Sequential degradation of polypeptides from the carboxyl ends I. Specific cleavage of the carboxyl-end peptide bonds

BIOCHIMICA ET BIOPHYSICAACTA

3o~

BBA 35701 SEQUENTIAL DEGRADATION OF P O L Y P E P T I D E S FROM T H E CARBOXYL ENDS I. SPECIFIC CLEAVAGE OF T H E CARBOXYL-END P E P T I D E BONDS

SABURO YAMASHITA

Hoshi College of Pharmacy, Shinagawa-ku, Tokyo (Japan) (Received June I6th, 197 o) (Revised manuscript received August 3Ist, 197 o)

SUMMARY

A direct chemical method of amino acid sequence determination from the C-terminal amino acid is proposed. The method involves an improved thiohydantoin formation which was applied to elucidate the amino acid sequence around the carboxyl termini of polypeptides and proteins. The conditions employed in this method are mild enough for biological materials, such as ribonuclease A which was elucidated by this method up to the fourth amino acid from the carboxyl terminus. The main features of the method are the following. I. Thiohydantoin was formed in a non-aqueous medium--a mixture of acetic anhydride or trifluoroacetic anhydride, acetic acid and sodium thiocyanate. 2. Cleavage of peptidyl thiohydantoin was made with an acidic form of a cation-exchange resin. 3. Separation of the cleaved thiohydantoin and the parent peptide less one amino acid moiety was made by chromatography on a Sephadex G-25 column. 4. The peptide fraction was concentrated by freeze-drying.

INTRODUCTION

Sequential analyses of polypeptides and proteins from the N-termini have been well established 1. On the contrary, the primary structure around the carboxyl ends of polypeptides and proteins could not be determined from the carboxyl ends owing to lack of a satisfactory method. Hydrazinolysis and tritium-labeling methods have been successfully employed for characterization of the C-terminal amino acid residues, but these methods cannot be applied for successive determination of the second amino acids at the carboxyl termini of the native polypeptides. Other qualitative C-terminal amino acid determinations made either by chemical or enzymic methods were reviewed by GREENSTEIN AND WINITZ ~. Biochim. Biophys. Acta, 229 (1971) 3Ol-3O9

302

S. Y A M A S H I T A

Recently, S'I'ARKa and (;ROMWF1A+ AND .%TARKla reported the determination of the sequence of some peptides ff()m the (]-termini by making the thiohydantoin derivatives of amino acids. However, a careful examination of the reactions described by these authors was fl)und t() be too drastic for application of the method to biological materials; that is, after the formation of peptidyl thiohydantoin, CROMWELl. ANI) STARKla used 12 M HC1 to cleave the peptidyl thiohydantoin bond, which seemed to cleave not only the peptidyl thiohydantoin bond but also the parent peptide bonds, and the availability of this method seemed to be limited to qualitative determinations o f at most up t o the sec(md or third amino acid from the C-terminus. The present paper deals with improved thiohvdantoin methods including selective cleavage of peptide bonds only at the C-termini of polypeptides under nmch milder conditions, which do not hydrolyze the residual peptide bonds. Tile reactions are essentially carried out according to the following scheme. ~2 0.,19 H RI

CH-C- N-CH-C-OH

(CHAD) 20

I

NH2

J

NHCOCHIi

I SCN,", R2

I ?t

CH--C

CH-C-N

R2

191 I

i

CH-.C-~,4

Nil

~

CH--C

I II II

N

S

H+ (Amberl~te IR-120)

~, CH-C-OH

,

NHCOCH3

o

I I

HN

NH

S

EXPERIMENTAL

Preparation of reference standard crystalline thiohydantoin derivatives of amino acids General procedure. IO mmoles of amino acid were dissolved in a mixture of 15 ml of acetic anhydride and 5 ml of acetic acid. When a homogeneous solution was obtained, 1. 5 g of crystalline sodium thiocyanate were added to the solution in small portions with vigorous shaking, After the thiocyanate had dissolved the solution was kept at 80 ° for 3° rain. Volatile solvent was evaporated to dryness, or to oil, under reduced pressure at 4 o°. A minimal amount of concentrated HC1 was added to dissolve the residue and the solution was allowed to stand for 3° rain at room temperature; 20 ml of water were then added and again evaporated to dryness under reduced pressure. Adequate water was again added, and crystals were precipitated on standing. The crystals were separated by filtration, and were recrystallized from water.

