The CD, absorption, and fluorescence spectra and fluorescence lifetimes of three highly homologous, basic cytotoxic proteins isolated from wheat (α1-, α2-, and β-puro-thionins) and a moderately homologous protein isolated from Crambe abyssinica (crambin) have been measured. The purothionins each contain a single tyrosine, while crambin has two tyrosine residues. At neutral pH in buffered solution or in water, β-purothionin showed a single fluorescence emission peak maximal at 345 nm; α1 and α2-purotmonins gave a double-humped emission (λmax 308 and 345 nm), while crambin emitted only at 303 nm. Under acid pH conditions (<pH 3) or when denatured in 6 M guanidine hydrochloride, the spectra of the α- and (β-purothionins showed predominantly the 303-nm emission (τ = 3.1 ns) while at pH >10.0 only the 345-nm emission was evinced by all three proteins. Crambin showed typical tyrosine emission in the pH range 3-9 and weak tyrosinate fluorescence at pH >10.5. From these features, and from the absorption and CD spectra, we infer that the 345-nm fluorescence emission of either α1-or β-purothionin is from tyrosinate moieties. The purothionin emission spectra appear to be generated by excited-state proton transfer rather than from tyrosinate species in the ground state.
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