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Proceedings Paper

Time-resolved circularly polarized phosphorescence studies of horse-liver alcohol dehydrogenase
Author(s): Joseph A. Schauerte; Ari Gafni; Duncan G. Steel
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Paper Abstract

Using a combination of time-resolved room temperature phosphorescence and time-resolved circularly polarized phosphorescence (TR-CPP, demonstrated in proteins for the first time), we provide strong evidence supporting the existence of conformational heterogeneity in the apo form of horse liver alcohol dehydrogenase (LADH). Using extensive photon counting and correspondingly improved statistics, we show a clear biexponential decay in the phosphorescence of deoxygenated LADH in contrast to earlier studies where monoexponential decay was reported. Under extensively deoxygenated conditions, the protein shows long lifetime components of 510 and 765 milliseconds (pre-exponentials vary with conditions). TR- CPP studies of LADH show that the emission anisotropy factors (a measure of the local chirality associated with the excited state) associated with the two decay components are identical (gem equals 0.004; where the fractional anisotropy is given by gem equals {2*[Ileft-Iright)/Itotal]}. This indicates that the chirality of the environment of the phosphorescent tryptophan is the same for both lifetimes. Upon binding the coenzyme fragment adenosine-diphosphoribose (ADPR), LADH phosphorescence decay becomes essentially monoexponential (1285 milliseconds) with a time independent gem comparable to the gem of apo LADH. The quenching rates of the long lifetime components of apo-LADH by the electron acceptor TEMPOL were found to be similar, suggesting that the conformational states of LADH have nearly equal susceptibility to oxidation by TEMPOL.

Paper Details

Date Published: 1 April 1992
PDF: 6 pages
Proc. SPIE 1640, Time-Resolved Laser Spectroscopy in Biochemistry III, (1 April 1992); doi: 10.1117/12.58222
Show Author Affiliations
Joseph A. Schauerte, Univ. of Michigan (United States)
Ari Gafni, Univ. of Michigan (United States)
Duncan G. Steel, Univ. of Michigan (United States)

Published in SPIE Proceedings Vol. 1640:
Time-Resolved Laser Spectroscopy in Biochemistry III
Joseph R. Lakowicz, Editor(s)

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