ANALYTICAL BIOCHEMISTRY 102, 380-383 (1980)

Analysis of Dimethylaminoazobenzene-Thiohydantoins of Amino Acid by High-Pressure Liquid Chromatography

J. Y. CHANG', A. LEHMANN, AND B. WITTMANN-LIEBOLD

Max-Hawk Institut fiir Molekulare Genetik, Abt Wittmann D-1000 Berlin-Dahlem. Germany

Received August 28, 1979

Dimethylaminoazobenzene-thiohydantoins of amino acid can be quantitatively analyzed by high-pressure liquid chromatography at picomole level. As little as 5 to 10 pmol of dimethylaminoazobenzene-thiohydantoins of amino acid can easily be detected in the visible region (436 nm) against a stable baseline. Three amino acid pairs, namely glutamine and threonine, methionine and proline, and leucine and isoleucine, have not yet been separated. This new technique provides a sensitive and efficient tool for measuring the recovery of amino terminal amino acids using the dimethylaminoazobenzene-isothiocyanate method and the repetitive yield of sequence determination using the dimethylaminoazobenzene-isothiocya- nate phenylisothiocyanate double-coupling method.

Stepwise degradation of polypeptide by the dimethylaminoazobenzene-isothiocya- nate (DABITC)2 phenylisothiocyanate (PITC) double-coupling method (1,2) has proven to be a very useful method for micro- sequence analysis of proteins. The sensi- tivity and efficiency of this new method re- lies on the identification of the released N-terminal amino acid as colored dimethyl- aminoazobenzene-thiohydantoin derivative (DABTH). So far, only thin-layer chroma- tography on polyamide sheets (1,3,4) and silica gel plates (5) were developed for identification of DABTH-amino acids. Al- though these techniques are simple, ef- ficient, and perfectly adequate for quali- tative identification of all DABTH-amino acids at picomol level, they are not suitable for quantitative identification of DABTH- amino acids at the same sensitivity level.

’ Present address: Department of Immunology, Mayo Medical School, Rochester, Minnesota 55901.

* Abbreviations used: DABITC, dimethylaminoazo- benzene-isothiocyanate; PITC, phenylisothiocyanate; DABTH, dimethylaminoazobenzene-thiohydantoin; hplc, high-pressure liquid chromatography.

We report here the use of high-pressure liquid chromatography (hplc) for quanti- tative analysis of DABTH-amino acids at picomol scale.

MATERIALS AND METHODS

DABITC was synthesized by the method described (6,7) or purchased from Fluka (recrystallized from boiling acetone before use). Solvents used in this report were commercial analytical grade.

Preparation of standard DABTH-amino acids. A solution of 1 mg of amino acid (ca. 5 pmol) in 100 ~1 triethylamine-acetate buffer (0.4 ml triethylamine + 5 ml 0.2 M acetic acid + 50 ml water + 50 ml acetone, pH 10.4) was treated with 50 ~1 of DABITC solution (2 nmoVp.1 in acetone) and heated at 52°C for 1 h. The mixture was dried in a vacuum dessicator (care must be taken that samples do not bump) and redissolved in 20 ~1 of water and 40 ~1 of acetic acid saturated with HCl (4). The solution was heated with constant agitating at 52°C for 50 min and then dried in a vacuum dessicator. The DABTH-amino acid residue was dis-

0003-2697/80/040380-04$02.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

380

ANALYSIS OF COLORED AMINO ACID THIOHYDANTOINS 381

solved in 2 ml of 80% ethanol and used as standard sample (1 nmol/M ~1) for hplc.

Most DABTH-amino acids thus pre- pared were free from other colored by- products except for asparagine, glutamine, serine, and threonine which were con- taminated by trace amounts of deaminated product or minor amounts of dehydro- product (1,8). The presence of excess amino acid did not interfere with the chromato- graphic separation of DABTH-amino acid.

Performance of hplc. High-pressure liq- uid chromatographic analyses were per- formed with a Waters Associate Model 204 Liquid Chromatograph which comprises a Model 440 uv (254 nm) and visible (436 nm) dual wavelength detector, a Model 6000 A solvent delivery system, and was equipped with a Model 660 solvent programmer. DABTH-amino acids (dissolved in 5 to 20 ~1 of ethanol or 80% ethanol) were in- jected automatically with a Model 710 A sample processor. Column (RP8,7pm) was purchased from Kanuer (West Berlin).

Manual sequence analysis ofpolypeptide.

Manual sequence analysis of peptide and protein (2 to 8 nmol) were carried out by using the liquid-phase DABITC-PITC method (1) or solid-phase DABITC-PITC method (2). The released DABTH-amino acids were dissolved in ethanol and applied on hplc.

