ANALYTICAL

195,183-196

BIOCHEMISTRY

(1991)

REVIEW Chemical Procedures for C-Terminal Sequencing of Peptides and Proteins Adam S. Inglis Pacific Biotechnology

Received

January

Ltd., 74 McLachlan

Avenue, Rushcutters

Bay, New South

Wales 2010, Australia

3, 1991

The search for a chemical method for stepwise re- pounds, the remainder giving relatively weak sequence data only. None of the models included aspartic acid or moval of amino acids from the C-terminal end of proteins began early in this century when Schlack and proline. Likewise, more recently Shively and co-workers (30-33) claimed that an organic isothiocyanate was suKumpf (1) applied the method of Johnson and Nicolet perior to ammonium thiocyanate, but its use did not (2), for conversion of acylamino acids to acylthiohydanlead to greatly improved sequence data, and it did not toins, to a small peptide, and then cleaved the thiohydantoin from the molecule with 1 M sodium hydroxide. solve problems of reaction with aspartic acid and proWhile other alternative chemistries have been proposed line, for example. In addition, reagents used instead of (3-14), seeTable 1, over the years scientists have periodsodium hydroxide for cleavage were found to be a source ically attempted to utilize or modify the Schlack and of poor repetitive yields-12 M HCl (30) being abanKumpf degradation procedure for C-terminal sequence doned in favor of acetohydroxamic acid (32) and then analysis (15-38). None of the procedures proposed has triethylamine (33) which itself had some shortcomings. led to an accepted routine determination, and, although Not surprisingly, when the question of C-terminal sehe was probably too harsh on the prospects for chemical quencing was aired at the 7th International Conference sequencing methods, Tarr (13) quite rightly pointed to on Methods in Protein Sequence Analysis in Berlin the dearth of sequence information that has come from (1988), differing views were expressed as to the likelithem in the past. hood of success of the chemical methods (13,31,32). The severity of the reaction conditions has been Perhaps propitiously, some new promising findings blamed for the lack of success with the Schlack and were also reported (31) for the Schlack-Kumpf-type Kumpf reaction (l), but-based on results achieved degradation using HSCN coupling. First, good sequence with alternative conditions-without a great deal of jus- data were obtained for up to nine residues for a peptide tification. Using a reaction temperature of 50°C for containing hydrophobic amino acids (in nanomole thiohydantoin formation with ammonium thiocyanate, amounts), thereby establishing that these amino acids Stark (21) and Cromwell and Stark (22) were not able to react virtually quantitatively and that there are no maadvance the art materially, although they made valu- jor interfering side reactions associated with the chemisable contributions to the subject. They were sceptical of try. Second, the degradation proceeded successfully the possibilities of the procedure working for aspartyl through an aspartyl residue in a peptide. Third, in supand prolyl residues. Intriguingly, the encouraging se- port of earlier work of Kubo et al. (25), a stable derivaquencing results published subsequently by Yamashita tive of proline was obtained after reaction with acetyl(23) and Yamashita and Ishikawa (24), Kubo et al. (25) proline. The sequence data were obtained by using more Rangarajan and Darbre (26), and Williams and Kassell vigorous reaction conditions (80 vs 55°C) than had been (27) were apparently not reproducible by others. After a proposed previously (29) for thiohydantoin formation prolonged study in which they promoted the use of thiowith much larger samples. Rapid cleavage at room temcyanic acid (HSCN) rather than a thiocyanate salt for perature occurred on treatment with 0.5 M potassium thiohydantoin formation, Gurd and co-workers (2829) hydroxide in aqueous methanol. Subsequent analyses obtained good results for only one of their model comwith this procedure established that all polar amino 0003.2697/91$3.00

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

183

184

ADAM S. INGLIS TABLE

Derivatives Thiohydantoin Formation Cleavage References Amine/Amide Formation Cleavage Cycle 2 References AminelAlcchcl Formation Cleavage Cycle 2 References Acyl Urea Formation Cleavage Reference Oxazolidone Formation Cleavage Reference Cyanamide Formation Cleavage Reference Oxazole Formation Cleavage Reference Hydrazide Formation Cleavage Reference

of to

of C-Terminal Amino Acid Proposed for CTerminal Sequence Analysis Thiohydantoin Amino acid (1,2,15-38)

thiohydantoin;

