title: The role of aromatic side chain residues in micelle binding by pancreatic colipase. Fluorescence studies of the porcine and equine proteins.
abstract: Fluorescence techniques have been employed to study the interaction of porcine and equine colipase with pure taurodeoxycholate and mixed micelles. Nitrotyrosine-55 of porcine colipase is obtained by modification with tetranitromethane (low excess, in the presence of taurodeoxycholate) of the protein followed by gel filtration and ion-exchange chromatography. Verification of the residue modified was obtained by h.p.l.c. peptide purification and sequence analysis. Reduction and quantitative reaction with dansyl chloride yields a fluorescent derivative that is twice as active in conjunction with lipase as is native colipase and that exhibits a strong emission band at 550 nm. Addition of micellar concentrations of taurodeoxycholate causes a 4.3-fold increase in the emission maximum as well as a 70 nm blue shift to 480 nm. Inclusion of oleic acid to form a mixed micelle reduces these spectral effects. Scatchard analysis of the data yield a Kd of 6.8 X 10(-4) M and a single colipase-binding site for taurodeoxycholate micelles. The data, by analogy to a phospholipase system, are consistent with a direct insertion of dansyl-NH-tyrosine-55 into the micelle. The presence of a single tryptophan residue (Trp-52) in equine colipase provides an intrinsic fluorescent probe for studying protein-micelle interaction. The emission maximum of horse colipase at 345 nm indicates a solvent-accessible tryptophan residue which becomes less so on binding of micelles. A blue shift of 8 nm and a 2-fold increase in amplitude is indicative of a more hydrophobic environment for tryptophan induced by taurodeoxycholate micelles. There is also a decrease in KSV for acrylamide quenching in the presence of micelles, which further supports a loss of solvent accessibility. The most dramatic pH effects are observed with KI quenching, and may indicate the presence of negative charges near Trp-52.
authors: Department of Biochemistry and Molecular Biology, University of Cincinnati College of Medicine, OH 45267.
authors: J C
authors: JC
authors: McIntyre
authors: P
authors: P
authors: Hundley
authors: W D
authors: WD
authors: Behnke
mesh_terms: D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002586:Cesium / Q000494:pharmacology; D002712:Chlorides; D003089:Colipases / Q000378:metabolism*; D003840:Deoxycholic Acid / Q000031:analogs & derivatives*; D006736:Horses; D007700:Kinetics; D008823:Micelles; D010179:Pancreas / Q000378:metabolism*; D011193:Potassium Iodide / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013552:Swine; D013657:Taurodeoxycholic Acid / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives / Q000032:analysis* / Q000378:metabolism
chemical_list: D000178:Acrylamides; D002712:Chlorides; D003089:Colipases; D008823:Micelles; D011506:Proteins; D003840:Deoxycholic Acid; D011193:Potassium Iodide; D002586:Cesium; D020106:Acrylamide; C016952:N-acetyltryptophanamide; D013657:Taurodeoxycholic Acid; D014364:Tryptophan; C028019:cesium chloride