分光光度法研究EDTA对小牛肠碱性磷酸酶的不可逆抑制动力学

运用邹氏酶活性不可逆改变动力学 ,在不同酶底物浓度及不同抑制剂浓度下 ,用分光光度法监测酶底物水解产物浓度随时间变化的过程 ,研究了抑制剂EDTA对小牛肠碱性磷酸酶活性的不可逆抑制作用。结果表明 :EDTA对小牛肠碱性磷酸酶抑制反应机理为 :EDTA与小牛肠碱性磷酸酶发生络合作用 ,形成中间态的酶 EDTA络合物 ,此络合物的形成 ,导致该酶活性中心微环境构象发生变化 ,使酶的催化活性丧失 ,随后EDTA将酶中金属离子拉出 ,使酶发生不可逆失活。实验测定了 3 7℃下EDTA对小牛肠碱性磷酸酶不可逆抑制作用的微观速率常数ki 为 0 0 5 2 9s-1及EDTA与酶结合的平衡常数KI 为 4 0 0mmol·L-1。     

Nd-四甘醇醛缩苯丙氨酸Schiff碱配合物与DNA相互作用的光谱研究

研究了Nd-四甘醇醛缩苯丙氨酸Schiff碱配合物(NdL)与天然和变性DNA的相互作用.在DNA存在下,测定了NdL的紫外吸收光谱(UV)和荧光激发光谱(FL).利用紫外滴定法,测定了NdL与DNA的结合常数.在20mmol/L Tris-HCl缓冲溶液(pH=7.0),Nd-四甘醇醛缩苯丙氨酸Schiff碱配合物与小牛胸腺DNA的结合常数为4.11×103L/mol,化合物本身的UV和FL峰强度减小.NdL与DNA的作用模式为"静电引力".     

苯丙氨酸银溶胶表面增强拉曼光谱的研究

研究了银溶胶与L-苯丙氨酸溶液体系的表面增强拉曼光谱(SERS),增强效果明显,L-苯丙氨酸在银溶胶中的SERS光谱与苯丙氨酸固体常规拉曼光谱相比,主要峰位置基本一致,但某些峰发生了频移,相对强度也发生了一定变化。探讨了三种不同的激发光源对SERS光谱强度的影响。用不同光源测定,其SERS光谱图中各峰位置基本不变,但峰强度有明显变化。在实际工作中应根据需要选择合适的光源,一般情况下以514.42 nm为佳。不同浓度的苯丙氨酸在银溶胶中产生的表面增强拉曼光谱有明显的差别,浓度太大或太小都不利于SERS光谱的产生,溶液浓度在1×10-3mol·L-1时SERS最强,增强效果最好。体系的pH对增强效应亦有较大的影响,在pH为8时增强效应最强,这是pH对银溶胶的凝聚状态和苯丙氨酸分子存在状态综合影响的结果。     

L-苯丙氨酸构象和电离能的理论研究

我们对L-苯丙氨酸进行了全势能面搜索,采用B3LYP方法优化了L-苯丙氨酸的648种可能构象,最终得到了37种稳定存在的构象.分别采用B3LYP、B3PW91、M06-2X、MP2和CCSD(T)计算了L-苯丙氨酸最稳定的10种构象的相对能量,其中M06-2X和MP2方法能够给出较好的结果.对比不同的基组,说明采用aug-cc-pVDZ已经接近达到基组收敛极限.用电子传播子理论P3近似方法计算稳定构象外价壳层轨道的垂直电离能与光电子能谱实验符合的很好;根据构象的相对能量以及理论模拟与实验的光电能谱的比对,说明对气相光电子能谱至少四种构象有贡献.     

L-苯丙氨酸构象和电离能的理论研究

摘要: 我们对L-苯丙氨酸进行了全势能面搜索,采用B3LYP方法优化了L-苯丙氨酸的648种可能构象,最终得到了37种稳定存在的构象。分别采用B3LYP、B3PW91、M06-2X、MP2和CCSD(T)计算了L-苯丙氨酸最稳定的10种构象的相对能量,其中M06-2X和MP2方法能够给出较好的结果。对比不同的基组,说明采用aug-cc-pVDZ已经接近达到基组收敛极限。用电子传播子理论P3近似方法计算稳定构象外价壳层轨道的垂直电离能与光电子能谱实验符合的很好;根据构象的相对能量以及理论模拟与实验的光电能谱的比对,说明对气相光电子能谱至少四种构象有贡献.     

