Self-association accompanies inhibition of Ca-ATPase by thapsigargin.

Recent studies have demonstrated a relationship between the activity of the Ca-ATPase of sarcoplasmic reticulum and its state of self-association. In the present study, the effects of thapsigargin (TG), a toxin that specifically inhibits the Ca-ATPase of rabbit skeletal muscle sarcoplasmic reticulum membrane, were studied by detecting the time-resolved phosphorescence anisotropy (TPA) decay of the Ca-ATPase that had been labeled with the phosphorescent probe erythrosin-isothiocyanate (ErITC). Anisotropy decays were fit to a function that consisted of three exponential decays plus a constant background, as well as to a function describing explicitly the uniaxial rotation of proteins in a membrane.

Detection of intermediate protein conformations by room temperature tryptophan phosphorescence spectroscopy during denaturation of Escherichia coli alkaline phosphatase.

The reversible denaturation of Escherichia coli alkaline phosphatase (AP) was followed by monitoring changes in enzymatic activity as well as by measurements of the time-resolved room temperature phosphorescence from Trp 109. It is well known that the denaturants, ethylene diamine tetraacetic acid (EDTA), acid and guanidine hydrochloride (GdnHCl) inactive AP by different mechanisms as reflected by differences in the time dependence of inactivation. However, further information about structural changes that result during inactivation is obtained by measurement of the phosphorescence intensity and radiative decay rate.

Consideration of dipole orientation angles yields accurate rate equations for energy transfer in the rapid diffusion limit.

Dipole-dipole energy transfer between suitable donor and acceptor chromophores is an important luminescence quenching mechanism and has been shown to be useful for distance determination at the molecular level. In the rapid diffusion limit, where the excited-state lifetime of the donor is long enough to allow the donor and acceptor to diffuse many times their average separation before deexcitation, it is usually assumed that the relative dipolar orientation is completely averaged due to rotational Brownian motion. Under this simplifying assumption, analytical expressions have been derived earlier for the energy transfer rate between donor and acceptor characterized by different geometries.

Quenching of tryptophan phosphorescence in Escherichia coli alkaline phosphatase by long-range transfer mechanisms to external agents in the rapid-diffusion limit.

Quenching of the room-temperature phosphorescence of Escherichia coli alkaline phosphatase by several freely diffusing molecules was studied, each of whose absorption spectrum overlaps the long-lived emission of this protein and which therefore can quench the excited triplet state by diffusion-enhanced Förster energy transfer. The presence of additional nonresonance transfer mechanisms was also detected, from a lack of linear dependence of quenching rate on spectral overlap. The quenching agents used were the dye molecules methyl red, methyl orange, and 2-[(4-hydroxyphenyl)azo]benzoic acid, as well as the embedded heme groups of myoglobin, metmyoglobin, and the reduced and oxidized forms of cytochrome c.