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Partial purification and characterization of a peroxidase from neonatal rat skin

Thesis/Dissertation ·
OSTI ID:5564116
Peroxidase activity was partially purified from neonatal 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 and hydrogen peroxide as substrates. Subcellular distribution studies indicated the specific activity to be highest and comparable in the 800 {times} g and 8000 {times} g pellets, lowest in the 100,000 {times} g pellet, and absent in the 100,000 {times} g supernatant. The peroxidase activity was partially purified by affinity chromatography on concanavaline-A-sepharose 4-B and by gel filtration using Bio-gel P-150. The apparent molecular weight of the native enzyme as determined by Bio-gel P-200 gel filtration was approximately 42,500 {plus minus} 2,300 daltons. 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{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 ({sup 3}H)-benzo(a)pyrene-7,8-dihydrodiol and ({sup 3}H)-2-aminofluorene to DNA and protein.
Research Organization:
Michigan Univ., Ann Arbor, MI (USA)
OSTI ID:
5564116
Country of Publication:
United States
Language:
English