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Bioehimica et Biophysica Acta, 578 (1979) 188--195 © Elsevier/North-Holland Biomedical Press

BBA 38165

MANUAL SOLID PHASE SEQUENCE ANALYSIS OF POLYPEPTIDES USING 4-N,N-DIMETHYLAMINOAZOBENZENE 4'-ISOTHIOCYANATE

J.Y. CHANG

Max-Planch-Institut fiir Molekulare Genetih, A bt. Wittmann D-I O00 Berlin-Dahlem (Germany)

(Received August 30th, 1978)

Key words: Dimethylaminoazobenzene isothiocyanate; Solid phase analysis; Polypeptide; Amino acid sequence

Summary

A manual solid-phase method for sequence analysis of polypeptides is described. The immobilized polypeptide was subjected to stepwise degradation by Edman-type reagent, using the 4-N,N-dimethylaminoazobenzene 4'-isothi- ocyanate phenyllisothiocyanate double coupling method. The N-terminal amino acids were released (after conversion reaction) as 4-N,N<timethylamino- azobenzene 4'-thiohydantoin (identified by thin layer chromatography) and phenylthiohydantoin derivatives. The method required 2--10 nmol polypep- tide.

Introduction

The combined use of solid-phase Edman degradation [1] with radioactive phenylisothiocyanate is a useful method for micro-sequence analysis of poly- peptides [2]. The major advantage of this technique is that excess reagent and by-products can be removed without endangering the extraction losses of poly- peptide, which persist when using the liquid-phase Edman method [3]. The utilization of radioactive phenylisothiocyanate, despite its high sensitivity, is rather time consuming.

Preliminary results of using DABITC (4-N,N-dimethylaminoazobenzene 4'- isothiocyanate) in place of radioactive phenylisothiocyanate for high-sensitiv- ity, solid-phase sequencing was reported [4]. Some imperfections, such as the

Abbreviations: DABITC, 4-N,N-dimethylaminoazobenzene 4 '- isothiocyanate; DABTC, 4-N,N-dimethyl- aminoazobenzene 4 '- thiocarbamyl; DABTH, 4-N,N-dimethylaminoazobenzene 4 ' - th iohydantoin; DITC, p- phenylene diisothiocyanate.

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strong overlapping and mechanical loss of the solid support during vacuum dry- ing, remain ye t to be improved. This paper reports refinements in the use of DABITC in the solid-phase sequencing technique.

Materials and M e t h o d s

Ribosomal proteins S l l , L29 and L30 were provided by Dr. H.G. Wittmann (Max-Planck-Institut fiir Molekulare Genetik, Berlin). The peptide L24-T10 (tryptic peptide from L24) was kindly supplied by Dr. B. Wittmann-Liebold. The peptide L13-SP3-T1 (tryptic peptide of L13) was a gift from L. Mende. DABITC was prepared as described in [5,6] or purchased from Fluka (recrys- tallized from boiling acetone). The sources and purities of other sequencing materials were as described previously [ 7 ].

Amination of the glass beads (CPG-75, Serva, Heidelberg) [8] and their acti- vation with DITC (p-phenylene diisothiocyanate, Eastman) were described previously [9].

Attachment of peptide and protein to the DITC-activated amino glasses and DABITC-phenylisothiocyanate degradation cycle.

These were performed as outlined in Scheme 1. The coupling of polypept ide

(A) (B)

Polypept ide 5--10 nmol $+ 0.8 ml 0.2 M NaHCO3/NaOH, pH 8.9

~+ 20 ~ 25 mg DITC glass $N 2, 52°C, 1 h $+ 20 /Jl ethanolamine SN 2, 52°C, 15 rain $H20 and CH3OH wash Sdried in vacuo $+ 600/~1 67% aq. pyridine $+ 50 #l PITC SN 2, 52°C, 30 min $CH30I-1 wash Sdried in vacuo $+ 200 ~1 trlfluoroacetic acid

