Scattering Spectral Assay Of Some Enzymes Activities

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.