The Analysis On The Fluorescence Spectrum Of Labeled Lipase By Fluorescent Probe In The Phase Of Liquid And Polymer Carrier

A new method is established to investigate the relationships among the fluorescence spectrum, the activity of lipase and the change of conformation in this dissertation, after the lipase was labeled with the fluorescent probe (Fluorescein isothiocyanate or Rhodamine B) and the fluorescence spectrum reflected the conformation change of lipase in the liquid and polymer carrier. At first, the four fluorescent probes were screened that FITC (Fluorescein isothiocyanate) and RhB (Rhodamine B) were suitable for lipase. Then the relationship between fluorescence spectrum and activity of enzyme was investigated under the various conditions of denaturation. Eventually the fluorescence inspection was taken to the polymeride of labeled lipase, in order to researching the effect of polymer carrier, induction of ligand, extraction of ligand and other conditions of denaturation to the conformation of lipase.The first part of the dissertation emphasizes on the investigated of lipase labeled with FITC, RhB, Rh123 (Rhodamine 123) and Rh6G (Rhodamine B). The results demonstrated that FITC and RhB labeling would reduce the activity of lipase in the optimal temperature, but in the other temperature the stability of lipase was increased because of the effect of steric hindrance. The fluorescence spectrum showed that the intensity of fluorescence increased with the rise of temperature. The phenomena illustrated that the conformation of lipase changed continuously, so the FITC and RhB could function well in the aspect of inspecting lipase conformation. However, the labeled lipase with Rh123 or Rh6G became deactivated, so these two probes weren't suitable for labeling lipase.The labeled lipase with FITC and RhB were further researched in the condition of denaturation in liquid. The results demonstrated: FITC and RhB labeling would reduce the activity of lipase in the optimal temperature, and the decreasing amplitude of FITC labeling was smaller than RhB labeling, and in the other temperatures the thermal stability of labeled lipase were increased because of the effect of steric hindrance. In the meanwhile, the labeled lipase became complete deactivated in 70℃, compare to being deactivated in 60℃of unlabeled lipase, which rised up 10℃. The fluorescence spectrum showed that the intensity of fluorescence increased with the rise of temperature. In the temperature of 50℃, 60℃and 70℃, the f_U of FITC labeled lipase were 0.63, 0.91 and 1, and the f_U of RhB labeled lipase were 0.65, 0.71 and 1. In the effect of pH, the activity of labeled lipase would decrease with the pH deviated from 7.4, and the tolerance to acid and alkali were also raised. From the analysis of fluorescence spectrum, FITC was liable to be influenced by value of pH, so the FITC wasn't suitable for detecting the change of lipase conformation in different pH, but probe RhB was stable in such condition, so it had advantage over FITC. In the pH of 3.2, 4.7, 7.4, 8.7 and 11, the f_U of RhB labeled lipase were 1, 0.67, 0, 0.47 and 0.79. With the increasing GuHCl concentration from 0 mol/L to 5 mol/L, the activity of labeled lipase decreased, and the intensity of fluorescence spectrum also decreased. The f_U of FITC labeled lipase were 0.38, 0.68, 0.84, 0.95 and 1, and the f_U of RhB labeled lipase were 0.48, 0.72, 0.80, 0.88 and 1. With the increasing urea concentration from 0 mol/L to 8 mol/L, the activity of labeled lipase decreased, and the intensity of fluorescence spectrum also decreased, just like the tendency of GuHCl condition. The f_U of FITC labeled lipase were 0.21, 0.30, 0.58, 0.79 and 1, and the f_U of RhB labeled lipase were 0.23, 0.46, 0.69, 0.88 and 1.The labeled lipase with the probe of FITC and RhB were polymerized in the experiments, and the polymer carriers used were polyethylene glycol 200-dimethacrylate and polyethylene glycol 400-dimethacrylate, with the inducing of ligand (lauric acid). The polymerization was initiated by ultraviolet irradiation. After polymerization the ligand was extracted using Sherwood oil, resulting in four lipase polymers (FITC labeled LP-200, FITC labeled LP-400, RhB labeled LP-200 and RhB labeled LP-400). By inspecting the activity of LP and their fluorescence spectrum, the effect of polymer carriers, induction of ligand and extraction of ligand to LP were investigated. The results demonstrated that polyethylene glycol 400-dimethacrylate could be more suitable for polymerization because of its structure, and adding ligand could change some parts of lipase conformation to express its higher activity, and the active centre of lipase could be occupied by the ligand if without extraction, so the activity might decrease a lot.Through the effect of different denaturation condtion to four labeled LP, such as temperature, value of pH, dissolution denaturant and sediment denaturant, and with the renaturation of LP in low concentration of GuHCl, the change of activity and fluorescence spectrum were investigated. The results demonstrated that in the effect of different temperature and pH, four labeled LP still could maintain high activity, and the fluorescence spectrum could reflect the LP activity because of the change of conformation. In the effect of dissolution denaturant and sediment denaturant, the activity of four LP firstly decreased, and then became steady, and their steady activity were all higher than 70%, which showed that the four LP had good resistance to denaturant, and the fluorescence spectrum which reflected the LP conformation had excellent relation with the change of LP activity. With the increasing GuHCl concentration from 0 mol/L to 5 mol/L, the activity of FITC labeled LP decreased, and the change of fluorescence spectrum were in correspondence with the change of LP activity. The labeled LP200 was renaturated by using low concentration of GuHCl, and the results showed that the change extents of LP activity and fluorescence spectrum were very small.