Biochim. Biophys. Acta, 229 (1971) 3oi-3o9

PEPTIDE CLEAVAGE AT THE CARBOXYL END

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TABLE I PROPERTIES

OF

5-SUBSTITUTED 2-THIOHYDANTOINS

Thiohydantoin derivative of Melting point

(nm)

Yield (approx.) (%)

263 280*** 263

IO 80 7°

292 (free 265) 264 281"**

60 IO 30

262

60

263

80

265

7°

264 263 272-276"** 265

20 5° 4° 3°

264*** 262 26o

20 3° 8o

263

IO

269, 318

4°

Amax

Found

Literature

G l u t a m i c acid Isoleucine Valine

115.5-116 131"** 138.5-139

Serine (i-acetyl) . . . . Arginine Methionine M e t h i n i n e sulfone Alanine

144-145 149-15o 93~)4 *** 160.5-161

Leucine

174.5 -175

Phenylalanine

178 . 5-179

Glutamine Lysine Tryptophan Tyrosine

189-19 ° 186--i 87 I64-I66"** 206-207

Histidine A s p a r t i c acid Glycine

277*** 220 228-229.5

Asparagine

248

Threonine

238-242

115-116" 132-133",** 137-14o* 139-14o** -148-15 °* I47-I49",** 233-236* 165-166 * 159-161"* 177 -178 * 173-175"* 178-18o* 182-183"* 189-191 * 189-191 * I9O-I92" 206-208* 205-207** 220* 221-222"* 229-231 * 228-229** 246** 252* 264*

* Ref. 13. ** Ref. 9. *** O b t a i n e d in crystalline f o r m only as I-acetyl d e r i v a t i v e in t h i s e x p e r i m e n t . . . . . N e w l y o b t a i n e d as crystalline d e r i v a t i v e in this e x p e r i m e n t .

Yields and melting points of the crystals are listed in Table I.

Application of the method to dipeptide. About o.I mmole of a dipeptide was added to I ml of acetic anhydride and 0. 5 ml of acetic acid. When the solution of dipeptide was obtained, IOO mg of crystalline sodium thiocyanate were added with vigorous shaking. The mixture was kept at 60 ° for 6 h. The solvent was evaporated to dryness, water was added to dissolve the residue and the solution was desalted by passing through a column ofAmberlite MB-3, 0. 9 cm × 15 cm. The eluate was concentrated under reduced pressure. To the aqueous solution of the thiohydantoin derivative of dipeptide, I g of Amberlite IR-I2O (H + form), was added and the peptidyl thiohydantoin was hydrolyzed according to the Method of hydrolyzing peptidyl thiohydantoin described below. Some of the results are shown in Figs. Ia-Ie. Application of the method to polypeptides. About I/,mole of a polypeptide (e.g. 13 mg of ribonuclease A (EC 2.7.7.16)) was dissolved in a mixture of 0. 5 ml of acetic anhydride, 0. 5 ml of trifluoroacetic anhydride and 0. 5 ml of acetic acid. When Biochim. Biophys. Acta, 229 (1971) 3Ol-3O9

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.'-;. YAMA.qHITA

Fig. l. a. Chromatographic behavior of thiohydantoin derived from serine. (See DISCUSSION). b. il'hiohydantoin derivatives derived from phenylalanine (Phe TH), glycine (Gly TH), and C terminus of (;lv-l'he. c. Thiohydantoin derivatives derived from glycine (Gly TH), alanine (Ala TH), and ~2-termini of Ala A|a and Gly-Gly. d. Thiohydantoin derivatives derived from tvrosinc (Tyr TH), glycine ((;ly TH), and C-terminus of Gly-Tyr. e. Thiohydantoin derivatives (ferived from proline (l'ro TH), glycine (Gly TH), and C-terminus of Gly- Pro. Biochim. Biophys. ~4cta,-'-'9 (197 t) 3° t 3°9

