Pseudo-native motifs in the noncovalent heme-apocytochrome c complex. Evidence from antibody binding studies by enzyme-linked immunosorbent assay and microcalorimetry.
When beef heart apocytochrome c is unfolded, it folds upon noncovalent heme binding (Dumont, M. E., Corin, A. F., and Campbell, G.A. (1994) Biochemistry, 33, 7368-7378). Here, the conformation of the heme-apocytochrome noncovalent complex is compared with that of holocytochrome c. A purification method was designed for obtaining in large amounts apocytochrome c that was shown by amino acid analysis and mass spectroscopy to be chemically intact. The apoprotein and its noncovalent complex were characterized by absorption, fluorescence, circular dichroism, and sedimentation velocity, confirming previous reports. Sedimentation-diffusion equilibrium showed that the apoprotein and its noncovalent complex with heme were monomeric. Surprisingly, whereas apocytochrome c was quite soluble, the noncovalent complex slowly formed heavy aggregates, thus precluding experiments at the concentrations needed for structural studies. Two monoclonal antibodies that bind strongly to distinct antigenic sites on native holocytochrome were used to probe the noncovalent complex conformation. For both antibodies, the affinity for the noncovalent complex was only about 5-10-fold smaller than that for native holocytochrome c, and about 50-100-fold larger than that for apocytochrome c. These results indicate that the noncovalent complex, although not entirely native, carries some pseudo-native structural motifs.
Goldberg
M E
ME
Unité de Biochimie Cellulaire, CNRS URA 1129, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris, Cedex 15, France. goldberg@pasteur.fr
Schaeffer
F
F
Guillou
Y
Y
Djavadi-Ohaniance
L
L
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001059:Apoproteins / Q000302:isolation & purification / Q000378:metabolism*; D002151:Calorimetry; D002417:Cattle; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000302:isolation & purification / Q000378:metabolism*; D045304:Cytochromes c; D004797:Enzyme-Linked Immunosorbent Assay; D006418:Heme / Q000378:metabolism*; D006736:Horses; D008024:Ligands; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D011487:Protein Conformation; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet
D000911:Antibodies, Monoclonal; D001059:Apoproteins; D003574:Cytochrome c Group; D008024:Ligands; D046911:Macromolecular Substances; D006418:Heme; D045304:Cytochromes c