The preparation of fully N-epsilon-acetimidylated cytochrome c

We have re-examined the acetimidylation and subsequent deprotection of cytochrome c by published methods in the light of recent findings on the tendency of protein acetimidylation reactions to yield side products of differing net charges. We find that the protection methods do indeed yield a mixture of products, some of which have considerably diminished biological activity. Our observations support a postulated mechanism for the generation of side products, and we have been able to identify the major factor responsible for their formation by published methods. The deprotection method appears to be free of side reactions. We describe a new procedure for acetimidylation that will produce fully N-epsilon-acetimidylated cytochrome c. This derivative, lacking detectable side products and having good biological activity, is useful for structure-function studies and as an intermediate in the semisynthesis of cytochrome c analogues.