Reversed-phase high-performance liquid chromatographic separation of bovine kappa-casein macropeptide and characterization of isolated fractions
Reversed-phase high-performance liquid chromatographic separation of bovine kappa-casein macropeptide and characterization of isolated fractions
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DOI：
10.1016/0021-9673(96)00122-7
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发表时间：
1996-08-30
影响因子：
4.1
通讯作者：
Visser, S
中科院分区：
文献类型：
--
作者：
Minkiewicz, P;Slangen, CJ;Visser, S
从非糖基化和不同糖基化酪蛋白巨肽（CMP；κ-酪蛋白片段 106-169；M(r) 大约 7000）的复杂混合物中分离出各种级分，并通过反相 HPLC 进一步纯化。通过质量测定和成分分析对馏分进行表征，并用于凝胶渗透色谱和核磁共振研究，以研究其分子大小行为与 pH、离子强度、肽浓度和糖基化程度的函数关系。没有发现任何证据表明任何 CMP 分数与所应用的实验条件有关，这与文献中提出的建议相反。增加的分子尺寸（表观分子质量约 30-45 kDa）可以通过由于内部静电和空间排斥而产生的分子种类较大来解释。此外，研究了一些非糖基化和糖基化 CMP 级分对来自金黄色葡萄球菌 V8 的 Glu 特异性内肽酶的酶攻击的敏感性，该酶的初始蛋白水解速率与糖基化。只有在高度糖基化的 CMP 的情况下，进一步水解成更小的片段才受到抑制。通过电喷雾电离和快原子轰击质谱法鉴定水解产物。
From complex mixtures of non-glycosylated and differently glycosylated caseinomacropeptides (CMP; kappa-casein fragment 106-169; M(r) approximate to 7000) various fractions were isolated and further purified by reversed-phase HPLC. The fractions were characterized by mass determination and composition analysis and also used in gel-permeation chromatography and NMR studies to investigate their molecular size behaviour as a function of pH, ionic strength, peptide concentration and degree of glycosylation. No evidence was found for association of any CMP fraction as a function of the experimental conditions applied, which is in contrast with suggestions made in the literature. The increased molecular size (apparent molecular mass approx. 30-45 kDa) is rather explained by a large voluminosity of the molecular species due to internal electrostatic and steric repulsion, Furthermore, the susceptibility of some non-glycosylated and glycosylated CMP fractions to enzymic attack by the Glu-specific endopeptidase from Staphylococcus aureus V8 was studied, Initial rates of proteolysis by this enzyme were independent of the degree of glycosylation. Only in the case of highly glycosylated CMP was further hydrolysis to smaller fragments inhibited. Hydrolytic products were identified by electrospray ionization and fast-atom bombardment mass spectrometry.