Rapid Simultaneous Determination of Tryptophan and Tyrosine in Synthetic Peptides by Derivative Spectroscopy

JACOPO BERTINI*’, CARL0 MANNUCCI*, ROBERTO NOFERINI*, ANDREA PERICO*, AND

Received November 27, 1991 , from *A. Menarini lndustrie Farmaceutiche Riunite S.r,l., Analytical Research Depaftment, Peptide Synthesis Laboratory, Via Set& Santi 3, 50131 firenze, Italy, and *CNR. lstltuto Mutagenesi e Differenziamento, Pisa, Italy. June 22, 1992.

PAOLO ROVERO*

Accepted for publication

Abstract 0 A method for the simultaneous quantitative determination of tryptophan and tyrosine in synthetic peptides by second-order derivative diode-array spectroscopy is reported. The method does not require hydrolysis of the peptides or a derivatization reaction; the sample is dissolved in 0.1 N NaOH and directly scanned between 262 and 264 nm to detect tyrosine and between 304 and 306 nm to detect tryptophan. From these results the peptide content of the synthetic sample c a n be easily calculated in a very simple and fast way. The results obtained by the described method with several peptides are compared with those obtained by classical high-performance liquid chromatographic analysis of amino acids after peptide hydrolysis. A very good correlation was found both for the tryptophanhyrosine molar ratio and the peptide content.

Quantitative determination of tryptophan in peptides and proteins involves methodological and analytical problems that have been the object of many investigations because this residue plays a crucial role in a number of peptides and proteins of biological interest. Most of the commonly used methods analyze tryptophan by chromatography, after hy- drolysis of the peptide. The high lability of tryptophan residues under the normal reaction conditions (6 N HCl at 110 “C1) requires special treatments (e.g., the addition of antioxidant agents2 or the use of enzymatic hydrolysis9, but even these conditions do not always assure good results. Moreover, this type of hydrolysis often leads to nonquantita- tive recovery of the other amino acids? with inevitable imprecision in the determination of the peptide content.

Another class of methods is based on the quantitative spectrophotometric measurement of tryptophan after direct color reactions of hydrolyzed or intact proteins with a variety of reagents, such as p-dimethylamin~benzaldehyde,~ 2-hy- droxy-5-nitrobenzylbromide,6 HNO3,6 HNO,,7 acid ninhy- drin,S and hydroxylamine and ceric cations.9 These methods, particularly those applicable to intact peptides,7-9 are indeed more rapid than the methods based on hydrolysis and chro- matography. However, these methods remain quite tedious and time-consuming because they require carefully con- trolled reaction conditions, long reaction time, and sometimes thermostatization.

Direct spectroscopic determination of tryptophan in the 250-300-nm region is strongly affected by the overlap of the absorption bands of tyrosine. Nevertheless, several direct methods have been proposed that are based on absorption measurements at two wavelengths at neutral pH10 or on the observation that tyrosine is present as the ionized form in alkaline solution (0.1 N NaOH) and shows a shift of the maximum of absorbance to higher wavelengths, thus increas- ing the spectral differences of the two amino acids.llJ2 All these methods need a considerably high amount of protein and are quite affected by other W absorbing components,

including other amino acids like phenylalanine. More re- cently, a method based on second-derivative spectroscopy has been proposed for the determination of aromatic amino acids in proteins.13J4 The mutual interference between the second- derivative bands of tyrosine and tryptophan has also been used16 for the determination of the tryptophanltyrosine ratio, with the possibility to obtain the absolute values by spectro- photometric titration.

We report here a faster method for the simultaneous quantitative determination of tryptophan and tyrosine in intact peptides by “zero-crossing,’’ second-order derivative spectroscopy, a technique recently developed and applied with success for quantitation of binary mixtures of pharmaceutical interest.16-23 Synthetic problems encountered during the study of new peptide antagonists of neurokinin A,24 contain- ing as many as three D-tryptophan and one tyrosine residues out of seven total residues (see Table I) required fast and reliable determination of the molar ratios tryptophan/ tyrosine in the synthetic samples. The present work was aimed at verifying the feasibility of a simultaneous spectro- photometric determination of tryptophan and tyrosine in synthetic peptides, with the goal of a rapid evaluation of their molar ratio and of the peptide content in synthetic peptides containing these amino acids.

