Interaction of cytochrome c with liposomes

Covalent labeling of externally bound protein by the fluorescent probe, azidonaphthalenedisulfonic acid, enclosed in the inner aqueous compartment of unilamellar vesicles

J. Szebeni, Gordon Tollin

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

The photoreactive fluorescent probe, 3-azidonaphthalene-2,7-disulfonic acid (ANDS) was encapsulated in the inner aqueous compartment of small unilamellar liposomes, prepared from egg phosphatidylcholine (PC) ± 20 mol% dihexadecylphosphate (DHP). After adding cytochrome c externally to a suspension of these vesicles, the probe was activated by ultraviolet irradiation, and the protein was separated from the lipids. When negatively charged (egg PC/DHP) vesicles at low ionic strength were used, which form an electrostatic complex with cytochrome c, the protein was labeled by ANDS. This process depended on irradiation time, and was inhibited by increasing the ionic strength of the medium. Labeling was not observed with isolelectric (egg PC) vesicles. These observations suggest that electrostatic binding of cytochrome c to the bilayer is accompanied by intramembrane penetration to such a depth that the protein can communicate with the inner membrane-water interface.

Original languageEnglish
Pages (from-to)153-159
Number of pages7
JournalBBA - Bioenergetics
Volume932
Issue numberC
DOIs
Publication statusPublished - 1988

Fingerprint

Unilamellar Liposomes
Cytochromes c
Phosphatidylcholines
Fluorescent Dyes
Liposomes
Labeling
Ovum
Ionic strength
Static Electricity
Osmolar Concentration
Acids
Electrostatics
Irradiation
Proteins
Suspensions
Membranes
Lipids
Water

Keywords

  • Azidonaphthalene-2,7-disulfonic acid
  • Cytochrome c
  • Fluorescent labeling
  • Liposome

ASJC Scopus subject areas

  • Biophysics
  • Medicine(all)

Cite this

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title = "Interaction of cytochrome c with liposomes: Covalent labeling of externally bound protein by the fluorescent probe, azidonaphthalenedisulfonic acid, enclosed in the inner aqueous compartment of unilamellar vesicles",
abstract = "The photoreactive fluorescent probe, 3-azidonaphthalene-2,7-disulfonic acid (ANDS) was encapsulated in the inner aqueous compartment of small unilamellar liposomes, prepared from egg phosphatidylcholine (PC) ± 20 mol{\%} dihexadecylphosphate (DHP). After adding cytochrome c externally to a suspension of these vesicles, the probe was activated by ultraviolet irradiation, and the protein was separated from the lipids. When negatively charged (egg PC/DHP) vesicles at low ionic strength were used, which form an electrostatic complex with cytochrome c, the protein was labeled by ANDS. This process depended on irradiation time, and was inhibited by increasing the ionic strength of the medium. Labeling was not observed with isolelectric (egg PC) vesicles. These observations suggest that electrostatic binding of cytochrome c to the bilayer is accompanied by intramembrane penetration to such a depth that the protein can communicate with the inner membrane-water interface.",
keywords = "Azidonaphthalene-2,7-disulfonic acid, Cytochrome c, Fluorescent labeling, Liposome",
author = "J. Szebeni and Gordon Tollin",
year = "1988",
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T2 - Covalent labeling of externally bound protein by the fluorescent probe, azidonaphthalenedisulfonic acid, enclosed in the inner aqueous compartment of unilamellar vesicles

AU - Szebeni, J.

AU - Tollin, Gordon

PY - 1988

Y1 - 1988

N2 - The photoreactive fluorescent probe, 3-azidonaphthalene-2,7-disulfonic acid (ANDS) was encapsulated in the inner aqueous compartment of small unilamellar liposomes, prepared from egg phosphatidylcholine (PC) ± 20 mol% dihexadecylphosphate (DHP). After adding cytochrome c externally to a suspension of these vesicles, the probe was activated by ultraviolet irradiation, and the protein was separated from the lipids. When negatively charged (egg PC/DHP) vesicles at low ionic strength were used, which form an electrostatic complex with cytochrome c, the protein was labeled by ANDS. This process depended on irradiation time, and was inhibited by increasing the ionic strength of the medium. Labeling was not observed with isolelectric (egg PC) vesicles. These observations suggest that electrostatic binding of cytochrome c to the bilayer is accompanied by intramembrane penetration to such a depth that the protein can communicate with the inner membrane-water interface.

AB - The photoreactive fluorescent probe, 3-azidonaphthalene-2,7-disulfonic acid (ANDS) was encapsulated in the inner aqueous compartment of small unilamellar liposomes, prepared from egg phosphatidylcholine (PC) ± 20 mol% dihexadecylphosphate (DHP). After adding cytochrome c externally to a suspension of these vesicles, the probe was activated by ultraviolet irradiation, and the protein was separated from the lipids. When negatively charged (egg PC/DHP) vesicles at low ionic strength were used, which form an electrostatic complex with cytochrome c, the protein was labeled by ANDS. This process depended on irradiation time, and was inhibited by increasing the ionic strength of the medium. Labeling was not observed with isolelectric (egg PC) vesicles. These observations suggest that electrostatic binding of cytochrome c to the bilayer is accompanied by intramembrane penetration to such a depth that the protein can communicate with the inner membrane-water interface.

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