{"title":"The denatured states of cytochrome c","authors":"John P Harrington","doi":"10.1016/0005-2795(81)90097-0","DOIUrl":null,"url":null,"abstract":"<div><p>The unfolding of horse ferricytochrome <em>c</em> 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 NaClO<sub>4</sub> and LiClO<sub>4</sub>. 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 CaCl<sub>2</sub> resulted in an overall decrease in the Soret region with a blue-shift to 401 and 400 nm, respectively. Visible spectra of ferricytochrome <em>c</em> exhibited new bands at 633 nm (9.0 M LiCl) and 636 nm (4.5 M CaCl<sub>2</sub>) 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 (<span><math><mtext>D</mtext><msub><mi></mi><mn><mtext>1</mtext><mtext>2</mtext></mn></msub></math></span>) at 4.0 and 8.6, and 2.6 and 6.4, respectively. A determination of <em>ΔG</em><sub>U</sub><sup>H<sub>2</sub>O</sup> and <em>m</em>, 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.</p></div>","PeriodicalId":100165,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Protein Structure","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"1981-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0005-2795(81)90097-0","citationCount":"8","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochimica et Biophysica Acta (BBA) - Protein Structure","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/0005279581900970","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
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 () 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.