Anal. Chem. l W 6 , 58,2375-2379 His A
2375
such as biopolymers and fine supports.
LITERATURE CITED
Figure 7. Standard curves for SIBr-PC derivatives of amino acids.
hydrolysis was much slower than the formation of urea derivatives, the determination of amino acids was performed without any influence of the hydrolysis (Figures 6 and 7). The resulting aromatic amine generated from the derivatization reagent gave a single peak that was widely apart from the peaks of amino acid derivatives (Figures 6 and 7). When SINC was used in the same aqueous media, excess reagent was also hydrolyzed giving fluorescent naphthylamine, which was also observed as a single peak. Coefficients of variation of the peak height given by 10 ng of each amino acids was in the range of 2.2-2.8 (n = 6). Detection limits of amino acids in this procedure were 0.15-0.3 ng ( S I N = 2). Many attempts (27-30) have recently been made to accomplish amino acid analysis by using precolumn derivatization with isothiocyanate type reagent. The present methods are also promising tools for the same purpose. Activated carbamate reagent(s) was found to have enormous value in the liquid chromatographic analysis of amino compounds. Further, the reaction proceeding through an activated carbamate compound as an intermediate, as it is one of the bifunctional reactions should be applicable to the introduction of an appropriate probe to desired location of macromolecules
(1) Spackman, D. H.; Stein, W. H.; Moore, S. Anal. Chem. 1958, 3 0 , 1190-1205. (2) Roth. M. Anal. Chem. 1971, 43, 880-882. (3) Benson, J. R.; Hare, P. E. R o c . Natl. Acad. Sci. U . S . A . 1975, 72, 619-662. (4) Samejima, K.; Dairman, W.; Udenfriend, S.Anal. Biochem. 1971, 4 2 , 222-236. ( 5 ) Stein, S.;Bohien, P.; Dairman, W.; Udenfriend, S. Arch. Biochem. Biophys. 1973, 155, 203-212. (6) Bohien, P.; Meiiet, M. Anal. Biochem. 1979, 9 4 , 313-321. (7) Ishida, Y.; Fujita, T.; Asai, K. J. Chromatogr. 1981, 204, 143-148. (8) Weber, G. Blochem. J. 1952, 5 1 , 155-167. (9) Tapuhi, Y.; Schmidt, D. E.; Lindner, W.; Karger, B. L. Anal. Biochem. 1981, 715, 123-129. (10) Lin. J. K.; Chang, J. Y. Anal. Chem. 1975, 4 7 , 1634-1638. (1 1) Einarsson, S.;Josefsson, B.; Lagerkvist, S. J. Chromatogr. 1983, 282, 609-618. (12) Einnarsson, S.J. Chromatogr. 1985, 348, 213-220. (13) Sanger, F. Blochem. J . 1945, 3 9 , 507-515. (14) Ghosh, P. B.; Whitehouse, M. W. Biochem. J. 1968, 108, 155-156. (15) Imai, K.; Watanabe, Y. Anal. Chim. Acta 1981, 130, 377-383. (16) Deiany, R. Anal. Biohem. 1972, 4 6 , 413-420. (17) Bjorkqvist, B. J. Chromatogr. 1981, 204. 109-114. (18) Edrnan, P. Acta Chem. Scand. 1950, 4 , 283-293. (19) Chang, J. Y.; Creaser, E. H. Blochem. J. 1978, 153, 607-611. (20) Mawda, H.; Kawauchi, H. Biochem. Biophys. Res. Commun. 1988, 3 1 , 188-192. (21) Chang, J. Y.; Creaser, E. H. J. Chromatogr. 1977, 132, 303-307. (22) Nambara, T.; Ikegawa, S.;Hasegawa, M.; Goto, J. Anal. Chim. Acta 1978, 101, 111-116. (23) Nimura, N.; Ogura, H.; Kinoshita, T. J. Chromatogr. 1980, 202, 375-379. (24) Kinoshita, T.; Kasahara, Y.; Noriyuki, N. J. Chromatogr. 1981, 270, 77-81. (25) Takeda, K.; Akagi, Y.; Saiki, A.; Tsukahara, T.; Ogura, H. Tetrahedron Lett. 1983, 2 4 , 4569-4572. (26) Ogura, H.; Kobayashi, T.; Shimizu, K.; Kawabe, K.; Takeda, K. Tetrahedron Len. 1979, 2 0 , 4745-4746. (27) Koop, D. R.; Morgan, E. T.; Tarr, G. T.; Coon, M. J. J. Bioi. Chem. 1982, 257, 8472-8480. (28) Heinrikson, R. L.; Meredith, S. C. Anal. Biochem. 1984, 136, 65-74. (29) Bidlingmeyer, B. A.; Cohen, S.A.; Tarvin, T. L. J. Chromatogr. 1984, 336, 93-104. (30) 'fang, C. Y.; Sepulveda, F. I. J. Chromatogr. 1985, 346, 413-416.
