Rapid preparation of native alpha and beta chains of human hemoglobin.
Rapid preparation of native alpha and beta chains of human hemoglobin.
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DOI：
10.1016/0020-711x(92)90109-e
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发表时间：
1992-06
期刊：
影响因子：
--
通讯作者：
K. M. Parkhurst;L. Parkhurst
中科院分区：
文献类型：
--
作者：
K. M. Parkhurst;L. Parkhurst
1. 目前分离血红蛋白天然链的方法为每条链使用两个离子交换柱，结果是容易自氧化的链，可测量Hb和Hg的污染。在新程序中，改变第一柱的缓冲条件可将Hb污染从2 - 5%降低到小于1%，即可检测的极限。3. 对α链，用DTT孵育1分钟，对β链，用DTT孵育三次，用凝胶过滤分离，取代第二柱和长时间的巯基乙醇洗涤。汞残留量小于0.1%。4. 在以前的方法中，氧化导致了低收率和不可靠的结合汞的光谱评估。新方法可以对无汞链进行简单的紫外分析。5. 由这些氧链重组的血红蛋白在氧结合平衡和激光光解后CO结合动力学方面与天然血红蛋白相同。
1. Current procedures for the isolation of native chains of hemoglobin employ two ion exchange columns for each chain and result in readily autoxidizable chains with measurable contamination by Hb and Hg. 2. In the new procedure, altered buffer conditions on the first column reduce Hb contamination from 2 to 5% to less than 1%, the limit of detectability. 3. The second column and lengthy washes with beta mercaptoethanol are replaced by incubation with DTT for 1 min for alpha chains and, for beta chains, three incubations with DTT and separations by gel-filtration. The residual Hg is less than 0.1%. 4. Oxidations in the previous procedure resulted in low yields and unreliable spectroscopic assessments of bound Hg. The new procedure resulted in a simple UV assay for Hg-free chains. 5. Hemoglobin reconstituted from these oxy-chains was identical to native Hb in oxygen binding equilibria and in the kinetics of CO binding following laser photolysis.