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 °C. In contrast, the CaCl2-extracted enzyme lost much, of its activity under these conditions by a process which could be 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.