Fluorescence Reaction for Amino Acids Marc Roth Laboratoire Central, Hzpital Universitaire, CH-1211 Genefie 4, Switzerland 0-Phthaldialdehyde reacts with amino acids in alkaline medium in the presence of a reducing agent such as 2-mercaptoethanol, by giving rise to strongly fluorescing compounds. Optimal wavelen ths are A,, = 340 nm and if,= 455 nm. This permits h o r i m e t r i c assay of amino acids down to the nanomole range. No heating i s necessary, and the fluorescence may easily be measured 5 min after mixing of the reagents. The reaction is well suited to the automatic determination of amino acids after ion exchange fractionation. The sensitivity is much better than with ninhydrin procedures. The imino acids proline and hydroxyproline, however, are not detected by the method.
AT PRESENT, the most widely used reagent for a-amino acids is ninhydrin. Intense colors are produced which serve as basis of numerous analytical procedures, both qualitative and quantitative. Although the sensitivity of the ninhydrin reaction is quite sufficient for many purposes, situations often occur in biochemistry where a more sensitive test for amino acids is needed. As fluorometry is known to be about one hundred times more sensitive than colorimetry, I tried to develop a fluorescent test for amino acids. On the basis of the older literature ( I ) o-diacetylbenzene was considered as a possible model reagent. After many unsuccessful trials, I found that addition of a strongly reducing agent to a solution of alanine and o-diacetylbenzene produced a bright blue fluorescence. This occurred also with some other amino acids. An investigation of the reaction showed that if o-diacetylbenzene was replaced by o-phthalaldehyde, a fluorescence was obtained with all common amino acids except cysteine and the imino acids proline and hydroxyproline. A sensitive technique of detection of amino acids has been developed on this basis (2) and is reported here. EXPERIMENTAL Apparatus. Fluorescence was measured on a Farrand spectrofluorometer. The slit width 'was 10 nm. The possibility of using a filter fluorometer was checked with an Aminco fluoromicrophotometer. Reagents. o-Diacetylbenzene was purchased from Schuchardt (Munich, Germany) and o-phthalaldehyde, potassium borohydride, and 2-mercaptoethanol from Fluka (Buchs SG, Switzerland). Amino acids (analytical grade) were racemic, except those which were more easily available in the L form. BORATEBUFFER(approx. 0.05M). A 0.05M solution of sodium tetraborate is adjusted to pH 9.5 with concentrated NaOH or to pH 9.0 with concentrated HC1. BUFFERED REAGENT.Mix 1.5 ml of o-phthalaldehyde solution (10 mg/ml in ethanol) with 90 ml of borate buffer of pH 9.5. Add 1.5 ml of a solution of 2-mercaptoethanol (5 Nl/ml in ethanol) and mix. The reagent is stable one day at room temperature. Procedure. To assay amino acids in aqueous solution, mix 100 pl of the unknown (concentration: approx. to lO-3M) with 3 ml of buffered reagent. Measure the fluorescence within 5 and 25 min after mixing. If a spectro(1) G. Hillmann, 2.Physiol Chem., 277,222 (1943). (2) M. Roth, Swiss patent application filed Feb. 17, 1970. 880
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fluorometer is used, set the monochromators at A,, = 340 nm and irl= 455 nm. A filter instrument with appropriate filters may also be used. Run a blank without amino acid and a standard solution (5 X 10-4Mamino acid) simultaneously. RESULTS o-Diacetylbenzene, which had been described by Hillmann (1) as a fluorogenic reagent for proteins, was investigated as a
possible reagent of amino acids. Indeed, when an aqueous solution of this compound was added to a solution of glycocoll in pyrophosphate buffer of pH 8.8 (0.05M), a fluorescence appeared. Other amino acids giving a fluorescence were ornithine, histidine, cysteine, and lysine. With ornithine, the fluorescence was strongest when the pH of the reaction mixture was 10.3. Neutral aliphatic amino acids gave practically no fluorescence. Further trials showed, however, that if potassium borohydride was added to the reaction mixture, a fluorescence was given by such amino acids as alanine. Best results were obtained when o-phthalaldehyde, instead of o-diacetylbenzene, was mixed with a solution of an amino acid in the presence of a strong reducing agent like potassium borohydride or 2mercaptoethanol. Table I gives