Protein stabilization. Some methods and mechanisms
Research output: Thesis › Doctoral Thesis (compilation)
The effect of different additives on protein stability was studied, and the basis for stabilization was investigated at molecular level. The addition of sorbitol enhanced thermal stability of hen egg white lysozyme in aqueous solution. NMR spectroscopy revealed changes in the relaxation properties of Ala and Thr methyl groups in lysozyme, indicating compression of the enzyme as a consequence of preferential exclusion of the polyol from the enzyme surface together with enhanced water structure. Displacement of water from the enzyme surface was also observed, indicating presence of close contacts between sorbitol and lysozyme besides exclusion. The polycation, poly(ethyleneimine) (PEI), increased the shelf life of number of proteins at temperatures above ambient. Porcine muscle lactate dehydrogenase (LDH) retained its activity completely for one month in the presence of PEI at pH 7.2 while in its absence, more than 50% of the activity was lost already after two weeks. Circular dichroism confirmed substantial preservation of secondary structure during the period of storage, however, PEI did not have any effect on the denaturation temperature of LDH. Sorbitol, on the other hand improved the thermodynamic stability of LDH but not the storage stability. Freeze-drying LDH in the presence of PEI also resulted in significantly improved storage stability of the dried preparation. The interactions between PEI and LDH were studied using dynamic light scattering and intrinsic tryptophan fluorescence. Aggregation of LDH was seen to decrease substantially with the polymer present in solution. Formation of a complex between PEI of different molecular weights and LDH was observed, the interactions being rather weak. The stabilizing effect of sorbitol and PEI and also several other additives against protein oxidation was investigated using LDH as a model. Oxidation of the enzyme was performed using CuSO4 and H2O2, either individually or together. PEI was shown to be the best stabilizing agent, followed by EDTA, against both metal ion and hydrogen peroxide mediated oxidation. The protective effect of PEI is attributed to its chelating properties and the close contacts between the polymer and enzyme. The combination of PEI and sorbitol was shown to protect against both oxidation and thermal denaturation. Immobilization of trypsin to a thermo-responsive reversibly soluble-insoluble polymer, poly-N-isopropylacrylamide, was performed and stability of the enzyme conjugate was studied. The immobilization increased the resistance of trypsin towards autolysis. Stability of the enzyme towards heat and organic solvents was improved. The enzyme conjugate could be recycled several times without activity loss.
|Research areas and keywords||
Subject classification (UKÄ) – MANDATORY
|Award date||1999 Dec 3|
|Publication status||Published - 1999|
Defence details Date: 1999-12-03 Time: 10:15 Place: Room C, Chemical Center, Sölvegatan 39, Lund External reviewer(s) Name: Cabral, Joaquim S. Title: Prof Affiliation: Instituto Superior Tecnico, Lisboa, Portugal ---