C-l

peptide

of

Azide; isocyanate; p-methoxybenzyl Aldehyde; C-l peptide amide Amide to amine and repeat cleavage (3-5)

of

Cerboxylic ester; alcohol; amine Amino alcohol; C-l peptide alcohol Peptide alcohol to amine and repeat K-9)

to

to

of to

of to

of to

of to

of to

1

Acyl urea (with N,-O-substituted (10)

carbodimides) amino acid;

Alcohol; 2-oxizolidone 2-Oxizolidone of amino (11) Isothiourea; Iminohydantoin (12,131

cyanamide of amino

acid;

acid;

Carboxylic ester; oxazole Amino acid ester; C-l peptide (14)

C-l

C-l

ester;

amine

cleavage

peptide

peptide

C-l

peptide

orthoester

Hydrazide; diacylhydrazide; phenythiocarbamyl Phenylthiohydantoin of amino acid; hydrazide of C-l peptide (13)

ALTERNATIVE THIOHYDANTOIN

CHEMICAL APPROACHES FORMATION

Except for recent work (5) on a direct C-terminal procedure utilizing and extending the chemical approach of Loudon and Parham (4), alternatives to thiohydantoin chemistry for C-terminal sequencing (Table 1) currently remain as academic studies with potential for future development. As indicated above, perhaps further examination of them with our modern technology and know-how could bring a realization of this potential. Khorana (10) directed his efforts to generating a substituted amino acid at the end of his two-step procedure, presumably for ease of identification, This comprised a reaction with an aromatic carbodiimide to produce an acyl urea, followed by cleavage of the C-terminal amino acid as an N,O-substituted amino acid. The procedure foundered because the acyl urea hydrolyzed also during the cleavage reaction. Fromageot et al. (6) developed a reduction procedure with lithium aluminum hydride. The ester of the carboxy1 group was first made, then reduced, then treated with an acyl chloride to effect a rearrangement to the 0-peptidyl amino alcohol via an N-O acyl shift. This was then reductively cleaved to yield the shortened peptide as an alcohol and the released amino acid as an amino alcohol: -CO-NH-CHR-COOH

+-CO-NH-CHR-COOEt-

CO-NH-CHR-CH*OH

+-CO-0-CH,-CHR-NH,

-CH,OH acids in peptides could be released in substantial amounts as 2-thiohydantoins and that automation of the procedure was feasible (34). Recently it has been shown that other isothiocyanate reagents (35,37) also provide satisfactory sequence data. When placed in the context of the additional advances in automation (35) and further advances in knowledge on the chemistry (36), they are important in lending credence to the belief that this approach to Cterminal sequencing still has a real future. It is timely then to review the thiohydantoin procedure critically, with a view to clarifying the situation that has arisen over the 64 years of its existence, assessing existing problems, and possibly encouraging further development of a valuable tool for biochemists. However, this is not to imply that some of the other chemistries that have been proposed are not viable alternatives. On the contrary, the recent developments with the thiohydantoin procedure suggest that it might also be appropriate and rewarding to reexamine them also in the light of our current knowledge and the availability of better analytical techniques.

TO

+ +

+ HOCH,-CHR-NH,

The reduction was studied further by others (7-g), and it was found that treatment with aqueous sodium borohydride and triethyl oxonium fluoroborate overcame problems of nonspecific cleavage (9). In another variation, Saund et al. (ll), who used sodium dihydrobis(2-methoxyethoxy)aluminate as the reductant, converted the alcohol to an N-peptidyl-2-oxazolidone derivative with carbonyldiimidazole. This could be cleaved from the chain with 0.3 M HCl at room temperature. Previero and Coletti-Previero (14) also began by converting the carboxyl group to the ester, but they then treated it with phosphorous trichloride to produce an oxazole at the C-terminus. This breaks down in alcoholic acid solution to give the shortened peptide (as an orthoester) and the C-terminal amino acid ester. Side reactions are said to be a problem with this procedure. The foregoing procedures require development of a method for analysis of the C-terminal amino acid which, unlike the thiohydantoins, do not have a characteristic uv absorption to facilitate detection. Such development should in general be straight forward.

