A case of spurious product formation during attempted resynthesis of proteins by reverse proteolysis. Some batches of 'pure' glycerol contain cross-linking agents.

In cases where enzyme-catalysed synthesis of a peptide bond is being used to re-form a protein from two large peptide fragments, the organic co-solvent chosen has so far been glycerol, for most solvents in use in small-molecule systems are potent protein denaturants. We have found, however, that impurities contaminating certain batches of glycerol are effective in cross-linking the complexes formed by these peptide fragments, thus mimicking the enzyme-catalysed process. In one such case, the reported re-formation of cytochrome c from a two-fragment complex system, cytochrome c-T, the extent and rate of conjugate formation duplicates that reported for enzymic resynthesis. We observed no difference between mixtures containing or lacking enzyme. We warn of the danger of confusion possible to those engaged in studies of resynthesis, and suggest a simple control of purchased glycerol to avoid it. We recommend similar caution to those (X-ray crystallographers and others) who seek to stabilize protein solutions by adding large quantities of glycerol.