Isolation, characterization and immunochemical studies of proteins from the shell of cypraea moneta (Linnaeus, 1758) (Cowry Shell)

Balogun, Rodiat Olusola (2012)

xviii,82p

Thesis

Cowry shells have recently become the focus of several research efforts because of their high content of chitin and chitosan, which have many important biomedical potentials. However many of these applications are potential sources of immune activation. Hence, there is a need to characterize the protein content and investigate the immunological properties of cowry shell. Proximate analysis of the powdered shell was carried out according to the procedure of the Association of Official Analytical Chemistry (A.O.A.C 2006). Protein was extracted with Phosphate Buffered Saline (PBS) or Citrate buffer according to standard procedure. Protein concentration was determined using Bradford method. The crude protein extract was separated and purified by gel filtration techniques. Degree of purity was ascertained by using non-denaturing polyacrylamide gel electrophoresis (PAGE). The native and subunit molecular weights were determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively. Immunological studies were performed with serum containing cowry shell antibody obtained from rabbits using Ouchterlony and Immunoelectrophoresis techniques. Group B, C, and D of mice was injected intraperitoneally with cowry shell protein of 10, 100, and 1000mg/kg body weight doses, respectively and the control with citrate buffer (group A). Changes in body weight, packed cell volume (PCV) and white blood cell (WBC) counts were monitored daily for two weeks, after which histopathological studies were performed on the organs liver, kidney, spleen, and lungs. The results showed the proximate analysis of powdered cowry shells as follows: Moisture content (0.74%); Nitrogen content (0.30%); Ash content (88.82%); Crude fibre (4.38%); Crude protein (1.91%) and Crude fat (0.18%). Gel filtration of the crude extract of powdered cowry shell on Sephadex G-150 showed two widely separated peaks that were also resolved in the process. The native molecular weights of the two peaks, determined by gel filtration, were 87,000 Da and 31,000 Da, respectively. Non-SDS- polyacrylamide gel electrophoresis (PAGE) of the crude extract showed two bands. Each of the two peaks pooled from gel filtration showed single bands on SDS- polyacrylamide gel electrophoresis (PAGE) with 19,000 Da and 19500 Da respectively. The serum containing cowry shell antibodies obtained from rabbits immunized with the crude extract precipitated the antigen in double immunodiffusion test. Immunoelectrophoresis of the crude extract showed precipitation of the protein only at the point of application, without any separation into bands. There were no significant differences between control mice and those injected with different concentrations of proteins, for PCV, WBC counts but there was significant difference in body weight of group B mice, compared to control. By physical observation no morphological or behavioral changes and no death of mice were recorded throughout the experimental period. Histopathological studies revealed visible damaging effects of the cowry shell protein on the liver, kidney, lung and spleen. It could be concluded that cowry shells contain low protein contents which did not manifest toxicity at low doses, but causes visible organ damage to some organs at high concentrations, suggestive of possible organ damage without concurrent physical manifestations in the mice.

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