CHARACTERISTICS OF TYROSINATE FLUORESCENCE EMISSION IN ALPHA-PUROTHIONINS AND BETA-PUROTHIONINS

The CD [circular dichroism], absorption and fluorescence spectra and fluorescence lifetimes of 3 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) were measured. The purothionins each contain a single tyrosine, while crambin has 2 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 (< pH 3) or when denatured in 6 M guanidine hydrochloride, the spectra of the .alpha.- and .beta.-purothionines showed predominately the 303-nm emission (.tau. = 3.1 ns) while at pH > 10.0 only the 345-nm emission was evinced by all 3 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, the 345-nm fluorescence emission of either .alpha.1- or .beta.-purothionin is apparently 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.