HSA的分频荧光及DNA和HSA的共振散射光谱研究
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摘要
第一部分：在生理pH(7.43)条件下，人血清白蛋白(human serum albumin，简称HSA)和色氨酸(tryptophane,简称Trp)在344nm和360nm分别产生荧光峰；在688nm和720nm分别产生1／2分频荧光峰。考察了分频荧光峰与荧光峰的半峰宽之比和峰高之比，发现它们分别接近一常数。两者具有相似的荧光特性。加入在色氨酸荧光峰波长处有强吸收的物质，荧光峰和分频荧光峰强度均同时减弱；当入射光波长大于荧光峰波长时，荧光峰和分频荧光峰均不出现。依据非线性光学原理和分频原理，探讨了半峰宽与中心波长(荧光峰波长)的关系及分频荧光峰产生的原因。
第二部分：在生理pH(7.43)条件下，DNA在470nm和350nm处分别产生最强共振散射峰和次强共振散射峰。固定激发波长，扫描发射波长，DNA溶液将产生瑞利共振散射峰、分频共振散射峰和倍频共振散射峰。研究了pH、离子强度、表面活性剂等对共振散射光强度的影响，由此可推测DNA分子结构与构象的变化。依据量子电动力学原理，探讨了由普通单色光引起的各种共振散射峰产生的机理，分析了各种共振散射峰强度不同的原因。
第三部分：利用金属配阴离子与蛋白质结合成超分子离子缔合物及界面的形成，采用共振散射技术建立蛋白质定量测定方法。在2.0×10'3mol.dm'3HCl介质中，蛋白质与[Fe(CN)‘]·通过静电引力为主的分子间作用力结合成超分子离子缔合物，在350nm处产生最强共振散射光信号。基于这一现象可以测定低至0.3μg.ml'~的人血清白蛋白。工作曲线在0-12μg.ml'’范围内呈线性关系。这一新方法的灵敏度比Coomassie
广西师范大学硕士论文：HSA的分频荧光及洲A和HSA的共振散射光谱研究
亮蓝法高 3倍．研究T pH、离子强度和厂e（CN）61s－浓度对反应体系
的作用。将这一方法用子合成样品的微量蛋白质测定，结果满意．
Part l:At physiological pH ( 7.43 ) human serum albumin(HSA) and tryptophane(Trp) exhibit their fluorescence peaks at 344nm and 360nm,respectively, while their 1/2 fraction frequency fluorescence peaks at 688nm and 720nm. The ratio of half peak width and the ratio of peak height (or fluorescence intensity) between 1/2 fraction frequency fluorescence peak and the fluorescence peak approach a constant respectively. The two fluorescence peaks have similar fluorescence feature and their intensities weaken at the same time while strong absorbing substance in the wavelength of Trp (or HSA) fluorescence peak is added to; the two fluorescence peaks don't appear if the excitation wavelength is longer than that of fluorescence peak . According to the principle of nonlinear optics and fraction frequency, the relationship between half peak width and center wavelength (fluorescence peak wavelength) and the cause of fraction frequency fluorescence peak have been considered.
Part II: At physiological pH(7.43),DNA exhibits the strongest and the second strong resonance scattering peaks at 470nm and 350nm,respectively. Rayleighx fraction frequency and double resonance frequency scattering peaks were found in the emission spectrum of DNA. Influence of pH?ionic
strength and surfactant on the intensity of light-scattering was studied. By this way, the variation of structure or conformation of DNA molecule has been inferred. According to the principle of quantum electrodynamics,the mechanism of all kinds of resonance scattering peaks caused by ordinary monochromatic light was
discussed systematically and the intensity of various resonance scattering light was also analysed.
PartIII:Based on the formation of supramolecular ion-association complexes resulting from the combination of metal complex anions with proteins and the interface formation, a new method for the determination of human serum albumin (HSA) by using the technique of resonance scattering has been founded. In the medium of 2. 0 ICT'mol. dm'3 HC1, proteins may combine with [Fe(CN)6]3~ by intermolecular forces(mainly by electrostatic force) to supramolecular ion-association complexes, causing a light signal of the strongest resonance scattering at 351nm. Based on this, no less 0.3 lAg-ml"1 HSA can be determined . Its calibration curve is near over the range of Q-12\ig.mTl .The sensitivity of this method is three-fold higher than that of Coomassie brilliant blue protein assay. Influence of experimental conditions such as pH, ionic strength and concentration of [Fe(CN)6] * on the intensity of resonance scattering light was studied. Effects of surfactants (cationic, anionic and nonionic) > ami no acids and metal ions on the combination of [Fe(CN)6J3" with protein were also investigated .This method has been used for the determination of synthetic samples with satisfactory results .