RECEIVED for review March 3,1986. Resubmitted May 9,1986. Accepted June 2, 1986.
Liquid Chromatographic Determination of Amino Acids after Gas-Phase Hydrolysis and Derivatization with (Dimethy1amino)azobenzenesulfonyl Chloride &ne Knecht and Jui-Yoa Chang* Pharmaceuticals Research Laboratories, Ciba- Geigy, Ltd., Basel CH-4002, Switzerland
The level of amino acld background In the blank sample Is one of the llmltlng factors for high sensitivity amlno acld analysts. By use of the (dlmethylamlno)arobenzenesulfonyl chlorlde (DABS-CI) precolumn labellng/HPLC method, the potentlal sources of contamlnants during hydrolysls and derlvatlzatlon were systematlcally lnvestlgated. The data Indlcate that the most llkeiy source of amlno acld contamlnants Is aqueous 6 N HCI. Gasphase acld hydrolysls can reduce the background generated by normal liquld-phase hydrolysls to about 25%. A typical blank sample, after gas-phase hydrolysis, yields an average of 0.5-3 pmd of Asp, Glu, Ser, and Gly. This low level of background together wlth the Improved DABS-CI method permlts routine and reliable amino acid analysis of low nanogram quantltles of protein hydrolysates. 0003-2700/86/0358-2375$01 SO10
The sensitivity limit of amino acid analysis depends upon the background of contaminants. When nanogram quantities of proteins were isolated and hydrolyzed for amino acid composition analysis, the background could result from the contaminants introduced in protein purification, in 6 N HC1, or in the glasswares etc. Despite strenuous pruification and cleaning of the solvents and the glasswares, there are low picomole levels of Gly, Ser, and Asp that are very difficult to remove (1-5). With the emerging new technique of precolumn derivatization/RP-HPLC, which is now capable of detecting femtomole levels of amino acid derivatives (6-26), these low picomole amounts of amino acid contaminants have become the major obstacle for high sensitivity amino acid analysis. In order to address this problem, we have investigated the 0 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
source of contaminants, the advantage of gas-phase acid hydrolysis, and the practical sensitivity limit of the (dimethy1amino)azobenzenesulfonyl chloride (DABS-C1) method (18-20). In the course of this work, we have also evaluated the stability of DABS-amino acids, and refined the conditions for derivatization and chromatographic separations of DABS-amino acids. I t is our opinion that together with the gas-phase acid hydrolysis, the DABS41 method can be used as a routine method for amino acid analysis of low nanogram quantities of proteins.
