碩士 / 國立臺灣海洋大學 / 水產養殖學系 / 96 / A protease inhibitor, α2 macroglobulin（α2 M）, was purified from cobia （Rachycentron canadum）plasma by a four-step procedure：poly ethylene glycol fractionation, concanavalin A sepharoseTM 4B chromatography, hydrophobic interaction chromatography and ion exchange chromatography on Fast Protein liquid chromatography system. It migrated as one band and its molecular mass was about 360 kDa in the native state, whereas in SDS PAGE it was about 180 kDa under nonreducing condition. This result revealed that the native protein was a dimer. In addition, it was cleaved into two different fragments of molecular mass about 93 and 87 kDa when reduced by dithiothreitol（DTT）. The α2 M was a glycoprotein as stained a Glycoprotein Staining kit. It was a purified protein as further checked Western blot and crossed immuno- electrophoresis（CIE）.
The protease inhibitory activities of cobia plasma or purified α2 M were sensitive to the addition of 0.2 M methylamine for 30 min. When incubated with methylamine, α2 M could not bind to other proteases. The purified α2 M（0.07 mg protein／ml）exhibited more than 50％ inhibitory activity against Vibrio alginolytius ECP（0.364 mg protein／ml） while it exhibited more than 50％ inhibitory activity against Streptococcus iniae ECP（0.097 mg protein／ml）. It seems that the ECP protease activity may affect the inhibitory activity of the α2 M.
The anti-protease activity of the purified α2 M was apparently decreased as temperature elevated. The α2 M exhibited highest inhibitory activity at pH 9. The results indicate that the α2 M is a heat-labile and alkaline protease inhibitor.
| Identifer | oai:union.ndltd.org:TW/096NTOU5086010 |
| Date | January 2008 |
| Creators | Chia-Yu Hung, 洪佳鈺 |
| Contributors | Kuo-Kau Lee, 李國誥 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 76 |
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