强度与AFP浓度的关系、方法的灵敏度等,进行了比较研究。发现不同的杂多酸对于甲胎蛋白的检出限(3σ)在5.2~78μg.L-1之间,其中以磷锑钼酸体系灵敏度最高,对于甲胎蛋白(AFP)的检出限为2.5μg.L-1。该方法具有操作简便、灵敏度高、线性范围宽等优点。考察了共存物质的干扰影响,获得了满意的结果。     

血清蛋白与4,5-二溴荧光素相互作用及其分析应用的研究

在 0 .10mol/mL的醋酸溶液中 ,4,5 二溴荧光素能与血清蛋白形成稳定的复合物 ,最大吸收波长 482nm ,与试剂比较 ,红移了 12nm。据此建立了测定血清蛋白的方法 ,用于BSA和HSA的测定 ,分别在 2～ 14mg·L-1有线性关系 ,表观摩尔吸光系数分别为 3.12× 10 5L·mol-1·cm-1和 3.2 7× 10 5L·mol-1·cm-1。应用该法测定了人血清样品总蛋白含量 ,结果令人满意。     

在不同物理化学条件下天花粉蛋白的圆二色性研究

用圆二色谱研究了天花粉蛋白在酸性和碱性溶液中的构象,并观察了在十二烷基磺酸钠(SDS)溶液中天花粉蛋白分子构象的变化,用Chen,Yang与Martinez等人的方法计算了各种条件下天花粉蛋白二级结构含量.结果表明:在很宽广的pH范围内(从pH2到pH12)分子构象基本上是稳定的.但是,在强碱性条件下即pH>12(室温)或pH11.5(40℃)螺旋含量显著减少.在碱性条件下,于250nm附近出现一新的CD带,它的强度与离解的酪氨酸残基个数呈线性关系,说明这一新带主要是由离解的酪氨酸残基产生的.     

基于SCX/SAX混合填料的集成化蛋白质组学样品前处理方法

本文建立了一种基于强阳离子交换填料/强阴离子交换填料(SCX/SAX)混合填料的集成化蛋白质组学样品前处理方法.本工作将前期已发展的离心式集成化蛋白质组学样品前处理技术SISPROT中的SCX填料替换成SCX和SAX混合填料,以溶菌酶和牛血清白蛋白为模型蛋白,研究了3种pH(3,7.4,12)条件下蛋白质在SCX/SAX混合填料上的保留行为;应用BCA方法对SCX/SAX混合填料的比例进行了优化,并应用SDS-PAGE法对酶解步骤中pH变化引起的蛋白质丢失情况进行了系统的考察.实验结果发现,在pH 7.4条件下,质量比为1:1的SCX/SAX混合填料的富集效率最佳,蛋白质富集容量为180μg,是SCX填料富集容量的两倍左右;且酶解步骤的pH变化过程不会影响蛋白质在SCX/SAX混合填料上的保留.最后应用改进的SISPROT方法对少量肠癌组织样品进行了集成化蛋白质组学样品前处理和分析,并与传统的基于SCX填料的SISPROT方法进行了对比研究.结果表明,蛋白质鉴定量、特异性肽段数量和肽段谱图匹配数量均与基于SCX填料的SISPROT技术相当,证实了该方法作为蛋白质反应器的可靠性.鉴于该方法可以实现在生理pH条件下进行样品前处理,且显著提高了上样容量和减少了样品损失,该方法有望成为一种更为通用的集成化蛋白质组学样品前处理技术.     