305

PEPTIDE CLEAVAGE AT THE CARBOXYL END

solution was complete, IOO mg of crystalline sodium thiocyanate were added with vigorous stirring. The mixture was kept at 60 ° for 6 h, during which insoluble sodium thiocyanate disappeared and the colorless solution turned to yellow. The solvent was evaporated by lyophilization, water was added to dissolve the residue, and the solution was desalted by dialysis. The aqueous polypeptidyl thiohydantoin was again subjected to lyophilization, and it was hydrolyzed according to the Method ofhyrolyzing peptidyl thiohydantoin given in the following paragraph. The solvent and the washings of the resin which was used for hydrolysis of peptidy] thiohydantoin were combined and concentrated by lyophilization*, and the concentrate was subjected to chromatography on a column of Sephadex G-25. The peptide was eluted immediately after the void volume, and the thiohydantoin, which showed a strong ultraviolet absorption at 26o nm, followed. The combined peptide fractions were lyophilized and used for the next cycle of the reaction. The recovery of the peptide was approx. 70-750/0 . The thiohydantoin fractions were concentrated under reduced pressure at 4 o°. The result is shown in Fig. 2.

Method of hydrolyzing peptidyl thiohydantoins The hydrolytic cleavage of thiohydantoin from the parent polypeptidyl thiohydantoin was performed by an improved modification of the method of COLLINS 4, who has reported the selective hydrolysis of the amide bond of I-benzoyl-I,2-dihydroquinaldamide yielding I-benzoyl-I,2-dihydroquinaldic acid by utilizing an ionexchange resin as a reagent. H

CONH2 I

COC6H5

aq~ a c e t o n e

~-

H

COOH I

COC6H5

After a peptidyl thiohydantoin had been formed and inorganic salts had been removed, it was dissolved in the minimal amount of water, or in o.I M HC1 if the hydrolysis were difficult, and i g ofAmberlite I R - i 2 o (H + form) (2.03 mequiv H+ available per g) was added for 0 . I - I mmole of sample. The mixture was vigorously shaken on 8 testtube shaker for 2 or 6 h at room temperature, and the liquid phase was collected * E x t r a c t i o n o f t h e t h i o h y d a n t o i n d e r i v a t i v e s w i t h e t h y l a c e t a t e a n d acetone f r o m t h e a q u e o u s solution w a s n o t satisfactory.

Biochim. Biophys. Acta, 229 (1971) 3Ol-3O9

.~,)1)

S. YAMASHITA

Vig. 2. T h i o h y d a n t o i n d e r i v a t i v e s derived from valine (Val TH), serine (Ser T t t ) , a l a n i n e (Ala TI t), a n d aspartic acid (Asp TIt) as t h e s t a n d a r d s ; a n d s e q u e n t i a l d e g r a d a t i o n p r o d u c t s of ribonucleasc \ from t h e ( ' - t e r m i n u s . N m n / m r s in p a r e n t h e s e s indicate t h e s e q u e n c e from t h e C-tcr]niilus.

by filtration. The ion-exchange resin was washed with water 3 times. The resin was washed with 5 ml each of ethyl acetate and water successively. The combined washings were concentrated to dryness under reduced pressure, the residue was dissolved in 2 nfl of 5o% acetic acid and the solution was subjected to chromatography on a colunm of Sephadex.

Thin-layer chromatography Plates for thin-layer chromatography were DC-Alufolien Kieselgel t: 254 with inorganic fluorescent indicator, on which the position of the thiohydantoin derivatives of amino acid became visible under ultraviolet irradiation at 253 •7 nm. The silica gel corresponding to the spot of the sample was scraped from the plate. The thiohydantoin derivative was extracted 4 times with I ml of methanol. The extracts were combined and the absorbance was deternfined at 260 nm. The detectable amount of a thiohydantoin derivative was about zoo nmoles on a thin-layer chromatographic plate. The solvent system used was I heptane I-butanol formic acid (IO:7:3, by w,1.). RESULTS AND DISCUSSION

Reaction of a-amino acids with thiocyanate in the presence of acetic anhydride to form 5-substituted (except with glycine) 2-thiohydantoins was introduced by JOHNSON AND NmoLl~:'r5 as early as I 9 I I . The amino acids that can react with thiocyanate leading to thiohydantoin derivatives are those having a tree carboxyl group and a free or monosubstituted a-amino group. This thiocyanate method has since been used to determine the C-terminal amino acids of various polypeptides and proteins. Successful examples of the method are exemplified by the determination of the Cterminal amino acids of glutathione 6, insulin 7 and ovomucoid8. However, the method seemed to be limited to determine only the C-terminal amino acid. The sequential degradation from the C-termini of proteins and peptides by this method could not be achieved, since severe conditions for hydrolyzing the peptidyl thiohydantoin lead to the destruction of the parent proteins or peptides. The author circumvented such disadvantage by using the acidic fi)rm of an BiockDn. Biopkys. ,4 eta, 220 (t971 ) 3o 1-3o9

