Study On The Paralytic Shellfish Poisoning Binding Protein In Crassostrea Hongkongensis And Its Structure-activity Analysis

Paralytic Shellfish Poisoning(PSP) is a kind of poison which can combine with the membrane ion channels to inhibite the nerve conduction to paralysis. Mainly produced in cyanobacteria. PSP is one of currently known the most frequently reason which caused harmful red tide algae toxin poisoning events, and a kind of the most dangerous poison to human. Shellfishes are one of the marine animals. During eating algaes, they can also accumulate the poisnon in the aglaes, such as PSP. And hunmans were poisoned by PSP after eating those shellfishes. With the harmful algal bloomed and PSP poisoning occurring more and more frequently, PSP pollution has becomed a global issue.Based on the Alexander minutum cultured and PSP extracted, economic shellfish in Guangdong and Guangxi China, Crassostrea hongkongensis were accumulated of PSP by artificial. Then its liver’s PSP binding protein was extracted. Then the separated line:exclusion chromatographyâ†'ion exchange chromatographyâ†'hydrophobic chromatography was used to separate PSP binding protein, and the biological method(MBA) and high liquid phase method(HPLC-FLD) were used as a means to detecte the location of the PSP binding protein and the composition of PSP. And the compositons of proteins during purification were detected through SDS-PAGE. The structure-activity analysis was identified by HPLC-MS-MS and silisco annalysis. Finally, the absorbtion dynamics between PSP and the PSP binding protein was tested. The main results were:(1) For preparing poison oysters and PSP samples, different wall-breaking methods were tested in this paper. They were: ultrasonic method, microwave method and freezing and thawing method, for the PSP extraction. And the extracted PSP was tested by the MBA and HPLC-FLD. To determine the freezing and thawing optimum condition, the response surface optimization test was done, and the quadratic regression equation was obtained as follows:Y=193.8+13.75A-35.35B+12.58C-20.2AB+6.95AC-13.35BC-38.75A2-78.05B2-51.70C2. Combined with the verification experiment, the best condition of the freezing and thawing method was known: freeze-thaw 3 times, thawing temperature 37℃,pH7.8, and the virulence was up to 6616±101 MU.(2) The effect of different pH buffer on protein extraction was firstly studied in the PSP binding protein purification experiment.The extraction was under the pH=8, Tris-HClbuffer, and then determined ammonium sulfate saturation was 40%-70% in ammonium sulfate precipitation to preliminary purification. After that, gradually separation was adopted: S-100 to obtain PSP binding protein peak; and then made the PSP binding protein peak obtained from S-100 to do the purification by Q-FF to obtain PSP binding protein peak; finally, made the PSP binding protein peak obtained from Q-FF to do the purification by RP-HPLC to obtain the PSP binding protein.The extracted PSP binding protein molecular weight was 33.5kDa(tested by SDS-PAGE).(3) The HPLC-MS-MS was tested to kown the the structure and homologous of the PSP binding protein. And modeled based on prediction: â‘  after the DNATSAR analysis,it was concluded that the molecular weight was 36.08 kDa which was agreed with SDS-PAGE detection. The protein was contained with 335 amino acids. The hydrophobic amino acid and polar amino respectively were 35.35% and 38.82% of the protein molecular weight, and the isoelectric point pI was 7.25. BLATS homology analysis of the protein showed that GAPDH was the mostly similarity with the PSP bingding protein with homology of 78% or so. â‘¡ using Predictprotein, NCBI, RPS-BLAST and ConSurf knowable its secondary structure was kown: in the alpha helix was occupied 28.06%, beta fold was 30.75%,and no rules curly was 41.19%. The protein had two domain structure:GADPH C area family and GADPH NAD area family, and the active site was between the148 to 155, which played an important rule in the ecolution of the sequence.â‘¢ GAPDH had affinite to the glutamine polymer parts. PSP also contains a large number of amide,which provided the protein was PSP binding protein.(4) The PSP and the PSP binding protein’s adsorption dynamic model(PSP binding protein purity was 67.47%) was studied. Single factor experiment was kown the optimum condition: pH was 9, protein concentration was 3mg/mL, and the time was 24 h. After fited to the first order reaction equation model and dynamic second order reaction equation model, the result proved that the adsorption reaction was conformed to the speed limited secondary reaction kinetics equation model.