In this study, hirudin, a protease inhibitor from the salivary gland of bloodsucking leech, was purified by immobilized metal affinity chromatography. Divalent metal ions such as copper, nickel, and zinc were used as ligand. Metal ions were immobilized on the gel, sepharose and then chelated with hirudin. The binding strength between metal ion and hirudin was dependent on the secondary structure of the chelation site on hirudin. Several types of hirudin were prepared by gene cloning of yeast, Sacaromyces cerevisiae. Four wild type hirudin were obtained : wild type, MH7, MH9, and MH13. The wild type hirudin has only one histidine residue at 51st site in its amino acid sequence. Hirudin MH7 has two histidine residues at 51st and 48th sites, while MH9 at 51st and 54th sites. MH13 has three histidine residues at 48th, 51st, and 54th sites.
The metal ion affinity of hirudin variants were tested. With copper ion all hirudin variants had affinity, and they were eluted after the pH gradient were started. The wild type hirudin was eluted at pH 5.5, MH7 at pH 4.4, MH9 at pH 5.2, MH13 type at pH 4.0. Thus, it was concluded that MH13 type had the strongest affinity of all hirudin variants. The specific activity of the wild type hirudin is 13,000 ATU/mg. But, those of the others were lower than this ; MH7 : 11,000 ATU/mg; MH9 : 9,700 ATU/mg; MH13 : 4,300 ATU/mg. As more histidines were substituted, the specific activity of the hirudin variants decreased.
The adsorption mechanism of wild type hirudin was studied and a mathematical model. The surface reaction constant was so large that the adsorption reaction at the surface was considered very fast. Since the coefficient for mass transfer through the liquid film was quite small, this step was considered to be the rate-limiting step.
Several purification processes for the wild type and MH7 hirudins were tested. Each process was composed of an ultrafiltration with molecular weight cut-off of 30,000 an ultrafiltration with molecular weight cut-off of 3,000 an anion exchange chromatography and a copper ion metal ion affinity chromatography. Descriptions on the performance of each process has been presented in detail.