The conventional approach to spectrophotometric analysis of a two-component mixture requires the measurement of the absorbance of the two components at two wavelengths to obtain the respective molar absorptivities. Because the ab- sorbance of the mixture at either wavelength will be the sum of the absorbances of both components, the mixture is mea- sured at the two wavelengths and the resulting simple equations are solved to find the concentration of each com- ponent. This approach is effective when the components have significantly different spectra. But, for components with similar spectra, like the aromatic amino acids, this approach is very sensitive to random errors in the measurement of both standards and mixtures.

Such a problem can be solved in two ways. As shown before, by scanning an alkaline solution of peptide it is possible to obtain a shift of the maximum of absorbance of tyrosine to

Table I-Sequences of the Peptides Analyzed by the Spectrophotometrlc Method

Peptide Sequence -

1 2 3 4 5 6 7 8

~ ~~ ~

Tyr-DTrp-Val-DTrp-DTrp-Orn-NH, Asp-Tyr-DTrp-Val-DTrp-DTrp-Orn-NH, Asp-Tyr-DTrp-Val-DTrp-DTrp-Nle-NH, Asp-Tyr-DTrp-Val-DTrp-DTrp-Ala-NH, Asp-Tyr-DTrp-Val-DTrp-DTrp-Leu-NH, Asp-Tyr-DTrp-Val-DTr p-DTrp-Glu-NH, Tyr-Pro-Trp-Thr-Gln-OH TrD-His-TrD-Leu-Gln-Leu-Lvs-Pro-Gly-Gln-Pro-Met-Tyr-OH

0022-3549/93/02OO-O 1 79$02.50/0 0 1993, American Pharmaceutical Association

Journal of Pharmaceutical Sciences I 179 Vol. 82, No. 2, February 1993

higher wavelengths. Moreover, it is well known that deriva- tive spectroscopy (first to fourth order) has been used to detect hidden peaks because resolution of overlapping peaks is enhanced by the derivative process. However, with each successive derivative, the noise is increased and the number of satellites next to the derivative centroid peak is increased, which makes the pattern more complex. In the present work we used the second-order derivative, which provides a good compromise between resolution enhancement and increased noise. Subsequently, to evaluate the limits of sensibility and the linearity of the responses, separate scannings of standard solutions of tryptophan and tyrosine in 0.1 N NaOH at different concentrations were performed. The responses were linear in a concentration range from -50 to -400 nmol/mL for both amino acids. The method was then tested with a series of test solutions containing tryptophan and tyrosine at known concentrations in different molar ratios from 1 5 to 5: 1. The results (Table 11) indicate that recoveries are quite good (within 2 2.2%) for each composition tested.

Finally, a number of synthetic peptides prepared in our laboratory or purchased from a commercial source were analyzed by the spedrophotometric method and by a classical method, namely hydrolysis of peptides by 6 N HC1 (containing 0.2% thioglycolic acid) and high-performance liquid chro- matographic (HPLC) analysis of amino acids. The results are in very good agreement both for the molar ratio tryptophan/

0.02017-

N

0.01090-

Y = - c 0.00162- = -

-.00765- e

Table 11-Simultaneous Spectrophotometrlc Determination of Tryptophan and Tyroslne In Binary Mixtures

-.01692.

Experimentally Original Determined % TAtir Amino Acid Concentration, Concentration,

nmol/mL nmol/rnL

1:l

1 :2

1 :3

1 :4

1 :5

2:l

2:3

2:5

3:l

3:2

3:4

3:5

4: 1

4:3

4:5

5: 1

5:2

5:3

5:4

237.7 262.3 158.4 349.7 11 8.8 393.4 95.1

419.6 79.2

437.1 31 6.9 174.8 190.1 314.7 135.8 374.7 356.5 131.1 285.2 209.8 203.7 299.7 178.2 327.8 380.3 104.9 271.6 224.8 21 1.2 291.4 396.1 87.4

