Stabilization and re-activation of trapped enzyme by immobilized heat shock protein and molecular chaperones.
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.
Yang
Yunhui
Y
Department of Chemistry, Chemical Sensors Group, University of Toronto at Mississauga, 3359 Mississauga Road North, Mississauga, ON, Canada L5L 1C6.
Zeng
Jiang
J
Gao
Chunguang
C
Krull
Ulrich J
UJ
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
D004793:Enzyme Reactivators; D004800:Enzymes, Immobilized; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D038201:alpha-Crystallins; D008156:Luciferases