deoxycholic-acid and chapso

The extent to which bovine cytochrome c oxidase (COX) dimerizes in nondenaturing detergent environments was assessed by sedimentation velocity and equilibrium. In contrast to generally accepted opinion, the COX dimer is difficult to maintain and is the major oligomeric form only when COX is solubilized with a low concentration of dodecylmaltoside, i.e., approximately 1 mg/mg protein. The dimer form is intrinsically unstable and dissociates into monomers with increased detergent concentration, i.e., >5 mg/mg protein. The structure of the solubilizing detergent, however, greatly alters detergent effectiveness by inducing either monomerization or aggregation. Triton X-100 is most effective at solubilizing COX, but it destabilizes COX dimers, even at low concentration. Undecylmaltoside, decylmaltoside, and octaethyleneglycolmonododecyl ether (C(12)E(8)) are less effective at solubilizing COX. Each prevents COX aggregation at high detergent concentration, but also destabilizes the COX dimer. Other detergents, e.g., Tween 20, sodium cholate, sodium deoxycholate, CHAPS, or CHAPSO, are completely ineffective COX solubilizers and do not prevent aggregation even at 10-40 mg/mL. The transition from dimers to monomers depends on many factors other than detergent structure and concentration, e.g., protein concentration, phospholipid content and pH. We conclude that the intrinsic dimeric structure of COX can be maintained only after solubilization with low concentrations of dodecylmaltoside at near neutral pH, and even then precautions must be taken to prevent its dissociation into monomers.

Topics: Animals; Cattle; Cholic Acids; Deoxycholic Acid; Detergents; Dimerization; Electron Transport Complex IV; Glucosides; Hydrogen-Ion Concentration; Octoxynol; Polyethylene Glycols; Polysorbates; Sodium Cholate; Solubility; Solutions; Structure-Activity Relationship

Enhancement of the fluorescence intensity of colchicine occurs in media of low polarity and appreciable viscosity; this is suggested to be the basis of the intensification of its fluorescence when it is bound to and immobilized in tubulin. We show here that the tubulin-bound fluorescence features of colchicine are largely reconstructed upon solubilizing it in chosen micellar aggregates that offer optimal polarities and microviscosities. Triton X-100 and bile salt micelles intensify the colchicine emission but the maximal effects are obtained with tetrameric aggregates of the peptide melittin. Estimates of the polarity, microviscosity and binding-site dimensions of colchicine are obtained using this mimetic approach. Our results suggest that well chosen micellar systems act as good models to reconstruct and analyze the spectral properties of molecules immobilized in their binding sites.

Topics: Binding Sites; Cetrimonium; Cetrimonium Compounds; Cholic Acids; Colchicine; Colloids; Deoxycholic Acid; Dioctyl Sulfosuccinic Acid; Fluorescence; Fluorescence Polarization; Melitten; Micelles; Octoxynol; Polyethylene Glycols; Sodium Dodecyl Sulfate; Solubility; Surface-Active Agents; Tubulin; Viscosity