小檗碱与牛血清白蛋白相互作用的光谱研究

利用紫外光谱和荧光光谱研究了中药有效成分小檗碱与牛血清白蛋白(BSA)的相互作用机制。利用荧光猝灭反应测得它们之间结合常数K=1.49×105L/mol,结合位点数n=9.77,依据F rster非辐射能量转移机制,测得供体 受体间结合距离R=3.09nm和能量转移效率E=0.443。认为小檗碱在BSA的位置阻断了酪氨酸残基与色氨酸残基之间的能量转移,导致BSA的荧光猝灭。     

蛋白质与刚果红结合反应的研究

研究了酸性溶液中蛋白质(BSA)与刚果红(CGR)的结合反应。BSA与CGR相互作用形成复合物,溶液颜色由蓝色变为红色,最大吸收波长紫移,表明在蛋白质正电荷作用下,CGR由游离态酸色型变为结合态碱色型。测得CGR与BSA的结合最大结合数约为43,符合Scatchard模型。荧光光谱表明,CGR与BSA作用导致BSA荧光猝灭,结合位点为BSA第212位色氨酸残基,相互作用为非辐射能量转移。     

十二烷基硫酸钠增敏荧光光谱法测定盐酸小檗碱

基于十二烷基硫酸钠(SDS)对盐酸小檗碱荧光有显著增敏作用,建立了测定盐酸小檗碱的荧光光谱法。在pH 7.0的Tris-HCl缓冲溶液中,盐酸小檗碱与0.01mol·L-1的SDS溶液反应15min后,体系在530nm处的荧光强度达到最大。盐酸小檗碱的浓度在1.0~10mg·L-1范围内与体系荧光强度呈线性关系,检出限(3s/k)为0.23mg·L-1。方法应用于制剂中盐酸小檗碱含量的测定,结果与药典法测定结果一致。加标回收率在94.9%~106%之间,测定值的相对标准偏差(n=6)在1.9%~3.7%之间。     

纳米金光度法测定药物中盐酸小檗碱

氯金酸用葡萄糖还原制得纳米金,最终获得浓度为2.4×10-4mol.L-1且呈酒红色的纳米金悬浮溶液。试验证实纳米金颗粒被均匀地分散在悬浮溶液中,其吸收光谱中在525 nm波长处出现一吸收峰。当在纳米金悬浮溶液中加入盐酸小檗碱,由于两者间的静电结合导致溶液在525 nm波长处的吸光度降低,且其降低的幅度与盐酸小檗碱浓度在2.0×10-7～3.0×10-6mol.L-1范围内呈线性关系,其检出限(3S/N)为1.7×10-8mol.L-1。应用此方法分析了3个批号的药物盐酸小檗碱或片剂样品,所测得的结果与按药典方法测得结果相符。     

荧光各向异性结合同步荧光法研究1-羟基芘与牛血清白蛋白的相互作用

在模拟生理条件下,应用荧光各向异性法研究了多环芳烃(PAHs)代谢标志物1-羟基芘(1-OHP)与牛血清白蛋白(BSA)的相互作用,并结合同步荧光法研究作用过程中BSA的构型变化,初步探讨了二者的结合方式.研究结果表明,1-OHP与BSA有较强的结合作用,形成1∶1复合物,平均结合平衡常数为3.63×106L/mol,且其结合作用强弱随着BSA浓度大小发生变化.1-OHP可与BSA的色氨酸残基结合,使BSA构型发生变化,进而使色氨酸残基周围环境的疏水性降低.     

低场核磁共振技术测定顺磁性钆(Ⅲ)和钕(Ⅲ)

利用长弛豫低场核磁共振技术分别测定了六水合氯化钆水溶液、N,N-二甲基甲酰胺(DMF)溶液和六水合硝酸钕水溶液在不同浓度下的横向弛豫时间(T_2)。3种溶液体系中氢质子的横向弛豫速率(1/T2)与稀土离子的浓度均呈现出良好的线性关系,提出了利用低场核磁技术测定溶液中Gd~(3+)和Nd~(3+)浓度的方法。     

