78

Amino Acid Analysis Using Precolumn Derivatization with Phenylisothiocyanate

G. Brent Irvine

1. Introduction The method described in this chapter is based on derivatization of amino acids,

produced by hydrolysis of peptides or proteins, with phenylisothiocyanate. This forms phenylthiocarbamyl amino acids, which are then separated by reversed-phase high-performance liquid chromatography and quantified from their UV absorbance at 254 nm.

Quantitative amino acid analysis, based on separation by ion-exchange chroma- tography followed by postcolumn derivatization using ninhydrin for detection, was developed during the 1950s (1) and remained the predominant method for 20 years. With the advent of reversed-phase high-performance liquid chromatography, how- ever, rapid separation of amino acid derivatives became possible. Precolumn derivatization also avoids dilution of peaks and so increases sensitivity. Methods involving derivatization with fluorogenic reagents, such as dansyl chloride (2) and o-phthaldialdehyde (3), were the first to be developed, and these enabled detection of <1 pmol of an amino acid. These methods have some disadvantages, however, including instability of derivatives, reagent interference, and lack of reaction with secondary amino acids. Derivatization using phenylisothiocyanate, the reagent used in the first step of the Edman method for determining the sequence of proteins, avoids many of these problems. The reaction, shown in Fig. 1, is rapid and quantitative with both primary and secondary amino acids.

The products are relatively stable, and excess reagent, being volatile, is easily removed. Sensitivity, at about the level of 1 pmol, is more than adequate, since it is difficult to reduce background contamination below this level. Quantitative analysis of phenylthiocarbamyl amino acids was first described by Knoop and coworkers (4). Full details of the application of this method to the analysis of protein hydrolysates were published in 1984 (5). A similar procedure was published (6) by employees of the Waters Chromatography Division of Millipore Corporation, and the equipment that they developed is commercially available as the Waters Pico-Tag | system (Millipore, Milford, MA). The method described in this chapter was carried out using Waters' equipment, but equivalent instrumentation could be used. A review (7) describes the application of the method to the analysis of free amino acids in physiological fluids, amino acids in foodstuffs, and unusual amino acids.

From: The Protein Protocols Handbook Edited by: J. M. Walker Humana Press Inc., Totowa, NJ

467

468 Irvine

~_~ -N=C=S + H2N-CHR-CO0-

I pH 9-10 S U -NH-C-NH-CHR-CO0-

Fig. 1. Reaction of phenylisothiocyanate with an amino acid to form a phenylthiocarbamyl amino acid derivative.

2. Materials 2.1. Apparatus

Two pieces of equipment are required. The first is for hydrolysis of proteins and for derivatization of the resulting amino acids. This can be carried out using the Pico-Tag work station, which comprises an oven that can accommodate up to four vacuum vials, and a vacuum/purge manifold with vacuum gage and a cold trap. Also required are a nitrogen line and a vacuum pump. Up to 12 tubes (6 x 50 mm, Waters), each containing a sample of protein for hydrolysis, can be inserted into a vacuum vial. The vial can be sealed with an air-tight plastic screw cap that has a drilled-through Teflon TM

valve fitted with a slider control that can be pushed to seal or release the vacuum. To the bottom of the vacuum vial is added HC1. The top is screwed on and inserted into the vacuum manifold, and the vial is evacuated using a vacuum pump and cold trap. The valve is then sealed, and the vial is removed and heated in the built-in oven, causing hydrolysis of protein by HC1 in the vapor phase. The vial has a bulge in the middle to prevent condensing HC1 from running down the inside into the sample tubes. After removal o f HC1 under vacuum, phenylthiocarbamyl amino acids are formed by addi- tion of phenylisothiocyanate. Excess reagent is then removed under vacuum.

The second piece of equipment is a reversed-phase high-performance liquid chro- matography system for separation and quantification of the phenylthiocarbamyl amino acids. This requires two pumps, a gradient controller, an automated injector, a C18 column and column heater, a UV detector set at 254 nm, and an integrator.

2.2. Chemicals (see Note 1) 2.2.1. Hydrolysis and Derivatization

1. HC1/phenol: add melted crystalline phenol (10 pL) to hydrochloric acid (constant boiling at 760 mm) (1.0 mL) (Pierce, Rockford, IL).