339.5 149.9 297.1 196.7 264.1 233.1

247.3 260.0 162.2 351.2 122.5 396.2 99.7

41 9.5 82.2

437.9 321.4 175.7 203.3 306.2 137.3 379.6 356.1 135.6 290.2 21 1.7 209.7 300.3 181.9 331.4 380.2 108.5 275.3 228.6 215.7 294.7 395.3 91.4

341.1 153.6 299.6 201 ‘2 267.5 235.2

104.1 99.1

102.6 100.5 103.1 100.7 104.8 100.0 103.7 100.2 101.4 100.5 106.9 97.3

101.1 101.3 99.9

103.4 101.7 100.9 102.9 100.2 102.1 101.1 100.0 103.4 101.4 101.7 102.1 101.1 99.8

104.6 100.5 102.5 100.8 102.3 101.3 100.9

tyrosine and for the peptide content of the synthetic sample (Table 111). The undestructive character of the spedrophoto- metric method allows accurate recoveries of the acid-labile tryptophan residues, thus enabling a more reliable determi- nation of the peptide content.

Experimental Section Materials and Reagents-Acetonitrile and tetrahydrofuran were

HPLC grade; constant-boiling 6 N HC1, L-tryptophan, and L-tyrosine standard for spectrophotometric analysis and HPLC analysis of amino acids were analytical grade; and thioglycolic acid, sodium acetate trihydrate, sodium a i d e , borate buffer (0.4 M), o-phthalal- dehyde reagent, and 9-fluorenylmethylchloroformate reagent were analytical grade. The synthetic peptides 1-6 (Table I) were prepared in our laboratory as previously described24; the others were from Novabiochem.

UV Analysis-Absorbance measurements were recorded by sec- ond-order derivative spectroscopy with a photodiode array spedro- photometer (Hewlett-Packard 8452A). Peptides were dissolved in 0.1 N NaOH at a concentration of -200 nmoVmL and scanned in comparison with separate standard solutions of tryptophan and tyrosine at the same concentration in 0.1 N NaOH. The reference was

Table Ill-Molar Ratio Tryptophan/Tyrosine and Peptide Content In Synthetic Peptldes Analyzed by Spectrophotometrlc and Classical Methods

Spectrophotometric Method Hydrolysis/HPLC

Peptide Peptide Peptide

Trpnyr Content, % Trpnyr Content, %

1 2.94 2 2.93 3 3.10 4 2.96 5 3.22 6 3.16 7 0.90 8 1.79

59.8 72.2 77.2 82.1 75.5 79.2 83.5 86.0

3.01 2.96 3.07 3.10 2.87 2.72 1.04 1.73

57.8 68.3 76.1 77.7 69.7 74.9 83.0 87.0

n B

I

P -‘-5931 I1 \ I 250 300 350 4m

UeVELENGTH ~

-.016W , , , . 200

Flgure 1-Second-derivative spectra of (A) tryptophan and (B) tyrosine solutions at 200.94 and 200.54 nrnol/mL in 0.1 N NaOH, respectively.

180 I Journal of Pharmaceutical Sciences Vol. 82, No. 2, February 1993

1

UUVELEWGTH

0.01901, n

0.02008, 1

A

Figure 2-Second-derivative spectra of mixtures of tryptophan and tyrosine solutions in 0.1 N NaOH at the respective concentrations of (A) 289.6 and 295.6 nmol/mL, (B) 434.6 and 147.2 nmol/mL, and (C) 482.9 and 98.5 nmol/mL.

0.1 N NaOH. The second-derivative spectra of peptides and standard solutions were then recorded. The absolute values of the derivatives between 262 and 264 nm for determination of tyrosine and between 304 and 306 nm for determination of tryptophan were measured.

Procedure for Peptide Hydrolysis-Peptide (-0.3 nmol) was dried in a hydrolysis tube and the tube sealed under reduced pressure after the addition of 1 mL of constant-boiling 6 N HC1 containing 0.2% thioglycolic acid and maintained at 110 "C for 24 h. At the end of hydrolysis, HCl was removed by lyophilization and the resulting residue was dissolved in 1 mL of 0.1 N HC1. For precolumn deriva- tization, 50 pL of 0.4 M borate buffer were mixed with 10 pL of o-phthalaldehyde/3-mercaptopropionic acid solution, 10 pL of 9-flu- orenylmethylchloroformate in acetonitrile solution, and 10 pL of sample solution. The resulting solution was mixed thoroughly and 1 pL was injected after 1 min.