SN 2 , 52°C, 15 min

$CH3OH wash Sdried in vacuo Ssubject to DABITC-PITC degradat ion

DABITC-PITC cycle 4+ 400 #1 50% aq. pyridine

$+ 200 ~1 DABITC (15 nmol/~l in pyridine) $N2, 52°C, 45 min 4+ 50 pl PITC

4N 2, 52°C, 30 min Sremove supernatant by gentle centrifugation

4wash with 2 ml pyridine and 2 X 2 ml CH3OH

~lried in vacuo $+ 200 pl trifluoroacetic acid SN 2, 52°C, 15 min ~dried trifluoroacetic acid in vacuo 4extract thiazollnones with 300 pl CH 3 OH $CH3OH wash Sdried in vacuo Ssubject to next cycle

* Col lected f o r DABTH-amino acid identification, see the text. PITC, phenyllsothiocyanate.

Scheme 1. Sequence of steps for preparation of b o u n d po lypept ide (A) and DABITC-PITC degradation cycle (B).

to the solid support and their subsequent sequence analysis were carried out in the same tube (1 cm internal diameter × 5 cm, fi t ted with a C-10 glass stopper and a 2 mm × 6 mm magnetic stirring bar). Reactions were performed by plac- ing the tube in a heating block (52°C) over a magnetic stirrer.

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A C-10 glass s topper fit ted with a D2 sinter was used at every vacuum drying to prevent the loss of glass beads.

The thiazolinone extract was evaporated and converted into its thiohydantoin derivative in the aqueous acid as described [7]. Identification of DABTH-amino acids was performed as described previously [6,7,11,12]. Quantitative analysis of DABTH-amino acids was carried out by the method described in the accom- panying paper [21]. In cases where DABTH-amino acid appeared without con- tamination (such as residues 2, 6, 11 of insulin B chain, Fig. 2). the yield could be determined by measurement of the absorbance of an ethanol solution of the derivative.

Results

Attachment of polypeptide to the solid support and performance of sequence determination

Attachment of polypeptide through lysine side chain to the DITC-activated solid support [13] has been the most useful technique in solid-phase sequenc- ing. The NaHCO3 buffer [10] was used for both peptides and proteins in the at tachment reaction. Yields were found to be over 65% for the ribosomal pro- teins and insulin B chain and 60% for the peptide L24-TI0. The present method used 5--10 nmol polypeptide as the starting material, but this could be scaled down to approx. 1 nmol (the sensitivity of detecting DABTH-amino acid is at the 10 pmol level). This DABITC-phenylisothiocyanate degradation can be applied to polypeptides immobilized by methods other than the DITC method.

Sequence analysis of peptides and proteins The identification of DABTH-amino acids relies on the running of the small

polyamide sheet chromatography [ 11,12] and interpretation of the developed sheet [6,7]. The sequence of a decapeptide L24-T10 was determined by both this DABITC-phenylisothiocyanate double-coupling method (Fig. 1, top row) and a DABITC single coupling method [4] (Fig. 1, bo t tom row). The double- coupling method eliminated much of the overlapping which was presented in

Fig. 1. P h o t o g r a p h o f t h e p o l y a m i d e shee t s ( 2 - - 9 res idues ) resu l ted f r o m t h e s e q u e n c e analys is o f p e p t i d e L24-T10 ( V I V E G I N L V K ) b y the so l id -phase D A B I T C - p h e n y l i s o t h i o c y a n a t e m e t h o d d e s c r i b e d in th is t e x t ( top row) and by the sol id-phase D A B I T C m e t h o d [4] ( b o t t o m row) . Th e N- and C- termini w e r e a t t a c h e d to the sol id suppor t , One c a n n o t i c e the relat ive posi t ions of D A B T H - V a l and D A B T H - L e u / I I e to the b lue m a r k e r e (cyc led) at 2rid, 3rd and 9 t h res idues . T h e so lvent s u s e d for TLC separa t ion were : so lven t 1, wa te r / ace t i c acid (2 : 1, v /v) ; so lvent 2, t o l u e n e / n - h e x a n e / a c e t i c acid (2 : 1 : 1, by vo lume) .

