TIME-RESOLVED FLUORESCENCE OF THE SINGLE TRYPTOPHAN RESIDUE IN RAT ALPHA-FETOPROTEIN AND RAT SERUM-ALBUMIN - ANALYSIS BY THE MAXIMUM-ENTROPY METHOD

The time-resolved fluorescence emissions of the lone tryptophan residues in rat α-fetoprotein (RFP) and rat serum albumin (RSA) were studied. The total fluorescence intensity decays in both proteins were multiexponential. Analysis of the data by nonlinear least squares as a sum of discrete exponentials showed that four exponentials were needed for a satisfactory fit for both proteins. Analysis by the maximum entropy method using 150 logarithmically equally spaced exponentials yielded four well-resolved excited-state lifetime classes with barycenters and relative amplitudes values (ci) that corresponded to those obtained from the nonlinear least-squares method. Changing the temperature affected the relative amplitudes of the lifetime classes but had little effect on the lifetime values themselves. This suggests that the four classes reflect local conformational substates that exchange slowly with respect to the time window of observation defined by the longest lifetime. The internal rotational dynamics of the tryptophan in each protein was monitored by fluorescence anisotropy decay measurements. The mobility of the tryptophan appeared to be larger and faster in RFP than in RSA. The nonlinear least-squares analysis suggests the existence of three rotational correlation times of 0.1, 3, and 55 ns for this protein. As a function of temperature, the long correlation time did not follow the Perrin's law expected for a rigid rotating body. This suggests that this correlation time may reflect not only the Brownian rotation of the whole protein but also the flexibilities of domains in the protein. For RSA a two-component model with correlation times of 0.4 and 31 ns was sufficient to describe the data. The variation of the long correlation time with temperature followed Perrin's law. The average angular displacement of the internal motion of the Trp residue was restricted in both proteins. In RFP, it increased from 15° to 20° over the temperature range 1-38 °C, in RSA it increased abruptly from 12° to 17° above 20 °C. A two-dimensional analysis of lifetimes and correlation times recently developed [Brochon, J. C., & Livesey, A. K. (1988) in Light in Biology and Medicine (Douglas, R. H., Moan, J., & Dall'Acqua, F., Eds.) pp 21-29, Plenum Publishing Corp., New York] was performed. The analysis confirmed the complexity of motion of the Trp residue in RFP, but it was not possible to attribute specific mobilities to the different excited-state populations. In RSA, the transition around 20 °C corresponds to the occurrence of a fast flexibility affecting the short-lifetime population. © 1990, American Chemical Society. All rights reserved.