PROTEIN

CHEMICAL

C-TERMINAL

In an attempt to rationalize the various chemical approaches, Tarr (13) grouped his cyanamide degradation with the acyl urea and thiohydantoin procedures and, in view of problems encountered in the cyanamide work, regarded the other chemistries with some skepticism. That this is too simplistic is exemplified by the cleavage of valine and isoleucine which is very slow in his procedure, but fast with the thiocyanate procedure (31) using a different reagent. Loudon and Parham (4) made a peptidyl azide by reaction of the carboxyl group with p-nitrophenylphosphoryl azide. This was converted successively to the isocyanate, the p-methoxybenzyl ester, and then to the amine. This derivative decomposes on heating in aqueous solution to release elements of the C-terminal amino acid: -CO-NH-CHR-NH,

+ -CO-NH,

+ RCHO

+ NH,

Reaction of the amide with bis(trifluoracetoxy)iodobenzene regenerates the amine which can be subjected to another degradation cycle. Le and Tatemoto (5) report a modification of the method in which the aldehyde released is reacted with 2-aminothiophenol. The benzothiazolidine adduct formed is stable and absorbs at 310 nm. The method was applied successfully to the hexapeptide amide (Ala’*) secretin 22-27, and is an interesting advance.

PROPOSED REACTION PATHWAYS SCHLACK-KUMPF DEGRADATION

FOR

THE

Very little definitive work has appeared on the mechanism of the thiocyanate degradation reaction. A plausible pathway, postulated by Stark (38), is shown in Fig. 1. This neither indicates the rates of the different reactions involved nor the temperatures and reagents required for quantitative reaction, but rather it provides a reasonable framework for discussion of research findings on the degradation procedure.

Current Terminology In the past the Schlack and Kumpf procedure has been described as a thiocyanate degradation because the reactions include a coupling step with either thiocyanate salts or HSCN. However, in the proposed pathway a peptidyl isothiocyanate (III) is formed. Since isothiocyanates are known to react to form the thiohydantoin (19,30,35,37), it may also be regarded as an isothiocyanate degradation (37). Since all of the variations give a peptidylthiohydantoin which on cleavage yields a C-ter-

SEQUENCING

185

minal amino acid thiohydantoin (TH),’ it was deemed appropriate here to refer to them all as thiohydantoin procedures. Acylation

Reactions

Although Stark (21) did not use a separate step for initial activation of the carboxyl group with acetic anhydride, others have (29,33), and there is an assumption implicit in the reaction pathway of Fig. 1 that the formation of I and III could proceed sequentially. There is little evidence to support the efficacy of doing this, apart from ensuring that the sample is dry for the subsequent coupling reaction. Bailey and Shively (33) found some improvement for the trimethyl isothiocyanate reaction, however, and pretreatment increased the coupling yield by 35% to give a total yield of 89%. For the HSCN procedure (29), extending the activation with acetic acid and acetic anhydride from 5 to 30 min did not help the overall yield, furthermore, apparently very little activation had occurred because omission of the acetic acidacetic anhydride mixture from the subsequent HSCN coupling solution was most detrimental to the results (author, unpublished work). It may be concluded that, if the mixed anhydride (I) is an essential intermediate, it is not formed substantially in the absence of HSCN. The latter then may be required to catalyze the formation of I as well as react with it to form III. Extra peaks appear on the HPLC traces of the coupling solution on standing, which raises the possibility that a new acylating species could be formed, for example, an acylated form of the reagent (CH,CONCS). In earlier studies Waley and Watson (15) assumed that an unstable oxazolinone intermediate (II) was formed in acetic anhydride solution and could react with HSCN to generate a peptidyl isothiocyanate (III). Their attempts to bypass this pathway by using a reagent such as benzoyl isothiocyanate (BITC) which could react directly with the carboxyl carbon were unsuccessful. Recent work of Hawke and Boyd (37) also showed that BITC in acetic anhydride was not effective in producing thiohydantoin; however, the reaction did proceed when BITC was dissolved in a polar solvent, acetonitrile containing pyridine (lo%), a result which suggests that a mixed anhydride need not be involved at all. Although the carbon of carboxylic acids is generally more vulnerable to attack by nucleophiles in the presence of acid, in this case the relatively unstable nature of the acyl link to the isothiocyanate nitrogen could probably enhance the nucleophilicity of the latter for attack on the elec-

1 Abbreviations used: TH, thiohydantoin; cyanate; PVDF, polyvinyl idene difluodde; isothiocyanate; DTT, dithiothreitol; AM, ._ trlauoroacetic acid.

