ß-Galactosidase Langmuir Monolayer at Air/X-gal Subphase Interface.

This article investigates the surface chemistry properties of the ß-galactosidase monolayer at the air-subphase interface at the vicinity of its substrate, X-gal. We have demonstrated that the ß-galactosidase in the monolayer form remained active and performed hydrolysis of the X-gal in the subphase. We investigated the ß-galactosidase Langmuir monolayer in absence and presence of X-gal in the subphase of varying concentration of X-gal with the sodium chloride solution. It was found that the limiting molecular area as well as the collapse surface pressure kept on decreasing with the increasing concentration of X-gal. In accordance to the data obtained from the isotherm it was also found that ß-galactosidase forms a stable monolayer that does not aggregate at the air-subphase interface. The stability of the monolayer at the air-subphase interface was studied by using compression-decompression cycles with and without X-gal at varying concentration and different surface pressures. The infrared reflection-absorption spectroscopy (IRRAS) and Brewster angle microscopy (BAM) of ß-galactosidase Langmuir monolayer was also investigated for pure and mixed ß-galactosidase at the air-subphase.

Surface chemistry and spectroscopy of the ß-galactosidase Langmuir monolayer.

The changes of interfacial properties of ß-galactosidase introduced into different pH environments are investigated through surface chemistry and in situ spectroscopy. Conditions for an optimal Langmuir monolayer formation were firstly obtained by varying the subphase salt concentration and the surface-pressure area isotherm was used to extrapolate the limiting molecular area of the enzyme monolayer to be around 42,000 Å(2) molecule(-1). Surface pressure stability measurements held at 20 mN/m for 90 min along with compression-decompression cycles revealed no aggregate formation at the air-water interface. Consistent with the data obtained from the isotherm, in situ UV-Vis and fluorescence spectroscopy shows a steep rise in absorbance and photoluminescence intensity correlating to with a switch from a liquid-expanded to a liquid-condensed phase. A decrease in subphase pH increased the electrostatic repulsion as the enzyme was protonated, leading to an expanded monolayer. Infrared absorption-reflection spectroscopy demonstrates that the enzyme adopts mainly ß-sheet conformation at the air-water interface before and during the compression.