PEPTIDE CLEAVAGE AT THE CARBOXYL END

307

ion-exchange resin, which was originally reported by COLLINS4 for the hydrolysis of I-benzoyl-I,2-dihydroquinaldamide. The procedure hydrolyzing the peptidyl thiohydantoin bond introduced here was sufficient and successful for hydrolyzing the bond between peptide and thiohydantoin molecules selectively, but not the other peptide bonds. An anion-exchange resin o f - O H - type was also successfully used to cleave the peptidyl thiohydantoin bond, but because of the relatively unstable nature of the thiohydantoin derivatives in alkaline media TM, the acidic form of an ion-exchange resin was consistently used in this experiment. Heat-labile biological material was treated at a temperature as low as possible, the thiohydantoin formation being performed at 60 ° for 6 h, according to a previous report that the incorporation of radioactive thiocyanate with amino acid residues was almost complete at IOO° in 3 ° min although at lower temperature the reaction proceeded rather slowly9. Hydrolysis of the peptidyl thiohydantoin bond was carried out at room temperature. The thiohydantoin liberated was concentrated under reduced pressure at 4 o°, and the peptide moiety exposing a new C-terminal amino acid was dried by lyophilization. To make thiohydantoin derivatives of amino acids, ammonium thiocyanate was employed in most hitherto reported work3,~, TM, but the NH4 + may be undesirable for the present purpose; that is, it may react with the COO- to form ammonium salt, which on heating may be converted to the corresponding amide; or it may disturb the elementary analysis of nitrogen if a trace amount cannot be removed. Therefore, in these experiments, sodium thiocyanate was extensively used. The method was applied to known peptides, ribonuclease A and egg white lysozyme (EC 3.2.I.I7), whose amino acid sequence around the C-termini are already known to be -Pro-Val-His-Phe-Asp-Ala-Ser-Val (ref. IO) and - A l a - T r p - I l e - A r g Gly-Cy~-Arg-Leu (refs. i i , I2), respectively. In a typical experiment, I-IO #moles (13-13o mg) of the intact peptide were weighed, and the amino acid sequence was determined according to the method described in EXPERIMENTAL.In the case ofribonuclease A, recovery of valine from the C-terminus was approx. 85%, of serine 80%, of alanine 80% but the recovery of aspartic acid, the fourth amino acid residue from the C-terminus, was found to be markedly less, approx, lO%. Intact lysozyme was also used for an analysis. The thiohydantoin derivative from leucine was recovered in good yield, from arginine less, and from thiohydantoin no derivative was obtained from the cystine residue, though peptidyl thiohydantoin which was formed after removal of leucine and arginine seemed to be formed judging from its strong ultraviolet absorption. Quantitative determination of the C-terminal amino acids was carried out with the corresponding thiohydantoins. Thiohydantoin derivatives of amino acids were quantitatively extracted from the thin-layer plate with methanol. The absorbance of the thiohydantoin derivatives was determined at 260 nm by using a molar extinction coefficient, 1.7 • lO4 in 0.005 M HC1. As for the preparation of standard thiohydantoins from amino acids, it has been pointed out that crystalline thiohydantoins derived from serineg, 13 and threonine TM could not be prepared. A thiohydantoin derived from threonine was recently prepared in crystalline form by CROMWELLAND STARKTM as the 5-ethylidene-2-thiohydantoin. The crystalline thiohydantoin derivative originating from serine was prepared Biochim. Biophys. Acta, 229 (1971) 3Ol-3O9