第二部分：在生理pH(7.43)条件下，DNA在470nm和350nm处分别产生最强共振散射峰和次强共振散射峰。固定激发波长，扫描发射波长，DNA溶液将产生瑞利共振散射峰、分频共振散射峰和倍频共振散射峰。研究了pH、离子强度、表面活性剂等对共振散射光强度的影响，由此可推测DNA分子结构与构象的变化。依据量子电动力学原理，探讨了由普通单色光引起的各种共振散射峰产生的机理，分析了各种共振散射峰强度不同的原因。
第三部分：利用金属配阴离子与蛋白质结合成超分子离子缔合物及界面的形成，采用共振散射技术建立蛋白质定量测定方法。在2.0×10'3mol.dm'3HCl介质中，蛋白质与[Fe(CN)‘]·通过静电引力为主的分子间作用力结合成超分子离子缔合物，在350nm处产生最强共振散射光信号。基于这一现象可以测定低至0.3μg.ml'~的人血清白蛋白。工作曲线在0-12μg.ml'’范围内呈线性关系。这一新方法的灵敏度比Coomassie
广西师范大学硕士论文：HSA的分频荧光及洲A和HSA的共振散射光谱研究
亮蓝法高 3倍．研究T pH、离子强度和厂e（CN）61s－浓度对反应体系
的作用。将这一方法用子合成样品的微量蛋白质测定，结果满意．
Part l:At physiological pH ( 7.43 ) human serum albumin(HSA) and tryptophane(Trp) exhibit their fluorescence peaks at 344nm and 360nm,respectively, while their 1/2 fraction frequency fluorescence peaks at 688nm and 720nm. The ratio of half peak width and the ratio of peak height (or fluorescence intensity) between 1/2 fraction frequency fluorescence peak and the fluorescence peak approach a constant respectively. The two fluorescence peaks have similar fluorescence feature and their intensities weaken at the same time while strong absorbing substance in the wavelength of Trp (or HSA) fluorescence peak is added to; the two fluorescence peaks don't appear if the excitation wavelength is longer than that of fluorescence peak . According to the principle of nonlinear optics and fraction frequency, the relationship between half peak width and center wavelength (fluorescence peak wavelength) and the cause of fraction frequency fluorescence peak have been considered.
Part II: At physiological pH(7.43),DNA exhibits the strongest and the second strong resonance scattering peaks at 470nm and 350nm,respectively. Rayleighx fraction frequency and double resonance frequency scattering peaks were found in the emission spectrum of DNA. Influence of pH?ionic
strength and surfactant on the intensity of light-scattering was studied. By this way, the variation of structure or conformation of DNA molecule has been inferred. According to the principle of quantum electrodynamics,the mechanism of all kinds of resonance scattering peaks caused by ordinary monochromatic light was
discussed systematically and the intensity of various resonance scattering light was also analysed.
PartIII:Based on the formation of supramolecular ion-association complexes resulting from the combination of metal complex anions with proteins and the interface formation, a new method for the determination of human serum albumin (HSA) by using the technique of resonance scattering has been founded. In the medium of 2. 0 ICT'mol. dm'3 HC1, proteins may combine with [Fe(CN)6]3~ by intermolecular forces(mainly by electrostatic force) to supramolecular ion-association complexes, causing a light signal of the strongest resonance scattering at 351nm. Based on this, no less 0.3 lAg-ml"1 HSA can be determined . Its calibration curve is near over the range of Q-12\ig.mTl .The sensitivity of this method is three-fold higher than that of Coomassie brilliant blue protein assay. Influence of experimental conditions such as pH, ionic strength and concentration of [Fe(CN)6] * on the intensity of resonance scattering light was studied. Effects of surfactants (cationic, anionic and nonionic) > ami no acids and metal ions on the combination of [Fe(CN)6J3" with protein were also investigated .This method has been used for the determination of synthetic samples with satisfactory results .
