Preparation and electron paramagnetic resonance characterization of spin labeled monoderivatives of horse cytochrome c

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• 作者：Turyna ; Bohdan ; Osyczka ; Artur ; Kostrzewa ; Anna ; Blicharski ; Wojciech ; Enghild ; Jan J. ; Froncisz ; Wojciech
• 关键词：Cytochrome c ; Spin label ; Electron paramagnetic resonance
• 刊名：Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular Enzymology
• 出版年：1998
• 出版时间：July 28, 1998
• 年：1998
• 卷：1386
• 期：1
• 页码：50-58
• 全文大小：306 K

文摘

Horse cytochrome c was reacted with the spin label (succinimidyl-2,2,5,5-tetra-methyl-3-pyrroline-1-oxyl-carboxylate) using optimized conditions and the reaction products were separated by a combination of cation-exchange chromatography and HPLC. The purified cytochrome c derivatives were digested with TPCK treated trypsin and the resulting peptides were separated by reverse phase HPLC. The modified Lys residues were subsequently characterized by Edman degradation and mass spectrometry. These analyses showed that five distinct cytochrome c derivatives had been produced which were modified at the specific Lys residues including Lys⁸, Lys²⁵, Lys⁷², Lys⁸⁶ or Lys⁸⁷, respectively. The electron paramagnetic resonance (EPR) spectra for each cytochrome c derivative revealed that for the spin label attached to Lys⁸ and Lys⁸⁷ only one component contributes to the spectrum whereas for Lys²⁵, Lys⁷² and Lys⁸⁶ the spectrum consists of two components. The highest mobility with the rotational correlation time, τ_B, of 0.38 ns was observed for Lys⁸⁷. The longest τ_B of 1.84 ns was obtained for Lys⁷². An attempt to correlate the spin label mobility with the local protein structure is presented. These mono derivatized cytochrome c molecules provide a unique tool for EPR studying the interaction between cytochrome c and the lipid bilayer, as well as cytochrome c oxidase and reductase.