The denatured states of cytochrome c

John P Harrington
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引用次数: 8

Abstract

The unfolding of horse ferricytochrome c in the presence of several inorganic salts has been studied under a variety of denaturing conditions and followed by means of absorbance changes in the Soret region (390–450 nm) and visible region (450–750 nm), as well as by viscosity measurements. Changes in the Soret region, usually sensitive to the heme environment, were characterized by gradual increases in absorbance at 409 nm for low concentrations of NaClO4 and LiClO4. As denaturant concentrations were increased, the low-spin state of the ferric heme is altered, as seen by a maximum shift to shorter wavelengths (402–407 nm) accompanied by a further increase in absorbance in the Soret region. Unlike the effect of several organic denaturants and the above salts, denaturation in the presence of LiCl and CaCl2 resulted in an overall decrease in the Soret region with a blue-shift to 401 and 400 nm, respectively. Visible spectra of ferricytochrome c exhibited new bands at 633 nm (9.0 M LiCl) and 636 nm (4.5 M CaCl2) indicative of a change in the spin state of the iron upon displacement of methionine 80. LiCl and LiBr produced intermediate states of protein unfolding with midpoints (D12) at 4.0 and 8.6, and 2.6 and 6.4, respectively. A determination of ΔGUH2O and m, the free energy of unfolding in the absence of denaturant and the dependence of free energy of denaturation on denaturant concentration, was used to analyze the relative effectiveness of these denaturants.

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细胞色素c的变性状态
在多种变性条件下,通过索氏区(390-450 nm)和可见光区(450-750 nm)的吸光度变化以及粘度测量,研究了马铁色素c在几种无机盐存在下的展开。Soret区域的变化通常对血红素环境敏感,其特征是低浓度NaClO4和LiClO4在409 nm处吸光度逐渐增加。随着变性剂浓度的增加,铁血红素的低自旋态发生了变化,从波长最大位移到更短的波长(402-407 nm),并伴随着Soret区域吸光度的进一步增加。与几种有机变性剂和上述盐的作用不同,LiCl和CaCl2存在下的变性导致Soret区域的整体下降,蓝移分别达到401和400 nm。铁细胞色素c的可见光谱在633 nm (9.0 M LiCl)和636 nm (4.5 M CaCl2)处出现了新的谱带,这表明在蛋氨酸80取代后,铁的自旋态发生了变化。LiCl和LiBr的蛋白展开处于中间状态,中间点D12分别为4.0和8.6,2.6和6.4。通过测定无变性剂时的展开自由能ΔGUH2O和m,以及变性自由能与变性剂浓度的关系,分析了这些变性剂的相对有效性。
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