;I
STANDARD
DABS-ONa
K
r
5 pMol
I DE
00005 Au
EXPERIMENTAL SECTION Materials and Instruments. The DABS-C.1was obtained from Fluka (Switzerland) and recrystallized (19). Double recrystallized DABS-Cl could also be purchased from Pierce. The reagent was prepared in stock in small portions (800 nmol/Eppendorf tube) (19) and each sample was redissolved in 200 pL of acetonitrile shortly before derivatization. The 6 N hydrochloric acid (no. 24309) and amino acid standard H (no. 20088) were purchased from Pierce. The Pierce amino acid standard, supplied with a concentration of 2.5 nmol/pL in 0.1 N HCl (except for cystine which was 1.25 nmol/pL), was diluted 50 times to 50 pmol/pL with water (triple distilled). Myoglobin was from Beckmann and eel calcitonin was from Bachem (Switzerland). The gas-phase hydrolysis vessel (no. 07363) was a product of Waters Associates. The vessel is a flat-bottom glass tube (2.7 cm i.d. X 9 cm) fitted with a heat-resistant plastic screw cap. The cap is equipped with a Teflon valve, which is closed after vacuumizing the vessel. For HPLC, two pumps (6000A, Waters) and one system controller (720, Waters) were combined with a variable wavelength detector (Spectroflow730, Kratos) and an integrator (3390A) from Hewlett-Packard. The column was Merck Lichrosphere 100 CH-l8/2 (5 pm). Sample glasswares were preheated at 500 "C for 6 h. All solvents including acetonitrile were from Merck (min purity 99.7%) without further purification. The individual DABS-amino acid standard was prepared as described (19). It is important to mention, however, that those standards were used only at the early stage of setting up this new technique. They were used to check and establish the chromatographic separation of DABS-amino acids. For quantitative analysis of unknown samples, the DABS-amino acid standards have to be obtained from an amino acid standard mixture that has been hydrolyzed and dabsylated in parallel with unknown samples under identical conditions (see hydrolysis and dabsylation). Gas-Phase Acid Hydrolysis. Approximately 0.05-2 pg of protein samples (preferably salt free) were placed in hydrolysis tubes (4 mm id. X 50 mm) and were dried in a vacuum centrifuge. Twelve samples can be simultaneously placed in a Waters gasphase hydrolysis vessel. At least one Pierce standard (500 pmol) was included in each batch for hydrolysis. In cases when less than 0.1 pg of protein was hydrolyzed, one or two blank samples were included for hydrolysis. This is to control the level of background and substract them from the unknown samples if necessary (see blank samples hydrolyzed with gas-phase 6 N HCl in the Results section). About 400 pL of 6 N HCl was then placed in the bottom of the vessel. The vessel was purged with argon for 10 s and was finally sealed through the control of the Teflon valve with a good vacuum of lower than 0.1 mbar. Gas-phase hydrolysis was carried out at 110 O C for 24 h. After hydrolysis, the tubes were dried again under vacuum prior to dabsylation. Liquid-phase 6 N HCl hydrolysis was carried out as described (19). Twenty-five microliters of 6 N HC1 was added to each sample tube (4 cm i.d. x 10 cm). A standard mixture and a blank sample were also included for hydrolysis. Dabsylation and HPLC Analysis of DABS-Amino Acids. Hydrolyzed samples, standards, and blank samples were dissolved in 20 pL of 50 mM sodium bicarbonate, pH 8.1. Next, 40 pL of DABS-Cl solution was added to each sample (4 nmol/pL in acetonitrile, freshly prepared shortly before dabsylation; see Materials). The samples were sealed with silicon-rubberstoppers and were heated at 70 'C for 10 min (the mixture will become completely soluble after heating). After dabsylation, the samples were directly diluted with a diluting solution (50 mM sodium phosphate pH 7.0/ethanol, 1:1, v/v) t o suitable volumes and
I 5
10
15
20
25
30
35
MIN Figure 1. Original chromatograms of HPLC separation of DABS-amino acids. Pierce standard (500 pmol, except for cystine which was 250 pmol) was dabsylated. Five picomoles (top) and 0.5 pmol (bottom) were injected. NH, stands for DABS-",. Three major ghost peaks are marked by an asterisk. Solvent A was 25 mM sodium acetate, pH 6.5 containing 4 % dimethylformamide (degased with helium). Solvent B was acetonitrile. The gradient was 1 5 % B to 4 0 % B in 20 min, 4 0 % B to 70% B from 20 to 32 min, kept at 70% B from 32 to 34 min, then back to 15% B from 34 to 36 min. The cycle time from injection to injection was 44 min. The column temperature was 40 'C. The flow rate was 1 mL/min. The detector wavelength was 436 nm.
aliquots were injected directly for HPLC analysis. The final volume of each diluted sample is roughly dependent upon the amount of proteins. When 0.5 pg of proteins (or 500 pmol of standards) were hydrolyzed and dabsylated, they were usually diluted to 1 mL and 20 pl was injected for analysis (Figure 1). The improved standard conditions for the DABS-amino acids analysis are described in Figure 1. The quantity of each DABS-amino acid can be calculated from either the peak area integration or peak height. The peak heights generally give more accurate results when small peaks are quantified.
