To elucidate the contribution of tryptophan side chains to the vacuum-ultraviolet (VUV) circular dichroism (CD) of Escherichia coli dihydrofolate reductase, we measured the VUVCD spectra of eight tryptophan mutants down to 175 nm. The difference spectra between the wild-type and the mutants clearly demonstrated that the contribution of tryptophan side chains extends to the high-energy peptide CD in the VUV region. These results should be useful for a theoretical study on improving protein secondary-structure analysis by VUVCD spectroscopy. (C) 2013 Elsevier B. V. All rights reserved.