Determination of the sialylation pattern of human fibrinogen glycopeptides with fast atom bombardment.
Determination of the sialylation pattern of human fibrinogen glycopeptides with fast atom bombardment.
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用快原子轰击测定人纤维蛋白原糖肽的唾液酸化模式。
DOI：
10.1021/bi00321a016
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发表时间：
1984
期刊：
影响因子：
2.9
通讯作者：
Lee,YC
中科院分区：
文献类型：
--
作者：
Townsend,RR;Heller,DN;Fenselau,CC;Lee,YC
材料与方法糖肽制备。先前表征的纤维蛋白原糖肽（汤森等人，1982）进行了分析，没有化学修饰（表I和II）。为了获得仅含Asn的糖肽用于标准混合物分析（图1和2），将上述纤维蛋白原糖肽（50 µ）用链霉蛋白酶（5 mg）（Cal-biochem-Behring）在0. 05 M 4-（2-羟乙基）-1-哌嗪乙磺酸缓冲液，pH 7.5（消化缓冲液）（10 mL），含2 mM氯化钙。在无菌条件下，在37 ℃下继续消化5天，每天加入相同剂量的链霉蛋白酶。Sephadex G-50（2.5 × 200 cm）层析后，将材料溶解于含有1 mM氯化镁和5.5单位氨肽酶M（Boehringer-Mannheim）的消化缓冲液中。使消化在37 ℃下进行24小时;然后再次加入酶并继续孵育24小时.糖肽经Sephadex G-50柱层析纯化后，用40 U羧肽酶Y（Boehringer-Mannheim）在10 mL醋酸缓冲液（pH5.0）中消化24 h。再次加入羧肽酶，将pH升至6.0，然后再温育24小时。由于在此阶段糖肽的氨基酸分析显示存在Glu，因此用氨基肽酶进行进一步消化（48 h），导致90%的糖肽仅含Asn。单唾液双触角的
Materials and MethodsGlycopeptide Preparation. The fibrinogen glycopeptides previously characterized (Townsend et al., 1982) were analyzed without chemical modification (Tables I and II). To obtain glycopeptides with only Asn for standard mixture analysis (Figures 1 and 2), the above fibrinogen glycopeptides (50 µ) were further digested with Pronase (5 mg)(Cal-biochem-Behring) in0. 05 M 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer, pH 7.5 (digest buffer)(10 mL), containing 2 mM calciumchloride. The digestion was continued for 5 days at 37 C under sterile conditions with daily additions of the same dose of Pronase. After Sephadex G-50 (2.5 X 200 cm) chromatography, the material was dissolved in the digest buffer containing 1 mM magnesium chloride and 5.5 units of aminopeptidase M (Boehringer-Mannheim). The digestion was allowed to proceed for 24 h at 37 C; then another addition of enzyme was made and the incubation was continued for 24 h. The glycopeptides were purified by Sephadex G-50 chromatography and further di-gested with 40 units of carboxypeptidase Y (Boehringer-Mannheim) in acetate buffer (10 mL, pH 5.0) for 24 h. Another addition of carboxypeptidase was made, and the pH was raised to 6.0, followed by incubation for another 24 h. Since amino acid analysis of the glycopeptides at this stage revealed the presence of Glu, further digestion with amino-peptidase (48 h) was performed, which resulted in 90% of the glycopeptides containing only Asn. Monosialobiantennary