Spectroscopic properties of organophosphorus hydrolase (OPH) and organophosphorus acid anhydrolase (OPAA).

摘要

The research described in this dissertation explores the spectroscopic properties of organophosphorus (OP) compounds degrading enzymes, including organophosphorus hydrolase (OPH) and organophosphorus acid anhydrolase (OPAA).; OP compounds are probably the best known and most widely used agents in recent years as pesticides, insecticides, and chemical warfare (CW) agents. However, they are also a major global cause of health problems. Due to their critical consequences, there is a need for improving sensors or techniques to detect OP compounds in the environment. OPH and OPAA are two bacterial enzymes which have been identified and characterized for use in the detoxification of the OP compounds.; The secondary structure of OPH in aqueous solution was studied with circular dichroism (CD) spectroscopy. The effect of pH on the secondary structure of OPH aqueous solution indicated that the secondary structure of OPH was well defined when the pH value was near the isoelectric point (7.6); however, it was destroyed when the pH values were increased or decreased further. Comparative study of the thermal treatment on the secondary structure of OPH in solution, Langmuir-Blodgett film, and dry film showed that the molecular arrangement played a dominant role in the thermal stability of OPH.; The Langmuir film organization of organophosphorus hydrolase (OPH) was monitored using polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). The shape and position of the amide I and amide II bands were used to estimate the surface conformation and orientation of OPH. From the amide I/amide II ratio and the band position, it can also be deduced that the enzyme adopted an organization which gave a higher occupied surface and rotated to a more vertical conformation at high surface pressure. The decompression of the monolayer indicated that the behavior of the secondary structure at the air-water interface was reversible. PM-IRRAS was also used to investigate the pH effect of the subphase on the secondary structure of OPH monolayer. The secondary structure of OPH at the air-water interface was well defined when the pH of the subphase was near its isoelectric point (IP, pH 7.6). However, it adopted a different orientation when the subphase pH values were higher or lower than the IP with formation of random coil structure.; Molecular interaction between the enzymes and their substrates in aqueous solution and at the air-water interface was investigated utilizing different spectroscopic techniques, that is, CD, fluorescence in aqueous solution and PM-IRRAS at the air-water interface. The characterization of the enzymes and their secondary structure in aqueous solution and as a monolayer at the air-water interface in the absence and presence of their substrates dissolved in aqueous solution or in the aqueous subphase demonstrated significantly distinctive feature. The research described here demonstrated that the two enzymes can be used in enzyme-based biosensors for OP compounds detection.