Tryptophan, fluorometric determination

Tryptophan was determined fluorometrically by the method of Dugan and Udenfriend (9). 

Amines and amino acids were extracted as described by Tachiki and Aprison (1975). Tissues were homogenized in 2 ml of acidified-n-butanol and centrifuged at 1500 x g for 30 min. (4 C). A 1.0 ml aliquot of supernatant was returned to a second set of tubes containing 4.0 ml n-heptane and 0.4 ml O.IN HCl. This mixture was shaken for 10 min. and the layers cleanly separated by centrifugation at 1000 x g for 5 min. The organic layer was removed and 5-HIAA content was determined by the OPT fluorometric method. Serotonin (in the O.IN HCL) was assayed by the OPT fluorometric method while tryptophan was determined fluorometrically as described by Denckla and Dewey (1967). Tyrosine was determined flurometrically by the method of Waalkes and Undenfriend (1957). 

Table IV gives the analytical results obtained for all of the amino acids except cystine, cysteic acid, and tryptophan for the various irradiation treatments given in Table I as well as for the frozen nonirradiated control beef. Table V gives the assay data for cystine plus cysteine, by the colorimetric procedure, and cysteic acid. The assay data for tryptophan in Table V were determined by the fluorometric procedure.

Determination of oxidized amino acids in urine is usually performed by isotope dilution gas chromatography-mass spectrometry (L9). DOPA is estimated by HPLC separation of acid protein hydrolysates with fluorescence detection (excitation 280 nm, emission at 320 nm) (A15). Other methods are based on borate-hydrochloric acid difference spectroscopy (this method suffers interference from tyrosine and tryptophan) (W2), derivatization of DOPA with nitrite and subsequent coulometric determination (W3), and fluorometric detection after derivatization with ethylenediamine (A15). 3-Hydroxylysine is quantitated by HPLC with 9-fluorenylmethyl chloroformate precolumn derivatization (M25) of amino acids obtained by gas-phase hydrolysis of proteins (F21). Other general methods to detect amino acid damage are mass spectometry methods applied to protein hydrolysates, such as tandem mass spectrometry (F6). 

One of the most useful applications of fluorescence is in the routine determination of certain important molecules in body fluids for diagnostic purposes. Some such molecules are naturally fluorescent, but others must be chemically treated to form fluorescent products. For example, the amino acids tyrosine, tryptophan, and phenylalanine are all measured fluorometrically. Both tyrosine and tryptophan possess aromatic rings that absorb intensely and therefore have an intense natural fluorescence. Tyrosine is excited at both 225 and 280 nm, and emits at 303 nm tryptophan is excited at 220 and 280 nm and emits at 438 nm [34]. 

The fluorometric method of Denkla and Dewey (97) for assay of tryptophan is the most suitable and the one most commonly used for the determination of tryptophan concentrations in tissue samples because of its sensitivity, specificity and simplicity. Tryptophan is converted to a fluorophore, norharman, by heating it in acid solution with formaldehyde and ferric chloride. A revised procedure of the original method has been also reported (46). 

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