Studies on some aspects of peroxidase from submaxillary gland

Abstract

A peroxidase has been purified 25- to 30-fold over crude homogenate from goat submaxillary gland, which shows a single band of protein on polyacrylamide gel electrophoresis at four different pH values (4.6-10.0). A molecular weight (M_r) of approximately 2 × 10⁴ per heme binding site has been found. The molecular weight of the enzyme determined by Sephadex-gel filtration method, appeared to be 4 × 10^4. The sedimentation pattern of the purified enzyme shows a symmetrical peak, although there was evidence of some small heterogenous material near the meniscus. The sedimentation coefficient of the enzyme (s^o 20^wat 0.4% of the enzyme concentration) was found to be 4.18, which indicates the molecular weight of the enzyme to be approximately 6 × 10⁴. The purified peroxidase can oxidize 9.60 μmoles of guaiacol/min/mg of protein, shows maximum activity at pH 4.6 and has an apparent K_m of 7.15 × 10^-3_M for H₂O₂. The enzyme is inhibited by CN^-, N₃^-, F^- and iodoacetic acid, and stimulated by FAD, DPN⁺, TPN⁺. The spectral properties of the peroxidase suggest that the enzyme is a typical hemoprotein. The prosthetic group of this peroxidase can be separated from the apoenzyme by acid butanone treatment and has been identified as ferric protoporphyrin IX from the spectral studies as well as by the analysis of the reduced alkaline pyridine hemochrome prepared both from the enzyme and the separated prosthetic group. The molecular and enzymic properties of the original enzyme can be fully restored by the addition of the prosthetic group (separated from the enzyme) or bovine heme to the apoenzyme.