Separation of 18 6-Aminoquinolyl-carbamyl-Amino Acids by Ion-Pair Chromatography

ANALYTICAL BIOCHEMISTRY ARTICLE NO.

249, 79–82 (1997)

AB972155

Separation of 18 6-Aminoquinolyl-carbamyl-Amino Acids by Ion-Pair Chromatography Noriko Shindo, Saiko Nojima, Tsutomu Fujimura, Hikari Taka, Reiko Mineki, and Kimie Murayama Division of Biochemical Analysis, Central Laboratory of Medical Sciences, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan

Received December 11, 1996

Eighteen 6-aminoquinolyl-carbamyl (AQC)-amino acids were separated by ion-pair chromatography, using tetrabutylammonium (TBA) as a counterion. Optimum separation was obtained on a C8 reverse-phase column using gradient elution with two mobile phases, A (5 mM TBA, 75 mM ammonium acetate, pH 7.5) and B (80% acetonitrile). The AQC-amino acids were detected by fluorescence with excitation at 250 nm and emission at 395 nm, and the analysis time was 65 min. The response factors of individual AQC-amino acids to AQCphenylalanine ranged from 0.42 to 1.08 (except for tryptophan at 0.01), with an average of 0.8. Detection limits by fluorescence ranged from 11.8 fmol (threonine) to 51.7 fmol (methionine), except for tryptophan (1.8 pmol). q 1997 Academic Press

ratio to phenylalanine ranging from 0.20 to 0.39 and notably that for cystine being 0.05), and the other is that tryptophan cannot be detected by fluorescence. The low response factors of hydrophilic AQC-amino acids can be improved by postcolumn mixing with organic solvent (5). However, this is complicated and requires another pump for mixing with the solvent. Here we describe the coupling of AQC-amino acids possessing one or two carboxylic groups with a counterion such as TBA to obtain hydrophobicity, thus allowing ion-pair chromatography for AQC-amino acid analysis.

MATERIALS AND METHODS

Chemicals A highly sensitive amino acid analysis method is sometimes needed to confirm the data obtained from a protein sequencer or mass spectrometer, and several methods employing fluorescence detection can be used for this purpose (1–4). Cohen and Michaud have developed an excellent separation method for AQC1-amino acids using a C18 reverse-phase column (Nova-Pak) with 140 mM sodium acetate buffer (pH 5.05) and 17 mM triethylamine as mobile phase A and 60% MeCN as mobile phase B (1). This facilitates sub-picomolar level detection and simple derivatization and requires no extraction of excess reagents, and there is no contamination after derivatization. However, the most important problem is the low response factors of all hydrophilic amino acids (the 1 Abbreviations used: AQC, 6-aminoquinolyl-carbamyl; TBA, trichlorobenzoic acid; AHC, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; a-ABA, a-amino-n-butyric acid.

6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AHC) was obtained from Millipore (Millipore, Milford, MA). Boric acid, acetic acid, ammonium acetate (AcONH4), and acetonitrile (MeCN, HPLC grade) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Borate buffer (pH 8.8) was prepared at 0.2 M. TBA as an ion-pair reagent for acid compounds and a-amino-n-butyric acid (a-ABA) as an internal standard were purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). The amino acid standard solution, Type H, was purchased from Wako Pure Chemical Industries, Ltd., and tryptophan was added when needed. Lysozyme (egg white) was obtained from Sigma Chemical Co. (St. Louis, MO), and chymotrypsinogen A (bovine pancreas) was from Boehringer-Mannheim (Mannheim, Germany). All aqueous solutions were prepared with ultrapure water (analytical grade) using a Milli-Q water purification system. 79

0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

AID

AB 2155

/

6m36$$$$21

05-13-97 19:21:08

abas

80

SHINDO ET AL.

Derivatization of AQC-Amino Acids

TABLE 1

Derivatization of standard amino acids and acid hydrolysates of proteins with AHC was carried out by the method of Cohen and Michaud (1). Ten microliters of standard solution (100 pmol/ml) and acid hydrolysates (10 pmol/ml) was mixed with 70 ml of 0.2 M borate buffer (pH 8.8). Then 20 ml of 10 mM AHC in MeCN was added. The reaction mixtures were kept at room temperature for 1 min and then heated at 557C for 10 min. Five picomoles of AQC-amino acids or acid hydrolysates of proteins was loaded onto the column. Separation of AQC-Amino Acids A Gilson auto-LC system (including uv monitor 117, column oven, and autosampler; Gilson, France) equipped with a fluorescence photometer (Hitachi F1150, Katsuta, Japan) was used for AQC-amino acid analysis. Five picomoles of AQC-derivatives was separated on a C8 reverse-phase column (Pegasil C8 , 5 mm, ˚ , 4.6 mm i.d. 1 150 mm; Senshu Scientific Co., 120 A Ltd., Tokyo, Japan) using gradient elution with two mobile phases: phase A consisted of 5 mM TBA and 75 mM AcONH4 and was adjusted to pH 7.5, and phase B was 80% MeCN solution. The gradient programs and the column temperature are shown in Table 1. Detection was done by fluorescence with excitation at 250 nm and emission at 395 nm. Acid Hydrolysis by Vapor Phase of Proteins One hundred picomoles (20 ml) of lysozyme or chymotrypsinogen A in each inner glass tube (5.2 mm i.d. 1 46.2 mm) was added with 1 nmol of a-ABA (200 ml) and dried, respectively. The tubes were placed in outer glass tubes (9.6 mm i.d. 1 150 mm) which contained

