Dipolar relaxation in proteins on the nanosecond timescale observed by wavelength-resolved phase fluorometry of tryptophan fluorescence.

J.R Lakowicz,H Cherek

doi:10.1016/s0021-9258(19)86104-5

Abstract

We used phase fluorometry to investigate the wavelength dependence of the fluorescence lifetimes of N-acetyl-L-tryptophanamide (NATA) in solvents of varying viscosity and the lifetimes of tryptophan in human serum albumin, melittin, and liver alcohol dehydrogenase. In highly fluid solvents, and in completely vitrified solvents, the lifetime of NATA was constant across its emission spectrum. In viscous solvents, such as propylene glycol at -9 degrees C, the lifetimes of NATA increased across its emission spectrum, with the values being 3.3, 5.5, and 8.1 ns at 317, 344, and 400 nm, respectively. These wavelength-dependent lifetimes appear to be a result of reorientations of solvent dipoles around the excited state dipole moment of the indole moiety. For the three proteins investigated, the fluorescence lifetimes of tryptophan increased with increasing wavelength in a manner comparable to that observed for NATA in propylene glycol. These observations indicate that these protein matrices can reorientation around their tryptophan residues on the nanosecond timescale, and illustrate the potential of phase fluorometry for quantifying the details of these dipolar relaxation processes.

Highlights

From the Gray Freshwater Biological Institute and Department of Biochemistry, University of Minnesota, Navarre, Minnesota 55392 around the increaseddipole moment of tryptophan in the excited state
In completely vitrified extremely small T~ values, thermal disruptionof dipole-dipole solvents, the lifetime ofNATA was constant across its orientations, and shiftosf the fluorescence emissions to higher emission spectrum.In viscous solvents, such as propyl- wavelengths [10]
These observations indicatethat these proteinmatricescanreorientate around their tryptophan residues on the nanosecond timescale, and illustrate the potential of phase fluorometryforquantifyingthe details ofthesedipolar relaxation processes

Summary

MATERIALS AND METHODS

Rechromatographed HSA was obtained from Worthington, a crystallinesuspension of liveralcohol dehydrogenase from Boehringer. Fluorescence lifetime measurements were performed by the phase method [16] using a modulation frequency of 30 MHz. The effects of light at theexcitation wavelength of 295 nm was transmitted rotational diffusion on the observedlifetimeswere eliminated by by the 317 nm filter combination.We tested for scattered light excitingwithvertically polarized light and observing the emission using a glycogen suspension which scattered an equivalent through a polarizer orientated 55" from thevertical position [17].For lifetime measurements, theemission of the proteins and threference eompound (p-terphenyl, see below) were observed through interference filters which were obtained from Melles Griot. Structure in the emission spectra of NATA at the lowest temperatures used in these studies (-55°C)

The fluorescence emission spectrum of HSA is shown in

Nanosecond Relaxation in Proteins