141 BIOCHIMICA ET BIOPHYSICA ACTA BBA 35228 DETECTION OF AROMATIC AMINO ACID DERIVATIVES ON THIN-LAYER CHROMATOGRAMS BY VARIOUS ELECTRON ACCEPTORS Y. B U R S T E I N , M. F R I D K I N AND M. S H I N I T Z K Y Department of Biophysics, The Weizmann Institute of Science, Rehovoth (Israel) (Received F e b r u a r y 2nd, 1968) SUMMARY A new method for the detection of the aromatic residues in aromatic amino acids and peptides on thin-layer chromatography by the apparent colour of their charge-transfer complexes with various electron acceptors, is described. The method was found most suitable for the detection of tryptophan- and tyrosine-containing peptides where amounts of 1-2/~g of the peptide could be detected. The intact amino acids and peptides can be released by treatment of the coloured spots with suitable aqueous solvents. INTRODUCTION The aromatic amino acids may act as ~-electron donors according to their side chains 1. The electron-donating power of these compounds varies in the order: tryptophan > histidine > tyrosine > phenylalaninel, 2. A series of charge-transfer complexes of the four aromatic amino acids with the same acceptor should exhibit four different colours characteristic of each of the aromatic amino acids. Thus, a peptide containing an aromatic amino acid residue can be identified by the colour it gives in the presence of an electron acceptor. Furthermore, since charge-transfer complexes are easily destroyed by solvation z, peptides should be recoverable after staining. In the following we describe a method of detection and identification of aromatic amino acids and their peptides by characteristic coloured spots appearing on developing thin-layer chromatography plates with solution of various electron acceptors. MATERIALS AND METHODS Reagents. 2,4,7-Trinitro-9-fluorenone (Fluka) was recrystallized from acetic acid-water. Chloranil (Fluka) was recrystallized from alcohol. 1,3,5-Trinitrobenzene (Eastman Organic Chemicals, white label); maleic anhydride (British Drug House Laboratory); 2,4-dinitrobenzene sulfonic acid (Eastman Organic Chemicals, white label). Biochim. Biophys. Acta, 16o (1968) 141-144 142 Y. BURSTEIN, M. FRIDKIN, M. SHINITZKY Solvents. Chloroform, methyl alcohol, n-butanol, acetic acid, pyridine, cyclohexane and dichloromethane were of analytical reagent grade. Thin layers. Plates were prepared (500 # in depth) with Quickfit applicator and were air dried for 30 rain, then dried at IOO° for i h and allowed to cool before use, The stationary phase was Kieselgel G (E. Merck) applied as a slurry in water. Solvent systems used in thin-layer chromatography. System A, n-butanol-acetic acid-water (4:1:1 by vol.); System B, n-butanol-acetic acid-pyridine-water (15:3: IO: 12, by vol.) ; System C, chloroform-methanol (9: I, v/v). Detecting reagents, i °/o Solutions of chloranil, 2,4,7-trinitro-9-fluorenone , 1,3,5 ~ trinitrobenzene, 2-4-dinitrobenzene sulfonic acid and maleic anhydride in dichloromethane, chloroform or cyclohexane. Peptides. Tryptophyl peptides were kindly supplied by Dr. M. W I L C H E K , of our department; tyrosyl, histidyl and phenylalanyl derivatives were purchased from Miles-Yeda, Ltd. METHODS Blocked peptides were dissolved in methyl alcohol or dimethylformamide. Unblocked peptides were dissolved in dilute HC1 before application to the plates. The plates were placed in chromatography tanks which had previously been allowed to equilibrate with the solvent system for 2 h at room temperature. The solvent front was allowed to run for about lO-15 cm. The plates were then removed and dried and then sprayed with the detecting reagents. In another system the irrigation solvent contained 3 % of the acceptor. Tryptophyl peptides could be detected while moving on the chromatographic plate. The other aromatic peptides yielded coloured spots after the plates were dried. RESULTS AND DISCUSSION Table I summarizes the results obtained from experiments with a series of tryptophyl, tyrosyl, histidyl and phenylalanyl derivatives. The peptides were separated by thin-layer chromatography in several solvent systems and detected by their coloured charge transfer complexes with various acceptors. Of all the five acceptors listed in Table I, 2,4,7-trinitro-9-fluorenone and chloranil were found to be the most effective. The tryptophyl and tyrosyl derivatives tested gave coloured spots immediately after spraying with all the developers used. However, histidyl and phenylalanyl derivatives gave coloured spots only after several hours. It is thus possible that the acceptors bound in the latter charge-transfer complexes undergo chemical reactions