Analysis of the biotin‐binding protein actinavidin using affinity capillary electrophoresis
Analysis of the biotin‐binding protein actinavidin using affinity capillary electrophoresis
复制标题
使用亲和毛细管电泳分析生物素结合蛋白激活素
DOI：
10.1002/elps.1150171024
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发表时间：
1996
期刊：
影响因子：
2.9
通讯作者：
U. Bilitewski
中科院分区：
文献类型：
--
作者：
V. Okun;U. Bilitewski
应用亲和毛细管电泳法(ACE)研究了微生物生物素结合蛋白放线亲和素与生物素配体受体相互作用的生物亲和力。ACE方法是基于短时间孵育放线亲和素的混合物和增加生物素化寡核苷酸(BIO-ON)的浓度，BIO-ON被发现是一种有效的亲和配体。在胶束相存在下，通过毛细管区带电泳法将中间负载形式的放线菌素从游离型配体中分离出来，从而能够定量测定游离态的生物素，从而可以在没有和存在十二烷基硫酸钠(SDS)的情况下评价放线菌素与生物素的结合能力。尽管在后一种情况下，放线菌素失去了部分结合能力(不超过12%)，但仍有可能利用胶束电动毛细管色谱(MEKC)和血管紧张素转换酶(ACE)相结合的方法，建立一种间接、非竞争性测定培养液中放线菌素的方法。由于亲和作用，样品中的放线亲和素减少了添加的生物素的量，从而能够对蛋白质进行定量。SDS是本实验中防止蛋白质吸附到毛细管壁所必需的，极大地提高了重复性和峰形。测定的放线菌素水平与通常使用的固相分析结果一致。该方法的检出限为500 ng/mL。因此，该方法非常适合于评价放线亲和素的亲和力并监测其在培养过程中的水平。
Affinity capillary electrophoresis (ACE) was applied to study the bioaffinity of ligand‐receptor interaction between the microbial biotin‐binding protein actinavidin and biotin. The ACE method is based on short time incubation of a mixture of actinavidin and increasing concentrations of biotinylated oligonucleotide (bio‐ON), which was found to be an effective affinity ligand. Separation of intermediate loading forms of actinavidin from unbound ligand in the presence of micellar phase and by capillary zone electrophoresis enabled the quantitation of free bio‐ON, permitting the evaluation of the biotin‐binding capacity of actinavidin in absence and presence of sodium dodecyl sulfate (SDS). Although in the latter case actinavidin lost a part of its binding capacity (not more than 12%), it was still possible to develop an indirect, noncompetitive assay for the determination of actinavidin in culture liquid, utilizing the combination of micellar electrokinetic capillary chromatography (MEKC) and ACE. Due to the affinity inteaction, actinavidin in the sample decreases the amount of bio‐ON added, enabling quantitation of the protein. SDS, which is required in this assay to prevent protein adsorption to the capillary wall, greatly enhances the reproducibility and peak shape. Actinavidin levels determined are in agreement with those obtained by commonly used solid‐phase analysis. The limit of detection was about 500 ng/mL. Thus the proposed method was found to be well suited for the evaluation of actinavidin affinity and monitoring of its levels in cultivation process.
影响因子：
7.4
作者：
SCHULTZ, NM;HUANG, L;KENNEDY, RT
通讯作者：
KENNEDY, RT