amino-terminal analysis of polypeptide using dimethylaminoazobenzene isothiocyanate

ANALYTICAL BIOCHEMISTRY 102, 384-392 (1980)

Amino-Terminal Analysis of Polypeptide Using Dimethylaminoazobenzene lsothiocyanate

J. Y. CHANG’

Max-Planck Institute fiir Molekulare Genetik, Abt Wittmann D-1000 Berlin-Dahlem (Germany) and Protein Biochemistry Unit, The Ausfralian National University, Canberra ACT Australia, 2600

Received August 28, 1979

A new method for qualitative and quantitative N-terminal analysis of polypeptide using dimethylaminoazobenzene-isothiocyanate is presented. The method can recover all naturally occurring N-terminal amino acids, including asparagine, glutamine, and tryptophane in a nearly quantitative yield. Less than 1 nmol of polypeptide is required for qualitative N- terminal analysis and 5 to 10 nmol of polypeptide is used for quantitative N-terminal analysis. Applications and expected limitations of this new N-terminal method are described.

To establish the identity and purity of a polypeptide, N-terminal amino acid analysis is one of the simplest and most direct methods. An ideal N-terminal method should be highly sensitive and capable of recovering all N-terminal amino acid in a quantitative yield.

The sensitive 5-dimethylaminonaphthyl- I-sulphonyl chloride (dansyi-CB2 (I ,2) has been one of the more successful N-terminal reagents. However, the dansyl-Cl method suffers from the intrinsic disadvantage of being unable to recover asparagine, glutamine, and tryptophane.

The isothiocyanate degradation of N- terminai amino acid (3-5) requires only acid catalysis to cleave the N-terminal derivative and gives nearly quantitative recovery of asparagine, glutamine, and tryptophane (6). The new N-terminal reagent dimethylaminoazobenzene-isothiocyanate (DABITC) (7,8), combining the merits of the isothiocy-

* Present address: Department of Immunology, Mayo Medical School, Rochester MN 55901.

2 Abbreviations used: DABITC, dimethylaminoazobenzene-isothiocyanate; DABTC, dimethylaminoazobenzene-thiocarbamyl; DABTH, dimethylaminoazo- benezene-thiohydantoin; Dansyl-Cl, 5-dimethylaminonaphthyl-I-&phony1 chloride.

anate degradation and the sensitive chro- mophore dimethylaminoazobenzene, provides an alternative to overcome most of the shortcomings encountered in the conventional N-terminal method, both in qualitative and quantitative aspects.

MATERIALS AND METHODS

DABITC (7) and the solvents used in N- terminal analysis were prepared as described (9). The commercial DABITC of Fluka was recrystallized from boiling ace- tone before use. Bovine insulin is a product of Sigma (St. Louis, MO.) Oxidized insulin A-chain, insulin B-chain, as well as other peptides and proteins used in this study were purchased from Serva (Heidelberg, Germany). Staphylococcal protease was from Miles Laboratories (Elkhart, Ind.) Polyamide sheets were purchased from Schleicher and Schiill (Germany) and silica gel plates (G-60, without fluorescent indi- cator, 0.25 mm) were obtained from Merck.

Qualitative N-Terminal Analysis

The peptide or protein (1 nmol) was dissolved in 80 ~1 of 50% aq pyridine and

OOO3-2697/80/040384-09$02.00/O Copyright 8 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

384

AMINO-TERMINALANALYSISOFPOLYPEPTIDE 385

treated with 40 ~1 of DABITC solution (4 mg/ml in pyridine, freshly prepared, see Ref.(9)). The coupling reaction was done at 52°C for 2.5 h. After coupling reaction, the excess reagent and by-products were ex- tracted by mixing the reaction mixture with three portions of 500 ~1 heptane:ethyl acetate (2:1, v/v) on a Vortex mixer and centrifuging. The organic phase was col- lected and tested for the presence of the cleaved DABTH-N-methyl-amino acid (see Discussion). The sample in the aqueous phase was dried in vucuo over P,O, and then dissolved in 40 ~1 of water and 80 ~1 of acetic acid saturated with HCl . The cleavage reaction was performed at 52°C for 50 min. The sample was dried in vucuo and redissolved in 10 to 15 ~1 of ethanol or 80% ethanol. One to two microliters of the sample is sufficient for thin-layer chromatography or high-pressure liquid chromatography identifications.