BITC, benzoyl isothioDITC, 1,4-phenylene diacetylation mixture; TFA,

186

ADAM

S. INGLIS

(CH3CO)zO

0

NH2CHR2CONHCHRlCOOH

-

dipeptide

CH3CONHCHR2CONHCHRlCz

/

A

oxazolinone

1' / N

OCOCH3 anhydride

1

(SCN) e CH~CONHCHRZ-!-~

mixed

I

CHRl

*

CH~CONHCHRZCOJH

II

J

YHRIY=O fl bC=S

peptidylisothiocyanate

III

peptidylthiohydantoin

IV

/

CH3CONHCHR2CON-YHRJ

Sk \ ,&o p;’ H

CH3CONHO'

J

/

I

[CH3CONHCHR$ONHCOCH3]

HCl/NaOH

CH3CONHCHR2COOH

+

HV--CHRl

i3 acylamino FIG. 1. Reaction release the amino

pathway for peptidyl thiohydantoin acid thiohydantoin and a residual

tropositive carbon with subsequent bond of the carboxyl group.

Coupling

acid

amino acid thiohydantoin

formation, and cleavage with either acetohydroxamic peptide minus the C-terminal amino acid (38).

cleavage of the C-O

Reactions

Yamashita (23) found that sodium thiocyanate in trifluoroacetic acid/acetic anhydrideiacetic acid, (l/l/l) gave better results than ammonium thiocyanate in acetic acid-acetic anhydride (21). Kubo et al. (25) used a strong acid, trifluoroacetic acid, acetyl chloride, and HSCN in dioxane. The results of later workers (29,31) indicated that the free acid, HSCN, was more effective than ammonium thiocyanate if the acid was dissolved in acetone (approx 1.4 M solution) and mixed with acetic acid-acetic anhydride (1:4). Recently, however, it was shown that if trifluoroacetic acid was added in slightly greater than equivalent amounts to this mixture, the HSCN could be replaced by ammonium thiocyanate with greatly improved results (36). This finding is in accord with the view that HSCN, being a strong pseudohalogen acid, will be highly ionized in solution and SO react faster than ammonium thiocyanate in a medium of low polarity (28). In agreement also, are some unpublished data of the author that better results were obtained with HSCN in acetone than in less polar solvents.

V acid or strong

acids and bases to

It has been suggested (30) that the thiocyanate reagents suffer from the disadvantage that they must isomerize at elevated temperatures to isothiocyanates before III can be formed. Mass spectral data indicated that this was accompanied by polymerization reactions and generation of side products. The conclusion is at odds with the authors’ own finding for the isothiocyanate reaction which was unsatisfactory at 50 and 70°C until a different solvent system incorporating pyridine was used, in addition, others found that the “thiocyanate” reaction can be fast at low temperatures (2528, 36). Thus, Kubo et al. (25) prepared acetylamino acid thiohydantoins by reacting the acetylamino acids with HSCN in dioxan for 60 min at 30°C; Dwulet and Gurd (28) found that the reaction of proteins and peptides was fast with HSCN in acetone (12 min at 38°C for Gly, Arg, Hser, Val, and Leu at the C-terminus). This followed pseudo-first-order kinetics. These results are convincing evidence that high temperatures are not essential for an active HSCN reagent. Assuming that the intermediate isothiocyanate (III) is involved as proposed in Fig. 1, one must conclude that the HSCN reagent has, at least, some isothiocyanate present in equilibrium with thiocyanate ion throughout. Nonetheless, with respect to the reaction temperatures required for routine sequencing of peptides, 8O’C was necessary for optimal results with HSCN (31). A new

PROTEIN

reagent, similarly

guanidine (35).