2. Redry solution: ethanol:water:triethylamine (2:2:1 by vol). 3. Derivatization solution: ethanol:water:triethylamine:phenylisothiocyanate (7:1" 1" 1 by

vol). Vortex and allow to stand for 5 min before using. Use within 2 h. Phenylisothio- cyanate (Pierce): Store at -20~ under nitrogen. After opening an ampule, it can be di- vided into aliquots that should be resealed under nitrogen. It is important to allow the container to come to room temperature before opening, since this reagent is sensitive to moisture. Each aliquot should be used within 3 wk of opening.

Amino Acid Analysis 469

4. Amino Acid Standard H (Pierce): This contains a solution of 17 amino acids (2.5 mM each, except cystine, which is 1.25 mM) in 0.1N HC1.

2.2.2. Chromatography 1. Sample buffer: Dissolve anhydrous sodium dihydrogen phosphate (0.71 g) in water (1 L).

Adjust the pH to 7.4 with 1% (v/v) orthophosphoric acid. Add 52.6 mL acetonitrile. Filter through a 0.22-1am filter (Millipore type GV).

2. Eluent A: Dissolve sodium acetate trihydrate (19.0 g) in water (1 L). Add triethylamine (0.5 mL). Adjust the pH to 5.7 with acetic acid (see Note 2). Add acetonitrile (63.8 mL). Add 1.07 mL of a solution of ethylenediaminetetra-acetic acid dipotassium salt (100 mg) in water (100 mL). Filter through a 0.22-1am filter (Millipore type GV).

3. Eluent B: Mix acetonitrile (600 mL) with water (400 mL). Filter through a 0.22-~tm filter (Millipore type GV).

3. Methods 3.1. Hydrolysis and Derivatization

1. To a pyrex tube (6 x 50 mm), add an aliquot (10 l.tL) of a solution (2.5 mM) of peptide or protein in a volatile solvent, such as methanol or water (see Note 3). Volumes in excess of 50 l.tL should not be used, since solutions may "bump" out of the tube. Place these tubes into the vacuum vial.

2. Dry under vacuum on the work station until the pressure has fallen to 65 mtorr. Drying times using the work station depend on the efficiency of the vacuum pump. With a good pump, this step should take <1 h.

3. To the bottom of the vial (not into the tubes), add HCl/phenol (200 laL). 4. Flush the vial with oxygen-free nitrogen gas, and then evacuate to 1-2 torr. Repeat this

twice, sealing the vial after the third evacuation step. 5. Place the vial in the heating block of the work station at 110~ for 22 h (see Note 4). 6. Remove the vial, and allow it to cool. Remove excess HC1 from the outside of tubes by

wiping with a tissue. 7. Add to the vial two tubes, each containing an aliquot (10 ~tL) of Amino Acid Standard H.

Dry under vacuum on the work station until the pressure has fallen to 65 mtorr (about 1 h). 8. Clean out the cold trap to remove any traces of acid that might react with cyanate in the

next stages. 9. To each tube, add redry solution (20 laL) and vortex.

10. Dry under vacuum on the work station (about 30 min). 11. To each tube, add derivatization solution (20 l.tL) and vortex. Leave at room temperature

for 20 min. 12. Dry under vacuum on the work station. After the pressure has dropped to 65 mtorr (about

2 h), leave for a further 10 min to ensure complete removal of phenylisothiocyanate.

3.2. Chromatography 1. To each tube, add sample buffer (200 laL) and vortex. The relatively large volume added

here facilitates the next (filtration) step. Only a small proportion of the filtered sample is analyzed. If sample quantity is limited, see Note 5.

2. Filter the samples through a 0.45-1am filter (Millipore type HV). Use a 1-mL plastic sy- ringe and needle to withdraw samples. Then place the filter over the syringe and add a fresh needle to ensure that the sample is placed at the bottom of the tube from which it will be injected for chromatography. This should be carried out as soon as possible, and cer- tainly within a few hours if samples are left at room temperature (see Note 6).

3. Place the Pico-Tag column in the heating module at 39.0~ and commence flow of Eluent B (in which the column is stored) at a rate of 1.0 mL/min.

