【摘要】 加热处理后的沙冬青提取液依次经过DEAE-cellulose A52层析,Sephacryl S300层析,高效液相Poros20HP2层析和高效液相Source 15Q层析,分离纯化到抗冻蛋白amAFP28.amAFP28能修饰冰晶形态.在SDS-PAGE和IEF中,纯化的amAFP28显示单一蛋白染色带,其分子量约为28 kDa,等电点是6.68.经Schiff试剂检验,amAFP28不含碳水化合物.氨基酸组成分析表明,amAFP28富含亲水性氨基酸.圆二色谱(CD)显示amAFP28的二级结构组成为:α-螺旋17.2%,平行β-折叠和β-转角4.1%,反平行β-折叠39.7%,无规卷曲和γ-转角34.4%,-S-S-和芳香氨基酸4.6%.DTT对amAFP28的二级结构没有影响.更多还原

【Abstract】 The purified amAFP28,a heat-stable antifreeze protein from A.mongolicus,was achieved by using a procedure consisting of a heat treatment step followed by consecutive chromatography including ion-exchange chromatography on DEAE-Cellulose A52,molecular exclusion chromatography on Sephacryl S300,hydrophobic chromatography on Poros20HP2,and followed by ion-exchange chromatography on Source 15Q.The molecular mass of amAFP28 as estimated by SDS-PAGE was 28 kDa and its isoelectric point(pI) was 6.68 by IEF analysis.The purified amAFP28 could modify the morphology of ice crystals.amAFP28 did not contain carbohydrate.The result of amino acid composition analysis showed that the AFP was relatively enriched in the hydrophilic amino acid residues.The reduction of the disulfide bonds did not influence the secondary structure of amAFP28.A well-defined secondary structure of 17.2% α-helix,4.1% parallel β-pleated sheet and β-turn,39.7% antiparallel β-pleated sheet,34.4% random coil and γ-turn,and 4.6% disulphide and aromatic amino acid is deduced from circular dichroism(CD) of AFP spectral data.更多还原