Temperature-, electric field- and solute-induced percolation in water-in-oil microemulsions.

We report investigations on the percolation of the aqueous phase in water-in-oil microemulsions, comparing systems stabilized by ionic AOT and non-ionic Igepal amphiphiles. First, we briefly review the opposite effect of temperature on the two systems and compare electric conductivity with viscosity data. In the second part, we show that percolation can be induced by high electric fields resulting in a shift of the percolation curve. The electric field measurements allow to investigate the dynamics of clustering of the water droplets to form a network of percolating channels. We examine the slow build-up and the fast decay of the percolating structure, monitoring simultaneously electric conductivity and electric birefringence. In the third part we discuss the effect of some solutes on the percolation curve, especially of small molecules which act as protein denaturants and of native and denatured proteins like methemoglobin, chymotrypsin and gelatin. The spectroscopic determination of the dimerization of hemin, released from denatured hemoglobin, reflects the incorporation of the hemin monomers in the surfactant monolayer. In the gelatin system time resolved electric birefringence shows that even at low concentrations it is the macromolecule which determines the structure of the aqueous domain. In the appendix, a simple estimate of the intrinsic Kerr-constant is given for microemulsion droplets deformed in an electric field.

Kinetics of quinine-deuterohemin binding.

The interaction of quinine with free hemin is of importance for the antimalarial effect of the drug in infected erythrocytes. We have investigated the kinetics of the complex formation of quinine with deuterohemin using the temperature jump relaxation method. We use ethyleneglycol-water mixtures as a solvent, where sufficient solubility for both species is provided and dimerization of the hemins, which involves mu-oxo bridges, can be controlled. Equilibrium and kinetic data for the dimerization of deuterohemin are given at 30 and 50 vol% ethyleneglycol. Binding of quinine is significantly slower than dimerization. Both processes are well separated on the time axis and yield a relaxation progress curve which is described with high accuracy by two exponential terms. The slow relaxation process is analyzed with respect to a 1:1 complex formation. This is the simplest mechanism which accounts for the present data, leading at 30 vol% ethyleneglycol, pH 7.5 to a binding constant of 10(4) M-1 and rate constants of 4.4 x 10(5) M-1 s-1 for the association and 44 s-1 for the dissociation step. However, there is evidence from the fast relaxation process that monomeric and dimeric hemin exhibit different reactivity. There is a strong decrease in rate with increasing pH. The implication of the results with respect to the proposed mechanisms of complex formation with quinoline drugs is discussed.

Ionic strength dependence of the electric dissociation field effect. Investigation of 2,6-dinitrophenol and application to the acid-alkaline transition of metmyoglobin and methemoglobin.

We present data on the ionic strength dependence of the dissociation field effect (2nd Wien effect) of the protolytic reaction of 2,6-dinitrophenol in aqueous solution, monitored through optical absorption changes in high electric fields. The results are in very good agreement with the Onsager-Liu theory. We then investigate the field strength dependence of the acid-alkaline transition, i.e., the hydrolysis reaction of the water coordinated to the heme iron, in metmyoglobin and methemoglobin. The true field effect of the reaction is determined from measurements in buffers which exhibit no field effect. We conclude that negative charges on the protein influence the field effect in methemoglobin but not in metmyoglobin. The Onsager-Liu theory is applied to estimate the number of charges involved.

Two-step interaction of alpha-chymotrypsin with a fluorescent inhibitor. A dynamic study by the temperature-jump method.

Bovine alpha-chymotrypsin interacts with a dansylated inhibitor, 1-dimethylaminonaphthalene-5-sulfonyl-D-tryptophan ethyl ester, to give a stable and strongly fluorescent derivative. The kinetics of the interaction of the enzyme with this inhibitor has been investigated by the temperature-jump method. It is shown that the complex formation proceeds in two steps, a binding step and a slow isomerisation, as deduced from a study of the concentration dependence of the relaxation times. The rate constants for the binding process are k12 = 7.9 x 10(5) M-1s-1 and k21 = 1.36 x 10(3) s-1, giving a binding constant of 585 M-1. This last value, compared to the overall binding constant determined statically of 1740 M-1, leads to the determination of the kinetic parameters for the slow isomerisation k23 = 19 s-1 and k32 = 9 s-1. Both values have the same order of magnitude as the rate constants found for the pH-induced relaxation of some pancreatic proteins. This slow isomerisation can be related to internal motions of the active center of the enzyme.