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193

the single~oupling method. The portion of DABTH-amino acid extract used for TLC identification was approx. 1/60 (2nd residue, Ile/Leu) to 1/40 (9th residue, Val). The complete sequence (except N- and C-termini) of an 18 resi- dues. tryptic peptide (L13-SP3-T,, derived from ribosomal protein L13) was also determined (Mende, L., unpublished data).

Fig. 2 shows the 2--28 residues of the insulin B chain, in which all of the amino acid residues including the cysteic acid at positions 7 and 19, were identified without ambiguity. The recoveries of leucine at residues 6, 11, 15 and 17 are 100%, 67%, 40% and 35%, respectively (assuming the recovery at residue 6 is 100%).

Fig. 3 shows the 2-30 residues of ribosomal protein L29, which contains 5 leucine residues between the residues 14--22. Due to the heavy contamination of DABTH-Leu, the sequence between residues 20--30 could only be estab- lished by careful examination of the newly-appearing spots. It is interesting to notice the appearance of a-DABTH-e-phenylthiocarbamyl-Lys (K3) at positions 2, 4 and 9 (the unattached lysine side chains were previously coupled with phenylisothiocyanate, see Methods). It is obvious that attachment of these lysine side chains to the solid support were not quantitative. The varied degree of attachment of individual lysine side chain probably reflects its acces- sibility to the DITC functional groups.

The first 24 amino acid residues of ribosomal proteins $11 and L30 were also analyzed. For $11, the sequence was established up to 23 residues [7,14] and residues 19--23 were assigned tentatively as Val-Ala-His- ( )-His. The increas- ing background of DABTH-Gly, DABTH-Ala, DABTH-Val and DABTH-Leu/Ile greatly decreased the accuracy of sequence determination beyond residue 23. For protein L30 [20], the sequence could be clearly determined up to 24 resi- dues. The 22nd residue (threonine) was contaminated by a nearly equal amount of DABTH-Ala and DABTH-Leu/Ile.

Discussion

The N-terminal sequences of proteins and larger peptides are usually sequenced by an Edman-Begg liquid-phase sequencer [3] and the complete sequences of small peptides (less than 30 residues) are usually analyzed by the manual Edman method [15], a dansyl-Edman method [16] or with the Laursen-type, solid-phase sequencer [1]. It is important to attempt to adapt these techniques as general methods for sequencing peptides and proteins, especially at high sensitivity (1--10 nmol). Recently, we developed a new manual sequencing method (liquid-phase DABITC-phenylisothiocyanate) which could be applied to 2--8 nmol peptides and proteins [7]. This was due to the use of a sensitive Edman-type reagent DABITC and a different extraction sol- vent, heptane/ethyl acetate ( 2 : 1 , v/v). The manual, solid-phase DABITC- phenylisothiocyanate method provides another useful choice for micro- sequence analysis of peptides and proteins. The advantages and disadvantages of this new method are as follows:

(1) Comparison o f the manual solid-phase method to the automated solid-phase method [1]

The manual mode of solid-phase sequencing is not significantly less efficient

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than the automated one [1] and the running costs are less. An additional advantage is that the manual, solid-phase sequencing method can be used on more than 4 samples simultaneously and requires only simple equipment.

(2) Comparison o f the use o f DABITC to the use o f radioactive phenyliso- thiocyanate in high-sensitivity sequence analysis

The sensitivity of this method is equivalent to that of the radioactive phenyl- isothiocyanate method [2], but greater than that of the conventional phenyl- isothiocyanate method [1]. The highly coloured DABITC derivatives and the colour change between DABTC and DABTH derivatives enabled the quick and accurate identification of the DABTH-amino acids. The use of radioactive PITC is, costly and time-consuming, requiring detection of radioactive phenylthio- hydantoin-amino acids and disposal of radioactive by-products. A new approach, using high-performance liquid chromatography to identify phenyl- thiohydantoin-amino acids [17], is another alternative for high-sensitivity sequence analysis [18,19].

