There is a striking disparity between the heart-shaped structure of human serum albumin (HSA) observed in single crystals and the elongated ellipsoid model used for decades to interpret the protein solution hydrodynamics at neutral pH. These two contrasting views could be reconciled if the protein were flexible enough to change its conformation in solution from that found in the crystal. To investigate this possibility we recorded the rotational motions in real time of an erythrosin-bovine serum albumin complex (Er-BSA) over an extended time range, using phosphorescence depolarization techniques. These measurements are consistent with the absence of independent motions of large protein segments in solution, in the time range from nanoseconds to fractions of milliseconds, and give a single rotational correlation time phi(BSA, 1 cP, 20 degrees C) = 40 +/- 2 ns. In addition, we report a detailed analysis of the protein hydrodynamics based on two bead-modeling methods. In the first, BSA was modeled as a triangular prismatic shell with optimized dimensions of 84 x 84 x 84 x 31.5 A, whereas in the second, the atomic-level structure of HSA obtained from crystallographic data was used to build a much more refined rough-shell model. In both cases, the predicted and experimental rotational diffusion rate and other hydrodynamic parameters were in good agreement. Therefore, the overall conformation in neutral solution of BSA, as of HSA, should be rigid, in the sense indicated above, and very similar to the heart-shaped structure observed in HSA crystals.

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The photophysics and polarization of the phosphorescence and delayed fluorescence of erythrosin in conditions compatible with the current biological applications of the dye (aqueous buffers at pH 7.4 at ambient temperatures) and in ethanol have been studied as a function of dye concentration (10(-7)-10(-5) M) and temperature (245-333 K). The emission decay is strictly single exponential and the detailed kinetic analysis of all the rate processes connected with the emitting T1 state showed that (1) the lowering of the emission lifetime at the higher temperatures is due to a very efficient self-quenching process, (2) the back intersystem crossing rate T1-->S1 is temperature dependent (delta ETS approximately 7 kcal mol-1) but the T1-->S0 is not (Ea < 0.1 kcal mol-1) and (3) both intersystem crossing processes are very sensitive to solvent polarity, which accounts for the solvent dependence of the phosphorescence yield and lifetime. The high value of the phosphorescence anisotropy (r0 = 0.25 +/- 0.006) is independent of the excitation and emission wavelengths, and its evolution in time accurately reflects the rotational restrictions in solid solutions. The relevance of these findings to studies with protein-dye conjugates is also outlined to facilitate the design and interpretation of phosphorescence depolarization experiments that probe the microsecond-ms dynamics of biomolecules and supramolecular systems.