Purification, Characterization of Protein from the Ventral Portion of Puffer Fish ( Tetraodon patoca Hamilton) and Comparison of Their Characteristics with Tetrodotoxin

Abstract

A lectin was isolated and purified from the ventral portion of the Potca fish, Tetraodon patoca.The method was accomplished by gel filtration of crude protein extract on Sephadex G-50 followed by Ion exchange chromatography on DEAE- cellulose and finally by affinity chromatography on ConA­Sepharose matrix. The molecular weight of the lectin as determined by the gel filtration and SDS-PAGE was about 82,000 and 80,000 respectively, but 42,000 and 38,000 were indicated by SDS-PAGE in the presence of 2-mercaptoethanol. The Lectin agglutinated rat red blood cells and in a haptein­inhibition test, the lectin was inhibited specifically by the O-mannose and mannose containing saccharides. The Lectin is glycoprotein with neutral sugar content of about 0.35%. Like tetrodotoxin, the purified lectin also showed strong cytotoxic effects, which was confirmed by brine shrimp lethality bio assay and histopathological examinations. The N- terminal amino acid sequences of both the sub units of the lectin were also identified and used a blast search on N- terminal amino acid sequences of the sub units revealed that the lectin showed significant homology with the homologous proteins in database. Among them. marine toxins, relevant for human intoxication, tetrodotoxin has been known as one of the most prejudicial. Potca fish is originally thought to be the only animal from which tetrodotoxin could be isolated. Five different parts of potca fish were analyzed for their toxicity using brine-shrimp lethality bioassay. Among the parts examined, the ventral portion and egg contained comparatively the highest amount of toxicity. Tetrodotoxin (TTX) was isolated and purified from the ventral portion including liver by thin layer chromatography (TLC) using the solvent system 1-butanol: acetic acid: water (8: l: l): Two spots with Revalues of0.76 and 0.65 were showed under UV light and after spraying with l 0% potassium hydroxide/ninhydrin reagent the toxicity of both the compound were further reconfirmed by brine-shrimp lethality bio assay. The TTX and Potca Fish Protein (PFP) have showed no activities against gram positive as well as gram negative bacteria. Sub-acute toxicity study showed that the TTX and PFP had pronounced effects on the hematological profiles, such as total RBC, total WBC, platelet, % of hemoglobin and ESR. The other biochemical parameters such as SGPT, SGOT, SALP, serum bilirubin, creatinine and urea were also affected by the toxins. The histopathological examinations showed that the tissues such as liver, lung, kidney and heart of rats were severely affected after treated with both TTX and PFP as compared to those of the control group rats. Again, although TTX and PFP showed also similar effects but PFP is slightly less effective than TTX.