HEAT-STABLE LANGMUIR-BLODGETT-FILM OF GLUTATHIONE-S-TRANSFERASE

Glutathione-S-tranferase (GST) is an enzyme able to conjugate glutathi one to electrophilic molecules such as 1-chloro-2,4-dinitrobenzene or some triazinic pesticides, such as atrazine, with production of proton s. The Langmuir-Blodgett (LB) technique was utilized and optimized to obtain stable monolayers of this enzyme. The film was then characteriz ed by means of circular dichroism spectroscopy, nanogravimetry, and sp ectrophotometry in order to determine the structure, surface density, and functional activity of the enzyme when packed in the monolayers. T he functional analysis of the LB film was also conducted as a function of the number of layers and of the temperature. While the kinetic res ponse as a function of layers suggested that only the external layer w as active, the film activity as a function of temperature indicated th at, as shown earlier for other proteins, GST in a LB film appears to s trictly preserve its functional activity and thus its structure up to 423 K. The enzymatic activity was finally tested in solution with the potentiometric alternating biosensor (PAB) system, with the minimum de tectable amount of 1 chloro-2,4-dinitrobenzene being 25 mu M. The anal ysis was highly encouraging for a biosensor application, considering t hat both structure and function appear largely preserved after the dep osition.