a comparison of the fluorescence intensities obtained for a number of amino acids and related compounds with either o-phthalaldehyde or o-diacetylbenzene as the aromatic o-dicarbonyl reagent, with and without mercaptoethanol. The reaction mixture contained (in the order of addition) 3 ml of borate buffer (pH 9.5), 100 pl of a lO-3M solution of amino acid or amino compound, 50 p1 of a methanolic solution of either o-phthalaldehyde (10 mg/ml) or o-diacetylbenzene (100 mgiml), and 50 pl of either ethanol or an ethanolic solution of 2-mercaptoethanol ( 5 pl/ml). All conditions of fluorescence measurement were kept constant, except wavelengths which were set to optimal values-i.e., A,, = 340 nm and Xrl = 455 nm for o-phthalaldehyde and A,, = 355 nm and A f l = 445 nm for o-diacetylbenzene. The results (Table I) show that high intensities are obtained with most natural amino acids after reaction with o-phthalaldehyde and 2-mercaptoethanol. Proline and cysteine do not react, and the basic amino acids lysine and ornithine give higher intensities with o-diacetylbenzene than with o-phthalaldehyde. Since a mixture of o-phthalaldehyde and a reducing agent appeared to be the most favorable reagent for amino acids, the combination of o-phthalaldehyde and 2-mercaptoethanol was tried with a more extensive list of a-amino acids and related compounds. The reagent contained 0.1 ml of o-phthalaldehyde solution (10 mg/ml in ethanol), 0.25 ml of 2-mercaptoethanol, and borate buffer (pH 9.0) to make 3 ml. TO this were added 10 p1 of an aqueous solution (100 pglml) of amino acid or amine, and fluorescence was measured as indicated in the experimental section, Results, which were calculated on a virtual equimolar basis, are shown in Table 11. Among other compounds investigated, N-substituted amino acids such as N-carbobenzoxy-leucine gave no reaction. With histidylleucine and leucyl-glycyl-glycine the fluorescence was very low, whereas it was bright with leucine methyl ester.
Influence of o-Phthalaldehyde Concentration. Increasing amounts from 10 to 600 p1 of a solution of o-phthalaldehyde in methanol (10 mg/ml) were mixed with borate buffer of pH 9.5 to make 3 ml. Fifty microliters of 2-mercapto-ethanol solution (5 pl/ml in ethanol) and 50 p1 of 10-aMalanine were added and the fluorescence was measured. After a sharp increase, the fluorescence was nearly constant over the range from 100 to 600 pl, a small decrease being observed as the quantity of phthalaldehyde was increased. A much sharper decrease was observed if the buffered amino acid was first mixed with o-phthalaldehyde and mercaptoethanol was added last. This indicates that in the absence of a reducing agent, o-phthalaldehyde is capable of reacting otherwise with amino acids with the formation of nonfluorescent products. Influence of Mercaptoethanol Concentration. Variation of the amount of 2-mercaptoethanol from 0.8 to 400 nanoliters (added as solutions in ethanol) does not markedly influence the fluorescence of a mixture containing 3 ml of borate buffer of pH 9.0, 10 pl of alanine solution in water (10 pg/ml), and 100 p1 of o-phthalaldehyde solution in ethanol (10 mgiml). Influence of Amino Acid Concentration. Increasing amounts from 5 to 500 1 1 of a lO+M solution of alanine in water were completed to 3 ml with a freshly prepared mixture of 90 ml of borate buffer of pH 9.5, 1.5 ml of o-phthalaldehyde solution (50 mg dissolved in 2 ml methanol and diluted with the above borate buffer to make 50 ml), and 1.5 ml of a solution of 2-mercaptoethanol in ethanol (5 pl/ml). After mixing and waiting between 2 and 20 min, the fluorescence was measured. A linear relationship between fluorescence intensity and concentration was observed within the range of samples containing from 5 to 100 p1 of lO-3M alanine solution in the final volume. Concentration quenching became important above 400 pl. The fluorescence of the sample containing the lowest amount of alanine was 2.5 times as high as that of the blank without amino acid. An attempt was made to reduce the blank fluorescence, since this is the main factor limiting the sensitivity of the method. It was found that it can be decreased by reduction of the quantities of o-phthalaldehyde and 2-mercaptoethanol. If the reaction is performed with a tenfold dilution of the above mentioned mixture of o-phthalaldehyde and 2-mercaptoethanol with buffer, the fluorescence