基于纳米金光学性质的分子检测与应用

纳米金颗粒是近年研究最为广泛的纳米材料之一,它具有良好的生物相容性、化学稳定性以及独特的光学性质,在生物分子检测、诊断和治疗方面具有很大的发展潜力。尤其是纳米金显示出特殊的表面等离子体共振现象,导致了粒子表面产生强电磁场,并最终增强了诸如吸收和散射的辐射特性,其散射光强与粒子的尺寸和团聚状态有密切关系。而由于共振现象而产生的纳米金对光的强烈吸收并高效转换为热效应也被用于检测和治疗。此外,与纳米金尺寸相关的局域表面等离子体共振光学特性,能够在粒子附近产生很强的电磁场增强,从而构成表面增强拉曼散射的基础。纳米金在强光照射下也表现出良好的抗光漂白的荧光现象,其特有的荧光寿命也成为检测的一种有效手段。与其他荧光物质作用时,又表现出表面增强荧光特性以及荧光共振能量转移。综述中,在介绍纳米金这些特殊光学性质的基础上,回顾了其在生物分子检测方面的应用进展。     

基于pH的反馈补料方法在谷氨酸发酵中的应用

为简化谷氨酸发酵补料工艺,提出了一种新型的基于pH的补料方式。考察谷氨酸发酵过程中氨消耗量 (x) 和糖消耗量 (y) 发现,两者之间存在较好的线性关系 (y=7.4744x,R2=0.9989),以此为pH反馈补料工艺中补料液中葡萄糖与氨的混合比例,能较好地将谷氨酸发酵过程中葡萄糖浓度稳定在12~21 g/L。比较恒定葡萄糖浓度补料工艺与pH反馈补料工艺发现,采用pH反馈补料工艺进行发酵,葡萄糖转化率、谷氨酸产酸速率分别提高了9.06％和17.5％左右,同时发酵周期缩短2 h以上。     

胰蛋白酶活性的定量测定方法

对甲苯磺酰基精氨酸甲酯(TAME)是胰蛋白酶的专一性底物. TAME经胰蛋白酶水解释放出的对甲苯磺酰基精氨酸与活性测定混合物中的NaOH反应, 导致溶液pH值的下降. 以酚红为指示剂, 通过测定555nm处光吸收值的降低可以监测pH的变化. 在0.001～0.3μg的范围内, 胰蛋白酶含量与555nm处光吸收值的降低呈线性关系.     

苋菜红在银基汞膜电极上伏安特性的研究

本文讨论了不同条件下苋菜红在银基汞膜电极上的伏安行为.发现在 pH=4时,苋菜红在-0,24V 处有很灵敏的检出信号,在此条件下,汞膜电极有良好的再现性和使用寿命;在10-100ppb 范围内,还原电流与浓度呈良好的线性关系.     

氮素形态对小白菜生长和碳氮积累的影响

水培条件下,研究不同氮素形态(硝态氮、铵态氮、甘氨酸、谷氨酰胺、丙氨酸、牛血清蛋白,以及甘氨酸与硝态氮、牛血清蛋白与硝态氮的混合氮源)对小白菜生长和碳氮积累的影响.结果表明:不同氮素形态对小白菜质量、碳氮积累量、可溶性蛋白质含量、可溶性糖含量和游离氨基酸含量的影响不同;硝态氮处理下小白菜地上部分和根的干质量与鲜质量均最大;甘氨酸对小白菜根系的生长及碳氮积累具有明显的促进作用;在3种氨基酸中,谷氨酰胺更有利于小白菜地上部分的生长和氮积累.聚类分析表明,9种氮素形态处理按营养效应大小分为:硝态氮、谷氨酰胺＞甘氨酸与硝态氮混合氮源、牛血清蛋白与硝态氮混合氮源、甘氨酸、铵态氮＞丙氨酸、牛血清蛋白、对照.有机氮源可以作为小白菜生长的氮源,不同的氮素形态对植物产生的生理效应不同.     

A new and simple resonance Rayleigh scattering method for human serum albumin using graphite oxide as probe

Graphite oxide (GO) was prepared by the Hummer procedure, and can be dispersed to stable colloid solution by ultrasonic wave. The GO exhibited an absorption peak at 313 nm, and a resonance Rayleigh scattering (RRS) peak at 490 nm. In pH 4.6 HAc‐NaAc buffer solution, human serum albumin (HSA) combined with GO probe to form large HSA‐GO particles that caused the RRS peak increasing at 490 nm. The increased RRS intensity was linear to HSA concentration in the range 0.50–200 µg/mL. Thus, a new and simple RRS method was proposed for the determination of HSA in samples, with a recovery of 98.1–104%. Copyright © 2012 John Wiley & Sons, Ltd.     