FIG. 1. Chromatogram of 5 pmol of the AQC-amino acid standard mixture. Mobile phase A was 5 mM TBA, 75 mM AcONH4 , pH 7.5, and mobile phase B was 80% MeCN. AQC-amino acids are shown by one-letter code for standard amino acids.

AID

AB 2155

/

6m36$$$$22

05-13-97 19:21:08

Elution Program Time (min)

Mobile phase B (%)

Time (min)

Column temperature (7C)

0.00 3.00 3.01 28.00 43.00 43.01 65.00 65.01 70.00 70.01

7.5 7.5 10.6 28.1 22.1 30 (35) a 30 (35) a 100.0 100.0 7.5

0.00

30

30.00

35

40.00

30

a

Separation for tryptophan.

200 ml of 6 N HCl including 0.2% phenol. They were then evacuated and sealed, and acid hydrolysis was carried out at 1657C for 25 min. The inner tubes were then dried using a CC-105 centrifugal concentrator (Tomy Seiko, Tokyo, Japan), and the hydrolysates were dissolved in 20 ml of 0.01 M HCl and then derivatized. Finally, each 5 ml of the acid hydrolysates was loaded on the AQC-amino acid analysis column. RESULTS AND DISCUSSION

AQC-Amino Acid Analysis by Ion-Pair Chromatography Figure 1 shows the pattern obtained with 5 pmol of the AQC-amino acid standard mixture by ion-pair chromatography. This was obtained using a special elution program which was part of the reverse slope for mobile phase B between 28 and 43 min in order to resolve tyrosine and valine completely (Table 1). Ammonia and aminoquinoline (AMQ) appeared between arginine and histidine when isocratic 7.5% mobile phase B was run initially for 3 min. The fluorescence intensity of AQC-amino acids in the method was higher than in Cohen’s method. He reported that the fluorescence intensity of AQC-amino acids increased at a higher concentration of MeCN and a higher pH (1). However, the maximal concentration of mobile phase B was 30% as shown in Table 1, in which the final concentration of MeCN was 24%. Despite the lack of TBA and AcONH4 in mobile phase B, the eluent was kept at pH 7.5 by the end of analysis. On the other hand, TBA as a counterion affected the sensitivity of ion-pair chromatography. However, the higher concentration of TBA (10 or 15 mM) gave poor chromatogram resolution because some of the AQCamino acids eluted faster. The optimum concentration of TBA was finally set at 5 mM. All AQC-amino acids

abas

81

SEPARATION OF 6-AMINOQUINOLYL-CARBAMYL-AMINO ACIDS

Calibration Curve and Detection Limit

TABLE 2

Response Factors of AQC-Amino Acids a Amino acid

Response factor

Arginine Histidine Serine Glycine Proline Threonine Alanine Aspartic acid Glutamic acid Tyrosine Valine Methionine Isoleucine Lysine Leucine Cystine Phenylalanine Tryptophan

0.59 0.81 1.01 0.98 0.59 1.08 0.99 0.76 0.91 0.49 0.85 0.53 1.02 0.72 1.07 0.42 1.00 0.01

a The response factor was calculated using the ratio of each amino acid peak area to the phenylalanine peak area.

were eluted within 65 min using the method, compared with an analysis time of within 40 min for Cohen’s method. This was because AQC-amino acids coupled with TBA, which has a bulky structure, formed molecules that were more hydrophobic and thus eluted slowly. The relative response factors for all AQC-amino acids were calculated using the ratio of each amino acid to phenylalanine (Table 2). The response factors of hydrophilic AQC-amino acids (arginine, histidine, aspartic acid, and glutamic acid) were even higher than those of hydrophobic amino acids. Lower response factors were obtained for cystine, tyrosine, and methionine (0.42, 0.49, and 0.53, respectively), and the lowest value was for tryptophan (0.01). However, the average response factor value was 0.8, including tryptophan, which was higher than for Cohen’s original method. Tryptophan and Cysteine As tryptophan and cysteine play an important functional role in many proteins, quantitation of these amino acids is important for analysis of protein hydrolysates. AQC-tryptophan was separated using the modified elution program, in which the maximal concentration of mobile phase B was 35% (Table 1), and detected by fluorescence, even though the detection limit was 1.8 pmol and the retention time was 58.8 min. Also, the response factor of cystine was similar to that of tyrosine. Analysis of cystine was greatly improved in comparison with Cohen’s method (Table 2).