Quantitative N-Terminal Analysis

Five to ten nanomol of polypeptide was subjected to DABITC coupling and cleavage as described for qualitative N-terminal analysis. Instead of drying the aqueous acid solution after cleavage reaction, the acid solution was first divided into two equal volumes (portion A and portion B) with a microsyringe. Portion A was dried and hydrolyzed with 5.7 N HCl for amino acid analysis (Durrum D-500). Portion B was dried and redissolved in 20 ~1 of 80% ethanol for quantitative analysis of DABTH-amino acids by either thin-layer chromatography or high-pressure liquid chromatography (see identification of DABTH-amino acid). For more accurate calculation of the recoveries, any possible sample lose of portion A and portion B should be carefully observed.

The recovery of N-terminal amino acid was determined by dividing moles of the DABTH-amino acid (obtained from portion B) by moles of polypeptide (obtained from portion A).

In quantitative N-terminal analysis of a peptide mixture, comparative recoveries can be obtained by assuming the recovery of one specific N-terminal amino acid is one (see legend of Fig. 4). In this case, the amino acid analysis of portion A can be omitted and quantitative N-terminal analysis can be applied on samples less than 1 nmol when high-pressure liquid chromatography is used for quantitative analysis of DABTH- amino acids.

Identijication of DABTH-Amino Acids

For qualitative analysis, thin-layer chromatography on polyamide sheets (7- 10) and silica gel plates (11) are adequate to identify all DABTH-amino acids. Silica gel plates are used only to discriminate between DABTH-leucine and DABTH-isoleucine. Analysis with thin-layer chromatography can be carried out with picomole amounts of DABTH-amino acid and one identification takes only 7 min.

For quantitative analysis of DABTH- amino acids, both manual and automatic methods were used. The manual method was carried out by recovering the DABTH- amino acid from thin-layer plates as described (12). The automatic analysis of DABTH-amino acid was done with high- pressure liquid chromatography (13). The manual method is simple but suitable for analyzing samples with single DABTH- amino acid component and requires 3 to 5 nmol of DABTH-amino acid for analysis. The automatic method (13) is suitable for analyzing samples with single or multiple components and requires less than 0.05 nmol of DABTH-amino acid for analysis.

RESULTS AND DISCUSSION

Chemistry of DABZTC Degradation

DABITC couples with the amino group of N-terminal amino acid in alkaline solution to form DABTC-peptide and then cyclize in the aqueous acid solution to give a red color

386 J. Y. CHANG

0.8

*. =....

1 I a I

270 350 nm

FIG. 1. The change of uv absorption during FIG. 2. Spectrum change at visible region when cyclization of DABTC-His-Gly to form DABTH-His. DABTC-Phe (-) completely cyclized to form Cleavage reaction was carried out in 1 N HCI at 52°C for DABTH-Phe (---). Reaction was carried out in water/ 3 min (-), 13 min (---) and 38 min (. . .). acetic acid saturated with HCI (1:2, v/v) at 52°C for 20 Concentration: 0.023 mM. min. Concentration: 0.023 mM.

DABTH-amino acid (12). In an attempt to achieve a quantitative DABITC degradation, conditions must be chosen to carry out both quantitative DABITC degradation, conditions must be chosen to carry out both quantitative coupling and quantitative cleavage. Quantitative coupling can usually be obtained by using the conditions described in this paper or at 75°C for 1.5 h (14). This could be checked by analyzing the homoge- neity of the new N-terminus of the shortened peptide. Quantitative cleavage can be judged by the complete conversion of blue-colored DABTC-peptide to red colored DABTH-amino acid on thin-layer chromatography. Measurement of kinetics was based on the spectral differences of DABTH-derivative and DABTC-derivative both in the uv region (Fig. 1) and in the visible region (Fig. 2). The half lifes (t& of the cleavage (cyclization) reaction of samples were given in Table 1.

