CHARACTERIZATION OF HYDROGEN-PEROXIDE REMOVAL ACTIVITIES IN MOUSE HEMOLYSATES - CATALASE ACTIVITY AND HYDROGEN-PEROXIDE REMOVAL ACTIVITY BYHEMOGLOBIN

Hydrogen peroxide removal activities in normal and acatalasemic mouse hemolysates were examined to determine the optimal temperature of cata lase. 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% in activation was observed after 10 min incubation at 47.2 +/- 0.5 degree s C for normal hemolysates and 34.0 +/- 0.8 degrees C for acatalasemic ones. The removal activity deactivated at a higher temperature remain ed after the addition of sodium azide, and the 50% inactivation was ob served at 63.5 +/- 1.4 degrees C. After separation of the removal acti vities by carboxymethyl-cellulose column chromatography, the removal a ctivity deactivated at higher temperature was attributed to the activi ty by hemoglobin. From Lineweaver-Burk plot analysis of the removal ra tes by hemoglobin at 37 degrees C, the Michaelis constant for hydrogen peroxide and the maximum velocity were 201 +/- 53 mu M and 5.37 +/- 1 .39 mu mol/s per g of Hb, respectively. Removal rates by hemoglobin in mouse hemolysates at 37 degrees C in 70 mu M hydrogen peroxide were 1 .32 +/- 0.12 mu mol/s per g of Hb. Catalase activity (k/g Hb: rate con stant related to the hemoglobin content) in normal mouse hemolysates w as 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 r esults indicate that activity of hydrogen peroxide removal by hemoglob in is substantial and the activity in acatalasemic hemolysates is pred ominant at low concentration of hydrogen peroxide. (C) 1997 Elsevier S cience B.V.