Cardiolipin binds nonyl acridine orange by aggregating the dye at exposed hydrophobic domains on bilayer surfaces.

10-N-Nonyl acridine orange (NAO) has been used at low concentrations as a fluorescent indicator for cardiolipin (CL) in membranes and bilayers. The mechanism of its selective fluorescence in the presence of CL, and not any other phospholipids, is not understood. The dye might recognize CL by its high pK (pK(2)>8.5). To investigate that, we established that NAO does not exhibit a pK in a pH range between 2.3 and 10.0. A second explanation is that the dye aggregates at hydrophobic domains on bilayers exposed by the CL. We found that a similar spectral shift occurs in the absence of CL in a concentrated solution of the dye in methanol and in the solid state. A model is proposed in which the nonyl group inserts in the bilayer at the hydrophobic surface generated by the presence of four chains on the phospholipid.

Evaluation of electrochemical parameters for an EC mechanism from a global analysis of current-potential-time data: application to reductive cleavage of methylcobalamin.

Simultaneous evaluation of electron-transfer rate constant, k degree, following chemical reaction rate constant, kf, electron-transfer coefficient, alpha, and standard potential, E degree', for electron transfer coupled to a following chemical reaction (EC mechanism) is described. A mathematical model for the current response to a potential step is developed by incorporating the appropriate concentration terms into the Butler-Volmer equation. Experimental current-potential-time (i-E-t) surfaces are fit to this model to evaluate the parameters. Fitting individual i-t or i-E curves did not yield unique parameter values whereas an i-E-t surface constituted by several i-t or i-E curves could be fitted to obtain unique values. A generalized kinetic zone diagram for the EC reaction is drawn by examining the limiting forms of the expression for current. Theoretical limits of measurable rate constants are estimated from the zone diagram. The three-dimensional electro-chemistry described above was used to study the reductive cleavage of methylcobalamin in dimethyl sulfoxide (DMSO) solvent and 0.1 M tetrabutyl-ammonium perchlorate supporting electrolyte. The parameters estimated are as follows: alpha = 0.552 +/- 0.004; k degree = 0.011 +/- 0.0015 cm s-1; kf = 1500 +/- 140 s-1; E degree' = -1.54 +/- 0.01 V. The rate constant for the following reaction, kf, in DMSO solvent is approximately 4000-fold faster than the similar process in aqueous medium. It is suggested that this enhancement is relevant to methyl group transfer in enzymatic reactions, e.g., methionine synthase, if the enzyme mechanism involves a reductive cleavage which produces a methyl radical.

Theoretical and experimental investigation of steady-state voltammetry for quasi-reversible heterogeneous electron transfer on a mercury oblate spheroidal microelectrode.

A mercury microelectrode formed by electroreduction of mercury on an inlaid gold microdisk is experimentally shown to be well modeled by oblate spheroidal geometry when the ratio of the semiminor axis to the semimajor axis of the protruding drop is less than 1. The validity of the geometry is established by comparison of the experimentally determined coefficient in the steady-state diffusion current equation with the theoretical value for oblate spheroidal geometry. Spherical cap geometry is also shown to be an equally valid geometric model; however, theoretical treatment for this system is more difficult. The theory of a quasi-reversible electrode process is developed and applied to the determination of the electrode parameters of the RuIII(NH3)6/RuII(NH3)6 electrode reaction on a mercury oblate spheroidal microelectrode. Results agree well with others found in the literature for the same process on a mercury electrode.