Proximity Relationships within the Fc Segment of Rabbit Immunoglobulin G Analyzed by Resonance Energy Transfer

Robert T. Luedtke, Jane M. Vanderkooi, Fred Karush, Charles S. Owen

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Resonance energy transfer analysis has been carried out with a noncovalent rabbit hybrid of immunoglobulin G (IgG) composed of normal rabbit IgG and rabbit anti-lactose IgG. The hybrid IgG was prepared from proteins in which the single inter-heavy-chain disulfide linkage was specifically reduced and alkylated. Normal rabbit IgG was alkylated with the iodoacetyl derivative of N-(aminoethyl)-5-naphthylamine-l-sulfonic acid while the rabbit anti-lactose IgG was alkylated with either iodoacetamide or the iodoacetyl derivative of p-[[p-(dimethylamino)phenyl]azo] aniline. Fractionation with an anti-lactose-specific immunoadsorbent yielded a population in which each fluorescent donor [N-[(acetylamino)ethyl]-5-naphthylamine-l-sulfonic acid] was adjacent to a nonfluorescent acceptor [N-acetyl-p-[[p-(dimethylamino) phenyl]azo]aniline]. Data on fluorescence quantum yield, excited-state lifetime, time-resolved emission anisotropy, and steady-state fluorescence polarization revealed a distribution of distances between the donor and acceptor. In the native molecule, the hinge regions are known to be covalently linked by a single disulfide bond. In the absence of this linkage, the hinge regions were separated such that for a majority of the molecules in solution (~60%) the separation was 50-60Å. In the context of current knowledge of the IgG molecule, it is evident that the principle forces maintaining the integrity of the native, functional Fc segment are the strong noncovalent interactions of the CH3 domains and the single inter-heavy-chain disulfide bond.

Original languageEnglish
Pages (from-to)2927-2936
Number of pages10
JournalBiochemistry
Volume20
Issue number10
DOIs
StatePublished - 1 Jan 1981

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Energy Transfer
Energy transfer
Immunoglobulin G
Rabbits
Lactose
Disulfides
Sulfonic Acids
Hinges
Molecules
Fluorescence
Derivatives
Iodoacetamide
Immunosorbents
Fluorescence Polarization
Anisotropy
Quantum yield
Fractionation
Excited states
Polarization
Population

Cite this

Luedtke, Robert T. ; Vanderkooi, Jane M. ; Karush, Fred ; Owen, Charles S. / Proximity Relationships within the Fc Segment of Rabbit Immunoglobulin G Analyzed by Resonance Energy Transfer. In: Biochemistry. 1981 ; Vol. 20, No. 10. pp. 2927-2936.
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Proximity Relationships within the Fc Segment of Rabbit Immunoglobulin G Analyzed by Resonance Energy Transfer. / Luedtke, Robert T.; Vanderkooi, Jane M.; Karush, Fred; Owen, Charles S.

In: Biochemistry, Vol. 20, No. 10, 01.01.1981, p. 2927-2936.

Research output: Contribution to journalArticle

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N2 - Resonance energy transfer analysis has been carried out with a noncovalent rabbit hybrid of immunoglobulin G (IgG) composed of normal rabbit IgG and rabbit anti-lactose IgG. The hybrid IgG was prepared from proteins in which the single inter-heavy-chain disulfide linkage was specifically reduced and alkylated. Normal rabbit IgG was alkylated with the iodoacetyl derivative of N-(aminoethyl)-5-naphthylamine-l-sulfonic acid while the rabbit anti-lactose IgG was alkylated with either iodoacetamide or the iodoacetyl derivative of p-[[p-(dimethylamino)phenyl]azo] aniline. Fractionation with an anti-lactose-specific immunoadsorbent yielded a population in which each fluorescent donor [N-[(acetylamino)ethyl]-5-naphthylamine-l-sulfonic acid] was adjacent to a nonfluorescent acceptor [N-acetyl-p-[[p-(dimethylamino) phenyl]azo]aniline]. Data on fluorescence quantum yield, excited-state lifetime, time-resolved emission anisotropy, and steady-state fluorescence polarization revealed a distribution of distances between the donor and acceptor. In the native molecule, the hinge regions are known to be covalently linked by a single disulfide bond. In the absence of this linkage, the hinge regions were separated such that for a majority of the molecules in solution (~60%) the separation was 50-60Å. In the context of current knowledge of the IgG molecule, it is evident that the principle forces maintaining the integrity of the native, functional Fc segment are the strong noncovalent interactions of the CH3 domains and the single inter-heavy-chain disulfide bond.

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