Structural requirements of rabbit IgG F(ab')2 fragment for activation of the complement system through the alternative pathway—II. Ionic and indole groups |
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Authors: | C Juarez JP Albar F Vivanco-Martínez R Bragado F Ortíz |
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Institution: | Department of Immunology, Fundación Jiménez Diaz, Universidad Autónoma de Madrid, Avda Reyes Católicos 2, Madrid 3, Spain |
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Abstract: | The structural features of rabbit IgG F(ab')2 fragments required for complement activation through the alternative pathway have been investigated by means of chemical modification of specific groups. These included ionic (amino and carboxyl) and indole groups.Potassium cyanate, glycine-ethyl ester in the presence of EDC,2 and NBB were used as modifying agents of the amino, carboxyl and indole groups respectively. Three different aspects of the modified protein were analysed: the conformational integrity of the protein (by CD and antigenic reactivity), the antibody activity (by the haemaglutination test and radiobinding to the erythrocyte surface) and the capacity to activate complement through-the alternative pathway (by direct haemolysis in conditions in which the classical pathway was blocked).Changes in protein conformation caused by the chemical treatment applied were not detected by the techniques used. An enhancement of the antibody activity of the F(ab')2 fragment against the sheep erythrocyte membrane was observed when it was amidated by glycine-ethyl ester and a decrease when it was carbamylated or two tryptophanyl residues were altered per molecule. The complement-activating capability was preserved after tryptophan modification. On the contrary, modification of amino groups produced a partial decrease in the complement-activating ability and almost total loss of the ability when 34 carboxyl groups were amidated. |
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Keywords: | PBS PBSG PBSA NRS normal rabbit serum NRS-PBSA PAGE polyacrylamide gel electrophoresis SDS-PAGE sodium dodecyl sulphate-PAGE IDD immunodiffusion CD circular dichroism EDC 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide TNBS 2 4 6-trinitrobenzenesulfonic acid NBB 2-hydroxy-5-nitrobenzyl bromide EGTA HSA human serum albumin |
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