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.
Zhang
Y Y
YY
Department of Physiological Chemistry II, Karolinska Institutet, Stockholm, Sweden.
Hamberg
M
M
Rådmark
O
O
Samuelsson
B
B
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