Purification and characterization of a novel abundant protein in rat bile that binds azo dye metabolites and copper.
Following intravenous administration to rats of the azo dye hepatocarcinogen 3'-methyl-N,N-dimethyl-4-aminoazo-[14C]benzene, 60-70% of the injected dose was recovered in bile in 2 h. Approximately 10% of bile radioactivity was trichloroacetic acid-precipitable, not extracted by n-butanol and non-dialyzable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bile followed by fluorography revealed two major and several minor proteins to which radiolabelled azo dye metabolites were bound; one of these major proteins (50 kDa) was purified from bile and shown to be homogeneous by SDS-polyacrylamide gel electrophoresis, isoelectric focusing (pI 7) under denaturing conditions and N-terminal analysis. The protein (MBP) comprises 15% of the total bile protein. Amino acid analysis revealed a preponderance of acidic and hydrophobic amino acids. The absorption spectrum of the native protein had a major peak at 280 nm and minor peaks at 345, 400, 600 and 650 nm. The fluorescence spectrum showed a major excitation maxima at 285 and 350 nm and corresponding emission maxima at 345 and 440 nm. Atomic absorption spectroscopy revealed 5 atoms of Cu per mol protein. Approximately 90% of the Cu was EPR silent. MBP did not react with antisera directed against rat serum IgA, albumin or ceruloplasmin; nor did these proteins react against antisera to MBP. Seven distinct peptide bands ranging from 5 to 18 kDa were obtained when MBP was subjected to CNBr cleavage and the digests were analyzed by SDS-polyacrylamide gel electrophoresis. The [14C]-Me-DAB derived radioactivity was present in only two of the peptides, indicating specific binding sites for azodye metabolites.
D000818:Animals; D001391:Azo Compounds / Q000378:metabolism*; D001646:Bile / Q000032:analysis*; D004578:Electron Spin Resonance Spectroscopy; D007525:Isoelectric Focusing; D008297:Male; D008749:Methyldimethylaminoazobenzene / Q000494:pharmacology; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence
D001391:Azo Compounds; D011506:Proteins; C002432:2-methyl-4-dimethylaminoazobenzene; D008749:Methyldimethylaminoazobenzene