Masked imidazole groups in cyanoferrihemoglobin and carbonylhemoglobin.
Masked imidazole groups in cyanoferrihemoglobin and carbonylhemoglobin.
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氰基铁血红蛋白和羰基血红蛋白中的掩蔽咪唑基团。
DOI：
10.1021/bi00907a005
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发表时间：
1962
期刊：
影响因子：
2.9
通讯作者：
S. Beychok
中科院分区：
文献类型：
--
作者：
J. Steinhardt;R. Ona;S. Beychok
早期的论文已经表明，铁血红蛋白和羰基血红蛋白在0和25的可逆酸变性释放出大约22个酸结合基团（可能是咪唑），这些基团不与天然蛋白质中的酸联合收割机结合。铁血红蛋白的证明依赖于（a）快速流动测量（2-3秒）的pH值在足够高的离子强度的溶液（0.3 M氯化物）抑制天然蛋白质和变性蛋白质之间的酸结合差异，这种差异仅仅是由于它们的带电基团的静电相互作用的变化;和（B）当pH降低时，滴定曲线中的差异不通过明显的窄最大值（如果只涉及静电效应的话），但在很宽的pH范围内保持在几乎恒定的水平。在0.3的离子强度下，该差异在pH 4以下不能维持，这可能是因为在较低的pH值下，部分变性发生在pH测量所需的2至3秒内。在本文件中追索权是滴定氰铁血红蛋白（CN-血红蛋白）和羰基血红蛋白（CO-血红蛋白）-这两者都是更稳定的酸比铁血红蛋白-扩展之间的恒定差异的滴定曲线的天然和变性蛋白质的高离子强度的解决方案，大大降低pH值，从而加强实际解蔽的解释。结果表明，pH恒定差的范围扩大到2个pH单位，并证实了早期的结论，即22个组氨酸中的咪唑基团的基本功能在天然马血红蛋白中被掩蔽，而其他碱性较低的基团，如羧酸盐，是完全反应性的。结果还表明，氰血红蛋白的解蔽率与用荧光光度法测得的这种蛋白质的变性率大致相同。（一氧化碳血红蛋白）（Steinhardt和Zaiser，1951）或高铁血红蛋白（Steinhardt和Zaiser，1953）在pH 4.5和7.5之间滴定的亲质子基团的数量超过了在相应的pH值范围之间滴定的数量约36。反应天然蛋白质。在最早的报告（Steinhardt和Zaiser，1951）中，提出了至少部分这些“掩蔽”基团存在于组氨酸残基中的可能性，但弱羧基也被认为是很有可能的。在
Earlier papers have shown that the reversible acid denaturation of ferrihemoglobin and carbonylhemoglobin at both 0 and 25 liberates approximately 22 acid-binding groups (presumably imidazole) which do not combine with acid in the native protein. The demonstration for ferrihemoglobin has depended (a) on rapid-flow measurements (2-3 seconds) of pH in solutions of sufficiently high ionic strength (0.3 m chloride) to suppress differences in acid-bindingbetween native and denatured protein which are due only to changes in the electrostatic interaction of their charged groups; and (b) on the fact that the difference in the titration curves does not pass through a well-marked narrow maximum as the pH is lowered (as it would if only electrostatic effects were involved), but remains at a nearly constant level over a wide range of pH. At an ionic strength of 0.3 the difference is not sustained below pH 4, presumably because atlower pH values partial denaturation occurs within the 2 to 3 seconds required for pH meas-urement. In the present paper recourse is had to titrating cyanoferrihemoglobin (CN-hemoglobin) and carbonylhemoglobin (CO-hemoglobin)—both of which are much more stable to acid than is ferrihemoglobin—to extend the constant difference between the titration curves of native and denatured protein in solutions of high ionic strength to considerably lower pH, and thus to strengthen the interpretation of actual unmasking. The results show an extension of the pH region of constant difference up to 2 pH units, and confirm the earlier conclusions that the basic function of theimidazole groups in 22 histidines is masked in native horse hemoglobin, and that other less basic groups, such as carboxylate, are completely reactive. The results also show that the rate of unmasking in cyanohemoglobin is approximately the same as the rateof denaturation of this protein measured spectrophotometrically.In acid-denatured carbonylhemoglobin (CO-hemoglobin)(Steinhardt and Zaiser, 1951) or ferrihemoglobin (Steinhardt andZaiser, 1953) the number of prototropic groups titrating between pH 4.5 and 7.5 exceeds by about thirty-six the number titrating between these limits of pH in the corre-sponding native proteins. In the earliest report (Steinhardt and Zaiser, 1951) the possibility was advanced that at least part of these “masked” groups were in histidine residues, but weak carboxyls were also considered a strong possibility. In