Diffusion-limited rates for monoclonal antibody binding to cytochrome c.
The kinetic and spectroscopic changes accompanying the binding of two monoclonal antibodies to the oxidized form of horse heart cytochrome c have been investigated. The two epitopes recognized by the antibodies are distinct and noninteracting: antibody 2B5 binds to native cytochrome c near a type II turn (residue 44) while antibody 5F8 binds on the opposite face of the protein near the amino terminus of an alpha-helical segment (residue 60). Antibody-cytochrome c binding obeys a simple bimolecular reaction mechanism with second-order rate constants approaching those expected for diffusion-limited protein-protein interactions. The association rate constants have small activation enthalpies and are inversely dependent on solvent viscosity, as expected for diffusion-controlled reactions. There is a moderate ionic strength dependence of the rate of association between the 2B5 antibody and cytochrome c, with the rate constant increasing about 4-fold as the ionic strength is varied between 0.14 and 0 M. Comparison of the rates for antibody-cytochrome c complex formation for binding to the reduced-native, oxidized-native, and alkaline conformations shows that for MAb 2B5 the forward rate constant depends slightly on cytochrome c conformation. Investigation of the pH-induced transition between the native and alkaline conformational states for free cytochrome c and for antibody-cytochrome c complexes shows that antibody binding stabilizes the native form of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Raman
C S
CS
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Jemmerson
R
R
Nall
B T
BT
Allen
M J
MJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000276:immunology / Q000378:metabolism*; D004058:Diffusion; D006736:Horses; D007700:Kinetics; D008433:Mathematics; D011487:Protein Conformation; D013053:Spectrophotometry; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D003574:Cytochrome c Group