HPLC Analysis-The liquid chromatograph used was a Hewlett- Packard 1090 L apparatus equipped with autoinjector, autosampler, work station (Hewlett-Packard 79994A with Hard Disk), printer, plotter, and diode array variable wavelength detector (Hewlett- Packard 1040 M) selected at 338 and 266 nm. The analytical column used was a Hypersil ODS column (5 pm, 200 x 2.1 mm) fitted with a guard column (RP-18 5 pm 30 x 2.1 mm).

Mobile PhaseSolvent A 0.03 M sodium acetate trihydrate (pH 7.5): 0.01 M NaN, (99:l) with the addition of 0.25% tetrahydrofuran; solvent B: 0.1 M sodium acetate trihydrate: 0.1 M NaN, (99:l): acetonitrile (20:80, v/v). The gradient program was as follows: 0% B for 1 min, linear step to 30% B in 7.5 min, linear step to 80% B in 3 min, linear step to 100% B in 1 min, isocratic step at 100% B for 4 min, and linear step to 4% B in 0.5 min. The flow rate was 0.44 mL/min and the oven temperature was 35 "C.

0.01169

0.00369

-.WP

A

V -.02013 , . . . . , . . . . I . . . . , , , . . 1

200 M iw U I U € L E I i G T H

0.02012, I

0.01205-

0.00397-

-.004LO-

-.01217-

-.0202i4 , V m0 250 3w X O iw

"I ,,EL E N 5 I"

Figure 3-Second-derivative spectra of mixtures of tryptophan and tyrosine solutions in 0.1 N NaOH at the respective concentrations of (A) 289.6 and 295.6 nmol/mL, (B) 144.8 and 443.6 nmol/mL, and (C) 96.6 and 473.0 nmol/mL.

Results and Discussion Spectrophotometric Measurement-Reliable results

were obtained by the second-derivative spectra method. To validate the present method, calibration graphs were pre- pared by measurements of the total second-derivative spectra of a mixture of tyrosine in 0.1 N NaOH (200.5 nmol/mL) with increasing amounts of tryptophan (from 50.2 to 401.9 nmol/ mL) and another mixture of tryptophan at 200.9 nmol/mL, with increasing amounts of tyrosine (from 50.1 to 401.1 nmol/mL) (Figures 1-4). It is important to note that the value of the second-derivative spectrum of tyrosine is near to zero between 304 and 306 nm. However, in this range, it is possible to take derivative measurements of the mixture proportional to the tryptophan concentration. Similarly, between 262 and 264 nm, the value of the second-derivative spectrum of tryptophan is near zero, whereas in the same range there is a very good linearity between derivative measurements of the mixture and tyrosine concentration.

The regression equation calculated for tyrosine and tryp- tophan by measurements described above are, respectively, y

Correlation coefficients are 0.998 for tyrosine and 1.000 for tryptophan. The minimum detectable level is 7.6 nmol/mL for tyrosine and 29.8 nmol/mL for tryptophan.

Accuracy and Precision-Relative standard deviations of 0.4% for tryptophan and 0.8% for tyrosine were calculated from a series of 10 measurements for replicate analysis of each amino acid at 200 nmol/mL. Twenty-one known mixtures of tryp-

= 3.09E-05X + 3.953-04 and y = 1.37E-05X - 7.133-05.

Journal of Pharmaceutical Sciences / 181 Vol. 82, No. 2, February 1993

A

0.01916-

N

0.01095-

w =- - - 0.MZW. =- - - Y -.@n541- -

-.01367-

-.m1887

200 250 300 350 +m W R V E L E M G I H

V , . . . . , . . .

tophan and tyrosine in ratios ranging from 1:5 to 5:l were prepared and analyzed. An average recovery of 102.2% with a relative standard deviation of 1.8% for tryptophan, and an average recovery of 101.0% with a relative standard deviation of 1.6% for tyrosine were obtained (Table 11).

