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本文以凤眼莲为模式植物,研究植物重金属螯合肽的分离纯化及其快速鉴定。凤眼莲放养在含2μg/ml Cd~(2+)的水中诱导13天。组织抽提液在低盐浓度下(0.01mol/L PBS)经Sephadex G-50柱层析,得到一个含Cd的巯基峰,凝胶过滤HPLC表观分子量为10000。将此巯基峰用过甲酸氧化,凝胶过滤HPLC表观分子量降至1300以下,经反相HPLC分析只有一个肽峰。G-50 Cd-巯基峰与反相HPLC肽峰的氨基酸组成相同,均为(Glu/Gln):Cys:Gly=2:2:1。依据重金属螯合肽的通式(γ-Glu-Cys)_n-Gly,可知n=2。过甲酸氧化后,反相HPLC和氨基酸组成偶联分析是鉴定是否为重金属结合肽以及其n值的快速有效的方法。
In this paper, Eichhornia crassipes as a model plant to study the separation and purification of plant heavy metal chelating peptides and its rapid identification. Eichhornia crassipes was induced in water containing 2 μg / ml Cd 2+ for 13 days. Tissue extracts were subjected to Sephadex G-50 column chromatography at a low salt concentration (0.01 mol / L PBS) to give a thiol-containing peak. The apparent molecular weight of the gel filtration HPLC was 10,000. The thiol peak was oxidized by formic acid and the apparent molecular weight of the gel filtration HPLC was reduced to below 1300 and only one peptide peak was analyzed by reverse phase HPLC. The amino acid composition of the G-50 Cd-thiol peak is the same as that of the reverse-phase HPLC peptide peak and both are (Glu / Gln): Cys: Gly = 2: 2: 1. Based on the formula (γ-Glu-Cys) _n-Gly of the heavy metal chelating peptide, it was found that n = 2. Coupling with reverse phase HPLC and amino acid composition after over-oxidation of formic acid is a fast and efficient method to identify whether it is a heavy metal-binding peptide and its n-value.