FLUORESCENCE HISTOCHEMICAL DEMONSTRATION OF PEPTIDES WITH NH2- OR COOH-TERMINAL TRYPTOPHAN OR DOPA BY CONDENSATION WITH GLYOXYLIC ACID

Abstract

Condensation with glyoxylic acid vapor, recently introduced as a highly sensitive method for fluorescence histochemical visualization of biogenic monamines, has been found to allow also the demonstration of certain tryptophan- or dopa-containing peptides. Thus, in model experiments, treatment with glyoxylic acid vapor induced fluorescence from di- and tetrapeptides with tryptophan or dopa in NH2-terminal or COOH-terminal position. Peptides without tryptophan or dopa in terminal positions gave no observable fluorescence. Differences were recorded between peptides with NH2-terminal and COOH-terminal tryptophan with respect to their fluorescence yields under different reaction conditions and the spectral characteristics of the fluorophores (excitation/emission maxima: 375-380/500-520 nm and 340-370/435 nm, respectively). In addition, the tryptophan-containing peptides could be distinguished microspectrofluorometrically from the dopa-containing peptides. Thus, the fluorophore of the NH2-terminal dopa peptide had excitation/emission maxima at 330 and 380/495 nm, and the corresponding values for the fluorophore of the COOH-terminal dopa peptide were 330 and 370/460 nm. The glyoxylic acid treatment produced intense fluorescence in certain endocrine cell systems in pituitary, thyroid and gastric mucosa, previously suggested to store peptides with NH2-terminal tryptophan. The present findings support this hypothesis.