Phosphorescence depolarization and the rotational dynamics of eosin-labeled purified (Na+, K+)-ATPase in phospholipid vesicles

Liqun Yang; Olga Lopina; Daniel McStay; A. A. Boldyrev; Alan J. Rogers; Peter J. Quinn

doi:10.1117/12.145251

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1 May 1993 Phosphorescence depolarization and the rotational dynamics of eosin-labeled purified (Na+, K+)-ATPase in phospholipid vesicles

Liqun Yang; Olga Lopina; Daniel McStay; A. A. Boldyrev; Alan J. Rogers; Peter J. Quinn

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Proceedings Volume 1890, Biomolecular Spectroscopy III; (1993) https://doi.org/10.1117/12.145251
Event: OE/LASE'93: Optics, Electro-Optics, and Laser Applications in Scienceand Engineering, 1993, Los Angeles, CA, United States

Abstract

Purified Na+, -adenosine triphosphatase (ATPase) in membrane vesicles has been covalently labelled with the triplet probe eosin S'-isothiocynate. The rotational mobility of the protein has been investigated by measurement of time-resolved depolarization of the emitted phosphorescence, induced by a laser pulse. The probe was specifically attached to a lysine residue of the protein located at the putative ATP binding site. The anisotropy of the emission was recorded over the temperature range 3°-42 °c. The total anisotropy was found to be temperature dependent with an initial value of about 0.2 at 3 °c and about 0.13 at 42 °C. The overall decrease of total anisotropy with temperature reversed in the temperature range around 13 °C where a peak value of total anisotropy was observed. The anisotropy decay curve was found to fit a double exponential decay process composed of a rapidly rotating component with a rotational correlation time of 20µs-10µs and a slower rotating component with a rotational correlation time of 400µs-100µs, over the temperature range of 3 °C-42 °C. These motions areindividually assigned to the motions of the monomer and oligomer states of the protein molecules, rotating about its axis normal to the plane of the membrane. It was also found that the associated weighting functions were also temperature-dependent; the weighting function of the slowly rotating species increases with increase in temperature, while the weighting function of the rapidly rotating species exhibits the opposite trend. It was estimated that about 80% of the total anisotropy signal was contributed by the fast rotation, and the remainder from slow rotation at 3°C and this reduced to 60% at 42°C.

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Liqun Yang, Olga Lopina, Daniel McStay, A. A. Boldyrev, Alan J. Rogers, and Peter J. Quinn "Phosphorescence depolarization and the rotational dynamics of eosin-labeled purified (Na+, K+)-ATPase in phospholipid vesicles", Proc. SPIE 1890, Biomolecular Spectroscopy III, (1 May 1993); https://doi.org/10.1117/12.145251

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