Partial purification and characterization of a peroxidase from neonatal rat skin.

Abstract

Peroxidase activity was partially purified from neonatal (3-6 days old) CFN rat skin. The membrane-bound peroxidase activity was extracted with 0.5 M calcium chloride and was monitored spectrophotometrically at 470 nm with 2-methoxyphenol (guaiacol) and hydrogen peroxide as substrates. Subcellular distribution studies indicated the specific activity to be highest and comparable in the 800 x g and 8000 x g pellets, lowest in the 100,000 x g pellet, and absent in the 100,000 x g supernatant. The peroxidase activity was partially purified by affinity chromatography on concanavalin-A-sepharose 4-B and by gel filtration using Bio-gel P-150. Purification factors from these two steps were about 25 and 4, respectively. The apparent molecular weight of the native enzyme as determined by Bio-gel P-200 gel filtration was approximately 42,500 $\pm$ 2,300 daltons. Spectral analyses of partially purified rat skin peroxidase revealed an absolute maximum of approximately 406 nm, dithionite reduced CO difference spectrum maximum of approximately 418 nm, and a CN complex maximum of approximately 421 nm. Peroxidase extraction in the presence of n-ethyl-, n-methyl-, or n-phenylmaleimide produced an approximate two-fold increase in activity. Peroxidase activity increased linearly with increases in protein concentration, time, and guaiacol concentration. Activity was inhibited approximately 75% by 0.1 mM potassium cyanide or 0.05 mM sodium azide. Pyrogallol, hydroquinone, p-cresol, catechol, benzidine, 3,3$\sp\prime$-dimethoxybenzidine, tetramethylbenzidine, and p-phenylenediamine were active as substrates for rat skin peroxidase. Rat skin peroxidase was also shown to mediate non-extractable binding of ($\sp3$H)-benzo(a)pyrene-7,8-dihydrodiol and ($\sp3$H)-2-aminofluorene to DNA and protein. Comparison of rat skin peroxidase properties with those of other peroxidase activities identified in the literature, suggest it is a previously undescribed activity.