The Study On Chemical Modification Of Chymopapain

Chymopapain is the most abundant cysteine proteinase with the highest proteolytic and milk clotting activities in the papaya latex. Besides widely employed in the food industries, it also has very important medical application, in which the treatmemt of prolapsed intervertebral disc is the most effective, deep and successful. However, because of its short half-life in vivo and antigenicity, its medical efficacy was reduced greatly. Therefore, The chemical modification of Cp with mPEG was researched. In this study, the Cp was purified from commercial spray-dried latex, and then the pure Cp was modified with activated monomethoxypolyethylene glycol(mPEG 5000). by the experiments, the optimum conditions for chemical modification were selected, the mPEG-modified Cp was deternined and analyzed, and the main properties of the Cp before and after the modification were also studied by comparison.In the research, the inactivated crud enzyme was applied on CMC ion exchange chromatography, and then through the ultra-filtration, vacuum freeze drying, the pure Cp was obtained with a specific activity of 3389.1U/mg, and the recovery of activity of 68.9%, the recovery of protein of 64.1%. The SDS-PAGE of the purified Cp showed a single band.It is necessary to determine antigenicity of various modification products by immunological techniques during the research on the chemical modification of mPEG. So, the preparation of polyclonal antibody of Cp and its immunological determine were studied. The antiserum of Cp was obtained by using inactivated Cp and by hypodermic injections combining with intravenous injection. The antiserum titre of Cp was 10⁶ by ELISA. While the optimum dilution of antigen and antibody were determined by ELISA, the optimum dilutions of antigen and antiserum were 1:400 and 1:500, respectively.Two types of mPEG derivative, which had been activated by cyanuric chloride, just mPEG₁ and mPEG₂ were prepared, which owned different modification effects. The comparison results on modification effect of mPEG₁ and mPEG₂ showed that the affinity and the catalytic activity of the mPEG₂-modified Cp for macromolecular substrate was reduced. Then, the activated mPEG₁ was chosen for optimum modifier.The best conditions for chemical modification with activated mPEGi were as...