Investigation Of Effects Of Fbinolytic Compounds On Protein Crystallization Characteristics

Proteins are an essential part of biological process and are the basis of life itself.Determination of proteins’ three-dimensional structure by X-ray diffraction has animportant role in understanding macromolecular structure, the mechanism of drug, thechange or modification of molecular structure. Thrombotic disease is a major threat tohuman’s health; most of the clinical use of thrombolytic drugs such as heparin,abciximab, urokinase, there will be bleeding, allergic reactions, poor specificity, etc.Therefore, drugs which have the low risk of bleeding, do not damage the blood factors,the activity can be simply regulated need to occur. GDG and FGFC1were low moleculeweight compound extracted from marine organism, they had fibrinolysis enhancingeffects. The premise of the protein structure analysis by X-ray diffraction is suitablesingle crystal, however, the high quality single crystal is difficult to determine. Hen eggwhite lysozyme (HEWL) has been widely researched as the template of the proteincrystallization theory by crystallization scientists due to its stability, solubility and easyto crystallize.In this paper, HEWL was illustrated for crystallization behavior to research the effectof fibrinolytic compounds GDG and FGFC1on crystallization characteristic and crystalstructure of HEWL and fibrinolysis factors. Explore the mechanism of low moleculeweight compounds action to high molecule weight proteins, the impact of smallmolecule drugs on protein, protein-protein interactions to build theory support forprotein structure-function relationship and new thrombolytic drug.In the first chapter, the thrombotic diseases research progress, the mechanism of newthrombolytic drugs, the relationship between bio-pharmaceutical and proteomics andthe determination of protein crystal structure by synchrotron radiation X-raydiffraction were concluded. Current clinical antithrombotic drugs were not usednormally in daily life for injection of inconvenience and untoward effect.Therefore, the development of effective, safe, suitable for use for a long time, or evenoral drugs has significant significance. Fibrinolytic compounds GDG and FGFC1had been studied for fibrinolysis effect enhancing, the purpose of this paperis to explore the effect of the GDG and FGFC1on crystal structure of lysozyme andfibrinolysis factors, supporting evidence for the establishment of marine fibrinolyticcompounds in fibrinolysis enhancing characteristic.The second chapter mainly described the principles and methods of the hangingdrop vapor diffusion. Selected the crystallization conditions of lysozyme and optimizedthe crystallization conditions using a microscopeto observe the lysozyme crystal appearance. Identified the optimal lysozymecrystallization conditions:15mg/ml lysozyme, containing5%(w/v) sodium chlorideand0.02%(w/v) sodium azide, pH5.2,0.1M sodium acetate buffer, which ensuredlysozyme crystals had smaller amounts and better size.In the third chapter, effects of fibrinolytic compounds GDG and FGFC1on crystallization characteristic of lysozyme based on theoptimal lysozyme crystallization condition were researched. Got microscopes analysisof lysozyme crystals added GDG and FGFC1, analyzed fibrinolytic compounds onthe characteristic of lysozyme crystals. The results showed that GDG might affectlysozyme crystals in the same crystallization conditions and the GDG could promote theformation of crystal nucleus.In the fourth chapter, effects of fibrinolytic compounds on lysozyme crystals structurewere researched. Based on the third chapter GDG and FGFC1of lysozymecrystallization characteristics affected by GDG and FGFC1, this chapter collectedlysozyme crystal added GDG and FGFC1diffraction data using synchrotron radiationX-ray diffraction. Analysed the is three-dimensional structure based on the role of smallmolecules in the mechanism of the macromolecular protein theoretical basis for theresearch of effects of fibrinolytic active compounds on lysozyme main chain, side chain,the catalytic domain, the active sites.In the fifth chapter, pilot study of fibrinolytic compound on crystal structure offibrinolysis factors was made. Our laboratory had found that fibrinolytic compoundGDG induced fibrinolytic factors conformational change and activity change and itpromoted thrombolysis characteristics, yet to be ascertained whether GDG increasedfibrinolytic factors’ activity, such as plasminogen (original), fiberplasminogen activator.In order to determine GDG whether reduced the activity of the plasminogen activator inhibitor, plasmin inhibitor, found the mechanism of GDG promoting fibrinolysis invivo. In this chapter, crystallization conditions and characteristics of GDG tourokinase-type plasminogen activator, streptokinase-plasminogen activator wereresearched, but crystallization condition was not confirmed, which needed be furtherstudied.