Stabilization of purified human 5-lipoxygenase with glutathione peroxidase and superoxide dismutase.
Human 5-lipoxygenase (5LO) becomes very unstable after purification. Commonly used methods for protein stabilization could not prevent this inactivation. However, addition of small amounts of glutathione peroxidase (0.15 micrograms/ml) and superoxide dismutase (1 microgram/ml) to the solution of purified 5LO (300-500 micrograms/ml) stabilized the enzyme during storage. The protected 5LO maintained full activity for at least 12 days at 25 degrees C, while 50% of the activity was lost within 10 h without protection. Glutathione peroxidase alone also preserved the activity of 5-lipoxygenase; however, the effect declined rapidly in the absence of superoxide dismutase. 2-Mercaptoethanol was the most efficient hydrogen donor substrate for glutathione peroxidase in the protection of 5LO. Catalase was less effective as a stabilizing agent, and ebselen, a synthetic glutathione peroxidase-mimicking compound, did not protect 5LO. Since many metal ion binding proteins are susceptible to H2O2 inactivation, this method could be useful also for the stabilization of other proteins.
Department of Physiological Chemistry II, Karolinska Institutet, Stockholm, Sweden.
Y Y
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Zhang
M
M
Hamberg
O
O
Rådmark
B
B
Samuelsson
D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry* / Q000302:isolation & purification; D001393:Azoles / Q000737:chemistry; D002256:Carbonic Anhydrases / Q000737:chemistry; D002374:Catalase / Q000737:chemistry; D004795:Enzyme Stability; D005979:Glutathione Peroxidase / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D054833:Isoindoles; D008054:Lipid Peroxides / Q000032:analysis; D016566:Organoselenium Compounds / Q000737:chemistry; D013482:Superoxide Dismutase / Q000737:chemistry*
D001393:Azoles; D054833:Isoindoles; D008054:Lipid Peroxides; D016566:Organoselenium Compounds; C042986:ebselen; D006859:Hydrogen; D002374:Catalase; D005979:Glutathione Peroxidase; D001094:Arachidonate 5-Lipoxygenase; D013482:Superoxide Dismutase; D002256:Carbonic Anhydrases