The effect of linoleic acid on pH inside sodium bis(2-ethylhexyl)sulfosuccinate reverse micelles in isooctane and on the enzymic activity of soybean lipoxygenase.

The effective pH of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelles (pHrm), containing buffers of different pH (pHst) and various amounts of linoleic acid, was studied within the range of compositions used to study the activity of soybean lipoxygenase in reverse micelles. Significant shifts of pHrm versus pHst were observed for the solutions of relatively higher pHst, dependent on linoleic acid and buffer concentrations. The effect diminished as pHst became closer to 7. When low-ionic-strength buffers were added to AOT solutions in isooctane, a significant buffering effect of linoleic acid in reverse micelles was observed. Solubilization of > 3 mM linoleic acid in micellar solutions containing 25 mM buffers gave the observed pHrm values almost independent of pHst. This effect diminished with the ionic strength of the buffering solution, but did not vanish even at 200 mM buffer. The observed effects result from the balance between ionization of linoleic acid and its partition between the water pool and the micellar interface. The enzymic activity of soybean lipoxygenase in the AOT reverse micellar solutions of the determined pHrm values was also studied. A significant reduction of the kinetics of the enzymic activity was observed, for all studied reverse micellar solutions. Changes of pHrm, caused by the presence of acidic substrate (linoleic acid) do not explain the observed reduction of activity directly through the effect on the enzyme. Due to unfavourable partition of the substrate between the microphases present in the systems, enhanced by reduction of pH at higher total concentrations of linoleic acid, the saturation of the enzyme with the substrate was not observed in the system and is difficult to attain experimentally in reverse micelles. A shift of the lipoxygenase activity/pHrm profile but negligible shift of the activity/pHst profile, with respect to aqueous buffer solutions, were observed. This indicates that either the information given by pH indicators used does not reveal the true pH of the enzyme in these reverse micelles, or that the sample of the enzyme is pH-insensitive over a broader pH range than results from the relationship observed for linoleic acid in aqueous solutions. The latter conclusion is consistent with the data on lipoxygenase activity towards linoleyl sulfate in aqueous solutions [Bild, G. S., Ramadoss, C. S. & Axelrod, B. (1977) Lipids 12, 732-735]. Conditions for measuring pH-independent lipoxygenase activity in reverse micelles are discussed.

Activity and spectroscopic properties of bovine liver catalase in sodium bis(2-ethylhexyl)sulfosuccinate/isooctane reverse micelles.

Spectroscopic properties such as ultraviolet-visible spectroscopy, circular dichroism, steady-state fluorescence and the catalytic activity of bovine liver catalase (BLC) have been studied in reverse-micelles microemulsions formed by sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. Activity measurements were carried out with the highly water soluble hydrogen peroxide as substrate as a function of temperature and pH, at various concentrations of substrate, enzyme, AOT and at different w0 values (w0 = [H2O]/[AOT]). The folding of catalase in reverse micelles is not drastically altered, although there are changes in the local surrounding of the prosthetic group. Kinetic measurements have been carried out under first-order conditions at low substrate concentration. They indicate that the measured substrate decomposition rate is limited by the exchange of substrate molecules between the substrate-filled and the enzyme-containing micelles. The specificity constant, ks = kcat/KM, as related to water pool concentrations, k(s,wp)rm, was, at all w0 values, considerably lower than ks measured in buffer (ksb). In contrast, overall values of ks in reverse micelles, k(s,ov)rm, were always 2-4-times higher than ksb. This apparent superactivity can, however, be ascribed to concentration effects. No 'bell-curve' to describe the w0 dependence of catalase activity in reverse micelles was found, k or ks increasing monotonously up to w0 = 50.