ROTATIONAL REORIENTATION KINETICS OF DANSYLATED BOVINE SERUM-ALBUMIN

Time-resolved fluorescence intensity and anisotropy decays of dansylated bovine serum albumin (BSA) are investigated by multifrequency phase and modulation fluorescence spectroscopy. We found that a double exponential decay law best describes the fluorescence intensity decay of covalently attached dansyl to BSA. The short lifetime component is attributed to dansyl located at the exterior surface of BSA. The longer-lived component reflects dansyl at the interior of BSA. This result indicates that there are two dansyl-BSA populations in the ground state. An associated model is then found to best describe the anisotropy decay kinetics of dansylated BSA. This result is consistent with the observed ground-state heterogeneity. The rotational reorientation kinetics of dansylated BSA are described by three distinct rotational correlation times. The longest is attributed to the global motion of the entire BSA molecule. The two shorter rotational correlation times are a consequence of local motions of dansyl at the exterior surface of BSA and within an internal hydrophobic site. The temperature effects on the anisotropy decay of this system were also studied and followed a simple Arrhenius rate law. The semiangle (theta) for the cone of rotational reorientation associated with two local motions indicates that the surface dansyl is moderately free to rotate (theta = 45-degrees) and that temperature does not affect its reorientational freedom. In constrast, dansyl bound within the BSA matrix shows restricted motion, but this restriction wanes (17-degrees greater-than-or-equal-to theta less-than-or-equal-to 30-degrees) with temperature.