Chromatofocusing profile of purified human alpha-fetoprotein and albumin differs from those of crude samples: effect of protein concentration of the elution of the sample.
Chromatofocusing profile of purified human alpha-fetoprotein and albumin differs from those of crude samples: effect of protein concentration of the elution of the sample.
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纯化的人甲胎蛋白和白蛋白的色谱聚焦曲线与粗样品的色谱聚焦曲线不同：样品洗脱的蛋白质浓度的影响。
DOI：
10.1016/0003-2697(91)90557-a
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发表时间：
1991
影响因子：
2.9
通讯作者：
Keel,BA
中科院分区：
文献类型：
--
作者：
Leal,JA;Eddy,KB;Keel,BA
利用色谱聚焦来表征纯化的人甲胎蛋白 (AFP) 和人血清白蛋白 (HSA) 的电荷微异质性。粗脐带血样本产生三种亚型：AFP-IA、IB 和 II，pIs 分别为 4,57 (52%)、4,27 (43%) 和 <4,00 (5%)。相比之下，10 μg 纯化的 AFP 或 250,000 cpm 的 125 I-AFP 完全作为亚型 AFP-II 洗脱。125 I-AFP 在粗制脐带血、羊水、成年男性血清或 25 mg 纯化 HSA 存在下聚焦，导致与粗制脐带血相似的洗脱曲线。纯 AFP 与 0.1、1.0、5.0 或 10 mg HSA 一起聚焦显示从 AFP-II 逐渐转变为 AFP-I。当 HSA ≥5 mg 时，亚型 I 进一步分解为 AFP-IA 和 IB。类似地，250,000 cpm 的 125 I-HSA（也完全以异构体 II 的形式洗脱）显示，当添加浓度增加的未标记 HSA 时，逐渐转变为异构体 I。当未标记 HSA ≥5 mg 时，HSA-IA、IB 和 IC 中异构体 HSA-I 的分辨率再次得到改善。当不同 pI 值的载体蛋白与纯化的 AFP 一起进行色谱聚焦时，观察到只有那些 pI 在 AFP 范围内的蛋白才会引起 AFP 相对分布的显着改变。我们得出的结论是，当将色谱聚焦用于纯化样品以及比较不同来源和不同蛋白质浓度的样品的洗脱曲线时，必须仔细考虑样品蛋白质浓度和组成。
Chromatofocusing was utilized to characterize charge microheterogeneity of purified human α-fetoprotein (AFP) and human serum albumin (HSA). Crude cord blood samples yielded three isoforms: AFP-IA, IB, and II, with pIs 4,57 (52%), 4,27 (43%), and <4,00 (5%), respectively. In contrast, 10 μg of purified AFP or 250,000 cpm of125I-AFP eluted entirely as isoform AFP-II.125I-AFP focused in the presence of crude cord blood, amniotic fluid, adult male serum, or 25 mg purified HSA resulted in elution profiles similar to those of crude cord blood. Pure AFP focused along with 0.1, 1.0, 5.0, or 10 mg HSA showed a gradual shift from AFP-II to AFP-I. With ≥5 mg HSA, isoform I was further resolve into AFP-IA and IB. Similarly, 250,000 cpm of125I-HSA, which also eluted entirely as isoform II, showed a gradual shift to isoform I when increasing concentrations of unlabeled HSA were added. The resolution of isoform HSA-I in HSA-IA, IB, and IC was again improved with ≥5 mg unlabeled HSA. When carrier proteins of varying pI values were chromatofocused along with purified AFP, it was observed that only those proteins withpIs in the range of AFP caused significant alteration in the relative distribution of AFP. We conclude that sample protein concentration and composition must be carefully considered when chromatofocusing is being used for purified samples and when the elution profiles of samples from different origins and varying protein concentrations are being compared.