PREPARATION AND ELECTRON-PARAMAGNETIC-RESONANCE CHARACTERIZATION OF SPIN-LABELED MONODERIVATIVES OF HORSE CYTOCHROME-C

Horse cytochrome c was reacted with the spin label 2,5,5-tetra-methyl- 3-pyrroline-1-oxyl-carboxylate) using optimized conditions and the rea ction products were separated by a combination of cation-exchange chro matography and HPLC. The purified cytochrome c derivatives were digest ed with TPCK treated trypsin and the resulting peptides were separated by reverse phase HPLC. The modified Lys residues were subsequently ch aracterized 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 Lys(8), Lys (25), Lys(72), Lys(86) or Lys(87), respectively. The electron paramagn etic resonance (EPR) spectra for each cytochrome c derivative revealed that for the spin label attached to Lys(8) and Lys(87) only one compo nent contributes to the spectrum whereas for Lys(25), Lys(72) and Lys( 86) the spectrum consists of two components. The highest mobility with the rotational correlation time, tau(B), of 0.38 ns was observed for Lys(87), Lys(87). The longest tau(B) of 1.84 ns was obtained for Lys(7 2). An attempt to correlate the spin label mobility with the local pro tein structure is presented. These mono derivatized cytochrome c molec ules provide a unique tool for EPR studying the interaction between cy tochrome c and the lipid bilayer, as well as cytochrome c oxidase and reductase. (C) 1998 Elsevier Science B.V. All rights reserved.