尾式金属卟啉配合物的研究Ⅸ.苯丙氨酸基尾式卟啉及其锌配合物的合成和荧光性质

本文合成了L-苯丙氨酸基尾式卟啉及其锌配合物,研究了它们的荧光性质,观察到苯丙氨酸与卟啉环之间存在激发态分子内能量转移过程,讨论了影响卟啉特征荧光峰位置的配位化学因素.     

环丙氟沙星荧光探针对小牛胸腺DNA相互作用的研究

使用紫外和荧光光谱法研究了环丙氟沙星(CIF)和小牛胸腺DNA(ctDNA)之间的相互作用。在pH 7.0磷酸盐缓冲液中,CIF在270 nm激发和在420 nm处有很强的荧光发射。它们间的沟槽作用导致小牛胸腺ctDNA对CIF的荧光存在着很强的猝灭作用,猝灭常数为2.64×104 mol-1L(25 ℃)。利用ctDNA对CIF的荧光猝灭作用建立了核酸测定的新方法,线性范围为80 nmol·L-1～45 μmol·L-1,对25 μmol·L-1ctDNA的相对标准偏差为4.2％。     

葫芦[7]脲与二茂铁及芳香族氨基酸的相互作用研究

以摩尔比率法研究了葫芦[7]脲与二茂铁以及芳香族氨基酸之间的相互作用,研究发现葫芦[7]脲与二茂铁及芳香族氨基酸(色氨酸及苯丙氨酸)均为1∶1进行包结,并利用核磁研究了pH值对葫芦[7]脲-苯丙氨酸相互作用的影响。     

一种新型肽核酸单体的FTIR,FT-Raman光谱及SERS研究

报道了S-胸腺嘧啶-L-半胱氨酸的FTIR光谱,及其固态、饱和液态的FT-Raman光谱.通过红外与拉曼光谱的结合,对其分子结构中各基团的振动情况进行了较为全面的解析;实验发现,该化合物在银胶上的最佳SERS的浓度为10-4mol·L-1,氨基峰在酸性条件下增强,羧基峰在碱性条件下增强,而其他基团峰随pH值的改变变化不大.依据SERS作用机理和规律,推测S-胸腺嘧啶-L-半胱氨酸在银胶表面的吸附是通过硫原子、羧基、氨基、胸腺嘧啶环和其中的氮原子与银原子配位,且胸腺嘧啶环是倾斜着与银纳米颗粒相吸附;该吸附模型的建立为拉曼光谱更深入研究PNA、多肽及其他生物分子提供了十分重要的信息和有益的参考.     

FT-SERS研究非极性R侧链氨基酸在银胶体系中的吸附状态

利用傅里叶变换表面增强拉曼光谱(FT-SERS)研究了以L-蛋氨酸为代表的非极性R侧链氨基酸在纳米银衬底上的吸附状态和相互作用的特性,并结合浓度、pH值的变化探讨了吸附作用的特点和规律。实验结果表明, L-蛋氨酸在银胶上的最佳FT-SERS的浓度范围为10-3～10-4 mol·L-1;其pH范围以酸性、等电点、碱性而分段,当pH值在等电点附近及酸碱度过大时的FT-SERS都较差;L-蛋氨酸与纳米银的作用是通过氨基、羧基、硫与银的物理、化学吸附,其吸附态随着pH值的变化而改变。结合文献,对非极性R侧链氨基酸与纳米银的吸附特性作了总结,以期有助于对氨基酸、蛋白质、多肽、酶等相关领域更深入的研究。     

有机化合物对Luminol-KIO4-H2O2体系化学发光的抑制和增强

研究了36种有机化合物对鲁米诺-高碘酸钾-过氧化氢(luminol-KIO₄-H₂O₂)体系化学发光的影响,发现其大部分能抑制或增强体系化学发光强度,并且抑制或增强化学发光强度的能力与化学发光体系的pH值以及有机化合物分子结构中芳香环上功能基(—OH和—NH₂)的数目、位置,取代基的电子效应、空间效应等有关。讨论了化学发光强度抑制或增强的机理。基于24种有机化合物对体系化学发光的抑制或增强考察了其分析应用的可能性,发现数个化合物的检测限可达ng·mL^-1水平。     

紫外吸收光谱积分法分析蛋白质浓度-以碱性磷酸酶为例

利用矿物(针铁矿,蒙脱石)和太湖沉积物吸附碱性磷酸酶(alkaline phosphatase, APase),测定吸附后上清液中剩余碱性磷酸酶浓度时发现其紫外吸收光谱发生了变化,利用传统280 nm处紫外吸收法无法直接准确测定其浓度值。基于对碱性磷酸酶252～305 nm处吸收峰面积积分方法可以消除影响,并准确分析测定碱性磷酸酶浓度。其测定结果与考马斯亮蓝法测定结果进行比较,表明了该方法可以方便,快速和准确地测定此类实验中碱性磷酸酶浓度。同时,该方法还可以扩展至其他蛋白质的定量分析,甚至其他类似实验中,一定程度上克服传统方法应用单波长进行定量分析中存在的易受干扰的缺点。     

Medial efferent effects on auditory-nerve responses to tail-frequency tones. I. Rate reduction.