中药黄连有效成分盐酸小檗碱与牛血清白蛋白的相互作用

从天然中药材黄连中提取分离并精制得到盐酸小檗碱(BC),采用UV光谱和荧光光谱(FS)研究其与牛血清白蛋白(BSA)的相互作用,解释了BC导致BSA的荧光发射光谱峰裂分的现象,其二重峰分别归属于色氨酸及酪氨酸残基.结果表明,静态猝灭和非辐射能量转移是导致BC对BSA荧光猝灭的两大原因,BC与BSA的表观结合常数K_A为8.66×10⁴L/mol(30℃)和8.72×10⁴L/mol(37℃),BC在BSA分子上的结合位点数为(3.1±0.2).BC与BSA分子中荧光性氨基酸残基之间的距离为3.75nm(30℃)和3.62nm(37℃),表明BC的部分片段能够插入BSA分子内部.热力学函数计算结果表明,该作用过程是一个熵增加、Gibbs自由能降低的自发超分子作用过程,并由此推断BC与BSA之间以疏水相互作用为主.     

牛血清白蛋白-Cu2＋结合过程中盐酸小檗碱的变构效应

用核磁共振(1H NMR)、圆二色谱(CD)、荧光光谱(FS)以及紫外光谱(UV)技术考察了中药有效成分盐酸小檗碱(BC)对牛血清白蛋白(BSA)-Cu2＋结合过程的变构效应, 得到分别表征BSA内源荧光猝灭、BSA-Cu2＋复合物稳定性以及Cu2＋在BSA分子上的结合位点发生变构的定量效应参数βQ (βA和βn)和效率参数γQ (γA和γn). 结果表明, BC对Cu2＋猝灭BSA内源荧光呈负变构效应(0＜βQ＜1), 而对BSA-Cu2＋复合物稳定性以及Cu2＋在BSA分子上的结合位点呈正变构效应(βA＞1, βn＞1); 变构效应随BC浓度增加而增强, BC对BSA-Cu2＋复合物稳定性的变构效率明显高于其对荧光猝灭和结合位点的变构; BSA分子构象转变是变构效应的主要原因.     

胶束增敏荧光光度法测定小檗碱的含量

在pH 10的磷酸盐缓冲介质中,十二烷基苯磺酸钠(SDBS)对小檗碱的荧光有明显的增敏作用,且其荧光的增强程度与小檗碱浓度在2.0×10-9～1.0×10-3mol·L-1之间呈线性关系.此反应对小檗碱的检出限(3S/N)为1.29×10-9mol·L-1.利用此反应作为测定小檗碱的方法并应用于小檗碱片剂的分析,测得结果与药典法测定结果相符,对药片的标示量而言,测定所得回收率的平均值为99.8%,测定结果的相对标准偏差(n=5)的平均值为0.17%.     

生物样品中小檗碱的电化学分析新方法

由于小檗碱氧化电位较高(>1.1 V vs. Ag/AgCl),进行生物样品中小檗碱的选择性电化学分析具有挑战性。基于溴百里酚蓝(BTB)和小檗碱的分子间非共价作用,通过单壁碳纳米管(SWNTs)将BTB修饰在电极表面并通过电化学聚合产生poly-BTB,利用聚BTB(poly-BTB)电极过程可逆以及氧化还原电位低的特点,以poly-BTB同时作为小檗碱的识别元件和电化学探针建立了小檗碱的电化学分析新方法。循环伏安和电化学阻抗谱结果表明,poly-BTB和小檗碱的分子间作用导致小檗碱结合在poly-BTB/SWNTs修饰电极表面,从而引起修饰电极的峰电流下降。在最优化的实验条件下,poly-BTB/SWNTs修饰电极的电流下降率和小檗碱的浓度在0.05～1μmol/L和1～100μmol/L范围内呈良好线性关系,检测限为0.022μmol/L。动物实验结果表明,该方法对生物样品具有很好的选择性,可用于血浆和肝脏匀浆中小檗碱含量的测量,为小檗碱相关的生理病理研究提供了一种简单有效的方法。     

Binding position of phenylbutazone with bovine serum albumin determined by measuring nuclear magnetic resonance relaxation time

