Conchie J, Hay A J
Carbohydr Res. 1983 Feb 1;112(2):261-79. doi: 10.1016/0008-6215(83)88291-3.
An ovomucoid variant free from sialic acid has been prepared in a pure state by ion-exchange chromatography on DEAE-cellulose. The purified glycoprotein contained 10-11 residues of mannose, 2-3 residues of galactose, and 21 residues of 2-acetamido-2-deoxyglucose. Glycopeptides have been prepared by exhaustive digestion with Pronase followed by ion-exchange chromatography on Dowex 50 (X2) resin. Three fractions were obtained, all with similar contents of mannose and hexosamine but with various contents of galactose. The sugar-aspartic acid ratios indicated that all of the fractions were heterogeneous, the major fraction having mannose-galactose-hexosamine-aspartic acid ratios of 2.6:0.5:5.8:1.0. Cleavage of asialo-ovomucoid with cyanogen bromide and proteolytic digestion of the isolated fragments gave two heterogeneous glycopeptide fractions of similar composition. Both asialo-ovomucoid and the principal glycopeptide fraction were degraded with beta-D-galactosidase, alpha-D-mannosidase, and beta-N-acetylglucosaminidase singly and in sequence. Removal of much of the carbohydrate from asialo-ovomucoid had no appreciable effect on its anti-tryptic activity. By sequential degradation of the glycopeptide, a pentasaccharide core alpha-D-Man-[alpha-D-Man]-beta-D-Man-beta-D-GlcNAc-beta-D-GlcNAc-Asn was obtained. Other structural features revealed by enzymic degradation are discussed.
通过在二乙氨基乙基纤维素上进行离子交换色谱法，已制备出一种不含唾液酸的卵类黏蛋白变体，并达到了纯态。纯化后的糖蛋白含有10 - 11个甘露糖残基、2 - 3个半乳糖残基以及21个2 - 乙酰氨基 - 2 - 脱氧葡萄糖残基。通过用链霉蛋白酶彻底消化，随后在Dowex 50（X2）树脂上进行离子交换色谱法，制备出了糖肽。得到了三个组分，它们的甘露糖和己糖胺含量相似，但半乳糖含量各不相同。糖与天冬氨酸的比例表明，所有组分都是异质的，主要组分的甘露糖 - 半乳糖 - 己糖胺 - 天冬氨酸比例为2.6:0.5:5.8:1.0。用溴化氰裂解去唾液酸卵类黏蛋白，并对分离出的片段进行蛋白水解消化，得到了两个组成相似的异质糖肽组分。去唾液酸卵类黏蛋白和主要糖肽组分分别依次用β - D - 半乳糖苷酶、α - D - 甘露糖苷酶和β - N - 乙酰氨基葡萄糖苷酶进行降解。从去唾液酸卵类黏蛋白中去除大部分碳水化合物对其抗胰蛋白酶活性没有明显影响。通过对糖肽的顺序降解，得到了一个五糖核心α - D - 甘露糖 - [α - D - 甘露糖] - β - D - 甘露糖 - β - D - N - 乙酰葡糖胺 - β - D - N - 乙酰葡糖胺 - 天冬酰胺。文中还讨论了酶促降解揭示的其他结构特征。