One way medial efferents are thought to inhibit responses of auditory-nerve fibers (ANFs) is by reducing the gain of the cochlear amplifier thereby reducing motion of the basilar membrane. If this is the only mechanism of medial efferent inhibition, then medial efferents would not be expected to inhibit responses where the cochlear amplifier has little effect, i.e., at sound frequencies in the tails of tuning curves. Inhibition at tail frequencies was tested for by obtaining randomized rate-level functions from cat ANFs with high characteristic frequencies (CF > or = 5 kHz), stimulated with tones two or more octaves below CF. It was found that electrical stimulation of medial efferents can indeed inhibit ANF responses to tail-frequency tones. The amplitude of efferent inhibition depended on both sound level (largest near to threshold) and frequency (largest two to three octaves below CF). On average, inhibition of high-CF ANFs responding to 1 kHz tones was around 5 dB. Although an efferent reduction of basilar-membrane motion cannot be ruled out as the mechanism producing the inhibition of ANF responses to tail frequency tones, it seems more likely that efferents produce this effect by changing the micromechanics of the cochlear partition.     

Effect of ultrasonically induced cavitation on inhibition behavior of polyethylene glycol on carbon steel corrosion

In this investigation the influence of 24 kHz ultrasound wave upon the corrosion of carbon steel in 3N sulphuric acid at 25 degrees C in the presence of inhibitors was studied. The inhibitors were polyethylene glycols (PEG) in different molecular weights (from 400 up to 10,000 gmol(-1)). The polarization and impedance spectroscopy results show the effectiveness of polyethylene glycols on the cavitation-corrosion inhibition of carbon steel in sulphuric acid. The inhibition efficiency is increased with increasing mean molecular weight of polymer and its concentration. The weight loss method has confirmed these results. The analysis of SEM images indicates that these inhibitors prevent propagation of pits on the eroded specimen. The inhibition effect of PEGs can be attributed to cushioning effect of adsorbed polymers on cavitation phenomenon produced by bubble collapse.     

Detection of Pyrophosphate and Alkaline Phosphatase Activity Based on PolyT Single Stranded DNA - Copper Nanoclusters

The determination of pyrophosphate and alkaline phosphatase activity plays a significant role in medical diagnosis. In this work, a label-free “ON-OFF-ON” fluorescence strategy is developed for the analysis of pyrophosphate and alkaline phosphatase activity. Using PolyT single strand DNA as templates to synthesize fluorescent copper nanoparticles, the coordination effect of pyrophosphoric acid on Cu²⁺ inhibited the generation of fluorescence. Afterwards, the addition of alkaline phosphatase into hydrolyze pyrophosphoric acid resulted in the release of Cu^2+, whereby the fluorescence intensity could be recovered. Thereupon enhanced-sensitivity for alkaline phosphatase was obtained (0.1 mU/L), much better than previously reported methods. Meanwhile, it could be performed directly in homogeneous solution, which was very close to the actual activity level of alkaline phosphatase under physiological conditions. Likewise, satisfactory results were also obtained in specificity assessment, which demonstrated its potential application in clinical diagnosis. Notably, a new, sensitive, low-cost, short-time, and high-sensitivity platform for alkaline phosphatase detection was constructed, and the design of biosensor using DNA-templated Copper nanoclusters (CuNCs) was instructed in this study.

     

Time-gated microscopic energy transfer measurements for probing mitochondrial metabolism

Spectroscopic and microscopic methods for probing mitochondrial malfunction were established using cultivated endothelial cells from the calf aorta and various inhibitors of the respiratory chain, which is located at the inner mitochondrial membrane. Time-gated fluorescence spectroscopy was used to measure autofluorescence of the coenzyme NADH as well as “energy transfer efficacy” from excited NADH molecules (energy donor) to the mitochondrial marker rhodamine-123 (energy acceptor). Autofluorescence usually exhibited a weak increase after specific inhibition of enzyme complexes of the respiratory chain. In contrast, a pronounced increase in energy transfer efficacy was observed after inhibition of the same enzyme complexes. The detection of donor (NADH) and acceptor (R123) fluorescence in different nanosecond time gates following the exciting laser pulses enhances selectivity and improves quantification of energy transfer measurements. Therefore, timegated energy transfer spectroscopy is suggested to be an appropriate tool for probing mitochondrial malfunction.     

