Further purification and properties of rat uterine peroxidase.

Peroxidase was purified 3700-fold from homogenates of estradiol-treated rat uteri by affinity chromatography on concanavalin A (ConA)- Sepharose followed by gel filtration on Bio-Gel P-150 with high recovery of enzyme. A single protein (molecular weight (MW) 45 000) staining for heme was shown by sodium dodecyl sulfate - polyacrylamide gel electrophoresis to be present in the peak fractions of enzymic activity eluted from the ConA-Sepharose column. This protein had the same mobility as bovine lactoperoxidase (MW 78 000) in a cationic gel electrophoretic system under nondenaturing conditions. Peroxidase activity in a NaCl extract of the uterus was lower than that in a CaCl2 extract but was unaffected by prolonged storage at - 20 degrees C. In contrast, the CaCl2-extracted enzyme lost much of its activity under these conditions by a process which could by prevented by the addition of glycerol. The sulfhydryl reagent, N-ethylmaleimide, which caused a marked increase in the activity of uterine peroxidase, provided only partial protection against inactivation during storage of CaCl2 extracts of this enzyme at low temperature.