EXCHANGE OF HEME AMONG HEMOGLOBIN MOLECULES
EXCHANGE OF HEME AMONG HEMOGLOBIN MOLECULES
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DOI：
10.1073/pnas.56.3.974
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发表时间：
1966-01-01
影响因子：
11.1
通讯作者：
JANDL, JH
中科院分区：
文献类型：
--
作者：
BUNN, HF;JANDL, JH
方法.使用两种容易分离的正常人血红蛋白F和A。血红蛋白A是从洗涤红细胞的水溶血产物中制备的，然后用艾伦及其同事的方法通过阳离子交换色谱法纯化。1所用的缓冲液在pH值和离子强度上与他们的”显影剂#2”相同，但不含氰化物。以类似的方式，从正常脐带血的红细胞制备血红蛋白F。将富含网织红细胞的脐带血细胞与结合至血浆转铁蛋白的Fe 59孵育后制备Fe 59血红蛋白F。Fe 59血红蛋白A类似地从网织红细胞水平升高的成人患者的红细胞制备。在平行实验中，C ′ 4 -2-甘氨酸用于标记卟啉和球蛋白部分，C ′ 4-亮氨酸用于单独标记球蛋白。在pH6.8的氧合血红蛋白中加入1.2摩尔铁氰化钾/血红素，然后用孵育缓冲液透析，制备高铁血红蛋白。除非另有说明，标记的血红蛋白F与等量的未标记的血红蛋白A在37 ℃的磷酸盐缓冲液（pH7.18，离子强度0.09）中孵育。发现所述反应可通过浸入冰水中或加入过量的中性氰化物来终止。在用含有0.01 M氰化物的”显色剂#2”冷透析后，通过阳离子交换色谱分离血红蛋白混合物。每一级分的血红蛋白浓度测定为氰化高铁血红蛋白。用井型闪烁计数器测量每个级分的Fe 59活性。C14活性用”低背景”气体流量计数器对小块干燥的血红蛋白、血红素、I和珠蛋白等分试样进行测量。3加入未标记的氰化高铁血红蛋白作为载体后，从每个组分中分离出血红素和球蛋白。“对恢复和自我吸收进行了适当的修正。从每个级分的活性和浓度计算比活性，并表示为cpm/mg血红蛋白。计算相邻血红蛋白富集级分的比活度的加权平均值。如果血红素在血红蛋白分子之间自由交换，那么在平衡时，新标记的血红蛋白的比活度将是最初标记的血红蛋白的初始比活度的50%。新标记的血红蛋白在时间t的比活度除以原始标记的血红蛋白在时间0的比活度的一半的商是血红素交换百分比的量度。因此，如果血红素中标记的血红蛋白F与等量的未标记的血红蛋白A一起孵育，则血红素交换百分比= 2spec.act。A，/spec. act.福
Methods. Two easily separable normal human hemoglobins, F and A, were used. Hemoglobin Awas prepared from a water hemolysate of washed red cells, and then purified by cation-exchange chromatography by the method of Allen and co-workers. 1 The buffer employedwas identical in pH and ionic strength to their" developer# 2," but lacked cyanide. In like manner, hemoglobin F was prepared from red cells of normal umbilical cord blood. Fe59 hemoglobin F was prepared after incubating reticulocyte-rich cord blood cells with Fe59 bound to plasma transferrin. Fe59 hemoglobin A was prepared similarly from the red cells of adult patients with elevated reticulocyte levels. In parallel experiments, C'4-2-glycine was used for labeling both the porphyrin and globin moieties, and C'4-leucine for labeling globin alone. Ferrihemoglobin was prepared bythe addition to oxyhemoglobin, at pH 6.8, of 1.2 moles potassium ferricyanide per heme, followed by dialysis against the incubation buffer. Unless stated otherwise, labeled hemoglobin F was incubated with an equal quantity ofunlabeled hemoglobin A at 370C in phosphate buffer, pH 7.18, ionic strength 0.09. It was found that the reaction to be described could be terminated by immersion in ice water or by adding an excess of neutral cyanide. After dialysis in the cold against" developer# 2" containing 0.01 M cyanide, the hemoglobin mixtures were separated by cation-exchange chromatography.'The hemoglobin concentration of each fraction was determined as cyanmethemoglobin. Fe59 activity of each fraction was measured with a welltype scintillation counter. C14 activity was measured with a" low-background" gas flow counter on planchet-dried aliquots of hemoglobin, heme, I and globin. 3 The heme and globin were isolated from each fraction after the addition of unlabeled cyanmethemoglobin as" carrier." Suitable corrections for recovery and self-absorption were made. The specific activity was calculated from the activity and concentration of each fraction, and was expressed as cpm/mg of hemoglobin. A weighted mean of the specific activities of neighboring hemoglobin-rich fractions was calculated. If hemes exchanged freely among hemoglobin molecules, then at equilibrium, the specific activity of the newly labeled hemoglobin would be 50 per cent of the initial specific activity of the originally labeled hemoglobin. The quotient of the specific activity of the newly labeled hemoglobin, at time t over one half the specific activity of the originally labeled hemoglobin, at time 0, is a measure of the per cent of heme exchange. Thus, if hemoglobin F labeled in the hemes is incubated with an equal amount of unlabeled hemoglobin A, the per cent heme exchange= 2 spec. act. A,/spec. act. Fo.