yielding coloured products 4. Detecting reagents 2,4,7-trinitro-9-fluorenone, chloranil and 1,3,5-trinitrobenzene were suitable for the visualization of small quantities of tyrosyl and tryptophyl derivatives with a lower detection limit of approximately 1-2/~g. When 2,4,7-trinitro-9-fluorenone was added to the irrigation solvent, the corresponding coloured charge-transfer complexes of several tryptophyl and tyrosyl derivatives could be detected while migrating on the thin-layer chromatographic plate. The RE values obtained under these conditions were slightly different from those Biochim. Biophys. Acta, 16o (1968) t 4 I - I 4 4 t~ ~4 o TABLE I RF VALUES AND COLOURS OF THE CHARGE TRANSFER COMPLEXES o C, colorless s p o t on b a c k g r o u n d . Peptide A ~. ~" o 7 i. B e n z y l o x y c a r b o n y l - L - t r y p t o p h y l - L - t r y p t o p h y l g l y c i n e e t h y l ester 2. L - T r y p t o p h y l g l y c y l - L - t r y p t o p h a n e 3. B e n z y l o x y c a r b o n y l - L - t r y p t o p h y l - L - a l a n i n e benzyl ester 4. L-Tyrosylglycine 5- L-Tyrosine e t h y l ester 6. L-Histidine 7. L-Histidylgly¢ine 8. B e n z y l o x y c a r b o n y l - L - p h e n y l a l a n i n e 9. B e n z y l o x y c a r b o n y l - L - p h e n y l a l a n y l - L - s e r i n e m e t h y l ester io. L - P h e n y l a l a n i n e a m i d e * Colour a p p e a r e d i m m e d i a t e l y . ** Colour a p p e a r e d a f t e r several h o u r s . Colours obtained with R F values in system B C Chloranil 2,4,7- Trinitro9=fluorenone r,3,5- Trinitrobenzene 2,4-Dinitrobenzene sulfonic acid Maleic anhydride c~ o.95 o.65 o.88 0.73 0.90 o.oo P u r p l e grey* P u r p l e b r o w n * P u r p l e grey* P u r p l e b r o w n * Yellow orange* Yellow orange* Yellow* Yellow * Pale yellow* Pale yellow* o.96 o.6o o.52 o.o9 o.iI 0.73 o.96 o.62 o.59 0.24 o.3I o.6i o.95 o.oo o.65 o.oo o.oo o.68 P u r p l e grey* Purple* Purple* C** C** C** Purple brown* O r a n g e yellow* O r a n g e yellow* Brown** Brown** Yellow brown** Yellow orange* Yellow* Yellow* L i g h t yellow** L i g h t yellow** C** Yellow* Yellow** Yellow** Pale yellow* o.85 0.62 o.85 o.75 0.72 0.04 Grey** Khaki** Yellow brown** C** Yellow brown** Yellow** m tz © t~ 144 Y. BURSTEIN, M. FRIDKIN, M. SHINITZKY T A B L E II /~N VALUES OF SEVERAL P E P T I D E S IN T H R E E SOLVENT SYSTEMS W I T H AND W I T H O U T 2,4,7-TRINITRO9-FLUORENONE Peptide RF value in system" I. B e n z y l o x y c a r b o n y l - L - t r y p t o p h y l - L - t r y p t o p h y l glycine ethyl ester 2. B e n z y l o x y c a r b o n y l - L - t r y p t o p h y l - L - a l a n i n e benzyl ester 3. L-Tyrosine e t h y l ester A A" B B" C C' 0.95 0.90 0.88 0.86 0.90 0.72 0.96 0.52 0.9o 0.57 0.96 0.59 0.95 0.67 0.95 0.65 0.86 o.41 * T h e s o l v e n t s y s t e m s A', B' a n d C' c o n t a i n 3 ~o 2,4,7-trinitro-9-fluorenone- obtained in the absence of 2,4,7-trinitro-9-fluorenone in the irrigation solvent as summarized in Table II. The electron affinities of the acceptors used as developers vary in the order chloranil > 2,4,7-trinitro-9-fluorenone > 1,3,5-trinitrobenzene > 2,4-dinitrobenzene sulfonic acid > maleic anhydride 5. For a series of charge-transfer complexes of the same donor with various acceptors, the stronger the electron acceptor, the more will the colour of its complex shift towards the blue end of the spectrum 3. Table I shows that in all tryptophan- and tyrosine-containing compounds, the apparent colours vary as predicted for charge-transfer complexes. Charge-transfer complexes can be easily separated into their components by solvation ~. In the cases of tryptophan and tyrosine derivatives, the tested compound could be extracted from the coloured spot by a suitable aqueous solvent in which the acceptor is insoluble. The fact that the detected compound is undamaged upon identification emphasizes the advantage of this method over the other known detection methods. ACKNOWLEDGEMENT This work was supported by U.S. Public Health Service Research Grant AM05098. REFERENCES I A. PULLMAN AND g . P U L L M A N , in P. O. LOWDIN, The Quantum Theory of Atoms, Molecules and Solid State, A c a d e m i c Press, New York, 1966, p. 345. 2 F. J. BULLOCK, in M. FLORKIN AND E . H . STOTZ, Comprehensive Biochemistry, Vol. 22, Elsevier, A m s t e r d a m , 1967, p. 81. 3 G. B R I E G L E B , Electronen-Donator-Acceptor Komplexe, Springer, Berlin, 1961. 4 E. M. KOSOWER, Progress in Physical-Organic Chemistry, Vol. 3, Wiley, New-York, 1965, p. 81. 5 G. B R I E G L E B , Angew. Chem. Intern. Ed. Engl., 3 (1964) 617. Biochim. Biophys. Acta, 16o (1968) 141-144