【作者】 黄国霞；

【导师】 蒋治良；

【作者基本信息】 广西师范大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 第一部分:绪论介绍了共振散射技术理论及其在生化分析研究中的应用。介绍了木瓜蛋白酶、糜蛋白酶等五种蛋白酶活力测定的方法。综述了溶菌酶、辣根过氧化物酶的分析进展。第二部分:酪蛋白缔合微粒的共振散射光谱研究及其在木瓜蛋白酶活力测定中的应用在最佳实验条件下,木瓜蛋白酶催化水解酪蛋白(casein),剩余底物casein分别与三氯乙酸(TCA)、十二烷基苯磺酸钠(SDBS)、十二烷基硫酸钠(SDS)结合形成缔合微粒。该微粒在360 nm、400 nm、420 nm、470 nm、520 nm处产生5个共振散射峰,其中最强峰均位于470 nm。对于TCA-Casein-Papain、SDBS-Casein-Papain、SDS-Casein-Papain三个体系,Papain的酶活力分别在0.024～4.8、0.048～4.8、0.096～7.2 USP/mL范围内与△I470nm之间呈现良好线性关系。检测限分别为0.00832、0.04068、0.0814 USP/mL。TCA共振散射光谱法(RSS)应用于嫩肉粉中木瓜蛋白酶活力测定,结果满意。第三部分:四种动物蛋白酶活力的催化共振散射光谱测定在所选实验条件下,糜蛋白酶、胰蛋白酶、弹性蛋白酶、胃蛋白酶均能催化水解酪蛋白生成小分子产物。加入三氯乙酸可与剩余的酪蛋白结合形成缔合微粒并中止酶催化反应。该微粒在360 nm、400 nm、420 nm、470 nm、520 nm处产生5个共振散射峰,其中最强峰位于470 nm处。糜蛋白酶、胰蛋白酶、弹性蛋白酶、胃蛋白酶的酶活力分别在0.0012～0.06 U/mL、0.00024-0.04 U/mL、0.0002～0.006 U/mL、0.0006-0.024 U/mL范围内与470 nm处的散射光强度降低值△I470nm之间呈良好线性关系,其检出限分别为0.001148 U/mL、0.000839 U/mL、0.000156 U/mL、0.000557 U/mL。该法应用于实际样品中糜蛋白酶活力测定,结果比较满意。第四部分:溶壁微球菌的共振散射光谱研究及其在溶菌酶活力测定中的应用溶壁微球菌在360 nm、400 nm、420 nm、470 nm和520 nm处产生5个共振散射峰,菌浓度在2.0×106～9.3×108个/mL范围内与共振散射光强度I470nm有良好的线性关系。基于溶菌酶对溶壁微球菌的催化水解作用导致体系I470nm降低,建立了一个检测0.24～40 U/mL(即0.012～2μg/mL)溶菌酶的共振散射光谱新方法,其检出限(3σ)为0.014 U/mL(即0.0007μg/mL)。该法测定唾液样品的分析结果与比浊法结果一致,两种方法结果的线性回归方程的斜率、相关系数和截距分别为0.9665、0.9973和-87.50。第五部分:辣根过氧化物酶活力的催化共振散射光谱测定在所选实验条件下,辣根过氧化物酶催化H2O2氧化KI生成I2,过量的I-与I2可结合形成I3-,而I3-分别与罗丹明S(RhS),罗丹明G(Rh6G),罗丹明B(RhB),丁基罗丹明B(b-RhB)结合形成(Rh+ -I3-)缔合微粒,在320 nm、424 nm、508 nm、和610 nm处产生较强的共振散射效应。但四体系在610 nm处的共振散射均分别受RhS 556 nm、Rh6G 556 nm、RhB 580 nm、b-RhB 580 nm处同步荧光峰的影响。对于RhS、Rh6G、RhB、b-RhB四体系,辣根过氧化物酶的浓度分别在3.2×10-12g～4.8×10-9g /mL、2×10-11g～3.2×10-9g /mL、1.6×10-11g～3.2×10-9 /mL、1.6×10-11g～4×10-9 g /mL范围内与其在424 nm处的共振散射强度成线性关系,其检出限分别为2.2×10-12g /mL、2.5×10-12g /mL、4.4×10-12g /mL、2.6×10-12g /mL。该法应用于酶联免疫乙肝试剂盒中辣根过氧化物酶活力的测定,结果比较满意。更多还原

