The binding of a neutral aromatic molecule to a negatively-charged lipid membrane. II. Kinetics and mechanism.
The kinetics of the binding of the fluorescence indicator N-phenyl naphthylamine to bilayer vesicles of C12-methyl-phosphatidic acid have been investigated by means of the temperature-jump relaxation technique utilizing fluorescence light detection. Single-exponential relaxation curves were observed, with time constants in the range 0.2-3 ms. The concentration dependence of the relaxation time yielded an apparent association rate constant (expressed in terms of monomeric phospholipid) of k(on) = 5 x 10(6) M(-1) s(-1) in aqueous solution at 25 degrees . The activation energy and viscosity dependence associated with the binding rate show that this process is actually diffusion-controlled. The theory of diffusion-controlled reactions then allows a determination of the average size of the bilayer vesicles and of the true rate constant for the association of the indicator molecules with the vesicles. Assuming spherical geometry for the vesicles, the values are: r(ves) = 190 A, which corresponds to 20000 lipid molecules per vesicle and k'(on) = 1 x 10(11) M(-1) s(-1) (25 degrees). The correctness of this size-determination was confirmed semi-quantitatively by electron microscopy. Since in fact a distribution of vesicle sizes must be present, a discussion is included of the relaxation function which the system is expected to take in the general case. Biological implications of diffusion control for the transport of non-polar substances and for lipid mixing are indicated.
Woolley
P
P
Magdalene College, Cambridge, England.
Diebler
H
H