470 Irvine

Table 1 Gradient Table Time, Flow, min mL/min %A %B Curve no. a

Initial 1.0 100 0 * 10.0 1.0 54 46 5 10.5 1.0 0 100 6 11.5 1.0 0 100 6 12.0 1.5 0 100 6 12.5 1.5 100 0 6 20.0 1.5 100 0 6 20.5 1.0 100 0 6

aThe column headed "Curve No." refers to the setting used on the Waters gradient controller. Number 6 is linear, whereas number 5 is a shallow convex curve (the rate of change is greater in the earlier part of the period).

4. Run a linear gradient from 100% Eluent B to 100% Eluent A during 2 min. Allow the column to equilibrate for 30 min in the latter mobile phase.

5. Inject an aliquot (5 ~tL) of standard using the gradient shown in Table 1. Run time should be set at 21.0 min. The use of an automatic injector, such as the WISP (Waters), is highly recommended (see Note 7).

6. The second and third injections should be of the same solution (standard) as the first. Invariably, poor chromatography is obtained with the first injection, but good separation should be achieved with the second and third injections. These chromatograms should be checked to ensure that this is the case before proceeding with further samples.

7. Inject an aliquot (5 laL) of each unknown sample, recording the absorbance at 254 nm. 8. After the run, peaks are integrated, and the amino acid composition of the unknown is

determined by comparison with peak areas given by the Amino Acid Standard H.

4. Notes 1. The highest quality of reagents should be used. Suitable water can be obtained using a Milli-

Q purification system (Millipore, Bedford, MA) fed by a supply of tap water that has been distilled once. For other chemicals, suppliers of some suitable grades are suggested in Sec- tion 2. This is particularly important if high sensitivity is required (see Note 5).

2. Other workers often adjust the pH of Eluent A to 6.35 (6) or 6.40 (7) rather than the lower pH of 5.7 described above. At this lower pH, the resolution of Asp and Glu is increased.

3. Protein samples should be free of salts, amines, and detergents, although it has been reported that salts at concentrations up to 4M do not affect derivatization or separation (8).

4. Rapid hydrolysis may be carried out at 150~ for 1 h (6). However, more satisfactory results are obtained using the lower temperature and longer time described in Section 3.1. Acid hydrolysis of proteins causes conversion of asparagine and glutamine to aspartic acid and glutamic acid, respectively. Serine, threonine, and, to a lesser extent, tyrosine are slowly destroyed (serine suffers about 10% loss during 22 h at 110~ threonine and tyro- sine rather less than this). Accurate quantification of these amino acids requires hydro- lysis of replicate samples for 24, 48, and 72 h and extrapolation back to zero time. The longer hydrolysis times can also give more accurate values for alanine, valine, and isoleu-

Amino Acid Analysis 471

A 2 5 4

D .,. E

,:,:,

R P A T

H ~ ~

G ,~ . . . . . S ~ ~

u4

Y

~. V ix.

~ L I

.... !/il f i

E l u t i o n T i m e

Fig. 2. Separation of a standard mixture of 17 amino acids after derivatization with phenylisothiocyanate. The peak owing to each phenylthiocarbamyl amino acid is identified using the one-letter code for that amino acid, but with C2 being cystine rather than cysteine. The number above each peak is the elution time in seconds. The absorbance at 254 nm of the largest peak (K) was 0.14.

Amino acid standard H (10 ~L) (Pierce) was treated as described in Section 3.1. The phenylthiocarbamyl derivatives were dissolved in sample buffer (200 ~L), and an aliquot (5 ~tL, containing 625 pmol of each amino acid derivative) was subjected to chromatography as described in Section 3.2. Injections were made using a WISP 712 with two Model 510 pumps and an Automated Gradient Controller onto a Pico-Tag column (3.9 x 150 mm) equilibrated at 39.0~ in a Column Heater controlled by a Temperature Control Module. Peaks were measured by absorbance at 254 nm using a 441 Absorbance Detector (all above equipment from Waters) linked to a Trio Chromatography Computing Integrator (Trivector Inc., West Chester, PA). It can be seen that all the amino acid derivatives have been eluted by 12.1 min. The peaks eluting between F and K and between 13 and 15 min are the result of side reactions of phenylisothiocyanate with nonamino acid material.

cine, since hydrolysis of peptide bonds between certain aliphatic amino acids is incom- plete after 24 h. Methionine, cysteine, and cystine are partially destroyed by acid hydroly- sis with HC1, and for accurate determination, require conversion to more stable derivatives. Tryptophan is totally destroyed by acid hydrolysis with HC1, but may be analyzed after hydrolysis with methanesulfonic acid (7).