400 so0 600 nm

DABTH-amino acid has two absorption maxima. In the uv region, the absorption maximum (at 269 nm) and its molar

TABLE 1

HALF-LIFE (MN) OF THE CYCLIZATION REACTIONS OF DABTC-DERIVATIVES OF THE INDICATED

AMINO ACIDS AND PEPTIDES AT 50°C UNDER THE ACID

CONDITIONS SPECIFIED

CH,COOH saturated with HCI + HZ0

DABTC-derivative 1 N HCI (2:1, v/v)

Glu Gln Thr GUY Phe Gly-Leu Leu- Leu His-Gly Phe-Gly

7.5 2.3 5.6 1.6 8.7 4.4

38.5 7.9 6.8 1.4

69.3 18.7 10.7 4.2 6.7 2.8 7.1 2.0

AMINO-TERMINAL ANALYSIS OF POLYPEPTIDE 387

TABLE 2

QUANTITATIVE N-TERMINAL ANALYSIS OF POLYPEPTIDE BY THE DABITC METHOD”

Polypeptide N-Terminus Recovery

Myoglobin (sperm whale) Val 0.90 Myoglobin (horse) GUY 1.01 Insulin (bovine) Phe 0.84

GUY 0.80 Bradykinin AJ-g 0.86 MEHFRWG Met 0.81 FDASV Phe 0.78 LLVY Leu 0.89 PFGK Pro 0.91 SA Ser 0.72 WL Trp 0.93

’ Recovery of N-terminal DABTH-amino acids were determined by manual thin-layer chromatography method (see under Materials and Methods). Recover- ies were the average value of three independent deter- minations.

extinction coefficient (about 29,000) are independent on the nature of solvent (12). In the visible region, both the absorption maximum and its corresponding molar extinction coefficient increase as a positive function of the acidity of solvent (12). The molar extinction coefficient of DABTH-

amino acid at 420 nm (in ethanol) is about 34,000 and at 520 nm (in ethanol:6 N HCl, 2: 1, v/v) is about 47,000. The absorption of DABTH-amino acid in the uv region is less sensitive in terms of quantitative analysis.

Qualitative N-Terminal Analysis

The method has been applied to a great number of peptides and proteins without encountering major difficulties except for N-terminal N-monomethylamino acid (22). The abnormal degradation of the N-terminal N-monomethylamino acid is discussed later in this paper.

Quantitative N-Terminal Analysis

Table 2 gives samples of quantitative N- terminal analysis which calculates the recovery of N-terminal DABTH-amino acid from polypeptide in a molar basis. Recoveries are ranging from 0.8 to 1. Possible sample loss during the manipula- tions should at least partly account for the deviations.

Quantitative N-terminal analysis of a peptide mixture can be used to measure the relative quantity of the individual peptide

FIG. 3. The N-terminal maps (developed on polyamide sheets) of tryptic peptide of pig (A) and human (B) very low density lipoprotein C-II determined according to the method described in the text. The red color DABTH-amino acid was represented by one letter abbreviation. “e” is a blue marker DABTC- diethylamine (10). NH, is DABTC-NH,. The areas of the DABTH-amino acid approximately represent their original intensities. Solvent 1 is 33% acetic acid. Solvent 2 is toluenezn-hexane:acetic acid (2: 1: 1, by volume). Proteins were gifts from Dr. N. E. Fidge (The Australian National University, Canberra).

388 J. Y. CHANG

presented. This technique serves some practical uses in peptide analysis: (a) In addition to peptide mapping, quantitative (or qualitative) N-terminal map of protein digest (derived from some specific cleavage, e.g. trypsin, staphylococcal protease or CNBr) provides useful information in looking into the sequence homology of proteins. Figure 3 shows the schematic results of the N-termini of the tryptic peptide mixture of human and pig very low density lipoprotein C-II (M, 9000). The structural diversity is obvious. The N- termini liberated from human C-II tryptic peptides was confirmed by its primary structure (15); (b) it could be used to study the kinetics of specific peptide bond cleavage. Tryptic cleavage of glucagon at two peptide bonds was compared in Fig. 4. The peptide bond Lys-Tyr (residues 12- 13) was quantitatively hydrolyzed within 20 min while the bond Arg-Arg-Ala (residues 18- 19) was only partially hydrolyzed (34%) after 8 h. It is known that repetitive sequences of lysine or arginine are attacked by trypsin at a considerably slower rate (16).