RESULTS Dabsylation and HPLC Analysis of DABS-Amino Acids. The previous conditions (19) have been improved. First, acetone has been replaced by acetonitrile for DABS-C1 solution in the derivatization step. Dabsylations of Asp, Glu, Ser, Thr, and Cys now give yields comparable to those of other amino acids (Figure 1). Acetonitrile is also less volatile than acetone. Second, the phosphate buffer has been replaced by acetate buffer for the HPLC separation of DABS-amino acids (Figure 1). A fine precipitate was generated when the phosphate buffer was mixed with a large volume of acetonitrile. The precipitate causes buildup of column pressure. This problem was solved by the use of acetate buffer, which is readily miscible with a high percentage of acetonitrile. The conditions described in the caption of Figure 1 have been used for 18 months without any recurring problems of pressure buildup in the column. Columns from different manufacturers such as Supelcosil LC-18 (23) and Vydac C-18 also give complete separation of DABS-amino acids. We have stayed with the Merck (2-18 column mainly because of its lower cost. Among different batches of columns from the same manufacturer, the only observable variation was the elution time of DABS-Arg. This can be adjusted between DABS-Ala and DABS-Pro by varying the concentration of the acetate buffer between 20 mM and 40 mM. Stability of DABS-Amino Acids. The stability of DABS-amino acids was tested with a dabsylated standard
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
2377
STABILITY OF DABS-AMINO ACIDS
Figure 2. Stability of DABS-amino acids at room temperature over a period of 4 weeks. Recoveries of DABS-amino acas were evaluated by their peak heights and referenced to the recovery of Leu in each run as 1.0. Lys, His, and Tyr are all bis-DABS derivatives and their peak heights are approximately doubled.
Table I. Amino Acid Contaminants (pmol) in Various Types of Blank Samples"
amino acid
type of blank sampleb Pierce 6 N HC1 Merck 6 N HCl b c d b c d '
a
ASP
Glu Ser Thr GlY
1
3
1
1 1 9
0.5 0.5 0.6
2 3 12 1
1
3
5
14 4
3
12
10
12
11
13
Ala
1
Arg
Pro Val Metc Ile Leu Phe Lyse
4
9
1
3
3
3
2
5
5
3
His TYr All blank samples were dabsylated with 20 p L of buffer and 40 pL of DABS-Cl solution, 10% were analyzed, the recoveries of each amino acid contaminant were then normalized by 10-fold. The data presented in each type of blank sample are averaged from the analysis of 25 samples. The deviations of type a and b blank samples are 10-15%. The deviations of type c and d blank samples are 20-50% except for Met and Lys which are 10-20%. *(a) Blank sample tubes. (b) Blank sample tubes added with 25 pL of 6 N HCl, dried without heat hydrolysis. (c) Blank sample tubes hydrolyzed with liquid-phase 6 N HCl (25 pL). (d) Blank sample tubes hydrolyzed with gas-phase 6 N HCI. cThe contaminants eluted at the positions of Met and Lys are probably non-amino acid byproducts. mixture from Pierce. The DABS-amino acid standards (500 pmol dissolved in 1mL of the dilution solution) were tightly sealed with a silicon-rubber stopper and left a t 25 "C for up to 4 weeks. Aliquots (20 pL) were analyzed weekly with the same Merck column and identical chromatographic conditions (Figure 1). Recoveries of DABS-amino acids were normalized by referring to that of DABS-Leu in each run as 1.0 (Figure 2). The relative recovery of DABS-Leu in each run was 1.0 (10 min), 1.03 (1week), 0.92 (3 weeks), and 0.94 (4 weeks), respectively. This variation was most likely due to the aging of the column or the injection variations of the automatic injector which was usually 2-3% and in some cases could be 5 % . The relative peak height of each amino acid to Leu in
Flgure 3. Amino acid analysis of myoglobin using the gas-phase hydrolysis and the DABS-CI method. (A, top) 0.05 p g of myoglobin was hydrolyzed and dabsylated; 10% was injected. (A, bottom) A blank sample was hydrolyzed and dabsylated; 10% was injected. (B, top) 0.5 p g of myoglobin was hydrolyzed and dabsylated; 1YO was injected. (B, bottom) A blank sample was hydrolyzed and dabsylated; 1% was injected. Chromatographic conditions are described in Figure
1.
