Determination of lipid asymmetry in human red cells by resonance energy transfer

Jerome Connor, Alan J. Schroit

Research output: Contribution to journalArticle

91 Citations (Scopus)

Abstract

This report describes the application of a resonance energy transfer assay to determine the transbilayer distribution of 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled lipid analogues. The validity of this technique was established by determining the relationship between the distance of separation of lissamine rhodamine B labeled phosphatidylethanolamine (N-Rho-PE) acceptor lipid and NBD-labeled donor lipid and energy transfer efficiency. By determination of the distance between probes at 50% transfer efficiency (R0), the distance between fluorophores distributed symmetrically (outer leaflet label) and asymmetrically in artificially generated vesicles was determined. Calculation of the average distance between probes revealed a 14-Å difference between NBD-lipid and JV-Rho-PE localized in the same leaflet and in opposing leaflets, respectively. Application of this technique to the study of the transbilayer distribution of NBD-lipid in human red blood cells (RBC) showed that exogenously supplied NBD-phosphatidylserine (NBD-PS) was selectively transported to the inner leaflet, whereas NBD-phosphatidylcholine remained in the outer leaflet. In contrast, pretreatment of the RBC with diamide (a SH cross-linking reagent) blocked the transport of NBD-PS. The absence or presence of NBD-PS in the outer leaflet was independently verified by employing "back-exchange", trinitrobenzenesulfonic acid derjvatization, and decarboxylation with PS decarboxylase experiments. These control experiments yielded results which confirmed the lipid distributions determined by the resonance energy transfer assay.

Original languageEnglish (US)
Pages (from-to)5099-5105
Number of pages7
JournalBiochemistry
Volume26
Issue number16
StatePublished - 1987

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Energy Transfer
Energy transfer
Cells
Lipids
Phosphatidylserines
lissamine rhodamine B
Assays
Blood
Erythrocytes
Trinitrobenzenesulfonic Acid
Cross-Linking Reagents
Diamide
Decarboxylation
Carboxy-Lyases
Fluorophores
Labels
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Determination of lipid asymmetry in human red cells by resonance energy transfer. / Connor, Jerome; Schroit, Alan J.

In: Biochemistry, Vol. 26, No. 16, 1987, p. 5099-5105.

Research output: Contribution to journalArticle

Connor, J & Schroit, AJ 1987, 'Determination of lipid asymmetry in human red cells by resonance energy transfer', Biochemistry, vol. 26, no. 16, pp. 5099-5105.
Connor, Jerome ; Schroit, Alan J. / Determination of lipid asymmetry in human red cells by resonance energy transfer. In: Biochemistry. 1987 ; Vol. 26, No. 16. pp. 5099-5105.
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