Digestion of insulin by staphylococcal

FIG. 4. Rates of tryptic cleavage of glucagon at peptide bonds Lys-Tyr (residues 12- 13) and Arg-Ala (residues 18- 19). Digestion was carried out in 0.1 M

ammonia bicarbonate at 37°C. Enzyme/substrate = I/ 40. Aliquots of hydrolyzate were removed and lyophilized at time intervals and N-terminal amino acids were quantitatively determined by the DABITC method (using high-pressure liquid chromatography). The recovery of the original N-terminus histidine was assumed to be 1 in each removed aliquot. Percentage of the peptide bond cleavage was then calculated by comparing the recovery of the newly released N- terminus with the recovery of DABTH-His.

FIG. 5. Rates of enzymatic cleavage of oxidized insulin A-chain by staphylococcal protease. Only one (Glu-Asn, residues 17- 18) of the two glutamoyl bonds was cleaved by the enzyme. Instead, a nonspecific partial cleavage, which produced a new peptide fragment with leucine as the N-terminus, occurred at a comparably slower rate. Digestion was performed in 0.1 M ammonia bicarbonate at 37°C. Enzyme/substrate = l/40.

protease (17) (an enzyme which is specific for the glutamoyl bond) reveals several interesting features. In 0.1 M ammonia bicarbonate, pH 8, two glutamoyl bonds of oxidized insulin B-chain were completely accessible to staphylococcal protease. How- ever, only one (Glu-Asn, residues 17- 18) of the two glutamoyl bonds in oxidized insulin A-chain was susceptible to staphylococcal protease cleavage (Fig. 5). The other glutamoyl bond (Glu-Gin, residues 4-5) was totally resistant to staphylococcal protease digestion. Instead, a nonspecific partial cleavage occurred to give a new peptide with leucine as N-terminus (Fig. 5). N-terminal sequence analysis of insulin A-chain digest indicates that this unexpected cleavage occurred at residues 12- 13 (Ser-Leu) (Fig. 6).

With native insulin, four glutamoyl bonds were all susceptible to staphylococcal protease digestion in pH 8, with the Glu-Gin bond (residues 4-5 of A-chain) being attacked at a comparably slower rate (Fig. 7). The unexpected cleavage which gave an addi- tional N-terminus leucine also occurred with native insulin. N-Terminal sequence analysis by the liquid-phase DABITC-phenylisothiocyanate method (9) again indicates that this cleavage was located between residues 12- 13 of the A-chain (Figs. 8 and 9). Whether this unexpected cleavage was due to the non-


FIG. 6. N-Terminal sequence analysis (by the DABITC-phenylisothiocyanate method, see Ref. (9)) of peptides resulted from staphylococcal protease digestion of oxidized insulin A-chain. Enzyme digestion was carried out in 0.1 M ammonia bicarbonate at 37°C for 24 h. The sequences of the first three amino acid residues indicate that the peptide mixture contains a fragment with the iv-terminal sequence Gly-Leu-Val (original N-terminal sequence), a fragment with the N-terminal sequence of Asn-Tyr (expected cleavage at the Glu-Asn bond) and a fragment with the N-terminal sequence of Leu-Tyr- Gin (nonspecific cleavage at the Ser-Leu bond). The blue marker DABTC-diethylamine was cycled. For the solvent systems used, see legend of Fig. 3. The original size of the polyamide sheets were 2.5 X 2.5 cm.

specificity of staphylococcal protease or due to the contamination of other protease requires further investigation.

In ammonia acetate (pH = 4), the action of staphylococcal protease was much slower. None of the two glutamoyl bonds in oxidized insulin A-chain was cleaved in a detectable amount after 24 h of staphylococcal protease

‘““I

FIG. 7. Rates of enzymatic cleavage of native insulin by staphylococcal protease. The four glutamoyl bonds as well as a nonglutamoyl bond (Ser-Leu, residues 12- 13 of the A-chain) were cleaved. Digestion was carried out in 0.1 M ammonia bicarbonate at 37°C. Enzyme/ substrate = 1140.

digestion. In oxidized insulin B-chain, the Glu-Ala bond was quantitatively cleaved within 24 h, but no more than 12% of the Glu-Arg bond was cleaved within the same period. Native insulin is not very soluble in ammonia acetate buffer and only trace amounts of the Glu-Ala bond (in B-chain) was cleaved within 24 h of staphylococcal protease digestion. Those findings are some- what varied from the results reported by Houmard and Drapeau (17).

