Nowak T P, Barondes S H
Biochim Biophys Acta. 1975 May 30;393(1):115-23.
We have purified an agglutinin from the hemolymph of Limulus polyphemus about 1500-3000-fold by adsorption to formalinized horse erythrocytes, elution with N-acetylneuraminic acid and subsequent fractionation on Sephadex G-200. Recovery was in the range of 50 percent. On ultracentrifugation the agglutinin behaves as an homogenous protein with a molecular weight of about 460 000. On polyacrylamide gel electrophoresis of the dissociated protein in sodium dodecylsulfate we found a single prominent diffuse band with an apparent molecular weight of 22 000 plus or minus 2000. This band contained carbohydrate as determined by periodic acid-Schiff staining. The intensity of staining compared with standards suggested a carbohydrate content of less than 4 percent. The protein contains a preponderance of acidic amino acids and has an isoelectric point of 4.83.5 residues per 1000 of glucosamine were detected on amino acid analysis. Agglutination of formalinized horse erythrocytes by the purified protein is inhibited not only by N-acetylneuraminic acid but also by D-glucuronic acid; but not by a number of other monosaccharides. D-Glucuronic acid may be used in place of N-acetylneuraminic acid as the eluting sugar in the purification procedure.
我们通过用甲醛处理的马红细胞吸附、用N-乙酰神经氨酸洗脱以及随后在Sephadex G-200上进行分级分离，从美洲鲎的血淋巴中纯化出一种凝集素，纯化倍数约为1500 - 3000倍。回收率在50%的范围内。超速离心时，该凝集素表现为一种分子量约为460000的均一蛋白质。在十二烷基硫酸钠中对解离的蛋白质进行聚丙烯酰胺凝胶电泳时，我们发现了一条单一的明显弥散带，其表观分子量为22000±2000。经高碘酸 - 席夫染色测定，这条带含有碳水化合物。与标准品相比，染色强度表明碳水化合物含量低于4%。该蛋白质富含酸性氨基酸，其等电点为4.8。氨基酸分析检测到每1000个氨基葡萄糖中有3.5个残基。纯化后的蛋白质对甲醛处理的马红细胞的凝集作用不仅受到N-乙酰神经氨酸的抑制，还受到D-葡萄糖醛酸的抑制；但不受其他多种单糖的抑制。在纯化过程中，D-葡萄糖醛酸可以代替N-乙酰神经氨酸作为洗脱糖。