observed with 1 pl of 10-3M alanine solution is twice as high as that of the blank. Thus, as low as 1 nanomole of alanine may be assayed, and the sensitivity could even be increased by the use of microcells. Use of the diluted reagent gives a linear calibration curve from the origin up to 10 p1 of lO-3M alanine solution incorporated in the final mixture. The quantity of unknown solution added can be increased at the expense of buffer without change of the final volume. The same results are obtained if one adds 100 p1 of a lO-4M alanine solution, or 1 ml of 1 0 - ~ ~ s o l u t i o ninstead , of 10 p1 of a 10-3Msolution. Effect of pH. A solution of alanine was made to react with o-phthalaldehyde and 2-mercaptoethanol at pH 9, after which aliquots of the reaction mixture were mixed with buffers of increasing pH. The fluorescence intensity of the resulting solutions was the same over the entire pH range from 6.0 to 11.5. On the other hand, the fluorogenic reaction itself is markedly influenced by pH. To 3 ml of 0.05M buffer were added 50 pl of a 10e3M solution of amino acid, 50 p1 of o-phthalaldehyde solution (10 mg/ml in ethanol) and 50 p1 of 2-mercaptoethanol solution (5 p l / d in ethanol). The fluorescence intensities are shown in Table 111. Ali-
Table I. Fluorescence Intensities (Relative Units) Obtained with Amino Acids and Related Compounds upon Reaction with o-Phthalaldehyde or o-Diacetylbenzene, in the Presence or Absence of 2-Mercaptoethanol o-Phthalaldehyde o-Diacetylo-Phthal- o-Diacebenzene Amino aldehyde tylbenzene mercapto- mercaptocompound alone alone ethanol ethanol None 2 2 25 10 Alanine 3 900 2 24 Leucine 2 870 12 3 Serine 2 3 lo00 20 Tyrosine 3 810 2 20 Glutamic acid 2 lo00 20 3 Glutamine 2 21 3 lo00 8 Cysteine 14 8 20 Cysteic acid 2 12 2 lo00 Taurine 3 9 960 620 Methionine 12 3 3 1100 Proline 2 25 2 10 Histidine 18 5 800 360 Histamine 10 33 390 500 Lysine 2 8 30 600 Ornithine 35 28 2 515 8 26 Arginine 4 480 Citrulline 3 935 49 20 820 365a 2 725 2,3-Diaminopropionic acid Ammonia 4 2 40 14 a 3800 at A , = 395 nm and A r l = 475 nrn.
+
+
Table 11. Molar Fluorescence Intensities (Relative Units) of Amino Acids and Related Compounds after Reaction with a Mixture of o-Phthalaldehyde and 2-Mercaptoethanol at pH 9.0 FluoresFluorescence cence Amino compound intensity Amino compound intensity Glycocoll Glutamic acid 1018 562 Alanine Cysteine 840 0 Valine 48 1105 Cystine 554 985 Norvaline Taurine 681 Leucine Methionine 1033 Isoleucine Proline 0 1076 Histidine Norleucine 745 1009 Histamine 73 Phenylalanine 858 53 Lysine Tryptophan 852 Ornithine Tyrosine 1162 91 Arginine 1270 Serine 998 1072 Citrulline Threonine 916 Aspartic acid 1085 1248 Asparagine
phatic, acidic, sulfur, and hydroxy amino acids produced the highest fluorescence when reacted at pH values between 8.0 and 11.0. Lysine behaved differently, having its maximum from pH 6.0 to 7.0. Histamine gave a peak at pH 6.0 and a plateau above pH 10.5. As shown by the results at pH 8.0, fluorescence was consistently stronger in borate buffer than in phosphate buffer. If a correction is made for this inhibiting effect of phosphate buffer, a pH maximum at 7.5 may be assumed for arginine. Influence of Time. At room temperature, the reaction proceeds readily. The maximum of fluorescence intensity occurs 5 min after mixing of the reagents. A slow decrease is then observed, the stability being sufficient to allow routine measurements to be made within 5 and 25 min after the reaction has been started. Precision. The precision of the results given in Table I1 was checked for four amino acids. Based on 10 or more ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
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Table 111. Fluorescence Intensity (Arbitrary Units) as a Function of the pH of Reaction. PH 8.0
8.0
(phos- (borCompound 5.0 6.0 7.0 7.5 phate) ate) 8.5 9.0 9.5 10.0 43 52 51 51 Alanine 0 2 51 53 24 36 35 42 Arginine 32 54 46 36 1 4 34 40 48 52 53 54 53 57 Cysteic acid 0 1 18 32 57 56 59 60 60 64 Glutamic acid 0 5 18 39 56 57 Glutamine 1 6 29 45 52 58 58 58 1 7 10 12 18 26 Histamine 12 30 6 2 Histidine 0 22 21 18 20 32 36 46 54 60 2 1 2 3 1 1 Lysine 1 10 10 3 58 59 0 2 27 47 54 60 60 58 Methionine 55 54 Serine 46 54 54 55 1 2 26 39 25 48 47 46 46 47 2 9 30 23 Taurine a Buffers: acetate (pH 5-6), phosphate (pH 7-8), sodium borate (pH 8-11), and 0.01N NaOH (pH 12).