Study on the resonance Rayleigh scattering and resonance non‐linear scattering spectra of ion‐association complexes of [PdI4]2−‐protein systems and their analytical applications

In an acid medium solution, proteins such as bovine serum albumin, human serum albumin, ovalbumin, hemoglobin, lysozyme, γ‐globulin, α‐chymotrypsin and papain could react with [PdI₄]^2? by virtue of electrostatic attraction and hydrophobic force to form ion‐association complexes. As a result, the resonance Rayleigh scattering (RRS) and resonance nonlinear scattering such as second‐order scattering (SOS) and frequency doubling scattering (FDS) intensities were enhanced greatly and new scattering spectra appeared. The maximum scattering peaks of RRS, SOS and FDS were at 367, 720 and 370 nm, respectively. The enhanced RRS, SOS and FDS intensities were directly proportional to the concentrations of proteins. The detection limits for the different proteins were 2.4–11.8 ng/mL for RRS method, 9.5–47.9 ng/mL for SOS method and 4.6–18.5 ng/mL for FDS method. In this work, the influences of the interaction of [PdI₄]^2? with proteins on spectral characteristics of RRS, SOS and FDS were investigated and the optimum conditions were tested. Meanwhile, the effects of coexisting substances were tested and the results showed that the method exhibited a good selectivity. Based on the above research, a highly sensitive, simple and rapid method for the determination of trace amounts of proteins by resonance light scattering technique has been developed. It can be applied to the determination of proteins in tablet, human serum and urine samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Probing protein colloidal behavior in membrane‐based separation processes using spectrofluorometric Rayleigh scattering data

One of the primary problems in membrane‐based protein separation is membrane fouling. In this study we explored the feasibility of employing Rayleigh light scattering data from fluorescence studies combined with chemometric techniques to determine whether a correlation could be established with membrane fouling phenomena. Membrane flux was measured in a dead‐end UF filtration system and the effect of protein solution properties on the flux decline was systematically investigated. A variety of proteins were used as a test case in this study. In parallel, the colloidal behavior of the protein solutions was assessed by employing multiwavelength Rayleigh scattering measurements. To assess the usefulness of Rayleigh scattering measurements for probing the colloidal behavior of proteins, a protein solution of β‐lactoglobulin was used as a base‐case scenario. The colloidal behavior of different β‐lactoglobulin solutions was inferred based on published data for this protein, under identical solution conditions, where techniques other than Rayleigh scattering had been used. Using this approach, good agreement was observed between scattering data and the colloidal behavior of this protein. To test the hypothesis that a high degree of aggregation will lead to increased membrane fouling, filtration data was used to find whether the Rayleigh scattering intensity correlated with permeate flux changes. It was found that for protein solutions which were stable and did not aggregate, fouling was reduced and these solutions exhibited reduced Rayleigh scattering. When the aggregation behavior of the solution was favored, significant flux declines occurred and were highly correlated with increased Rayleigh scattering. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Static and dynamic light scattering approach to the hydration of hemoglobin and its supertetramers in the presence of osmolites.