Determination of Tryptophan and Tyrosine in Pep- tides-Peptides were dissolved in 0.1 N NaOH at a concen- tration of -200 nmoVmL and scanned in comparison with separate standard solutions of tryptophan and tyrosine at the same concentration in 0.1 N NaOH. By the value of d2(A)/ d o 2 between 262 and 264 nm and between 304 and 306 nm, the amounts of tyrosine and tryptophan in peptides, respec- tively, can be determined using the following equation:

In eq 1, C,, is the amount of tryptophan (nmol/mL), CS,, is the known concentration of tryptophan (nmoVmL) in the standard solution, Ac is the absolute value of absorbance of the sample solution, As is the absolute value of absorbance of the standard solution, and W is the analytical wavelength (nm).

Peptide content can be determinated using the following equation:

cTrp(Tyr) 100 peptide content =

CPep

In eq 2, C,, is the measured concentration of tryptophan (nmol/mL) and C,, is the known concentration of the sample solution (nmol/mL).

Conclusions In conclusion, we have shown that our method for the

simultaneous quantitative determination of tryptophan and tyrosine in peptides by second-order derivative spectroscopy favorably compares with classical methods, with the clear advantage of utmost rapidity. Moreover, the undestructive character of the spectrophotometric determination guaran- tees a fast and reliable determination of the peptide content in synthetic samples of peptides containing tryptophan or tyrosine.

3.

4. 5.

6. 7. 8.

9. 10. 11. 12. 13.

14.

15.

16. 17. 18. 19. 20. 21. 22. 23. 24.

References and Notes 1. Chang, J. Y.; Knecht, R.; Braun, D. G. Methods Enzymol. 1983,

2. Jones, B. N.; Paabo, S.; Stein, S. J. Liq. Chromatogr. 1981, 4, 91, 41-48.

565-586. Simpson,R. J.;Moritz,R. L.;Nice,E. C.;Grego,B.;Yoshizaki,F.; Sugimura, Y.; Freeman, H. C.; Murata, M. Eur. J. Biochem. 1986, 157,497-506. Spies, J. R. Anal. Chem. 1967, 39, 1412-1416. Horton, H. R.; Koshland, D. E. J. Am. Chem. SOC. 1965, 87, 1126-1132. Hassan, S. S. M. Anal. Chem. 1975,47,1429-1432. Krishna, K. V.; Archana, J. Talanta 1988, 35, 35-39. Molnhr-Perl, I.; PintBr-Szakiics, M. Anal. Biochem. 1989, 177, 1619. Chrastil, J. Anal. Biochem. 1986, 158, 443446. Edelhoch, H. Biochemistry 1967,6, 1948-1954. Goodwin, T. W.; Morton, R. A. Biochem. J. 1946,40, 62M32. Bencze, W. L.; Schmid, K. Anal. Chem. 1957,29, 1193-1206. Balestrieri, C.; Colonna, G.; Giovane, A.; Irace, G.; Servillo, L. Eur. J. Biochem. 1978,90,433440. Balestrieri, C.; Colonna, G.; Giovane, A.; Irace, G.; Servillo, L. Anal. Biochem. 1980,106,49-54. Servillo, L.; Colonna, G.; Balestrieri, C.; Ragone, R.; Irace, G. Anal. Biochem. 1982,126, 251-257. Morelli, B. J. Pharm. Sci. 1988, 12, 1042. Morelli, B. Anal. Lett. 1988,21 (l), 43. Morelli, B. J. Pharm. Biomed. Anal. 1988, 6, 199. Morelli, B. Anal. Lett. 1988,Zl (5), 759. Morelli, B. Analyst 1988, 113, 1077. Morelli, B. J. Pharm. Sci. 1988, 7, 615. O’Haver, T. C.; Green, G. L. Anal. Chem. 1976,48, 312. O’Haver, T. C. Clin. Chem. 1979,25, 1548. Rovero, P.; Quartara, L.; Fabbri, G. Znt. J. Peptide Protein Res. 1991,37, 140-144.

Acknowledgments This work was supported in part by Istituto Mobiliare Italiano,

Roma (grant no. 462871.

182 I Journal of Pharmaceutical Sciences Vol. 82, No. 2, February 1993