While the importance of quantitative N-terminal analysis could be partly replaced by the technique of amino acid composition analysis or gel electrophoresis, the technique of comparative quantitative N-terminal analysis of peptide mixture should provide a useful tool in study of protein chemistry. The conventional way of measuring the enzymatic digestion is done by monitoring the totally released amino group (18) or by titrating the totally released carboxyl group (19). This new DABITC method can now enable us to study kinetics at individual

390 J. Y. CHANG

FIG. 8. N-Terminal sequence analysis (by the DABITC-phenylisothiocyanate method) of the peptide mixture derived from native insulin, which was digested by staphylococcal protease in 0.1 M ammonia bicarbonate at 37°C for 24 h. The sequences of the first three amino acid residues, again, indicates that a nonspecific cleavage resulted in a new peptide with the N-terminal sequence of Leu-Tyr-Gin. The blue marker DABTC-diethylamine was cycled. The original size of the polyamide sheets were 2.5 x 2.5 cm.

susceptible bonds. To this respect, the work reported by Duckworth et al. (20) serves a good example. Instead of isolating the frag- mented peptides, the exact peptide bond and rate of insulin cleavage by insulin protease (20) could be precisely determined in a sensitive way by this new technique.

However, the DABITC method still has some apparent limitations: (a) Certain amino acid side chains were partially destroyed during the cleavage stage (in aqueous acid). Normally, less than 5 to 10% of asparagine, glutamine, and threonine and less than 20% of serine side chains were destroyed (14). The solution for preventing these labile side chains from deamination and dehydration has not yet been found. The use of 50% trifluroacetic acid or 6 N HCl:acetic acid (1:2, v/v) has not produced better yields. It should be mentioned here that the cleavage procedure of DABITC degradation used in the single N-terminal analysis described in this text is different from the one used in extended sequence analysis (9, 21). In the extended sequence analysis, the DABTC- peptide was cleaved in anhydrous trifluroacetic acid to give thiazolinone first which was then separated from the shortened peptide and converted in aqueous acid solution to thiohydantoin (DABTH-amino acid). The

use of anhydrous acid was necessary in protecting the amide bond from hydrolysis, but has been found to be the main cause of serine and threonine destruction (12). In the single N-terminal analysis, the cleavage and conversion were carried out together in aqueous acid solution (12) as there is no worry about the other peptide bonds. This modifica- tion not only simplifies the DABITC degra-

FIG. 9. Quantitative analysis of DABTH-amino acid by high-pressure liquid chromatography. The DABTH- amino acids (represented by one-letter abbreviations) were obtained from the N-terminal analysis of the peptide mixture of native insulin digested by staphylococcal protease (in 0.1 M ammonia bicarbonate at 37°C for 24 h). For the detailed condition of high-pressure liquid chromatography, please refer to Ref. (13).


FIG. 10. N-Terminal analysis of Ser-Ala and Thr- Phe by the DABITC method described in this paper (A) and by the DABITC-phenylisothiocyanate method (9) (B). DABTH-amino acids were developed on polyamide sheets (2.5 x 2.5 cm). Extensive destructions of DABTH-Ser (S) and DABTH-Thr (T) with the DABITC-phenylisothiocyanate method was due to the use of anhydrous trifluoroacetic acid as the cleavage reagent (12). For the mechanism of those destructions and the nature of those by-products (S’, So, So, TA and TX), please refer to previous reports (9, 12).

dation cycle but also, most importantly, increases the yields of DABTH-Ser and DABTH-Thr to a great extent (Fig. 10); (b) It has been recently observed that a direct formation of thiohydantoin ring occurs in alkaline solution by reaction of DABITC or phenylisothiocyanate with N-monomethyl amino acid (22). This reaction resulted in the appearance of iv-terminal N-monomethylamino acid as well as the second amino acid residue after the first degradation cycle (scheme 1). In case that the direct cleavage of N-monomethylamino acid is quantitative, as in ribosomal protein S11 (22), the second amino acid residue can virturally be misinterpreted as the N-terminal amino acid. This abnormal isothiocyanate degradation has led to incorrect conclusions by assuming the existence of isopeptide bonds (23) or two polypeptide chains (24). There are, however, two remedies which could be taken to circumvent this problem. One could use a second N-terminal method to complement the DABITC method or one could collect the heptane/ethyl acetate extract (see under Materials Methods) after the DABITC coupling which possibly contains the cleaved DABTH-N-monomethylamino

~~:N@N=N+~:~:s

+ ck--k---

c Alkaline

!I T2 P c~~~=~Q-~~-~-N-CH-C-NH-CH- s- Nktw -

s&i3 6 0 0 (4

+

SCHEME 1. Direct formation of thiohydantoin ring in alkaline solution by reaction of DABITC with N-monomethyl-amino acid as N-terminus.

