Reactivity of trypsin in reverse micelles: neglected role of aggregate size compared to water-pool components

Abstract

The catalytic efficiency of trypsin was estimated in cationic reverse micelles as a function of the concentration of water-pool components and aggregate size to determine their independent influence on enzyme activity. The variation in the aggregate size/water-pool size was achieved by changing both the W 0 (mole ratio of water to surfactant) and the headgroup area of surfactant through introduction of hydroxyethyl groups at the polar head. The local molar concentrations of water present inside the water-pool ([H 2O] wp) of different cationic reverse micelles across varying W 0 was estimated using a modified phenyl cation-trapping protocol. The [H 2O] wp in cationic reverse micelles (surfactant/isooctane/ n-hexanol/water) increases with W 0 and attains the molarity of normal water beyond W 0 = 40 irrespective of the nature of headgroup. Concurrently, the catalytic activity of trypsin compartmentalized within the water-pool increases with the increase in [H 2O] wp upto an optimal W 0 = 40 in organized solutions of any surfactant. The aggregate size (determined by static light scattering) also increases expectedly with W 0 and noticeably with the area of the surfactant headgroup at similar W 0. Since the enzyme activity rises both with the increase in water-pool size and [H 2O] wp, trypsin's efficiency was compared with these two parameters across reverse micelles of varying surfactant headgroup size at similar W 0 to determine their probable independent influence in regulating the enzyme activity. Noticeably, the efficiency of trypsin rises two to ninefold in spite of the [H 2O] wp being distinctly lower in case of hydroxyethyl group substituted surfactants compared to cetyltrimethylammonium bromide w/o microemulsions at similar W 0. Thus, the influence of the aggregate size possibly plays an important role alongwith the [H 2O] wp in modulating the enzyme activity.