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采用丙酮分级沉淀、Superdex 75层析和Blue Sepharose CL-6B层析,从苦瓜籽中获得核糖体失活蛋白MAP30。纯化的MAP30经PAGE、SDS-PAGE、IEF和Periodate-Schiff分析均为单一蛋白着色带或单一糖蛋白着色带,根据SDS-PAGE计算其相对分子量为30kDa,经IEF-PAGE计算其pI为9.0,采用硫酸-苯酚法测得其含糖量为1.05%。N端测序结果为Asp-Val-Asn-Phe-Asp-LeuSer-。光谱学研究表明:最大紫外吸收峰为280nm,以278nm波长光激发时,该蛋白具有304nm荧光发射峰和316nm副峰,以295nm波长光激发时,该蛋白的最大荧光发射峰在317nm。体外试验表明其对小鼠黑色素细胞瘤B16细胞具有明显抑制作用,而对完整细胞毒性极小。
Ribosome inactivation protein MAP30 was obtained from bitter gourd seeds using acetone fractionation, Superdex 75 chromatography and Blue Sepharose CL-6B chromatography. Purified MAP30 was a single protein band or a single glycoprotein band by PAGE, SDS-PAGE, IEF and Periodate-Schiff analysis. The relative molecular weight was 30 kDa based on SDS-PAGE and the pI was 9.0 by IEF-PAGE. Using sulfuric acid - phenol method measured the sugar content of 1.05%. N-terminal sequencing results were Asp-Val-Asn-Phe-Asp-LeuSer-. Spectroscopic studies show that the maximal UV absorption peak is 280 nm. When excited with 278 nm light, the protein has 304 nm fluorescence emission peak and 316 nm secondary peak. When excited with 295 nm light, the maximum fluorescence emission peak of this protein is at 317 nm. In vitro tests show that it has a significant inhibitory effect on mouse melanoblastoma B16 cells, and minimal cytotoxicity to intact cells.