Self-association of adrenodoxin studied by using analytical ultracentrifugation - Analytical Methods & Tools for Lyophilization

Self-association of adrenodoxin studied by using analytical ultracentrifugation
Received 16 May 2006; revised 19 July 2006; accepted 19 July 2006. Available online 17 August 2006.
Joachim Behlkea, , , Otto Ristaua, Eva-Christina M?llera, Frank Hannemannb and Rita Bernhardtb
Biophysical Chemistry
ScienceDirect
Copyright ? 2006 Published by Elsevier B.V.
aMax Delbr?ck Center for Molecular Medicine, 13092 Berlin, FRG
bUniversit?t des Saarlandes, Fachbereich Biochemie, 66041 Saarbr?cken, FRG
Abstract
The mitochondrial steroid hydroxylase system of vertebrates utilizes adrenodoxin (Adx), a small iron?sulfur cluster protein of about 14 kDa as an electron carrier between a reductase and cytochrome P450. Although the crystal structure of this protein has been elucidated, the solution structure of Adx was discussed contrary in the literature [I.A. Pikuleva, K. Tesh, M.R. Waterman, Y. Kim, The tertiary structure of full-length bovine adrenodoxin suggests functional dimers, Arch. Biochem. Biophys. 373 (2000) 44?55; D. Beilke, R. Weiss, F. L?hr, P. Pristovsek, F. Hannemann, R. Bernhardt, H. R?terjans, A new electron mechanism in mitochondrial steroid hydroxylase systems based on structural changes upon the reduction of adrenodoxin, Biochemistry 41 (2002) 7969?7978]. Therefore, it was necessary to study the self-association of this protein by using analytical ultracentrifugation over a larger concentration range. As could be demonstrated in sedimentation velocity experiments, as well as sedimentation equilibrium runs with explicit consideration of thermodynamic non-ideality, the full-length protein (residues 1?128) in the oxidized state resulted in a monomer?dimer equilibrium (Ka ~ 3 ? 102 M- 1). For truncated Adx (1?108), as well as the reduced Adx, the association behavior was strongly reduced. The consequences of this behavior are discussed with respect to the physiological meaning for the Adx system.
Keywords: Sedimentation velocity; Sedimentation equilibrium; Self-association; Association constants; Thermodynamic non-ideality

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