(3) Comparison o f the solid-phase method to the liquid-phase method [7] The solid-phase DABITC-phenylisothiocyanate method was no more sensi-

tive than the liquid-phase DABITC-phenylisothiocyanate method [7]. In fact, the solid-phase method has the disadvantage of possible sample loss during attachment of the sample to the support and also being unable to detect the N- terminus (when using the DITC method). Further, the residues which are actually attached to the support cannot be determined. However, the solid- phase DABITC-phenylisothiocyanate method does possess some merits: (1) the detection of DABTH-cysteic acid is possible, see Fig. 2, (2) the through washing-out of by-product is achieved, (3) extraction losses can be eliminated (this is important for analyzing the sequences near the C-terminal end of a hydrophobic peptide).

In Summary, the manual, solid-phase DABITC-phenylisothiocyanate method described in this paper is a simple, sensitive and efficient technique. The com- bined use of this method with the liquid-phase DABITC-phenylisothiocyanate method [7] should provide a general method for micro-sequence analysis of polypeptides, especially for those groups who cannot afford to run automatic sequencers.

Acknowledgement

The author is grateful to Drs. H.G. Wittmann and B. Wittmann-Liebold for reading this manuscript.

References

1 Laursen, R.A. (1971) Eur. J. Biochem. 20, 89--102 2 Bridgen, J. (1977) Methods Enzymol. 47 ,321 - - 335 3 Edman, P. and Begg, G. (1967) Eur. J. Biochem. 1, 80--91 4 Chang, J.Y., Creaser, E.H. and Hughes, G.J. (1977) FEBS Lett. 78, 147--150 5 Chang, J.Y., Creaser, E.H. and Bentley, K.W. (1976) Biochem. J. 153 ,607- -611 6 Chang, J.Y. (1977) Biochem. J. 163, 517--520

7 Chang, J.Y., Brauer, D. and Wittmann-Liebold, B. (1978) FEBS Lett. 93 , 205 - -214 8 Robinson, P.J., Dunnhill , P. and Lilly, M.D. (1971) Biochem. Biophys. Acta 242, 659--661 9 Bridgen, J. (1976) Biochemistry 15, 3600--3604

10 Cl~ang, J.Y., Creaser, E.H. and Hughes, G.J. (1977) FEBS Lett. (1977) 84, 187--190 11 Chang, J.Y. and Creaser, E.H. (1976) Biochem. J. 157, 77--85 12 Chang, J.Y. and Creaser, E.H. (1977} J. Chromatogr. 132, 303--307 13 Laursen, R.A., Horn, M.J. and Bonner, A.G. (1972) FEBS Lett. 12, 62--70 14 Wittmann-Liebold, B. (1973) FEBS Lett . 36 ,247 - - 249 15 Tart, G.E. (1977) Methods Enzymol. 47 ,335 - - 357 16 Gray, W.R. and Hartley, B.S. (1963) Biochem. J. 8 9 , 3 7 9 - - 3 8 0 17 Zimmermann, C.L., Appella, E. and Pisano, J.J. (1977) Anal. Bioehem. 77 ,569- -573 18 Margolies, N.N. and Brauer, A.W. (1978) J. Chromatogr. 148, 429--439 19 Hunkapil ler , N.W. and Hood, L.E. (1978) Biochemistry 17, 2124--2133 20 Ritter , E. and Wittmann-Liebold, B. (1975) FEBS Lett . 60, 153--155 21 Chang, J.Y. (1979) Bioehim. Biophys. Acta 578, 175--187

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