We used static and dynamic light scattering for comparing the mass (MW) and hydrodynamic radius (R(h)) of several hemoglobin systems, namely human hemoglobin, bovine hemoglobin, human hemoglobin cross-linked with a sebacyl residue, and bovine hemoglobin cross-linked with an adipoyl residue. We measured the MW and R(h) of these systems in 0.1M phosphate buffer at pH 7.0 in the absence and in the presence of either betaine or glycerol up to 1.7 molal concentrations. The 90 degrees scattering was measured with a photon counting machine equipped with a diode laser at 783 nm. The Rayleigh ratio [R(theta)] of the instrument was estimated using R(theta) = 7.19E-6 cm(-1) for toluene at 783 nm. The refractive index increment of hemoglobin solutions was measured using a laser beam at 750 nm. We estimated a value dn/dc = 0.210 cm3/g in the absence and dn/dc = 0.170 in the presence of 1.7 molal osmolites. For all systems both in liganded and unliganded form, the static light scattering data showed a 16% mass increase with increasing concentration of osmolites. The hydrodynamic radii of all investigated systems in the presence and absence of osmolites were close to 3.17 nm. Assuming a partial specific volume nu = 0.739 for hemoglobin, and using spherical geometry, the estimated average hydration volume of hemoglobin was 32.6 L/mole in the absence of osmolites. It decreased to 23.5 L/mole in the presence of 1.7 molal osmolites. Assuming that the density of water in the hydration volume is D = 1.0 g/cm3, the hydration of Hb was 0.51 gH2O/gHb, with a surface density of 0.20 molH2O/A2. The hydration decreased to 0.33 gH2O/gHb and 0.14 molH2O/A2 in the presence of 1.7 molal osmolites. The decreased hydration was compensated by the increased mass (i.e., decreased surface area per unit volume) so that the thickness of the water shell around these proteins remained close to a single layer of water molecules. These findings indicate that the combination of static and dynamic light scattering offer unique means for investigating the relevance of water activity on the structure and function of biological macromolecules. In the case of hemoglobin, the data suggest that the decreased oxygen affinity in the presence of osmolites reported by Colombo et al. (M. F. Colombo, D. C. Rau, and V. A. Parsegian Science, 1992, Vol. 256, pp. 655-659), as due to ligand linked water binding on hemoglobin surface, is part of a complex phenomenon involving the hydration shell of hemoglobin and the formation of low affinity supertetrameric molecules.     

Resonance scattering detection of trace microalbumin using immunonanogold probe as the catalyst of Fehling reagent-glucose reaction

A novel and sensitive resonance scattering (RS) spectral immunoassay for the determination of microalbumin (Malb) was developed, based on the catalytic effect of immunonanogold (ING) probe on Fehling reagent-glucose reaction, and resonance scattering effect of Cu(2)O particles. Nanogold particles in size of 10nm were used to label goat anti-human microalbumin (GMalb) to obtain an ING probe (AuGMalb) for Malb. The probe produced unspecific aggregation in pH 5.0 citric acid-Na(2)HPO(4) buffer solutions. Upon addition of Malb, the dispersed ING complex formed. The ING complex in supernatant was obtained by centrifuging and was used as catalyst for the reaction between Fehling reagent and glucose to form the Cu(2)O particles to amplify the resonance scattering signal at 610 nm. With addition of Malb, the ING complex in the supernatant increased and the RS intensity at 610 nm (I(610 nm)) enhanced linearly. The enhanced intensity DeltaI(610 nm) was proportional to the Malb concentration in the range of 0.014-0.43 ng ml(-1), with a detection limit of 7.2 pg ml(-1). The proposed method was applied to detect Malb in human urine sample with satisfactory results.     

The resonance Raman spectrum of carotenoids as an intrinsic probe for membrane potential. Oscillatory changes in the spectrum of neurosporene in the chromatophores of Rhodopseudomonas sphaeroides.

The resonance Raman spectrum of the carotenoid neurosporene is shown to be a sensitive monitor of absorption shifts, and thus changes in membrane potential, in chromatophores of the GlC mutant of Rhodopseudomonas sphaeroides. For a Raman excitation wavelength at 472.7 nm, the intensities of the two most prominent resonance Raman features (v1 and v2) respond very differently to small shifts in the absorption maxima. Thus, the ratio intensity v1/intensity v2 is a sensitive probe for absorption shifts. Changes in this ratio of approximately 20% were observed during a valinomycin induced diffusion potential. At 5 degrees C changes in the average intensity ratio of +6, -4 and -14% were brought about by oligomycin, FCCP and sodium deoxycholate, respectively. The changes in intensity ratio were temperature dependent and, in addition, effects due to the laser beam acting as an actinic light could be detected. Oscillatory changes were observed in absolute Raman and Rayleigh scattering intensities for chromatophores at 5 degrees C and for intact cells under growing conditions.     