each run remained practically unchanged through the 4-week period. This experiment confirms that all DABS-amino acids are perfectly stable in the dilution solution at room temperature. I t also reflects the excellent reproducibility of the column and the chromatographic system. Background Contaminants of Blank Samples. In order to search for the source of background contamination, four kinds of blank sample tubes were dabsylated and analyzed (Table I). (a) Blank Samples without Hydrolysis (Direct Dabsylation of Blank Hydrolysis Tubes). These blank samples yield backgrounds that were derived mainly from the reagent and the dabsylation buffer. In addition to the hydrolyzed excess reagent (DABS-ONa), there are DABS-NH2 and three major ghost peaks (marked by an asterisk in Figure l),which do not interfere with any DABS-amino acids at all. Two very minor ghost peaks, which eluted a t 21.8 min and 22.3 min, partially coeluted with DABS-Arg and DABS-Pro (Figure 3A). Eluted at the DABS-amino acid positions are approximately 1pmol of Gly, 4 pmol of Met, and 3 pmol of Lys (Table I). However, we suspect that the latter two contaminants may be non-amino
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
Table 11. Amino Acid Composition of Myoglobin Determined by the DABS-Cl Method Using Both Gas-Phase Acid Hydrolysis and Normal Liquid-Phase Acid Hydrolysis 1 rg" 20 ngb
GH' ASP
Glu
Ser Thr GlY Ala Arg
Pro Val Met
Ile Leu
Phe LYS
His
Tyr
8.7 18.3 6.9 4.9 12.1 17.3 5.6 4.3 8.0 1.3 7.9 18.0 6.1 17.5 13.1 2.5
0.2 fig0 20 ngb
0.5 rga 25 ngb
LH' I
C
1.1
18.9 6.6 5.7 12.1 17.2 4.6 4.3 7.0 1.0 8.3 17.9 6.5 19.7 10.2
0.05 wg" 5 ngb
0.1 rga 10 ngb
GH
LH
GH
LH
GH
LH
GH
LH
9.4 18.5 6.7 4.9 11.7 17.8 5.1 4.6 7.6 1.2 7.3 16.8 6.1 17.7 13.3 2.5
8.0 19.2 6.5 5.9 12.1 16.9 4.9 5.0 7.1 2.4 8.8 18.3 6.9 19.2 9.4
8.4 18.3 6.9 5.3 13.9 17.4 5.7 4.5 7.5
10.3 20.7 6.9 5.9 13.9 16.1 4.9 4.7 7.2 1.1 7.6 17.1 6.2 16.0 9.3
9.0 18.9 8.3 5.4 13.5 17.2 6.0 4.1 7.0 1.4d 7.8 16.6 6.7 16.7d 12.3 1.6
13.0 20.6 5.7 6.8 14.8 14.1 5.7 5.3 8.2 2.5d 8.4 17.4 6.7 13.gd 9.6
9.0 18.5 9.1 5.9 13.1 17.4 6.8 4.2 7.1 1.7d 7.0 15.6 6.5 15.8d 12.8 2.0
15.1 20.4 3.6 8.2 13.4 12.0 6.4 7.7 9.6 5.6d 9.7 19.2 7.4 8.6d 6.5
7.4 16.7 6.5 17.0 11.4 2.8
amt expected 8 19 6 5 12 18 5 4 8 2 8 18 n
18 12 2
a Hydrolyzed. *Injected. GH, gas-phase hydrolysis. LH, liquid-phase hydrolysis. dThe yields of Met and Lys were corrected by substracting the background contaminants from the blank samde.
