Covalent and noncovalent modifications induce allosteric binding behavior in a monoclonal antibody.

Covalent and noncovalent modifications induce allosteric binding behavior in a monoclonal antibody. Research Abstract Details 

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Covalent and noncovalent modifications induce allosteric binding behavior in a monoclonal antibody. Abstract Text:

Robert C Blake,Xia Li,Haini Yu,Diane A Blake,

Detailed equilibrium binding studies were conducted on a monoclonal antibody (8A11) directed against UO22+ complexed with 2,9-dicarboxy-1,10-phenanthroline (DCP-UO22+). Covalent modification of 8A11 with amine-reactive derivatives of Cy5 or Alexa 488 altered the binding curves obtained with DCP-UO22+ from hyperbolic to sigmoidal, the latter characterized by Hill coefficients of 1.5-1.6. Binding curves obtained with DCP-UO22+ and the bivalent (Fab)2 or the monovalent Fab fragments derived from limited proteolysis of the covalently modified 8A11 were characterized by Hill coefficients of 1.2 and 1.0, respectively. Incubation of 8A11 with saturating concentrations of the Fab fragments of goat antibodies directed against the Fc portion of mouse IgG increased the affinity of the native 8A11 for DCP-UO22+ by 3-fold. Conversely, incubation of the 8A11-Cy5 covalent conjugate with saturating concentrations of protein G (which likewise binds to the constant regions of mouse IgG) decreased the affinity of the primary antibody for DCP-UO22+ by 4-fold. In addition, the binding curves obtained with 8A11-Cy5 and DCP-UO22+ species changed from sigmoidal to hyperbolic at high concentrations of protein G. The presence of the antigen had a reciprocal effect on the binding of protein G to the 8A11-Cy5 conjugate; incubation of the 8A11-Cy5 conjugate with saturating concentrations of DCP-UO22+ decreased the affinity of the conjugate for protein G by 20-fold. These complex binding data were interpreted in terms of a free energy binding model in which (i) 2 mol of DCP-UO22+ and 1 mol of protein G bind to each mole of the 8A11-Cy5 conjugate, (ii) binding of the first equivalent of DCP-UO22+ to the antibody promotes the binding of the second equivalent of antigen in the absence of protein G, and (iii) DCP-UO22+ and protein G oppose each other's binding to the antibody. This is the first detailed description of the energetic balance of reciprocal binding events among the antigen binding sites and distant points on the constant portion of an immunoglobulin.

Covalent and noncovalent modifications induce allosteric binding behavior in a monoclonal antibody. Publishing Authors By Initials

RC Blake,X Li,H Yu,DA Blake,

For similar inorganic chemicals: uranium compounds research abstracts see: inorganic chemicals: uranium compounds research

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Covalent and noncovalent modifications induce allosteric binding behavior in a monoclonal antibody. Journal Published:

PUBLICATION TYPE: Research Support, U.S. Gov't,

Journal: Biochemistry

VOLUME: 46

Page Numbers: 1573-86

Journal Abbreviation: Biochemistry

ISSN: 0006-2960

DAY: 17

MONTH: 01

YEAR: 2007

Covalent and noncovalent modifications induce allosteric binding behavior in a monoclonal antibody. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 370623

Covalent and noncovalent modifications induce allosteric binding behavior in a monoclonal antibody. Keywords Mesh Terms:

KEYWORDS: Uranium Compounds

MESH TERMS: immunology

Chemical & Substance for Abstract: Covalent and noncovalent modifications induce allosteric binding behavior in a monoclonal antibody. Information

Substance Name: uranium dioxide

Registry Number: 1344-57-6

Grant and Affiliation Information for Covalent and noncovalent modifications induce allosteric binding behavior in a monoclonal antibody.

AFFILIATION: College of Pharmacy, 1 Drexel Drive, Xavier University of Louisiana, New Orleans, Louisiana 70125, USA. rblake@xula.edu

Country: United States

AGENCY: United States NIGMS

GRANT: GM08008-26S1

ACRONYM: GM

MEDLINETA: Biochemistry

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