Conformational studies on the beta subunits of human hemoglobin and their arginyl-COOH peptides.
Conformational studies on the beta subunits of human hemoglobin and their arginyl-COOH peptides.
复制标题
人血红蛋白β亚基及其精氨酰-COOH肽的构象研究。
DOI：
10.1021/bi00691a017
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发表时间：
1975
期刊：
影响因子：
2.9
通讯作者：
E. Bucci
中科院分区：
文献类型：
--
作者：
C. Bucci;E. Bucci
血红蛋白的β亚基在半胱氨酰残基与碘乙酰胺烷基化后显示出S20 w的沉降速度，接近单体亚基的1.8。它们与α链反应，产生沉降常数接近4.4的四聚体血红蛋白。它们的CD光谱在270 nm以下与未处理的β链的CD光谱无法区分，否则它们显示出在Soret区域变得明显的一些偏差，其中烷基化亚基的光学活性肯定低于天然亚基的光学活性。去除血红素后，脱辅基β亚基在光谱的远紫外区显示出降低的光学活性，表明螺旋含量的大量损失。它们的沉降行为与大聚集体的存在一致，大聚集体在用氰血红素重构时解离成单体。脱辅基-β亚基可以用6 M盐酸胍复性。它们与血红素以1：1的摩尔比进行化学计量反应。在与血红素重组后，它们的光学活性变得与光谱的远紫外区域中的天然β链相似，但在近紫外和Soret区域中保持较低。用柠康酸酐酰化赖氨酰残基后，用胰蛋白酶消化apo-β亚基，并通过凝胶色谱法分离脱乙酰基-COOH肽β（1-30）、β（31-40）、β（41-104）和β（105-146）。除了在中性pH下不溶的肽β/105-146）之外，其它肽的沉降行为显示存在小聚合物。未测试肽β（31-40）的沉降行为。测量各种蛋白质和肽的α螺旋、β构象和无规卷曲（或无序结构）的百分比，用Y.H. Chen等人（（1974），Biochemistry 13，3350）和N.格林菲尔德和动力局Fasman（（1969），Biochemistry 8，4108）.以这种方式，天然和重构的烷基化β亚基的螺旋含量似乎接近76%，该值非常接近血红蛋白晶体中相同亚基中存在的螺旋含量。在pH 9.6的0.04 M硼酸盐缓冲液中，apo-β亚基的螺旋含量降低至接近45%的值。在电解质溶液中分离的肽的螺旋含量在任何情况下都接近10%，表明它们在血红蛋白晶体中所具有的结构几乎完全丧失。氰基血红素与肽β（41-104）反应，然而，反应不是化学计量的，表明血红素对肽的低亲和力。除肽β（31-104）外，所有其他肽在溶解于50%甲醇中时恢复了它们的一些螺旋结构。值得注意的是，脱辅基-β亚基也这样做，这表明在去除血红素时结构的损失可能部分是由于血红素口袋暴露于水。
The beta subunits of hemoglobin upon alkylation of the cysteinyl residues with iodoacetamide showed a sedimentation velocity with an S20w, near 1.8 as for monomeric subunits. They reacted with alpha chains to give a tetrameric hemoglobin with a sedimentation constant near 4.4. Their CD spectrum was indistinguishable from that of untreated beta chains below 270 nm, otherwise they showed some deviation that became pronounced in the Soret region, where the optical activity of the alkylated subunits was definitely lower than that of the native subunits. Upon removal of the heme the apo-beta subunits showed a decreased optical activity in the far-uv region of the spectrum indicating a substantial loss of helical content. Their sedimentation behavior was consistent with the presence of large aggregates, which dissociates into monomers upon reconstitution with cyanoheme. The apo-beta subunits could be renatured from 6 M guanidine hydrochloride. They showed a stoichiometric reaction with heme in the molar ratio 1:1. Upon reconstitution with the heme their optical activity became similar to that of the native beta chains in the far-uv region of the spectrum, but remained lower in the near-uv and Soret regions. After acylation of the lysyl residues with citraconic anhydride the apo-beta subunits were digested with trypsin and the arginyl-COOH peptides beta(1-30), beta(31-40), beta(41-104), and beta(105-146) were separated by gel chromatography. With the exception of the peptide beta/105-146), which was insoluble at neutral pH, the sedimentation behavior of the other peptides showed the presence of small polymers. The sedimentation behavior of the peptide beta(31-40) was not tested. The percentage of alpha helix, beta conformation, and of random coil (or unordered structure) of the various proteins and peptides was measured fitting their CD spectra in the far-uv region with the parameter published by Y.H. Chen et al. ((1974), Biochemistry 13, 3350) and by N. Greenfield and G.D. Fasman ((1969), Biochemistry 8, 4108). In this way the helical content of the native and reconstituted alkylated beta subunits appeared to be near 76%, a value very near to that present in the same subunits in the hemoglobin crystal. The helical content of the apo-beta subunits in 0.04 M borate buffer at pH 9.6 decreased to a value near 45%. The helical content of the isolated peptides in electrolyte solutions was in any case near 10% indicating an almost complete loss of the structure that they have in the hemoglobin crystal. Cyanoheme reacted with the peptide beta(41-104), however, the reaction was not stoichiometric indicating a low affinity of the heme for the peptide. With the exception of the peptide beta(31-104), all of the other peptides recovered some of their helical structure when dissolved in 50% methanol. Notably also the apo-beta subunits did so suggesting that the loss of structure upon the removal of the heme could be in part due to the exposure of the heme pocket to water.