Identification of the bile acid binding proteins in human serum by photoaffinity labeling.
The binding of conjugated and unconjugated bile acids to human serum lipoproteins was investigated by density gradient centrifugation and photoaffinity labeling studies. The binding of bile acids to high-density lipoprotein increased by substitution of the 3 alpha-hydroxy group in cholate and taurocholate by a photolabile 3-azido or 3-azi-function. The affinity of bile acid derivatives to HDL showed the following ranking: 3 beta-azido-7 alpha,12 alpha-dihydroxy-,3,3-azo-7 alpha,12 alpha-dihydroxy- > 3 alpha,7 alpha,12 alpha-trihydroxy-,11 xi-azido-3 alpha,7 alpha,12 xi-trihydroxy- > 11 xi-azido-12-oxo-3 alpha,7 alpha-dihydroxy- > 7,7-azo-3 alpha,12 alpha-dihydroxy-,3 alpha,7 alpha-dihydroxy-,3 alpha,12 alpha-dihydroxy- > 3 alpha-hydroxy-cholan-24-oic acid. Based on the actual serum concentrations of albumin and HDL, a preference of hydrophilic bile acids to HDL is evident, the 3-azido- and 3-azi-derivatives showing a 5-23-fold higher binding to HDL compared to soluble serum proteins. For the identification of the bile acid binding proteins in human blood, photoaffinity labeling with a variety of photolabile conjugated and unconjugated bile acid derivatives was performed with subsequent analysis of radiolabeled serum proteins by one- and two-dimensional gel electrophoresis. In addition to albumin and the apolipoproteins A-I and A-II of high-density lipoproteins (Kramer et al. (1979) Eur. J. Biochem. 102, 1-9), three further proteins in the lipoprotein free serum fraction of M(r) 41,000, 50,000 and 83,000 were specifically labeled. By two-dimensional electrophoresis and by immunoprecipitation these proteins were identified as alpha 1-acid glycoprotein (M(r) 41,000), alpha 1-antitrypsin (M(r) 50,000) and transferrin (M(r) 83,000). No binding of bile acids to haptoglobin, alpha 2-HS-glycoprotein, hemopexin or alpha 1-fetoprotein occurred. In conclusion, these studies show that bile acid derivatives bind to several serum proteins in addition to albumin and furthermore that the substituent in position 3 of the steroid nucleus greatly influences the affinity of bile acids to high density lipoproteins.
Kramer
W
W
Hoechst Aktiengesellschaft, Frankfurt am Main, Germany.
D000328:Adult; D000345:Affinity Labels; D001391:Azo Compounds; D001647:Bile Acids and Salts / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D008075:Lipoproteins, HDL / Q000097:blood*; D008077:Lipoproteins, LDL / Q000097:blood*; D008297:Male; D015394:Molecular Structure; D009961:Orosomucoid / Q000378:metabolism; D011233:Precipitin Tests; D014168:Transferrin / Q000378:metabolism
D000345:Affinity Labels; D001391:Azo Compounds; D001647:Bile Acids and Salts; D001798:Blood Proteins; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D009961:Orosomucoid; D014168:Transferrin