The preparation of rat heme oxygenase-1 mutant to reduce the level of bilirubin.
OBJECTIVE
To prepare rat heme oxygenase-1 (HO-1) mutants and to determine the activity and inhibition of this mutated enzyme.


METHODS
pcDNA3HO1 containing truncated native rat HO-1 cDNA and pcDNA3HO1 delta 25 carrying mutated rat HO-1 cDNA (His25Ala) were constructed, respectively. COS-1 cells transfected with pcDNA3HO1 and pcDNA3HO1 delta 25 were collected and their activities were analyzed.


RESULTS
Native rat HO-1 was highly expressed in transfected cells and its activity was 13,688-15,600 U/mg protein per hour. However, the enzyme activity of mutated HO-1 declined and the value was 1948-2160 U/mg protein per hour. When an equal amount of mutant was added to the enzyme reaction system, the level of bilirubin decreased by 42%.


CONCLUSIONS
The His25Ala mutant reduced the formation of bilirubin, suggesting that the mutant could completely bind the heme with native enzyme.
Xia
Z
Z
Department of Pediatrics, Rui Jin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Shao
J
J
Shen
Q
Q
Wang
J
J
Li
Y
Y
Chen
S
S
Yu
S
S
D000818:Animals; D001663:Bilirubin / Q000032:analysis*; D019556:COS Cells; D018076:DNA, Complementary / Q000032:analysis; D006419:Heme Oxygenase (Decyclizing) / Q000037:antagonists & inhibitors / Q000235:genetics*; D051547:Heme Oxygenase-1; D006932:Hyperbilirubinemia / Q000188:drug therapy*; D008970:Molecular Weight; D009154:Mutation; D051381:Rats
D018076:DNA, Complementary; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; D001663:Bilirubin