acid byproducts rather than authentic Met and Lys. ( b ) Blank Sample Tubes Containing 25 p L of 6 N HC1, Dried without Heat Hydrolysis. These blank samples were analyzed to check the free amino acid contaminants existing in the 6 N HCl. The patterns of contamination are similar to that of type a blank samples. However, there is a slight increase of DABS-Gly and a byproduct eluted a t the DABS-Met position. ( c ) Blank Samples Hydrolyzed with 25 pL of 6 N HC1 (Normal Liquid-Phase Hydrolysis). These blank samples were designed to check the protein or peptide contaminants that may be present in the 6 N HC1 and the hydrolysis tube. In addition to the non-amino acid background described in type a blank sample, considerable amounts of DABS-Gly and DABS-Ser contaminants appeared after hydrolysis. There are also 1-3 pmol of DABS-Asp, DABS-Glu, and DABS-Ala depending on the source of 6 N HCl employed. ( d ) Blank Samples Hydrolyzed by Gas-Phase 6 N HC1. Amino acid contaminants reduced significantly as compared to that of liquid-phase hydrolysis (Table I). However, the amount of contaminants eluted at positions of DABS-Met and DABS-Lys remain practically unchanged. Recoveries of these two byproducts are fairly reproducible (& 10%) in this type of blank sample. Whenever less than 0.1-0.2 pg of protein samples are hydrolyzed by gas-phase 6 N HC1, a blank sample should be included and recoveries of these two byproducts are substracted from the unknown samples.
CALCITONIN EEL
GASPHASE
CALCITONIN EEL
HYDROLYSIS
A
ACID ANALYSIS
AMINO
L I Q U I D PHASE HYDROLYSIS
B
A M I N O 9CIC ANALYSIS
i
r
11
Amino Acid Composition Analysis of Myoglobin and Calcitonin. Varying amounts (0.05-2 pg) of myoglobin and calcitonin were hydrolyzed by both liquid-phase and gas-phase 6 N HCI. They were dabsylated and diluted and aliquots (containing 5-20 ng) were analyzed for their amino acid compositions. The results are presented in Figure 3, Figure 4,and Table 11. By use of liquid-phase hydrolysis, reliable compositions of myoglobin and calcitonin were obtained only when the amount of proteins that were hydrolyzed were larger than 0.2 pg. When 0.1 pg and 0.05 pg of myoglobin were hydrolyzed, the deviation of many amino acids became significant and some of them cannot be accounted for by the background of the blank sample. However, with gas-phase hydrolysis, reasonably accurate compositions were obtained even when 0.05 pg of myoglobin and calcitonin were hydrolyzed (see Table I1 and Figure 4).
DISCUSSION Background Contaminants and the Advantages of
Flgure 4. Amino acid compositions of eel calcitonin obtained from both gas-phase hydrolysis (A) and liquid-phase hydrolysis (6). Expected compositions are expressed at 100% rather than number of residues for each amino acid (nominal values). Four different shaded columns included in each amino acid represent recoveries obtained from hydrolysis and analysis of varying amounts of calcitonin.
Gas-Phase Hydrolysis. In many cases of protein microanalysis, contaminations have already been introduced during the protein purification. However, this problem will be faced by any method that is subsequently used to characterize its
ANALYTICAL CHEMISTRY, VOL. 58,
amino acid composition. In this work, the potential source of contaminants associated with the manipulation of the DABS-Cl method were systematically investigated. Our data indicate that the reagent, the derivatization buffer, and the glassware were contaminated by negligible amounts of free amino acids. The major portion of amino acid contaminants arose only after acid hydrolysis. Normal liquid-phase hydrolysis consistently resulted in higher levels of background (notably, Gly, Ser, Asp, Glu, and Ala) than gas-phase hydrolysis. Furthermore, recoveries of these amino acids, unlike those derived from the reagent, are by no means reproducible (f20-50%) (Table I). The origin of these contaminants is not clear. We believe, however, that they are invisible airborne particles that somehow absorbed into the acid solution or onto the wall of hydrolysis tubes ( 1 , 5 ) . Indeed, we have carried out an experiment that showed that a barely visible, airfloating dandruff, after hydrolysis, generated approximately 5000 pmol of free amino acids. Therefore, in performing high-sensitivity amino acid analysis, precautions must be taken in storage of the glasswares (particularly the hydrolysis tubes) as well as the 6 N HC1. Gas-phase acid hydrolysis avoids the contaminants that were dissolved in the 6 N HC1. In addition, the gas-phase hydrolysis is also technically easier to perform than the liquid-phase hydrolysis. It does not require vacuum sealing of each sample tube because more than 12 sample tubes can be sealed under vacuum in the same hydrolysis vessel.
