title: Stabilization and re-activation of trapped enzyme by immobilized heat shock protein and molecular chaperones.
abstract: The potential of using immobilized Heat Shock Protein 70 (HSP 70) in combination with other molecular chaperones to ameliorate problems of enzyme denaturation was investigated. Firefly luciferase was used as a model enzyme due to its sensitivity to thermal denaturation, and the availability of a sensitive chemiluminescent assay method for determination of relative activity of this enzyme. Control experiments and development of effective combinations of HSP with other chaperones involved re-activation of enzyme in bulk solution. A combination of HSP 70, alpha-crystallin and reticulocyte lysate (RL) in bulk solution were found to re-activate soluble firefly luciferase to about 60% of the initial activity after the enzyme activity had been reduced to less than 2% by thermal denaturation. HSP 70 that was covalently immobilized onto glass surfaces was also able to re-activate denatured enzyme that was in bulk solution. Over 30% of the initial activity could be regained from heat denatured enzyme when using immobilized HSP in the presence of other chaperones. The activity of soluble enzyme decayed to negligible values in a period of days when stored at room temperature. In the presence of immobilized HSP and chaperones, activity stabilized at about 10% of the initial activity even after many weeks. The results suggest that immobilized molecular chaperones such as HSP 70 may provide some potential for stabilization and re-activation of enzymes that are trapped in thin aqueous films for applications in biosensors and reactors.
authors.affiliation: Department of Chemistry, Chemical Sensors Group, University of Toronto at Mississauga, 3359 Mississauga Road North, Mississauga, ON, Canada L5L 1C6.
authors.forename: Yunhui
authors.initials: Y
authors.lastname: Yang
authors.forename: Jiang
authors.initials: J
authors.lastname: Zeng
authors.forename: Chunguang
authors.initials: C
authors.lastname: Gao
authors.forename: Ulrich J
authors.initials: UJ
authors.lastname: Krull
mesh_terms: D000818:Animals; D001517:Coleoptera / Q000737:chemistry; D004793:Enzyme Reactivators / Q000737:chemistry*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D008156:Luciferases / Q000737:chemistry*; D018832:Molecular Chaperones / Q000737:chemistry*; D011489:Protein Denaturation; D011817:Rabbits; D012156:Reticulocytes / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D038201:alpha-Crystallins / Q000737:chemistry
chemical_list: D004793:Enzyme Reactivators; D004800:Enzymes, Immobilized; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D038201:alpha-Crystallins; D008156:Luciferases