Of Peg-hirudin And Its Activity Was Detected

In this paper, traditional analytical methods, such as 2,4,6-trinitrobenzenesulfonic acid(TNBS) spectrometry and sodium dodecyl sulfate-polyacrylamine gel electrophoresis (SDS-PAGE), were improved and used to optimize the conditions of reaction between activated mPEG and hirudin. The product was separated by size exclusion chromatography (SEC) and then measured the bioactivity. At last, the modification of hirudin by oxidized soluble starch and heparin was explored. The analytical results revealed that TNBS spectrometry could be accurate only if the reaction between hirudin and TNBS was stopped in appropriate time. In addition, the staining of mPEG with BaCla and la is more specific and sensitive than the Coomassie Brilliant Blue staining. The optimal reaction conditions were determined by the improved analytical methods: The reaction should be stopped in 10 hours in the 0.2mol/L borate buffer at pH8.5 and 4癈. Under such conditions, the modification extent of amino groups is 65% and an average molecular weight of PEGylated hirudin is 17000. The PEGylated hirudin was separated by ultrafiltration, Sephadex G50 and Superdex 75 from a mixture of unmodified hirudin and mPEG-hirudin. The exploration about the modification of hirudin by polysaccharides, such as starch and heparin, was made in phosphate buffer (pH7.4, O.lmol/L). It demonstrated that the amino groups of hirudin were linked covalently and specifically to aldehyde groups of activated polysaccharide, which were produced by the oxidation of NaIC>4. Moreover, the bioactivity of modified hirudin was measured. It was retained only 25% compared to unmodified hirudin, partly because the covalent site (Lys47) is the active site of hirudin.