AIM:To purify and identify heme oxygenase(HO)isomerswhich exist in rat liver,spleen and brain treated with hematinand phenylhydrazine and in untreated rat liver and toinvestigate the characteristics of HO isomers,to isolate andconfirm the rat HO-1 cDNA that actually encodes HO-1 byexpressing cDNA in monkey kidney cells(COS-1 cells),toprepare the rat heme oxygenase-1(HO-1)mutant and todetect inhibition of HO-1 mutated enzyme.METHODS:First,rat liver,spleen and brain microsomalfractions were purified by DEAE-Sephacel and hydroxylapatite.The characteristics including activity,immunity andinducibility of two isomers(HO-1 and HO-2),and theirapparent molecular weight were measured by detectingenzymatic activities,SDS-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting analysis,respectively.Second,plasmid pcDNA3HO1 containing native rat HO-1cDNA and pcDNA3HOID25 carrying mutated rat HO-1 cDNA(His25Ala)were constructed by site-directed mutagenesis.COS-1 cells transfected with pcDNA3HO1 and pcDNA3HO1D25were collected and disrupted by sonication,the microsomeswere prepared byultracentrifugation.Third,the inhibitionof rat HO-1 mutant was analyzed.RESULTS:Two isomers were purified and identified intreated rat liver,spleen,brain and untreated rat liver.HO-1was the predominant form with a ratio of 2.0:1 and 3.2:1 ofHO-1 and HO-2 in liver and spleen,respectively,but onlythe activity of HO-2 in the brain and untreated liver couldbe detected.The apparent molecular weights of HO-1 andHO-2 were about M_r 30 000 and M_r 36 000 under reducingconditions,respectively.The antiserum against liver HO-2was employed in Western blotting analysis,the reactivity ofHO-1 in the liver was not observed.The plasmid pcDNA3HO1was highly expressed in endoplasmic reticulum of transfectedCOS-1 cells.The specific activity was≈5-fold higher thanthat of the control.However,the enzyme activity of mutatedHO-1 declined.While an equal amount of mutant was addedto the enzyme reaction system,the levels of bilirubindecreased 42 %. CONCLUSION:The studies suggest that HO-I and HO-2exist in the hematin and phenylhydrazine treated rat liverand spleen,but only HO-2 in the brain and untreated liver.Two constitutive forms are different in molecular weight,inducibility and immunochemical properties.The activity ofexpressed HO-1 in COS-1 cells is higher than that of purifiedenzyme from rat spleen tissue.It suggests that this clone hasan insert of 1030 base-pairs encodes HO-1.His25Ala mutantreduced the formation of bilirubin and it suggests that themutant could competely bind the heme with native enzyme. AIM: To purify and identify heme oxygenase (HO) isomerswhich exist in rat liver, spleen and brain treated with hematinand phenylhydrazine and in untreated rat liver and to investigate the characteristics of HO isomers, to isolate and confirm the rat HO-1 cDNA that actually encodes HO -1 byexpressing cDNA in monkey kidney cells (COS-1 cells), toprepare the rat heme oxygenase-1 (HO-1) mutant and todetect inhibition of HO-1 mutated enzyme. METHODS: First, rat liver, spleen and brain microsomalfractions were purified by DEAE-Sephacel and hydroxylapatite. these characteristics including activity, immunity and impairment of two isomers (HO-1 and HO-2), and theirapparent molecular weight were measured by detectingenzymatic activities, SDS-polyacrylamide gel electrophoresis blotting analysis, respectively.Second, plasmid pcDNA3HO1 containing native rat HO-1 cDNA and pcDNA3HOID25 carrying mutated rat HO-1 cDNA (His25Ala) were constructed by site-directed mutagenesis. COS-1 cells transfected with pcDNA 3HO1 and pcDNA3HO1D25were collected and disrupted by sonication, the microsomes prepared byultrantrifugugation.Third, the inhibition of rat HO-1 mutant was analyzed .RESULTS: Two isomers were purified and identified intreated rat liver, spleen, brain and untreated rat liver. HO-1was the predominant form with a ratio of 2.0: 1 and 3.2: 1 of HO-1 and HO-2 in liver and spleen, respectively, but only the activity of HO-2 in the brain and untreated liver could be detected. 1 andHO-2 were about M_r 30 000 and M_r 36 000 under reducing conditions, respectively. The antiserum against liver HO-2 was employed in Western blotting analysis, the reactivity of HO-1 in the liver was not observed. Plasmid pcDNA3HO1was highly expressed in endoplasmic reticulum of transfected COS-1 cells. The specific activity was ≈5-fold higher thanthat of the control. Host, the enzyme activity of mutatedHO-1 declined.While an equal amount of mutant added to the enzyme reaction system, the level sof bilirubindecreased 42%. CONCLUSION: The studies suggest that HO-I and HO-2exist in the hematin and phenylhydrazine treated rat liverand spleen, but only HO-2 in the brain and untreated liver. Two constitutive forms are different in molecular weight, inducibility and immunochemical properties. The activity of expressed HO-1 in COS-1 cells is higher than that of purifiedenzyme from rat spleen tissue. It suggests this this clone hasan insert of 1030 base-pairs encodes HO-1.His25Ala mutantreduced the formation of bilirubin and it suggests that theyutant could competely bind the heme with native enzyme.