Soman Hydrolase Separation And Purification Of The Active Sites Of Partial Sequence

Organophosphorous acid anhydrolases(OPAA) are found in a variety of organisms,It catalyses the hydrolasis of G-type organophosphorous compounds,such as soman,sarin,DFP etc.In this project, we aimed to isolate,purify and sequence the enzyme from human liver.A fluoride-electrode assay was established for the determination of somanase in human liver on basis of the release of free -fuoricions(F-)from the substrate soman.Somanase was purified from the fresh liver tissue stored at -75℃from a male adult died of an accident. Liver was minced and homogenated. The soluble fraction obtained by centrifugation was then seqentially purified by ultrafiltration (30KD-150KD) preparative electro focusing plate electrophoresis, Sephadex-G50, Sephadex-G75 , DEAE-Sepharose CL 6B ion exchange gradient chromatography and Sephacryl S-200 HR gel permeation chromatography.The somanase was been purified for 213-fold with a specific activity of 1070 n mol / min / mg protein,and a molecular weight of 36KD(SDS-PAGE).Amino acid composition analysis revealed that it was an alkaline residue-riched protein. By means of HPLC and electrospary ionzation tandem electrospary source mass spectrometry( ESI-MS/MS) analysisthree peptide fragments have been sorted out and sequenced. By searching peptide mass fingerprinting in the protein datebase, It showed that the human liver somanase was a novel unknown protein.Compared the peptide framents of somanase with other somanases from different species source by Clustal.w analysis,we found that human somanase was very similar to the squid type DFPase. BLAST search indicated that the somanase exihibied similarities to certain (?)xtent with camodulin in primary structure,Swiss-Prosite search further showed that the fragment DGDGDGYLSAAELR was a Ca2+ binding site, similar with the Squid-Type DFPase conserved sequences. The DGDGDGDGY sequence of the fragment DGDGDGYLSAAELR was aphosphorylation site of tyrosine kinase. Computer simulation analysis of the two sequences showed relatively high similarity to the corresponding sequence of Squid-Type DFPase in 3D-structure. The fragment GTLTTK was an acylation site.According to the comparison result between the partial sequences of somanase obtained and the sequence of the Squid-type DFPase, and the active site of Squid-type DFPase reported,The catalytic mechisms of somanase may be postulated as follows:1. Substrate soman approaches to the active site ,The phosphonyl oxygen attracts and binds to the Ca2+ ion increasing the positive electricity of the phosphorus atom in favour of the nucleophilic attack of the hydroxyl radical of H2O.2. The imidazole ring of histidine attracts the proton of H2O to activate the water molecule,and thus facilitates the formed hydroxyl radical to attack the phosphorus atom and bind to it.3.The fluoric ion is released in the opposite direction of the attacking water and the Ca2+ is removed from the hydrolyzed substrate.Following the deprotonation of the histidine,the catalytic reaction goes on intothe next cycle.