Thermodynamic stability of the asymmetric doubly-ligated hemoglobin tetramer (alpha+CNbeta+CN)(alphabeta): methodological and mechanistic issues.
Thermodynamic stability of the asymmetric doubly-ligated hemoglobin tetramer (alpha+CNbeta+CN)(alphabeta): methodological and mechanistic issues.
复制标题
不对称双结扎血红蛋白四聚体（α CNbeta CN）（αβ）的热力学稳定性：方法学和机制问题。
DOI：
10.1021/bi971382x
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Huang,Y
中科院分区：
文献类型：
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作者：
Ackers,GK;Perrella,M;Holt,JM;Denisov,I;Huang,Y
自由能对人血红蛋白（Hb）的八种连接中间体的协同性的贡献已经使用六种氧合类似物进行了广泛的表征[cf. Huang等人（1996）Biophys. J. 71，2094 - 2105，表2]。这些前所未有的数据库有力地支持了Hb协同作用的分子密码机制[Ackers et al.（1992）Science 255，54−83]。目前的研究解决了最近反对这项工作的论点[Shibayama et al.（1997）Biochemistry 36，4375−4381]，该论点基于CN−met类似物的双连接物种的“自由能”测定。Shibayama等人（1997）声称，在用于测定不对称“物种[21]”四聚体自由能的杂交实验中，据称在与脱氧Hb孵育期间，一部分结合的氰化物从CN−met Hb中释放出来，用于实现杂交平衡。这些作者声称，氰化物的释放导致了大量的电子交换血红素网站的杂交样品，导致不正确的评价的平衡物种人口的低温技术已被采用。在本报告中，我们证明了我们两个实验室广泛使用的用于这些平衡测定的方法既没有发生明显的氰化物损失，也没有发生电子交换[Perrella等人（1990）Biophys. Chem. 35，97 - 103; Daugherty等人（1991）Proc. Natl. Acad. Sci. U.S.A. 88，1110 - 1114]。Shibayama等人（1997）进行的另一个实验是为了说明他们的主张，该实验没有评估物种[21]杂交的热力学平衡性质。因此，它们新估计的“自由能”的相关性尚不清楚。尽管如此，Shibayama等人（1997）声称他们提出的“自由能”（比我们两个实验室独立发现的-11.4千卡的自由能更正1.3千卡）使血红蛋白协同性的分子密码机制失效。这种表述是完全没有根据的，因为比Shibayama等人（1997）提出的更正的自由能将完全符合分子密码机制。
Free energy contributions to cooperativity by the eight ligation intermediates of human hemoglobin (Hb) have been characterized extensively using six oxygenation analogs [cf. Huang et al. (1996)Biophys. J. 71, 2094−2105, Table 2]. These unprecedented data bases have strongly supported themolecular code mechanismof Hb cooperativity [Ackers et al. (1992)Science 255, 54−83]. The present study addresses a recent argument against this work [Shibayama et al. (1997)Biochemistry 36, 4375−4381] based on “free energy” determinations for a doubly-ligated species of the CN−met analog. Shibayama et al. (1997) have claimed that, in the hybridization experiments that have been used to determine free energy of the asymmetric “species [21]” tetramer, a portion of the bound cyanide is allegedly released from CN−met Hb during the incubation with deoxy Hb that is used to achieve hybrid equilibrium. These authors have claimed that cyanide release has resulted in extensive electron exchange between heme sites of the hybridizing sample, leading to incorrect evaluation of the equilibrium species population by the cryogenic techniques that have been employed. In this report, we demonstrate that neither appreciable cyanide loss nor electron exchange occurs with the methods that have been used extensively by our two laboratories for these equilibrium determinations [Perrella et al. (1990)Biophys. Chem. 35, 97−103; Daugherty et al. (1991)Proc. Natl. Acad. Sci. U.S.A. 88, 1110−1114]. An alternative experiment, which Shibayama et al. (1997) have carried out to illustrate their claim, does not evaluate a thermodynamic equilibrium property of the species [21] hybrid. The relevance of their newly-estimated “free energy” is therefore unclear. Nevertheless, Shibayama et al. (1997) have claimed that their proposed “free energy” (which is ∼1.3 kcal more positive than the free energy of −11.4 kcal found independently by our two laboratories) renders invalid themolecular code mechanismof hemoglobin cooperativity. This representation is utterly without foundation since a free energy even more positive than suggested by Shibayama et al. (1997) would be fully consistent with themolecular code mechanism.