After hydrolysis, in order to prevent condensing HC1 from running down the side of the vial into the tubes, keep the tubes upright. With fewer than 12 samples, add blank tubes for support.

5. The amount of each amino acid present in the chromatogram shown in Fig. 2 is 625 pmol. This value is very much higher than the sensitivity limit of the method (about 1 pmol).

472 Irvine

However, if adequate quantities of protein are available, working at this sensitivity will avoid problems owing to background contamination of samples. If limited quantities of protein are available, say 200 pmol (2 lag of a protein of mol wt 10,000), the procedures described above will need to be modified by redissolving dried, derivatized material in less sample buffer and/or injecting a larger volume (maximum 25 ~tL). This will ensure that more of the sample goes onto the column. A corresponding adjustment in the treat- ment of standards will also be required. However, if it is necessary to carry out analyses in the low pmol range, special precautions must be taken, since background contamination (especially of serine and glycine, where it can reach several pmol per sample) is a com- mon problem. These precautions include: pyrolysing glassware at 500~ overnight or steeping in sulfuric acid (250 mL) containing sodium nitrate (1 g); handling with clean gloves (nontalc) and forceps; including a control blank that has been subjected to hydroly- sis. Of course, it is good practice to use these procedures in any case, even for lower sensitivity analyses.

6. Phenylthiocarbamyl amino acids in solution at neutral pH are relatively stable, with < 10% loss of the least stable derivatives (Leu, lie) during 10 h at room temperature. Losses are much reduced in the cold, with <5% loss during 48 h at 4~

7. The use of automatic injectors, apart from their labor-saving function, gives constant in- jection volumes and constant intervals between injections (when using the WISP, this interval is actually about 21.7 min for run time set at 21.0 min). An identical interval between injections is an important criterion for obtaining reproducible retention times for each amino acid in different chromatograms, since the column is not given sufficient time to reequilibrate in Eluent A.

Acknowledgment Thanks are owed to Adrienne Healy for expert technical assistance.

References 1. Spackman, D. H., Stein, W. H., and Moore, S. (1958) Automatic recording apparatus for

use in the chromatography of amino acids Anal. Chem. 30, 1190-1206. 2. Hsu, K-T. and Currie, B. L. (1978) High-performance liquid chromatography of Dns-amino

acids and application to peptide hydrolysates. J. Chromatogr. 166, 555-561. 3. Lindroth, P. and Mopper, K. (1979) High performance liquid chromatographic determina-

tion of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Anal. Chem. 51, 1667-1674.

4. Knoop, D. R., Morgan, E. T., Tarr, G. E., and Coon, M. J. (1982) Purification and charac- terization of a unique isoenzyme of cytochrome P-450 from liver microsomes of ethanol- treated rabbits. J. Biol. Chem. 257, 8472-8480.

5. Hendrikson, R. L. and Meridith, S. C. (1984) Amino acid analysis by reverse-phase high- performance liquid chromatography: precolumn derivatization with phenylisothiocyanate. Anal. Biochem. 136, 65-74.

6. Bidlingmeyer, B. A., Cohen, S. A., and Tarvin, T. L. (1984) Rapid analysis of amino acids using pre-column derivatization. J. Chromatogr. 336, 93-104.

7. Cohen, S. A. and Strydom, D. J. (1988) Amino acid analysis utilizing phenylisothiocyanate derivatives. Anal. Biochem. 174, 1-16.

8. Hendrikson, R. L., Mora, R., and Maraganore, J. M. (1987) A practical guide to the general application of PTC-amino acid analysis, in Proteins: Structure and Function (L'Italien, J. J., ed.), Plenum, New York, pp. 187-195.