Snails Hemocyanin Purification And Nature Of A Preliminary Study

Oncomelania hupensis is the only intermediate host species for the human parasite named Schistosoma japonicum, the Oncomelania hupensis distribution was basically the same as Schistosomiasis japonica epidemic spots. Hemocyanin is a respiratory protein of Oncomelania hupensis. At present, the experiments on the hemocyanin of Oncomelania hupensis had not been reported. Systematically researches on the hemocyanin based on the molecular level may provide new methods for the controlling of Schistosoma. In our experiments, isolation and purification of the hemocyanin were carried out, the properties and structures of the hemocyanin were bascially characterized.The hemocyanin was firstly isolated and purified by ultracentrifugation and gel filteration chromatography on Sepharose 4B column.The absorption spectrum of the protein revealed two isolated bands at 270nm and 340nm while irradiated by the UV. Then, the band at 340nm was abolished by the addition of KSCN. The contents of the copper was determined about 0.23%. The typical quaternary structure was revealed by the transmission electron microscopy, and the large hollow cylinder was observed in our experiments. Decamers, didecameric and multidecameric structures were all observed. SDS-PAGE was used to test the protein components. Two isolated bands were also observed in the SDS-PAGE gel, one near 400KDa, and another is near 250KDa compared with the protein marker.As reported previously, the hemocyanins (Hc) and phenoloxidases (PO) of mollusks differe in physiological functions They both have copper binding sites, and Hc can be functionally converted into a kind of phenoloxidase-like enzyme (hemocyanin-derived phenoloxidase, HdPO) under certain conditions, displaying PO activities. SDS, bovine pancreatic elastase type IV, and urea were utilized to stimulate the convertion of Hc into HdPO, it was found that bovine pancreatic elastase type IV and urea were the most effective stimulant. Since SDS behaved less effective than SDS. So in our experiments, the purified Hc was stimulated with 2M ureas, and the generated HdPO was investigated biochemically and enzymatically, o-diphenol was used as the specific substrate. The results indicated that the optimum pH value of HdPO was 7.0 and 9.0, the temperature was 40℃, and the apparent Km value of the HdPO was about 2.69 mmol/L. Specificity studies on the substrate revealed that the HdPO was capable of oxidizing not only the o-diphenol, but also tyrosine. The apparent Km value on tyrosine was about 9.52 mmol/L. As shown in the experiments, HdPO behaved oxidizing activities both on diphenol and monophenol, which implies that the HdPO was probably not only a type of tyrosinase-type PO but also a catechol-type. Besides, the HdPO was also effect by metal chelator and metal ions. The activities of the PO-like enzyme could be inhabitted by the EDTA effectively. Ca2+,Mg2+,K+,Na+ had less inhibited to the PO-like enzyme activity.Detailed data on a mutual epitope was shared by Hemocyanin from Oncomelania hupensis and Schistosomiasis japonica.The optimum concentration of coating antigen was 50ng/ml, the dilution rate of antibody was 1:1000, the best blocking effect was reached at the condition of 37℃for 2hrs. The optimum dilution rate of enzyme-labeled antibody was 1:20000, the reacting time was 1hr. The determined optimum reacting time of TMB was 15min.