determinations, relative standard deviation was i1.9 % (aspartic acid and methionine), and f1.5 % (alanine), * 2 . 5 (arginine). DISCUSSION
The new reaction described in this paper has a specificity resembling that of ninhydrin, except that the imino acids proline and hydroxyproline do not react. Cysteine gives a poor fluorescence, but can be oxidized to cysteic acid which reacts fairly well. Besides its high sensitivity, the method has the advantage over ninhydrin techniques of not requiring heating. These features make the reaction ideally suited to the automatic assay of amino acids fractionated by ion exchange chromatography. Investigations in progress in our laboratory show that it is now possible to use a fluorometric detection for amino acids separated by the classical technique of Spackman, Stein, and Moore (3) or similar procedures. The length of the tubing between column and detector may be enormously reduced, which provides much better peak resolution. The high sensitivity permits to reduce both column size and elution time. At the present time, we have no idea of the nature of the chemical reaction involved. Some similarity with the ninhydrin reaction may exist, and it is interesting that ninhydrin is also often used in combination with a strongly reducing agent. It is possible that a single fluorescing reaction product is formed with any amino acid, since the excitation and fluorescence spectra were always the same. The order of addition of reagents is not of essential importance but the order given in the proposed method (amino acid added last) best minimizes unwanted side reactions. A few years ago, Cohn and Lyle ( 4 ) presented a fluorometric assay of glutathione based on its reaction with o-phthalaldehyde at alkaline pH; this yields a product fluorescing maximally at A,, = 350 nm and Xrl = 420 nm. No fluorescence is given by related compounds lacking a sulfhydryl group. This reaction may well represent a particular case of the more general reaction described in the present paper. Glutathione would behave simultaneously as an amino compound and, by virtue of its sulfhydryl group, as a reducing agent, thus sharing the two functions otherwise assumed by distinct compounds. (3) D. H. Spackman, W. H. Stein and S. Moore, ANAL.CHEM., 30, 1190(1958). (4) V. H. Cohn and J. Lyle, Anal. Biochem., 14,434 (1966). 882
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10.5 52 43 57 63 55 30 61 2 61 55 50
11.0 53 44 57 64 48 30
60 2 62 54 50
12 53 44 49 63 29 32 50 2 60 56 51
Other investigators recently described fluorogenic reactions for amino acids. The fluorescence with 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole (9, however, is less intense. The same is true for the Hantzsch condensation with acetylacetone and formaldehyde as proposed by Sawicki et al. (6). On the other hand, these two reactions may be used for the assay of amines, in which case they should prove superior to our phthalaldehyde reagent. Another fluorometric assay of amino acids has been presented by Guilbault and Hiesermann (7). It uses oxidation by the enzymes D- or L-amino acid oxidase. It is fairly sensitive, but is applicable only to those amino acids which are attacked by the enzymes. Interestingly, this permits the assay of proline, which is not detected by the present method (8).
ACKNOWLEDGMENT I am greatly indebted to Miss Sabine Wiederhold for her excellent technical assistance. The help of Ayoub Hampa;' in preliminary trials with an automatic amino acid analyzer is gratefully acknowledged. RECEIVED for review November 5 , 1970. Accepted February 1,1971. (5) P. B. Ghosh and M. W. Whitehouse, Biochem. J . , 108, 155 (1968). (6) E. Sawicki and R. A. Carnes, Anal. Chinz. Acta, 41,178 (1968). (7) G. G. Guilbault and J. E. Hiesermann, Anal. Bioclzem., 26 1 ( 1968). (8) Note added in proof. Since submission of this manuscript, I found that treatment of proline and hydroxyproline with sodium hypochlorite or chloramineT converts them to compounds giving the fluorescence reaction with o-phthalaldehydeand 2-mercaptoethanol.
Correct ion
Two Methods for Separation of Surface and Bulk Gases in Vacuum-Fusion Analysis of Metals In this article by K. W. Guardipee [ANAL.CHEM.,42, 469 1970)] the author neglected to mention a report of a method which was very similar to the multiple-sample method described in his paper. At the April 1967 meeting of Division I, Committee E-3, ASTM, Miss Virginia Horrigan of the Anaconda American Brass Co., Waterbury, Conn., reported such a method. Reference to this work should have been included in the above mentioned paper.
