Characterization of hydrogen peroxide removal activities in mouse hemolysates: catalase activity and hydrogen peroxide removal activity by hemoglobin.
Hydrogen peroxide removal activities in normal and acatalasemic mouse hemolysates were examined to determine the optimal temperature of catalase. From thermal stability of the removal activities in hemolysates, the removal activities were divided into two activities. The removal activity deactivated at lower temperature was catalase, and the 50% inactivation was observed after 10 min incubation at 47.2 +/- 0.5 degrees C for normal hemolysates and 34.0 +/- 0.8 degrees C for acatalasemic ones. The removal activity deactivated at a higher temperature remained after the addition of sodium azide, and the 50% inactivation was observed at 63.5 +/- 1.4 degrees C. After separation of the removal activities by carboxymethyl-cellulose column chromatography, the removal activity deactivated at higher temperature was attributed to the activity by hemoglobin. From Lineweaver-Burk plot analysis of the removal rates by hemoglobin at 37 degrees C, the Michaelis constant for hydrogen peroxide and the maximum velocity were 201 +/- 53 microM and 5.37 +/- 1.39 micromol/s per g of Hb, respectively. Removal rates by hemoglobin in mouse hemolysates at 37 degrees C in 70 microM hydrogen peroxide were 1.32 +/- 0.12 micromol/s per g of Hb. Catalase activity (k/g Hb: rate constant related to the hemoglobin content) in normal mouse hemolysates was 104 +/- 12 at 25 degrees C and 117 +/- 10 at 37 degrees C, and that in acatalasemic hemolysates was 10.5 +/- 1.7 at 25 degrees C. These results indicate that activity of hydrogen peroxide removal by hemoglobin is substantial and the activity in acatalasemic hemolysates is predominant at low concentration of hydrogen peroxide.
Masuoka
N
N
Department of Biochemistry, Okayama University Medical School, Japan. suokan@med.okayama-u.ac.jp
Wakimoto
M
M
Ohta
J
J
Ishii
K
K
Nakano
T
T
D020642:Acatalasia; D000818:Animals; D002374:Catalase / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D006441:Hemoglobin A / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006461:Hemolysis; D006861:Hydrogen Peroxide / Q000032:analysis*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008297:Male; D008706:Methemoglobin / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D013696:Temperature
D006454:Hemoglobins; C028881:hemoglobin A(0); D008706:Methemoglobin; D006441:Hemoglobin A; D006861:Hydrogen Peroxide; D002374:Catalase