LIMITED PROTEOLYSIS OF CYTOCHROME-C IN TRIFLUOROETHANOL

Horse heart cytochrome c is cleaved by thermolysin in 50% aqueous TFE (v/v) at neutral pH (25 degrees C, 24 h) at the Gly(56)-Ile(57) peptid e bond of the 104-residue chain of the protein. Additional, but anyway minor, fragmentation at the Gly(45)-Phe(46) and Met(80)-Ile(81) pepti de bonds is also observed. On the other hand, in buffer only and in th e absence of TFE, cytochrome c is digested by thermolysin to numerous small peptides. Considering the broad substrate specificity of the TFE -resistant thermolysin, clearly the conformational state of the protei n substrate dictates the observed selective proteolysis. It is propose d that the highly helical secondary structure acquired by cytochrome c when dissolved in aqueous TFE hampers binding and adaptation of the p rotein substrate at the active site of the protease and that peptide b ond fission occurs at flexible chain segments characterized by a low a lpha-helix propensity.