Kunitz-type glycosylated elastase inhibitor with one disulfide bridge
A glycosylated Bauhinia rufa elastase inhibitor (gBrEI) was purified and characterized using acetone precipitation, affinity chromatography on concanavalin A-Sepharose, ion-exchange chromatography on a HiTrap Q column, size exclusion chromatography on a Superdex 200 column and reverse-phase chromato...
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Published in | Planta medica Vol. 72; no. 5; pp. 393 - 397 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
Germany
01.04.2006
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Subjects | |
Online Access | Get more information |
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Summary: | A glycosylated Bauhinia rufa elastase inhibitor (gBrEI) was purified and characterized using acetone precipitation, affinity chromatography on concanavalin A-Sepharose, ion-exchange chromatography on a HiTrap Q column, size exclusion chromatography on a Superdex 200 column and reverse-phase chromatography on a C18 column. gBrEI inhibited pancreatic porcine elastase with an equilibrium dissociation constant (K(i)) of 6.18 x 10(-8) M, but it did not inhibit human neutrophil elastase, bovine trypsin, human plasma kallikrein or porcine pancreatic kallikrein. On SDS-electrophoresis, gBrEI appeared as a single 20-kDa band, also after reduction. Schiff reagent staining indicated a carbohydrate portion in the protein, which was confirmed by mass spectrometry. The glycosylated site was Asn 38, and a carbohydrate portion of 1.17 kDa was identified. gBrEI was found to contain 144 amino acid residues, and a FASTA database analysis showed that it belongs to the plant Kunitz-type inhibitor family. Val66 was identified as reactive site P1 residue by comparison of conserved positions in the sequences. Since gBrEI harbors a single disulfide bridge, it may be considered a new type of Kunitz inhibitor, intermediate between the classical Kunitz inhibitors, which contain two disulfide bridges, and those from B. bauhinioides, which do not have such bridges. |
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ISSN: | 0032-0943 1439-0221 |
DOI: | 10.1055/s-2005-916237 |