isothiocyanate,

CHEMICAL

apparently

C-TERMINAL

reacts

Cyclization One of the reasons advanced by Dwulet and Gurd (28) for the increased reactivity of HSCN, as compared with thiocyanate salts, was that it provided an improved rate of cyclization of the intermediate peptidyl isothiocyanate (III), Their kinetic data was based on an increase in the uv absorption of proteins at 266 nm on reaction with HSCN. The need to use more vigorous reaction conditions in subsequent sequencing work (29,31) was not consistent with the earlier rate studies. As the original experiment required a cleanup step prior to the uv absorption measurements, the data could be reconciled if some cyclization occurred during the cleanup operation after the kinetics experiment. Cyclization reactions are well known to occur under acid conditions: cyclization of the phenylthiocarbamyl derivatives of amino acids; N-terminal blocking of proteins because of ring closure of N-terminal glutamine; and the formation of homoserine lactone from homoserine. Hence, the improved results obtained on adding pyridine to the isothiocyanates (30,37) are at first surprising. Miller and Shively (30) proposed that the pyridine reacts with peptidyl isothiocyanate (III) to produce a more reactive carbon atom. Possibly the isothiocyanates also show increased stability under weakly basic conditions. It is interesting that the basic conditions are closer to those used for coupling amino groups (and the imino group of proline) to the Edman reagent, phenyl isothiocyanate. They might, therefore, promote reaction of III, as proposed, but, alternatively, the question arises as to whether it is possible that the organic isothiocyanate, or an acyl isothiocyanate, reacts initially with this nitrogen atom and this is followed by nucleophilic attack on the carboxyl carbon and cyclization to a thiohydantoin. If such a pathway were important, the lack of reaction for proline would be understandable because the peptide bond has no hydrogen to displace and a quaternary product would be unstable. On the other hand, one could envisage proline reacting if the tertiary nitrogen were attacked after carboxyl reaction with the isothiocyanate because cyclization could occur concomitantly with peptide bond cleavage (25,34). Evidence that glutamine can cleave prematurely with HSCN (34) is consistent with this last sequence of events, but in the case of glutamine, formation of an intermediate containing a quaternary nitrogen atom would occur only if its side chain also reacted with the adjacent nitrogen (with release of ammonia) to form a pyroglutamyl derivative. That the cyclization reaction is the slow step under acidic conditions is consistent with the need for higher activation energies for amino

SEQUENCING

187

acids with side chains that might impede the reaction (e.g., valine, leucine, and asparagine) as compared with those, that would not (e.g., glycine and alanine). Hence, the literature suggests two approaches that appear to be suitable for formation of peptidyl thiohydantoins. Both require a polar solvent system; the system involving thiocyanate reagents has proved successful with acidic reagents (28,36). Guanidine isothiocyanate has also been successfully employed under acid conditions (35) but other isothiocyanate reagents require mildly basic conditions (30,37). The findings cast doubts on the generally accepted order and nature of events leading to the formation of a peptidythiohydantoin, and leave unsettled the question as to why thiocyanates and isothiocyanates apparently require different reaction conditions. From the viewpoints of both the mechanisms involved and the advancement of the sequencing methodology, it would seem to be worthwhile to carry out more comparative work with the two types of reagent for “easy” and “difficult” residues. Precleavage

of the Acylthiohydantoin

Stark (38) reported that there was some preview of amino acids during his sequencing operations. Prerelease of C-terminal alanine was particularly high when it was preceded by aspartic acid which presumably catalyzed cleavage of the peptide bond via its side chain carboxy1 group. Initially the reagent. solutions of Stark contained water, and he used long periods of reaction at 50°C both of which could be conducive to premature deacylation of the peptidyl thiohydantoin. Using “anhydrous” conditions with HSCN in acetone as reagent at 80°C for 30 min, precleavage often occurred to a limited extent (31). This was not traced conclusively to the presence of water, but it was more pronounced when the reagents were old. The effects of the concentration of HSCN and the temperature on this premature release of the thiohydantoin have not been studied carefully and these need to be addressed further, particularly when they may be important also for the reactions with proline and glutamine (34). Cleavage of Acylthiohydantoins Formation of the thiohydantoin ring considerably weakens the link to the adjacent amino acid residue. Accordingly it has been cleaved with sodium hydroxide (1,15,31), 12 M HCl (21,22,30), a cation-exchange resin (23), acetohydroxamic acid (21,29,32,36), and organic bases (21,25,29,31,33,35-37) to release the C-terminal amino acid thiohydantoin and leave the free acid of the acyl moiety. This reagent list obviously embraces compounds with a wide range of reactivities and, hence, differing propensities to cause either nonspecific cleavage of peptide bonds or decomposition of the thiohydantoins.