The binding of phenylbutazone (PB) to bovine serum albumin (BSA) was considered to be predominantly due to hydrophobic interaction based on the thermodynamic parameters obtained by an equilibrium dialysis method. Little variation of proton nuclear magnetic resonance (1H-NMR) chemical shift of PB was found with change in the concentration of PB (0.5-5 mM) or upon the addition of BSA (7.25 x 10(-5) M). The NMR spectrum of PB in 0.1 M phosphate buffer solution at pH 7 showed that PB existed as a mesomeric anion. The spin-lattice relaxation time (T1) of PB was almost concentration-independent, but decreased in the presence of BSA to 36-38% for the phenyl group and 48-100% for the butyl group. The spin-spin relaxation time (T2) of PB was also almost independent of concentration, but was remarkably decreased in the presence of BSA to ca. 2.5% for the phenyl group and ca. 6-9% for the butyl group. The ratios of the spin-spin relaxation rate (1/T2) of the free PB to that of the bound PB were ca. 5000-11000 for the butyl group and ca. 23000 for the phenyl group. The binding of PB to BSA was considered to involve primarily the phenyl group.     

Binding position of azathioprine with bovine serum albumin determined by measuring nuclear magnetic resonance relaxation time.

The interaction between azathioprine (AZ) and bovine serum albumin (BSA) is mainly due to hydrophobic binding according to the dependence of the binding constant on the ionic strength obtained by equilibrium dialysis. The binding constant and partition coefficient of AZ were smaller than those of warfarin, phenylbutazone and ibuprofen. Little variation in the proton chemical shift of AZ was observed whether there was an absence or presence of BSA (7.25 x 10(-5) M). The spin-lattice relaxation time (T1) of AZ decreased in the presence of BSA to 6-22%. The spin-spin relaxation rate (1/T2) of AZ increased 16-24 times for the methyl group and the imidazole ring and 8-13 times for the purine ring in the presence of BSA. The ratio of the spin-spin relaxation rate of the free AZ to the bound AZ ((1/T2)b/(1/T2)f) of the methyl group and the imidazole ring was 2-3 times larger than that of the purine ring. The binding of AZ to BSA was concluded to be mainly at the methyl group on the imidazole ring of AZ.     

Determining the sizes of micropores in activated charcoals by the pulsed NMR method

The pulsed NMR method was used to measure the nuclear spin-spin relaxation of protons of water adsorbed in micropores of activated charcoal (AC) samples with different porous structures. A correlation was found between the spin-spin relaxation time of water protons in AC with completely filled micropores and the volume density of water primary adsorption centers in the AC samples. An equation for approximating obtained dependences is proposed that allows us to determine the volume of micropores in AC.     

Cross relaxation effects on the longitudinal relaxation rate of water in protein solutions

Non-selective and selective longitudinal relaxation rates were measured for water protons in protein solutions. The perturbations on water spin-lattice relaxation from cross relaxation between water and protein protons were shown to be significant at low temperature and small isotopic dilution.     

High resolution 1H-NMR relaxation study of polyisobutylene in solution

From the temperature dependence of integrated intensities and from line widths in high-resolution ¹H-NMR spectra, the relaxation times T₁ and T₂ of protons in CH₂ and CH₃ groups of polyisobutylene in CCl₄ solution have been determined. Although the relaxation time T₁ of methylene protons is determined mainly by intragroup interactions, intergroup interactions of two methyl groups from each two consecutive monomer units were found to contribute considerably to T₁ of methyl protons. The Structure and mobility of polyisobutylene (PIB) molecules in solution is discussed on the basis of the relaxation time data.     

Hydrocarbon chain packing in the micellar core of surfactants studied by 1H NMR relaxation

 ¹H NMR spin–lattice and spin–spin relaxation of different types (cationic cetyltrimethyl ammonium bromide, anionic sodium dodecyl sulfonate and nonionic Triton X-100) of surfactants in water solution were studied. Simulation of the decay curves of proton relaxation shows that the spin lattice relaxation of all the samples exhibits exponentially, while the spin–spin relaxation for several protons on the hydrophobic chains forming the micellar core is bi-exponential. The fast relaxing component is attributed to the part of the segments of the hydrophobic chain, situated near or on the surface of the micellar core, while the slower relaxing component is attributed to the rest part staying in the interior. The latter exchanges with the former in equilibrium. Thus, a part of each certain segment of the hydrophobic chain has an opportunity to stay in the surface layer of the micellar core and spend some time on the interface experiencing hydrophilic environment. Generally, the protons on the methylene carbon of the hydrophobic chain nearest to the polar head have more chance to spend time in the hydrophilic environment. However, it seems to be dependent on the chemical structure of the surfactant molecule. Large size of the polar group of CTAB shows steric hindrance on the packing of the hydrophobic chain. Quantitative results are given. The fact, that the fraction of slow relaxing protons on the hydrophilic ethylene oxide long chain of Triton X-100 dominates over that of fast relaxing protons, and that their T ₂ values are larger than those of the protons on the hydrocarbon chain in the interior of the micellar core, suggests that the ethylene oxide chain does not participate in the formation of the micellar core. Received: 10 March 1998 Accepted: 19 June 1998     