392 3. Y. CHANG

acid. Identification of the extract on thin- 9. Chang, J. Y., Brauer, D., and Wittmann-Liebold,

layer chromatography (22) should unravel B. (1978) FEES Left. 93, 205-214.

the presence of N-monomethyl amino acid 10. Chang, J. Y., and Creaser, E. H. (1977) J.

as the N-terminus. On the other hand, by Chromatogr. 132, 303-307.

understanding the nature of this direct 11. Chang, J. Y., Creaser, E. H., and Hughes, G. J.

(1977) J. Cizromatogr. 140, 125- 128. cleavage, one can maximize it as an efficient 12. Chang, J. Y. (1979) Biochim. Biophys, Acta 578,

tool in identifying the existence of N-mono- 175- 187.

methylamino acid. 13. Chang, J. Y., Lehmann, A., Wittmann-Liebold, B. (1980) Anal. Biochem. 102, 380-383.

14. Chang, J. Y. (1977) Ph.D. thesis, The Australian

ACKNOWLEDGMENT National University, Canberra. 15. Jackson, R. L.. Baker, H. R.. Gillian. E. B.. and

The author thanks Drs. H. G. Wittmann, B. Gotto, A. M: (1977) Proc. Nat. Acah. Sci. ‘USA Wittmann-Liebold, and E. H. Creaser for support 74, 1942- 1945. during the course of this work. Technical assistance 16. Kasper, C. B. (1975) in Protein Sequence from Mr. A. Lehmann and C. Southern are acknowl- Determination (Needleman, S. B., ed.) pp. Il4- edged. 158, Springer-Verlag, Berlin.

17. Houmard, J., and Drapeau, G. R. (1972) Proc. Nar.

REFERENCES

1. Gray, W. R. (1972) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., eds.), Vol. 25, pp. 333-344. Academic Press, New York.

2. Hartley, B. S. (1970) Biochem. J. 119, 805-822. 3. Edman, P. (1950)Acta Chem. Stand. 4, 277-282. 4. Edman, P. (1956)Acta Chem. Stand. 10,761-765. 5. Edman, P., and Begg, G. (1967)Eur. J. Biochem. 1,

80-91. 6. Sjoquist, J. (1960) Biochim. Biophys. Acta 41,20-

30. 7. Chang, J. Y., Creaser, E. H., and Bentley, K. W.

(1976) Biochem. J. 153, 607-611. 8. Chang, J. Y., and Creaser, E. H. (1976) Biochem.

J. 157, 77-85.

Acad. Sci. USA 69, 3506-3600. 18. Kasper, C. B., and Smith, E. L. (1%6). J. Biol.

Chem. 241, 3754-3762. 19. Jacobsen, C. J., Leonis, J., Linderstrom-Lang, K.,

and Ottessen, M. (1955) in Method of Biochemi- cal Analysis (Glick, D., ed.) Vol. 4, 171, Wiley- Interscience, New York.

20. Duckworth, W. C., Stentz, F. B., Heinemann, M., and Kitabchi, E. (1979) Proc. Nat. Acad. Sci. USA 76, 635-639.

21. Chang, J. Y. (1979) B&him. Biophys. Acta 578, 188- 195.

22. Chang, J. Y. (1978) FEBS Lett. 91, 63-68. 23. Chen, R., and Chen-Schmeisser, U. (1977) Proc.

Nat. Acad. Sci. 74, 4905-4908. 24. Hermodson, M. A., Chen, K. C. S., and Buchanan,

T. M. (1978) Biochemistry 17, 442-445.

amino-terminal analysis of polypeptide using dimethylaminoazobenzene isothiocyanate

Documents