Enzyme histochemical localisation of alkaline phosphatase activity in osteogenesis imperfecta bone and growth plate: a preliminary study

At the ultrastructural level alkaline phosphatase has been studied in calcifying cartilage but not in bone. The aim of this study was to assess if there is an osteoblast dysfunction in Osteogenesis Imperfecta (OI) with respect to alkaline phosphatase activity. Specimens from three OI type II foetal femoral bones, two OI type II growth plates, one normal foetal femoral bone and growth plate, one OI type III femoral bone specimen and one normal juvenile bone specimens were examined using modified lead nitrate method to identify alkaline phosphatase reactivity. The electron dense reaction product (indicative of the presence of alkaline phosphatase) was demonstrable on the cell membrane of the osteoblasts, as focal concentrations in the collagen osteoid and on the mineralisation front of normal bone. In normal bone the intensity of the reaction seemed to be stronger than in OI bone and appeared as a continuous black line along the osteoblast cell membranes. In OI bone the reaction product only appeared as a few electron dense beads along the osteoblast cell membrane.

There appeared to be reduced and diffuse reaction product on OI osteoblasts, thus implying either a reduced level and/or altered activity of alkaline phosphatase and hence a dysfunction of osteoblasts. This confirms the findings of the previous report of the impaired activity of alkaline phosphatase in OI osteoblasts. Even in the OI growth plate, hypertrophic chondrocytes showed less intense reaction product than the chondrocytes in the normal growth plate.

The normal human growth plates used in this study showed a similar pattern, but in the OI growth plate even the hypertrophic zone, where the alkaline phosphatase activity is reported to be high, showed less intense reaction product. Biochemical reports indicate that alkaline phosphatase levels are normal in cultured OI cell lines, yet ultrastructural histochemical observations reported here, show reduced enzyme localisation and this may suggest reduced amounts of protein or reduced activity at the tissue level.     

溶液pH对羟基苯硫酚同分异构体脱羟基反应影响的SERS研究

金属纳米结构因表面等离激元(SPR)而产生光学增强和催化效应已成为表面科学研究热点之一。SPR和电化学联用可以诱导催化一些非常规反应,并且不同pH值电解质溶液可改变表面吸附分子的存在形式,影响SPR光催化反应。以羟基苯硫酚的同分异构体为探针,采用电化学表面增强拉曼光谱(SERS)研究了取代基羟基位置、溶液pH值等对其在银电极表面吸附和SPR催化反应行为。结果表明,不同羟基取代基位置的羟基苯硫酚SPR催化脱羟基反应对溶液pH值的敏感程度不同,邻羟基苯硫酚(OHTP)的C—O键谱峰强度的变化与溶液pH值相关,其O端更易与金属作用而吸附在表面,且随pH增大而增强。对羟基苯硫酚(PHTP)在碱性条件下被完全抑制的脱羟基反应在间羟基苯硫酚(MHTP)和OHTP中均可发生。MHTP在中性(pH 7)溶液中SPR催化脱羟基反应效率最高,约为酸性(pH 2)的1.36倍,碱性(pH 12)的2.70倍。OHTP在碱性(pH 12)溶液中SPR催化脱羟基反应效率最高,约为酸性(pH 2)的13.71倍,中性(pH 7)的4.95倍。SPR催化脱羟基主要源于非去质子化条件以及形成Ag—O键这两种途径。酸性条件下MHTP及OHTP的脱羟基反应主要是未去质子化的羟基反应,碱性条件主要因去质子化后形成Ag—O键所致。中性条件下,两种贡献同时发生。对MHTP而言,由于位阻效应仅部分分子去质子化后形成Ag—O键而促进SPR催化脱羟基,因此pH 7溶液中两种效应的同时作用导致催化效率最高。对于OHTP分子,去质子化状态的O端更易与电极表面发生作用,且pH升高羟基呈现的去质子化程度更加彻底,更有利于发生脱羟基反应,在pH 12溶液中脱羟基反应主要由于形成Ag—O键,其效率亦最高。同分异构体结构以及介质酸碱度对SPR催化脱羟基反应的研究对于拓宽SPR催化反应类型及从分子水平解析其机理具有重要意义。