AID

AB 2155

/

6m36$$$$22

05-13-97 19:21:08

The calibration curves of AQC-amino acids over the 1 range 0.5 to 1000 pmol as the derivatized amount (10 of the derivatized amount was injected) demonstrated excellent linearity, with correlation coefficients ranging from 0.9989 to 0.9998, as shown in Table 3. Table 3 also shows a list of the detection limits of 18 AQCamino acids, which ranged from 11.8 to 51.7 fmol, except for tryptophan (1.83 pmol). Quantitation of Protein Acid Hydrolysates There were some problems with the microanalysis of protein hydrolysates such as trace amounts of amino acids contaminated or degraded during sample preparation. The amino acid composition of chymotrypsinogen A, as one of the sample proteins, is shown in Table 4. This should have been corrected using a hydrolysate of a standard protein such as lysozyme, which has a known amino acid composition. These were hydrolyzed under the same conditions. The recoveries ranged from 38 to 108% for lysozyme and from 35 to 100% for chymotrypsinogen A, as shown in Table 4. However, the corrected recoveries of cysteine, methionine, proline, and tryptophan using lysozyme hydrolysate were largely improved without premodification and corresponded with the theoretical recoveries. All corrected values were close to the theoretical values.

TABLE 3

Linearity of Calibration Curves and Detection Limits Amino acid

Correlation coefficient r 2 a

Detection limit (fmol) b

Arginine Histidine Serine Glycine Proline Threonine Alanine Aspartic acid Glutamic acid Tyrosine Valine Methionine Isoleucine Lysine Leucine Cystine Phenylalanine Tryptophan

0.9997 0.9995 0.9998 0.9998 0.9996 0.9997 0.9994 0.9989 0.9992 0.9997 0.9996 0.9997 0.9997 0.9994 0.9997 0.9998 0.9997 0.9999

12.2 22.8 12.6 13.3 22.7 11.8 12.3 16.3 13.8 31.1 19.7 51.7 22.7 30.2 16.0 49.9 24.4 1830.0

a

Amino acids (the amounts of each amino acid ranged from 0.5 to 1000 pmol) were derivatized and loaded onto the HPLC column using 1/10 of the derivatized amount. b Detection limits were calculated from a 1-pmol (5 pmol for tryptophan) injection and based on S/N Å 3.

abas

82

SHINDO ET AL. TABLE 4

Amino Acid Composition of Chymotrypsinogen Aa Standard (lysozyme)

Sample (chymotrypsinogen A)

Amino acid

Theoretical value

Experimental value

Recovery (%)

Theoretical value

Experimental value

Recovery (%)

Corrected value b with lysozyme

Cor/Theo (%)

Alanine Arginine Aspartic acid Cysteine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

12 11 21 8 12 5 1 6 8 6 2 3 2 10 7 6 3 6

11.8 10.5 20.5 3.0 12.1 5.4 0.8 5.5 8.2 5.1 1.5 3.0 1.0 8.1 6.2 3.6 2.8 5.6

98 96 98 38 101 108 80 92 103 85 75 100 50 81 89 60 93 93

22 4 23 10 23 15 2 10 19 14 2 6 9 28 23 8 4 23

19.6 3.7 21.1 3.5 21.0 14.1 1.8 8.5 18.0 13.6 1.1 6.0 4.3 19.8 17.8 4.7 3.5 19.7

89 93 92 35 91 94 90 85 95 97 55 100 48 71 77 59 88 86

19.8 3.9 21.7 9.3 20.9 13.1 2.2 9.3 17.6 16.1 1.5 6.0 8.6 24.6 20.0 8.0 3.7 21.1

90 97 94 93 91 87 109 93 93 115 75 100 96 88 87 100 94 92

a b

Injected amount: 5 pmol of the hydrolysate of chymotrypsinogen A (100 pmol). [Theoretical value (lysozyme)/experimental value (lysozyme)] 1 experimental value (chymotrypsinogen A).

ACKNOWLEDGMENT We thank Dr. Yasuyuki Kurosu, JASCO Technical Research Laboratories Co., for useful suggestions and discussion.

REFERENCES 1. Cohen, S. A., and Michaud, D. P. (1993) Anal. Biochem. 211, 279–287.

AID

AB 2155

/

6m36$$$$22

05-13-97 19:21:08

2. Ou, K., Wilkins, M. R., Yan, J. X., Gooley, A. A., Fung, Y., Sheumack, D., and Williams, K. L. (1996) J. Chromatogr. A 723, 219– 225. 3. Gerogi, G., Pietsch, C., and Sawatzki, G. (1993) J. Chromatogr. Biomed. Appl. 613, 35–42. 4. Simmaco, M., deBiase, D., Barra, D., and Bossa, F. (1990) J. Chromatogr. 504, 129–138. 5. Ookawa, K., and Iwamatsu, A. (1995) Seikagaku 66, 640,1225. [Abstracts of papers presented at the 68th Annual Meeting of the Japanese Biochemical Society]

abas