FORMALDEHYDE-INDUCED FLUORESCENCE OF A TRYPTOPHYL TETRAPEPTIDE

The formaldehyde condensation according to Falck and Hillarp has found widespread use as a method for the fluorescence histochemical demonstration of catecholamines and indolamines. Recently, N-terminal tryptophan dipeptides were found also to react. with formaldehyde, giving strong fluorescence in aqueous solution (3), on silica gel thin layer (4) and in a dried protein matrix (protein droplet models) (2). C-terminal tryptophan peptides did not react. Conceivably, condensation of tryptophyl dipeptides with formaldehyde results in the formation of tetrahydronorharman derivatives that are further converted to dihydronorharman derivatives by dehydrogenation. The second step in the reaction requires oxidizing conditions. Thus, in aqueous solution and in protein droplet models, tryptophyl dipeptides emitted a strong formaldehyde-induced fluorescence only in the presensce of oxidants such as hydrogen peroxide or ozone (2, 3). From results obtained with tryptophyl dipeptides in protein droplet models, it was suggested that a combined formaldehyde-ozone treatment may permit the fluorescence microscopic demonstration of N -terminal tryptophan proteins and polypeptides in tissues (2). However, evidence was available only from studies on dipeptides and, hence, it could not be predicted whether fluorescence would result also from formaldehyde-ozone treatment of tryptophan-containing polypeptides and proteins. The gastrin C-terminal tetrapeptide (tetragastrin), which has an N-terminal tryptophan residue, could be expected to provide the missing information. Synthetic tetragastrin (L-tryptophyl-Lmethionyl-L-aspartyl-L-phenylalanine amide) was kindly supplied by Dr. H. J. Petersen, 1)epartment of Synthesis, Leo Pharmaceutical Products, Copenhagen. Aqueous solutions (10’ 211) of tryptamine hydrochloride, tryptophan and tetragastrin were prepared. Small volumes (1-5 l) of the stock solutions were added to 3 ml 0.1 N H2SO4 together with 0.1 ml formaldehyde (18%). After 20 mm at 100#{176}C 0.1 ml hydrogen peroxide (5%) was added and heating was continued for another 20 mm. The resulting fluorescence was read in a spectrophotofluorometer, equipped with an x-y recorder. The excitation and emission spectra of the tetragastrin fluorophore were found to be similar to those of the tryptamine and tryptophan fluorophores (Fig. 1). If either formaldehyde or hydrogen peroxide was omitted from the reaction mixture, no fluorescence resulted. Small amounts (0.01-1 /2g) of tryptamine, tryptophan and tetragastrin were spotted onto silica gel thin layers in volumes of 0.5 pl. After drying, the thin layers were exposed to formaldehyde gas at 80#{176}C for 1 hr. Strong formaldehyde-induced fluorescence, visible in ultraviolet light, resulted from all three compounds tested; the minimal detectable amount of tryptamine and tryptophan was 0.03 pg/cm2 and of tetragastrin, 0.3 pg/cm2. Microspectrofluorometric analysis of the formal dehyde-induced fluorescence was performed with a modified Leitz microspectrograph as described in detail elsewhere (1). The spectral properties of the tetragastrini fluorophore were remarkably similar to those of the tryptamine fluorophore but different from those of the tryptophan fluorophore (Fig. 2). Human serum albumimi (final concentration 2%) was added to the tryptamine, tryptophan and tetragastrin stock solutions. The solutions were sprayed as microdroplets onto glass cover slips, which were dried at room temperature. These protein droplet models were exposed at 80#{176}C for 1 hr to formaldehyde gas alone, to a mixttnre of formaldehyde gas and ozone or to a mixture of formaldehyde gas and HC1 vapor. The intensity of the resulting fluorescence was evaluated with a precision galvanometer: excitationn at 365 nip (stabilized direct current Hg lamp), emission at the emission maximum of each individual compound. The fluorescence spectra were analyzed by microspect rofluorometry. Treatment with formaldehyde alone gave weak fluorescence with all three compounds. Combined formaldehyde -HC1 treatment markedly improved the fluorescence yield of tryptamine, but only to a minor extent that of trypto-