Synthesis and application of sorbents for affinity chromatography of serine proteases

Кузнецова, А. В.; Bogacheva, Anna M.; Rudenskaya, G. N.; Stepanov, V. M.

doi:10.1007/bf02505535

Cited by 4 publications

(1 citation statement)

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“…Affinity chromatography was used for further separation of the enzyme from pigments and other admixtures. A sorbent that contained Ala‐Ala‐Leu‐mrp attached via p ‐benzoquinone to AH‐Sepharose (Sepharose‐AAL‐mrp) [4] as a ligand has proved its efficiency. The proteinase from dandelion roots possessed high affinity to this sorbent, and no loss of enzyme was observed during chromatography.…”

Section: Resultsmentioning

confidence: 99%

Taraxalisin – a serine proteinase from dandelion Taraxacum officinale Webb s.l

et al. 1998

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Latex of dandelion roots contains a serine proteinase that hydrolyzes a chromogenic peptide substrate Glp-Ala-AlaLeu-pNA optimally at pH 8.0. Maximal activity of the proteinase in the roots is attained in April, at the beginning of plant development after the winter period. The protease was isolated by ammonium sulfate precipitation of the root extract followed by affinity chromatography on a Sepharose-Ala-AlaLeu-mrp and gel filtration on Superose 6R performed in FPLC regime. Pure serine proteinase named taraxalisin was inactivated by specific inhibitors of serine proteinases, diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF). Its molecular mass is 67 kDa and pI 4.5. pH stability range is 6^9 in the presence of 2 mM Ca P+ , temperature optimum is at 40³C; K m =0.37 þ 0.06 mM. The substrate specificity of taraxalisin towards synthetic peptides and insulin B-chain is comparable with that of two other subtilisin-like serine proteinases, cucumisin and macluralisin. The taraxalisin N-terminal sequence traced for 15 residues revealed 40% coinciding residues when aligned with that of subtilisin Carlsberg.z 1998 Federation of European Biochemical Societies.

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Section: Resultsmentioning

confidence: 99%