RESULTS

Separation of 18 DABTH-amino acids on the Kanuer RP8 column were shown on Figs. 1 and 2. Resolution of three DABTH- amino acid pairs, namely threonine and glutamine, methionine and proline, and leucine and isoleucine, has not yet been achieved. Thirty to fifty picomol of DABTH- amino acid appears to be the optimum amount when the absorbance unit of the de- tector was set at 0.01 to 0.02. However, as little as 10 pmol of DABTH-amino acid can be detected (Fig. 2).

Compared with the thin-layer chroma- tography method (l-5), the hplc method has the distinct advantage of analyzing

1’ 4 8 12

FIG. 1. High-pressure liquid chromatography of standard DABTH-amino acids (ca. 100 pmol each). Chromatographic conditions are: Column, Kanuer RP8 7 pm; Column temperature, 40°C; Solvent A. 8 mM acetate pH 5: Solvent B, 90% methanol; Gradient, linear (No. 6) from 60% B to 85% B in 20 min; Flow rate, I.5 mllmin; Detector, visible region at 436 nm.

382 CHANG, LEHMANN, AND WITTMANN-LIEBOLD

FIG. 2. Separation of approximately 10 pmol each of DABTH-amino acid by hplc. Conditions are outlined in the legend of Fig. 1.

DABTH-amino acids quantitatively at the picomol scale, but has the disadvantage of being unable to resolve all the DABTH- amino acids (including the destroyed prod- ucts of DABTH-serine and DABTH- threonine, see Ref. (1) and (16)) as well as being unable to discriminate the color dif- ference between the DABTH-derivative and the DABTC-derivative.

Analysis of the DABTH-amino acid sam- ples from the manual solid-phase DABITC- PITC degradations (2) are presented in Figs. 3, 4, and 5. Repetitive yields are 91% for the N-terminal sequence of ribosomal pro- tein L29 (Fig. 3), 95.5% for the N-terminal

4

3

12

g

I

1 MK

A

K

sequence of myoglobin (Fig. 4), and nearly 99.4% for the hexapeptide (TLLNEK) from ribosomal protein L29 (9) (Fig. 5).

DISCUSSION

High-pressure liquid chromatography has been widely used for identification of amino acid phenylthiohydantoins (lo- 14) during the sequence analysis of proteins using automatic Edman degradation (15). The re- sults shown in this report indicate that hplc is also a valuable technique for analyzing DABTH-amino acids.

The sensitivity for detecting DABTH- amino acid at 436 nm (molar extinction

No. Residue

FIG. 3. N-Terminal sequence of ribosomal protein L29 determined by the solid-phase DABITC- PITC method (2). DABTH-amino acids were quantitatively analyzed by hplc using the conditions described in Fig. I. Residue 1 (met) 2, 4, and 9 (Lys) were attached on the solid support.

ANALYSIS OF COLORED AMINO ACID THIOHYDANTOINS 383

coefficient is 34,000 at 420 nm) is about two- fold higher than that for amino acid phenyl- thiohydantoin at 254 nm (molar extinction coefficient is 16,000 at 269 nm). The sen- sitivity of detecting DABTH-amino acid could be further increased by employing acidic solvent as, in acidic solvent, the molar extinction coefficient of DABTH-amino acid is 47,000 (16).

The upper limit of sensitivity of detecting both amino acid phenylthiohydantoin (in uv region) and DABTH-amino acid (in visible region) is dependent upon the noise of base- line. In the uv region, trace amounts of uv absorption impurities presented in the eluting solvent usually result in a significant rise of the background. Detection at the visible region avoids this interference and the ab- sorbance unit of the detector can routinely be set at 0.02 to 0.005, which permits the detection of 2 to 10 pmol of DABTH-amino acids.

Quantitative analysis of DABTH-amino acids by hplc has provided a sensitive and efficient tool for evaluation of the recovery of the iv-terminal amino acid (8), as well as the repetitive yield of sequence determina- tion (1,2). Moreover, the fact that as little as IO pmol of DABTH-amino acid can be detected against a stable baseline encour- ages the possibility of performing high sen- sitivity amino acid analysis by derivatizing amino acid (as DABTH-derivative) prior to column chromatography, an approach which is different from the conventional ion-ex- change chromatographic method (17- 19).

3

" 2

E

1

1

No. Residue

FIG. 4. Recoveries of DABTH-amino acids from the N-terminal sequence of myoglobin degraded by the solid-phse DABITC-PITC method (2).

3 L LNE

2 22 z c

1

::- 2 3 4 5

No. Residue

FIG. 5. Recoveries of DABTH-amino acids from the sequence analysis of a hexapeptide (TLLNEK, tryptic peptide T, of ribosomal protein L 29) deter- mined by the solid-phase DABITC-PITC method (2).

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