Characteristics of tyrosinate fluorescence emission in alpha- and beta-purothionins.

The CD, absorption, and fluorescence spectra and fluorescence lifetimes of three highly homologous, basic cytotoxic proteins isolated from wheat (alpha 1-, alpha 2-, and beta-purothionins) 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, beta-purothionin showed a single fluorescence emission peak maximal at 345 nm; alpha 1- and alpha 2-purothionins gave a double-humped emission (lambda max 308 and 345 nm), while crambin emitted only at 303 nm. Under acid pH conditions (less than pH 3) or when denatured in 6 M guanidine hydrochloride, the spectra of the alpha- and beta-purothionins showed predominantly the 303-nm emission (tau = 3.1 ns) while at pH greater than 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 greater than 10.5. From these features, and from the absorption and CD spectra, we infer that the 345-nm fluorescence emission of either alpha 1- or beta-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.