引文
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83 钟福新，蒋治良，李廷盛等，不同颜色银纳米粒子的共振散射光谱研究．中国化学会二零零年学术会议论文集，2000年11月，p171
84 钟福新，蒋治良，李廷盛等，黄色银纳米粒子的光化学制备及光谱研究．感光科学与光化学，2001，24(1)：17
85 梁宏，沈星灿，蒋治良等，共振Rayleigh散射研究Ⅰ与血清白蛋白的结合平衡．中国科学(B辑)，2000，30(5)：460-466
86 武建国主编，实用临床免疫检验，江苏：科学技术出版社，1990：393
87 郭尧君编著，荧光实验技术及其在分子生物学中的应用．北京：科学出版社，1979：113
88 Markolf H. Niemz著，张镇西译，激光与生物相互作用——原理及应用．西安交通大学出版社，1999：16
89 沈元壤，非线性光学原理(上册)[M]，北京：科学出扳社，1987：71-120
90 曾谨言，量子力学[M]，北京：科学出版社，2000．56
91 Li D.Q., Marks T.J.,Nonlinear optical phenomena in conjugated organic chromophores theorical investigation via π-electron formation.J.Phys. Chem., 1992,96:4323
92 Arecchi I. T. Schiz-DuBois E O. 激光的物理应用(第五分册)[M]，《激光手册》翻译组译，北京：科学出版社，1979：60-61
93 刘颂豪，赫光生，强光光学及应用．广洲：广东科技出版社，1995：2
94 孙志贤主编，现代生物化学理论与研究技术．军事医学科学出版社，1995，168
95 Turner P. C., Mclennan A. G., Bates A. D. and White M. R. H.. Instant Notes in Molecular Siology BIOS scientific publishers limited, 1997: 37
96 刘颂豪，赫光生，强光光学及应用．广州：广东科技出扳社，1995：39-40
97 李原芳，黄承志，胡小莉，共振散射技术的原理极其在生化研究和分析中的应用．分析化学，1998，26(12)：7510
98 魏永巨，童沈阳，李克安，生物活性物质与光谱探针反应的研究——血清白-蛋白与铬天青S的反应，化学学报，1995，53(1)：82-89
99 华中师范大学，东北师范大学，陕西师范大学编，分析化学(第二版)，高等教育出版社，1987：298
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4 马春琪，刘英，李克安等，瑞利散光散射及其在生物化学分析中的应用研究．科学通报，1999，44(7)：682
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14 蒋治良，冯忠伟，金纳米粒子的分频核倍频散射研究，广西师范大学学报，2000，18(4)：60-65
15 蒋治良等，淀粉超分子的共振散射研究，光散射学报，2001，26，待发表
16 蒋治良，刘庆业，刘绍璞，金原子团簇的制备及共振散射研究，西南师范大学学报，2001，24(2)：226
17 蒋治良 刘庆业，李芳，银原子团簇共振散射光谱的密度矩阵研究，广西科学，2001，13(3)，待发表
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56 黄承志，李原芳，李念兵等，耐尔蓝硫酸盐在核酸分子表面的长距组装及核酸的三波长共振光散射测定，分析化学，1999，27(11)：1241-1247
57 马春琪，刘瑛，李克安，童沈阳，瑞利光散射及其在生物化学分析中的应用研究．科学通报，1999，44(7)：683-690
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60 刘绍璞，刘忠芳，汞(Ⅱ)-硫氰酸盐-维多利亚蓝4R体系的共振发光和二级散射光谱及其分析应用．高等学校化学学报，1996，17(6)：887-892
61 刘绍璞，刘忠芳，李明，离子缔合二级散射光谱的分析应用：Ⅲ汞(Ⅱ)-硫氰酸盐-罗丹明染料体系．分析化学，1996，24(5)：501-504
62 刘绍璞，刘忠芳，阳离子表面活性剂-曙红Y体系的二级散射光谱及其在分析化学上的应用．分析化学，1996，24(6)：665-668
63 刘绍璞，分析化学新进展(汪尔康主编)，大原：山西科学出版社，1997：34
64 刘绍璞，刘忠芳，分析化学新进展(汪尔康主编)，太原：山西科学出版社，1997：390-391
65 刘芹，刘绍璞，分析化学新进展(汪尔康主编)，大原：山西科学出版社，1997：394-395
66 刘绍璞，周光明，共振瑞利散射法测定硫氰酸盐-碱性三苯甲烷染料体系，高等学校化学学报，1998，19(7)：1040-1044
67 刘绍璞，刘忠芳，硒(Ⅳ)-碘化物-维多利亚蓝4R体系共振瑞利散射、二级散射，，西南师范大学学报，2000，25(14)：408-412
