ROTATIONAL REORIENTATION KINETICS OF DANSYLATED BOVINE SERUM-ALBUMIN

Time-resolved fluorescence intensity and anisotropy decays of dansylat ed bovine serum albumin (BSA) are investigated by multifrequency phase and modulation fluorescence spectroscopy. We found that a double expo nential decay law best describes the fluorescence intensity decay of c ovalently attached dansyl to BSA. The short lifetime component is attr ibuted to dansyl located at the exterior surface of BSA. The longer-li ved component reflects dansyl at the interior of BSA. This result indi cates that there are two dansyl-BSA populations in the ground state. A n associated model is then found to best describe the anisotropy decay kinetics of dansylated BSA. This result is consistent with the observ ed ground-state heterogeneity. The rotational reorientation kinetics o f dansylated BSA are described by three distinct rotational correlatio n times. The longest is attributed to the global motion of the entire BSA molecule. The two shorter rotational correlation times are a conse quence of local motions of dansyl at the exterior surface of BSA and w ithin an internal hydrophobic site. The temperature effects on the ani sotropy decay of this system were also studied and followed a simple A rrhenius rate law. The semiangle (theta) for the cone of rotational re orientation associated with two local motions indicates that the surfa ce dansyl is moderately free to rotate (theta = 45-degrees) and that t emperature does not affect its reorientational freedom. In constrast, dansyl bound within the BSA matrix shows restricted motion, but this r estriction wanes (17-degrees greater-than-or-equal-to theta less-than- or-equal-to 30-degrees) with temperature.