【Abstract】 PartⅠIntroductionSummarized the application of resonance scattering technology in analytical chemistry. The analytical progress of five proteinase including papain, chymotrypsin, trypsin, elastase and pepsin was introduced. The analytical progress of lysozyme and horseradish proxidase activities was also introduced. One hundred and ninety-eight references were quoted.PartⅡResonance Scattering Spectral of Casein Association Particles and its Application to Assay of Papain ActivityUnder the optimal experimental conditions, papain catalyzes the hydrolytic reaction of casein. The remain substrate casein associate with trichloroacetic acid, dodecyl benzene sulfonic acid sodium salt, sodium dodecyl sulphate to association particles respectively, which produce five resonance scattering peaks at 360, 00, 420, 470 and 520 nm, and a strongest peaks at 470nm.There is a good linear relationship between papain activity in the range of 0.024～4.8 USP/mL for TCA-Casein-Papain system,0.048～4.8 USP/mL for SDBS-Casein-Papain system,0.096～7.2 USP/mL for SDS-Casein-Papain system and the resonance scattering intensity△I470nm with a detection limit of 0.00832, 0.04068, 0.0814USP/mL respectively. The resonance scattering spectral assay(RSS) of TCA-Casein is applied to the determination of papain activity which contained in meat tenderizer with satisfactory results.PartⅢA new resonance scattering spectral assay of chymotrypsin activityUnder the optimal experimental conditions, proteinase catalyzed the hydrolysis of casein. The remain substrate associate with trichloroacetic acid to form association particles, which produce five resonance scattering peaks at 360 nm, 400 nm, 420 nm, 470 nm and 520 nm, with a strongest one at 470 nm. There are good linear relationship between enzyme activity and the resonance scattering intensity△I470nm in the range of0.0012～0.06 U/mL for chymotrypsin, 0.00024-0.04 U/mL for trypsin, 0.0002～0.006 U/mL for elastase, 0.0006-0.024 U/mL for pepsin, with a lowest detection limit of 0.000156 U/mL elastase. The resonance scattering spectra (RSS) assay of chymotrypsin is applied to the determination of real samples with satisfactory results.PartⅣStudies on Resonance Scattering Spectra of M.Lysodeikticus and its Application to Assay of Lysozyme ActivityM. Lysodeikticus has five resonance scattering peaks at 360 nm, 400 nm, 420 nm, 470 nm and 520 nm. The concentration of M. Lysodeikticus in the range of 2×106～9.3×108 cell/mL is proportional to the resonance scattering intensity I490nm. According to the decreasing of I490nm because of the bacteria-dissolving function of lysozyme to M. Lysodeikticus, a new resonance scattering spectral assay for determination of 0.24～40 U/mL(0.012～2μg/mL)lysozyme activity is proposed,with a detection limit(3σ) of 0.014 U/mL (0.0007μg /mL). The results for Lysoyme activity in saliva is according to the results of immunoturbidimetry, and the slope, intercept and the correlation coefficient of regression equation for the two methods is 0.9665,0.9973, -87.50 respectively.PartⅤA new resonance scattering spectra assay of horseradish peroxidase activityUnder the optimal experimental conditions, the reaction between H2O2 and KI wascatalyzed by horseradish peroxidase (HRP) to from I3-.The I3-can combines respectively with Rhodamine S (RhS), Rhodamine 6G(Rh6G), Rhodamine B(RhB)and butyl-Rhodamine B(b-RhB) to form association particles that exhibit stronger resonance(RS) scattering effect at 320 nm, 424 nm ,508 nm and 610 nm. However, the RS peaks at 580 nm is interfered with its synchronous fluorescence peak at 556 nm for RhS, 556 nm for Rh6G, 580 nm for RhB, 580 nm for b-RhB, repectively. The concentration of HRPin the range of 3.2×10-12g～4.8×10-9g /mL, 2×10-11g～3.2×10-9g /mL, 1.6×10-11g～3.2×10-9g /mL and 1.6×10-11g～4×10-9g /mL is linear to its RS intensity at 423 nm, with detection limits of 2.2×10-12g /mL, 2.5×10-12g /mL, 4.4×10-12g /mL and 2.6×10-12g /mL. This assay was applied to determination of HRP in the solution of hepatitis B surface antigen labeling HRP, with satisfactory results.更多还原