3o8

s. YAMASHITA

in tile present study. The crystals obtained from serine melted at I44 I45". The ultraviolet absorption curve (~tmax 292 nm) and elementary analysis calculated: (', 38.29; H, 4.25. Found: C, 38.6o; H, 3.95.i showed the product to be I-acetyl-5hydroxymethyl-2-thiohydantoin. Upon hydrolysis of the crystals with Amberlite IR-I2O as in the hydrolysis of peptidyl thiohydantoin, the ultraviolet absorption curve was shifted to ~rnax 265 nm, and the thin-layer chromatogram of the product gave a single spot of thiohydantoin of serine (Fig. Ia). However, the product could not be crystallized. The shift of the ultraviolet absorption curve upon hydrolysis of I-acetyl-5-hydroxymethyl-2-thiohydantoin supported the cleavage of the I-acctvl group to give 5-hydroxymethyl-2-thiohydantoin, and there was no evidence ~,f acetylation of the hydroxymethyl group. CROMWFLLAND .qTARKla reported that aspartic acid and proline do not form the corresponding thiohydantoin derivatives. HAUROWITZ Cl a[. ~ reported melting points and ultraviolet absorption data, although they stated that aspartic acid did not react with thiocyanate at ioo '~ in acetic anhydride under the conditions they used. However, under the experimental conditions presented here, that is the mixed anhydride of amino acid and acetic acid is prepared first followed by the addition of thioeyanate to the activated amino acid C-terminus, a thiohydantoin derivative of aspartic acid was successfully prepared. Elementary analysis, for CsH6N2OaS calculated: C, 34.48; H, 3.45. Found: (', 34.12; H, 3.65. The author was successful in preparing thiohydantoin derivatives of proline and hydroxyproline, although they have not yet been crystallized. With the aid of the chromatographically pure reference standard of a thiohydantoin derivatiw~ of proline, the peptide bond of glycylproline was cleaved successfully from the carboxvl end (Fig. ie). This result may contribute to the sequential analysis of peptides bearing proline on the carboxyl termini, since such peptides are not susceptible to the action of carboxypeptidases. The chemistry of the thiohydantoin derivatives of various amino acids will be presented separately. The acetic anhydride employed in the formation of thiohydantoin should activate the terminal carboxyl group to react with thiocyanate to form the thiohydantoin derivative under mild conditions. In this step the presence of water which may split the mixed anhydride bond (see Scheme i) and decrease of the yield of thiohydantoin is undesirable. Acetic anhydride alone is not always a good solvent for larger peptides and proteins, but trifluoroacetic anhydride is often rather a good reagent. Therefore, in the present experiment, when any peptides were not soluble in acetic anhydride, a mixture of acetic acid, acetic anhydride and trifluoroacetic anhydride, up to 5o% of the acetic anhydride was used. Removal of acetic anhydride from the above mixture did not give a good recovery of the thiohydantoins. In addition to the excellent solvent property of trifluoroacetie anhydride, it is an effective dehydration reagent; for example, for the preparation of eyclic-2',3'-phosphates of nucleoside from nucleoside-2' or -3'phosphate 14. Thus, the yield of the mixed anhydride in the presence of acetic anhydride and trifluoroacetic anhydride may be improved under milder conditions.

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ACKNOWLEDGMENT

The author expresses his sincere thanks to Professor Tyunosin Ukita of the Faculty of Pharmaceutical Sciences, University of Tokyo, for his valuable discussion and suggestions, and for reading the manuscript. REFERENCES i P. EDMAN AND G. BEGG, European J. Biochem,, I (1967) 80. 2 J. P. GREENSTEIN AND H. WINITZ, Chemistry of the Amino Acids, Wiley, N e w York, 1961, p. 1576. 3 G. R. STARK, Biochemistry, 7 (1968) 1796, 4 R. F. COLLINS, Chem. Ind. London, (1957) 736. 5 T. B. JOHNSON AND B. H. NICOLET, J. Am. Chem. Sue., 33 (1911) 1973. 6 B. H. NICOLET, J. Biol. Chem., 88 (193 o) 389 . 7 S. G. WALEY AND J. WATSON, J. Chem. Sue., (i951) 2394. 8 R. A. TURNER AND G. SCHMERTZLER, Biochim. Biophys. Acta, I i (1953) 586. 9 F. HAUROWlTZ, M. ZIMMERMAN, R. L, HARDIN, S. G. LISlE, J. HOROWlTZ, A. LIETZE AND F. BURSA, J. Biol. Chem., 224 (1957) 827. IO D. G. SMYTH, W. H. STEIN AND S. MOORE, J. Biol. Chem., 238 (1963) 227. t i R. CANFIELD, J. Biol. Chem., 238 (I963) 2698. 12 R. CANFIELD AND A. K. LIU, J. Biol. Chem., 240 (1965) 1997, 13 L. D. CROMWELL AND G. R. STARK, Biochemistry, 8 (1969) 4735. 14 D. M. BROWN, I. D. MARRATH AND A. R. TODD, dr. Chem. Sue., (1952) 2708.

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