Determination of doxepin hydrochloride in spiked human plasma and capsule after derivatization with eosin Y using resonance Rayleigh scattering,second-order scattering and frequency doubling scattering methods

Doxepin hydrochloride (DOX) is a tricyclic antidepressant drug. Three sensitive spectrofluorimetric methods, namely resonance Rayleigh scattering (RRS), frequency doubling scattering (FDS) and second-order scattering (SOS), were developed and validated for their estimation of doxepin in spiked human plasma and formulation using liquid–liquid extraction method through the formation of an ion pair complex with eosin Y at a pH of 4.5. Various factors affecting fluorescence intensity were optimized, and the reaction kinetics was determined using the Arrhenius equation method. Different scattering methods such as RRS, FDS and SOS were developed at maximum scattering wavelengths λ_ex/λ_em = 567/567 nm for RRS, 720/360 nm for SOS and 260/520 nm for FDS, respectively. The methods exhibited high sensitivities, and the detection limits for DOX were found to be 0.82, 1.20 and 1.03 ng/ml for RRS, FDS and SOS methods, respectively. The FDS method exhibited the highest sensitivity. The methods were validated using the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines and applied to determine DOX in capsule and spiked human plasma samples.     

A simple and sensitive resonance Rayleigh scattering method for determination of As(III) using aptamer‐modified nanogold as a probe

A simple and selective aptamer (ssDNA)‐modified nanogold probe (AussDNA) was prepared for the determination of trace As(III) in HEPES buffer solution (pH 8.2) containing 0.05 mol/L NaCl. The method coupled the aptamer reaction of AussDNA–As(III) and the resonance Rayleigh scattering (RRS) of nanogold aggregations at 278 nm. When the As(III) concentration increased, the RRS intensity at 278 nm increased to form more nanogold aggregation and a stable As(III)–ssDNA complex. Under selected conditions, the increased RRS intensity (ΔI) was linear to the concentration of As(III) in the range 3.8–230.4 ng/mL, with a detection limit of 1.9 ng/mL. This RRS method was applied to detect As(III) in water samples, with simplicity, sensitivity and selectivity. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitative scattering of melanin solutions

The optical scattering coefficient of a dilute, well-solubilized eumelanin solution has been accurately measured as a function of incident wavelength, and found to contribute <6% of the total optical attenuation between 210 and 325 nm. At longer wavelengths (325-800 nm), the scattering was less than the minimum sensitivity of our instrument. This indicates that ultraviolet and visible optical density spectra can be interpreted as true absorption with a high degree of confidence. The scattering coefficient versus wavelength was found to be consistent with Rayleigh theory for a particle radius of 38 +/- 1 nm. Our results shed important light on the role of melanins as photoprotectants.     

Resonance light scattering technique for the determination of proteins with Congo red and Triton X-100.

Resonance light scattering (RLS) of Congo red (CR) was greatly enhanced by BSA (HSA) in the presence of Triton X-100 (TX-100). In sodium citrate-HCl buffer (pH 2.7-3.0), the enhanced intensity of resonance light scattering at 360 nm was in proportion to the concentration of proteins [corrected] The linear relationship was obtained between the resonance light scattering intensity and proteins in the range 5.0 x 10(-8)-8.0 x 10(-6) g/mL and 1.0 x 10(-9)-6.0 x 10(-6) g/mL for BSA and HSA, respectively. Their detection limits were 1.4 x 10(-8) g/mL and 2.8 x 10(-10) g/mL (S:N = 3), respectively. Synthetic and actual samples were analysed satisfactorily.