Synthesis and characterization of the dansyltyrosine derivatives of porcine pancreatic colipase.
Steady-state and time-resolved fluorescence techniques were used to study dansyltyrosine derivatives of porcine pancreatic colipase. Nitration, reduction, acylation, and dansylation reactions were utilized to synthesize two fluorescently labeled colipases: (o-aminodansyltyrosine 55 porcine colipase) (DNStyr55PC) and o-aminodansyltyrosine 59 porcine colipase (DNStyr59PC). DNStyr55PC was 200% active, while the DNStyr59 derivative maintained 80% activity in a pH stat assay. Emission spectra, lifetime analysis, acrylamide quenching, polarization, and anisotropy decay studies indicated that Tyr55 was located on the solvent-exposed surface of the protein, where the fluorophore experienced free rotation. Identical experiments done on DNStyr59PC indicated that Tyr59 was in a partially buried environment and the motion of the dansyl tyrosine group was hindered. The double-exponential decay of the fluorescence emission of N-acetyl-o-aminodansyltyrosine ethyl ester (DNStyr) and the DNStyr derivatives of colipase was investigated with pH, temperature, solvent, and emission-resolved-lifetime experiments. The existence of excited-state processes was eliminated in both pH and emission-resolved-lifetime experiments, whereas temperature studies indicated either a rotational isomer or a differential solvent quenching mechanism for multiple decay kinetics. These experiments also showed that DNStyr was a sensitive probe of solvent polarity and viscosity, but not of pH.
McIntyre
J C
JC
Department of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, University of Cincinnati, Ohio 45267.
Schroeder
F
F
Behnke
W D
WD
D000818:Animals; D001665:Binding Sites; D003089:Colipases / Q000138:chemical synthesis* / Q000378:metabolism*; D003619:Dansyl Compounds / Q000138:chemical synthesis* / Q000378:metabolism*; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D010179:Pancreas / Q000201:enzymology*; D011506:Proteins / Q000138:chemical synthesis* / Q000378:metabolism*; D013552:Swine; D013696:Temperature; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism*
C063804:2-aminodansyltyrosine 55 colipase; C063806:2-aminodansyltyrosine 59 colipase; D003089:Colipases; D003619:Dansyl Compounds; D011506:Proteins; D014443:Tyrosine