Purification and characterization of human salivary peroxidase.
Human salivary peroxidase (SPO) has been purified to homogeneity by subjecting human parotid saliva to immunoaffinity, cation exchange, and affinity chromatography. These procedures resulted in a 992-fold purification of the enzyme. When purified SPO was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), three Coomassie stainable bands were apparent, all of which stained positive for enzyme activity. The apparent molecular weights of the three bands were 78,000, 80,000, and 280,000 as analyzed by SDS-PAGE. Reduction with 2-mercaptoethanol resulted in a decreased mobility of these bands, and enzyme activity could no longer be detected on the gels. The SPO preparation had the characteristic peroxidase heme spectrum in the range 405-420 nm. The ratio between the absorbance of the Soret band (412 nm) and the absorbance at 280 nm was 0.81. The enzyme activity was inhibited by the classical peroxidase inhibitors cyanide and azide. Salivary peroxidase is similar to bovine lactoperoxidase (LPO) in amino acid composition, in ultraviolet and visible spectrum, in reaction with cyanide, in susceptibility to 2-mercaptoethanol inactivation, and in thermal stability. The two enzymes differ in carbohydrate composition and content. SPO contains 4.6% and LPO 7% total neutral sugars. The ratio of glucosamine to galactosamine is 2:1 in SPO and 3:1 in LPO. SPO contains mannose, fucose, and galactose in a molar ratio of 1.5:1.5:1.0, while the ratio was 14.9:0.5:1.0 in LPO. Glucose was present in both preparations in minor amounts. The concentration of azide required for 50% inhibition of enzyme activity was 20-fold greater for LPO than for SPO.(ABSTRACT TRUNCATED AT 250 WORDS)
Mansson-Rahemtulla
B
B
School of Dentistry, University of Alabama, Birmingham 35294.
Rahemtulla
F
F
Baldone
D C
DC
Pruitt
K M
KM
Hjerpe
A
A
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D006801:Humans; D007700:Kinetics; D007784:Lactoperoxidase; D008970:Molecular Weight; D010306:Parotid Gland / Q000201:enzymology; D010544:Peroxidases / Q000302:isolation & purification* / Q000378:metabolism; D012463:Saliva / Q000201:enzymology*
D000596:Amino Acids; D002241:Carbohydrates; D007784:Lactoperoxidase; D010544:Peroxidases