Reliability and Sensitivity of the DABS-Cl Method. The reliability of a precolumn derivatization method hinges critically upon the reproducibility of the derivatization and the stability of the derivatives. Though previous studies indicate that all amino acid derivatives of (dimethylamino)naphthalenesulfonyl chloride (DNS-C1) (6, 7), o-phthaldialdehyde (OPA) (8-13), 4-fluoro-7-nitrobenzo-2,3-oxadiazole (NDB-F) (14,15),phenylisothiocyanate (PITC) (16,17),and DABS-Cl(l8-26) can be reproducibly prepared and detected a t the low picomole level, their stabilities vary. The sulfonamide bonds of the DNS- and DABS-amino acids are comparably the most stable ones. In this paper, we have demonstrated that all protein DABS-amino acids can be left a t room temperature, under daylight, in the dilution solution for up to 1 month without any detectable degradations. This property ensures the reliability of the DABS-Cl method. DABS-amino acids are also unique because they are detected in the visible region. At low picomole level, a stable base line can be obtained with a large variety of solvents and gradient systems. Indeed, DABS-amino acids are readily detected at the femtomole level (Figure 1) and the detection limit can be further improved by using a photothermal refraction detector (26). The intrinsic detection limit of DABS-amino acids should in theory allow hydrolysis and subsequent composition analysis of picograms of proteins. However, before reliable methods are developed to isolate sub-nanogram quantities of proteins and ways are found to reduce the background contaminants of the blank samples down to the low femtomole level, it is only practical to hydrolyze 50 ng to 1 pg of proteins and analyze portions of them. This sensitivity level has been very useful to us since we have routinely received samples less than 1 pg for structure analysis. It is important that one need only 10% of these samples for
NO. 12, OCTOBER 1986
2379
amino acid determination before sequence analyses are undertaken. Limitations of the DABS-Cl Method. There are limitations of the DABS41 method which are inherent in the precolumn derivatization. First, it is necessary to know the approximate amount of protein samples that are hydrolysed and dabsylated. With a given dabsylation condition, the amount of DABS-Cl (in molar) should be at least 4-fold higher than the total amino acids generated from hydrolysis in order to achieve quantitative derivatization. For instance, when 20 pL of sodium bicarbonate buffer and 40 p L of DABS-Cl solution (160 nmol of DABS-Cl) are applied, the amount of hydrolyzed protein to be dabsylated should be kept under 4 pg. Naturally, one can hydrolyze more than 4 pg of protein and use a protion of the hydrolyzate for dabsylation. Second, the presence of an excess amount of salt, such as urea, SDS, phosphate, or ammonium bicarbonat, will alter the pH of the buffer and interfere with the dabsylation reaction. I t is preferable to prepare samples in salt-free form to ensure accurate amino acid composition analysis.
ACKNOWLEDGMENT The authors thank C. S. Liu for his comments on this manuscript. Registry No. Asp, 56-84-8;Glu, 56-86-0; Ser, 56-45-1; Thr, 72-19-5; Gly, 56-40-6; Ala, 56-41-7; Arg, 74-79-3; Pro, 147-85-3; Val, 72-18-4; Met, 63-683;ne, 73-32-5; Leu, 61-90-5; Phe, 63-91-2; Lys, 56-87-1; His, 71-00-1; Tyr, 60-18-4;DABS-Cl, 56512-49-3; HC1, 7647-01-0; calcitonin (eel), 57014-02-5.
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RECEIVED for review April 10,1986. Accepted June 19,1986.