CPS K 221 Model of proton longitudinal magnetic relaxation in hydrated crosslinked poly(methacrylic acid)

Proton longitudinal magnetic relaxation time (T₁) measurements have been made at 30 MHz over a wide range of temperature for crosslinked poly(methacrylic acid), PMA, hydrated with H₂O as well as with D₂O. From the point of view of nuclear magnetic relaxation, PMA hydrogel is a multiregion system in which three proton regions (a, b, c) can be distinguished. Region a is regarded as to be formed by the nonexchangeable polymer protons, region b by the protons of -COOH · H₂O combinations, and region c by the protons of remaining water molecules. Cross relaxation between polymer and water protons and a log normal distribution of correlation times have been assumed to take place. Temperature dependences of the T₁ time for the particular regions have been determined, from which the distribution width parameter, the second moment and the intramolecular proton-proton distance for sorbed water have been calculated.     

Recent developments in the characterization of water interacting with proteins by 17O NMR

Transverse and longitudinal ¹⁷O-water relaxation rates were detected in different samples of BSA solutions after one-quantum and triple-quantum-filtered NMR sequences. Another contribution other than quadrupolar relaxation was found for transverse relaxation, which did not change significantly with the concentration and hence could not correspond to agglomeration of proteins. This was interpreted as chemical exchange between different types of ¹⁷O-water in fast motion; probably free water and water weakly bound to the proteins. At lower BSA concentrations, two peaks were detected for water; this was in agreement with this hypothesis. The interactions between BSA and lactic acid were also studied. It was shown that at a sufficient concentration of lactic acid, the number of strongly bound water molecules detected diminishes. On the other hand, the weakly bound water properties do not change significantly.     

Kinetic study of ion exchange reaction between lysozyme and carboxymethyl Sephadex C-25

The ion-exchange reaction of lysozyme with carboxymethyl Sephadex C-25 was followed by conductivity change as a function of time just after the rapid mixing of the protein solution with the Sephadex suspension. A single relaxation process was observed; the conductivity increased exponentially with time in the 100 s scale. In this process, protons were released from the Sephadex C-25 in the same time scale. The relaxation process slowed down with an increase in the lysozyme concentration, but it quickened upon the addition of HCl. On the other hand, the potential on the Sephadex C-25 surface changed from a negative value to a positive one with an increase in the amount of lysozyme adsorbed on the surface. On the basis of these data, the relaxation process was attributed to the ion-exchange reaction of lysozyme with several protons of carboxymethyl groups of the Sephadex.     

Interaction of poly(vinylpyrrolidone) with cationic and nonionic surfactants in aqueous solution studied by <Superscript>1</Superscript>H NMR

Aqueous solutions of surfactant at various concentrations with 0.2% poly(vinylpyrrolidone) (PVP) were studied by ¹H NMR methods, including relaxation time and self-diffusion coefficient measurements and two-dimensional nuclear Overhauser enhancement spectroscopy. Two surfactants were concerned: cationic cetyltrimethylammonium bromide (CTAB) and nonionic Triton X-100 (TX-100). In the presence of 0.2% PVP, the variation of the T ₂ values of CTAB protons is similar to that in the absence of PVP. Relaxation times of PVP protons are not significantly affected by the increasing concentration of CTAB. This indicates that no interaction between PVP and CTAB could be detected. However, in the presence of 0.2% PVP, TX-100 micelles are formed at a concentration lower than its normal critical micellization concentration. According to the results of relaxation time measurement of water protons, the presence of 0.2% PVP also induces the contraction of the hydrophilic layer of the TX-100 micelle. This indicates some interaction between PVP and TX-100, but the mechanism of this interaction needs further investigation.