Isolation and purification of Oncomelania hupensis agglutinin and determination of its molecular weight

Objective To isolate and purify agglutinin from Oncomelania hupensis snail and determine its molecular weight.Methods Agglutinin was preliminarily isolated from snail tissue homogenate by 0%-40% saturated ammonium sulfate,and then successively purified with Sephadex G-75 gel filtration and Sepharose 4B affinity chromatography.Bradford assay was used to determine the protein content.The agglutination activity was determined by rabbit erythrocytes.The purity of agglutinin preparations was assessed by SDS-PAGE.The molecular weight of agglutinin subunit was determined by Sephadex G-75 gel filtration.Results The specific activity of snail tissue homogenate was 21.74 titer/mg.After ammonium sulfate precipitation,Sephadex G-75 gel filtration and Sepharose 4B affinity chromatography,the specific activity of snail agglutinin from the homogenate solution increased to 61.93 titer/mg,75.89 titer/mg and 963.86 titer/mg,respectively.SDS-PAGE analysis indicated that snail agglutinin(Mr 53 000) was purified by Sephadex G-75 gel filtration and Sepharose 4B chromatography.The molecular weight of the snail agglutinin produced by Sephadex G-75 gel filtration was Mr 78 000.Conclusion Combined use of salt fractionation,gel filtration and affinity chromatography can be efficient for extraction and purification of agglutinin from Oncomelania hupensis species.The snail agglutinin is characterized as monosubunit protein with a molecular weight of Mr 78 000.