68 刘绍璞，刘忠芳，李明，离子缔合二级散射光谱的分析应用，化学学报，1995，53：1178-1184
69 刘绍璞，刘忠芳，李明，离子缔合二级散射光谱的分析应用Ⅱ汞(Ⅱ)-硫氰酸盐-碱性三芳基甲烷染料体系，化学学报，1995，53：1185-1192
70 蒋治良，李芳，梁宏，磷钼杂多酸-罗丹明S体系的共振散射光谱研究．化学学报，2000，58(8)：1059-1063
71 蒋治良，金纳米粒子与光相作用的光散射方程，分析测试技术与仪器，2000，6(4)：98-102
72 蒋治良，金纳米粒子的分频散射光谱研究，光子学报，2001，31：396
73 黄永炎，蒋治良，王盛棉等，硅锑钼杂多酸罗丹明S体系的共振散射光谱研究，分析科学学报，2000，16：127-130
74 蒋治良，杨明媚，莫琪，银的光化学共振散射光谱分析，贵金属，200，21(3)：34-39
75 蒋治良，李芳，李廷盛等，金粒径与共振散射光强度的关系，高等学校化学学报，2000,21：1488-1491
76 蒋治良，冯忠伟，蒋毅民，刘庆业等，金纳米粒子的散射光强度函数研究，无机化学学报，2001，17(3)：396
77 蒋治良，李芳，梁宏，绿色银纳米粒子的共振散射光谱研究，化学学报，2001．59(3)：438-441
78 Jiang zhiliang, Lin xiaoming, Microwave synthesis of gold nanoparticle and its spectral properties. Chinese Chemical Letters,2001,13: 302
79 Jiang zhiliang, Feng zhongwei, Jiang Yimin, Microwave synthesis of TiO_2 nanoparticle and its spectral properties. Chemical Journal on Internet, 2001, 03(4):017ne
80 Jiang Zhiliang, Feng Zhongwei, Li Fang, Study on the resonance scattering spectum of gold nanoparticle, Science in China(B), 2001, 44(2): 175
81 谢济运，蒋治良，李廷盛等，人细胞的光谱特性研究．分析测试技术与仪器，2000，6(3)：98
82 谢济运，蒋治良，柑桔溃疡菌的共振散射光谱研究．物理化学学报，2001，19(5)：586
83 钟福新，蒋治良，李廷盛等，不同颜色银纳米粒子的共振散射光谱研究．中国化学会二零零年学术会议论文集，2000年11月，p171
84 钟福新，蒋治良，李廷盛等，黄色银纳米粒子的光化学制备及光谱研究．感光科学与光化学，2001，24(1)：17
85 梁宏，沈星灿，蒋治良等，共振Rayleigh散射研究Ⅰ与血清白蛋白的结合平衡．中国科学(B辑)，2000，30(5)：460-466
86 武建国主编，实用临床免疫检验，江苏：科学技术出版社，1990：393
87 郭尧君编著，荧光实验技术及其在分子生物学中的应用．北京：科学出版社，1979：113
88 Markolf H. Niemz著，张镇西译，激光与生物相互作用——原理及应用．西安交通大学出版社，1999：16
89 沈元壤，非线性光学原理(上册)[M]，北京：科学出扳社，1987：71-120
90 曾谨言，量子力学[M]，北京：科学出版社，2000．56
91 Li D.Q., Marks T.J.,Nonlinear optical phenomena in conjugated organic chromophores theorical investigation via π-electron formation.J.Phys. Chem., 1992,96:4323
92 Arecchi I. T. Schiz-DuBois E O. 激光的物理应用(第五分册)[M]，《激光手册》翻译组译，北京：科学出版社，1979：60-61
93 刘颂豪，赫光生，强光光学及应用．广洲：广东科技出版社，1995：2
94 孙志贤主编，现代生物化学理论与研究技术．军事医学科学出版社，1995，168
95 Turner P. C., Mclennan A. G., Bates A. D. and White M. R. H.. Instant Notes in Molecular Siology BIOS scientific publishers limited, 1997: 37
96 刘颂豪，赫光生，强光光学及应用．广州：广东科技出扳社，1995：39-40
97 李原芳，黄承志，胡小莉，共振散射技术的原理极其在生化研究和分析中的应用．分析化学，1998，26(12)：7510
98 魏永巨，童沈阳，李克安，生物活性物质与光谱探针反应的研究——血清白-蛋白与铬天青S的反应，化学学报，1995，53(1)：82-89
99 华中师范大学，东北师范大学，陕西师范大学编，分析化学(第二版)，高等教育出版社，1987：298