188

ADAM

Acetohydroxamic acid. This oxygen-containing nucleophile was used successfully by Stark (21) at approximately pH 8.2 for cleavage of the peptidylthiohydantoin. He found that these mild conditions gave results at least as good as HCl, not only for the 1st degradation cycle, but also for subsequent cycles, thus establishing that the free carboxyl group on the shortened peptide was generated by the cleavage. This compound was subsequently used successfully by others (29,32,36), although under some conditions it has led to premature termination of the sequencing experiment (33). It would therefore seem preferable to 12 M HCl which was not found to be satisfactory by later workers (29,32). Reactions of bases in organic solvents. Although Stark found that hydrolysis of the acyl group was faster in aqueous solutions of acetohydroxamic acid, for solubility reasons he used a solution containing 50% pyridine (38). Organic solvents were found to modify the reaction rates for a range of basic reagents with acetyl and propionyl amino acid thiohydantoins (31). In agreement with the findings of earlier workers (21,29), this work confirmed that the cleavage experiments on model amino acid derivatives gave much more encouraging results than did those on peptides; that is, reagents that were suitable for the former were often not suitable for the latter. The fastest and cleanest results were obtained for 0.5 M potassium hydroxide in 33% methanol. It reacted with proteins and peptides within 3 min at room temperature. Longer reaction periods caused losses of peptide from the insoluble support (porous glass activated with carbonyldiimidazole (29)), so these conditions are probably too critical and it is suggested (36) that the dilution of this reagent should provide better control over reaction conditions, improved thiohydantoin stability, and increased cleavage specificity. Waley and Watson (15) found that aqueous 0.1 M alkali reacted in a few minutes at room temperature, or 20 min at 0°C; and 0.01 M reagent took 1 h for cleavage. Reagents of 0.16 M have been tried successfully on nanomole amounts of protein with reaction times of 4.5 min using a manual procedure and a PVDF-DITC membrane as a solid support (36). Triethylamine (33,35) and piperidine (37) have been used successfully in recent work. Blocking reactions during cleavage. As mentioned above, acetohydroxamic acid was found to modify the carboxyl group of the shortened peptide under some reaction conditions (33). A similar situation arises on treatment with primary amines. Ethylenediamine (36) and propylamine (37) both cleaved the TH from a peptide but also rendered it unreactive to further degradation, presumably because of amidation of the carboxyl group: -CO-TH

+ NH,

CH, - CH,-NH, -CO-NH-CH,

--f - CH,-NH,

+ TH

S. INGLIS

The above reaction obviously proceeds to completion and, although of no further interest for C-terminal sequencing, it should be applicable to the labeling of proteins at the C-terminus and for covalent attachment of the new C-terminus to solid supports. Stability of released thiohydantoin. Thiohydantoins are slowly decomposed by acetohydroxamate in 50% pyridine (half-life of 4 h) (38). Molecular oxygen in 0.5 M NaOH can cause oxidation of thiohydantoins, and even in 0.05 M sodium bicarbonate at room temperature acetylthiohydantoins decompose gradually as measured by the change in uv absorption of the compound at 266 nm (38). The stabilizing effect of antioxidants on amino acid thiohydantoins in a number of basic reagents has been demonstrated clearly (31). This work showed just how very important it is when using a strong inorganic base such as NaOH not only to include dithiothreitol, for example, in the solution, but also to restrict the reaction time at room temperature. It is sobering to reflect that the reagents that were found to be effective for sequencing at the nanomole level (31) are not radically different to those originally proposed by Schlack and Kumpf over 60 years ago. The major changes are (for ammonium thiocyanate) addition of a small amount of a strong acid to the coupling solution, a shorter cleavage step in alcoholic KOH, and addition of an antioxidant to stabilize the thiohydantoin. On the other hand, the availability of good solidphase attachment chemistry, and excellent HPLC instruments for rapid identification and quantitation of the thiohydantoin, has proved to be a great boon to the more recent C-terminal sequencing research. EXPECTED LIMITATIONS PROCEDURE

OF THE

THIOHYDANTOIN

In reporting new positive findings on the Schlack and Kumpf degradation and the exciting sequence data that followed these, reference was made to analogies with the development of the Edman chemistry for N-terminal sequence determinations (31). Such comparisons are extended here, not only to keep the present limitations of the C-terminal method in perspective, but also because results for the automatic N-terminal procedure provide an excellent bench mark. Nowadays automatic sequence analyses are made routinely in laboratories throughout the world, but at the time Edman and Begg (39) reported the first sequenator (approximately 17 years after Edman’s initial manual work), the manual Edman procedure was only capable of giving a few residues of protein sequence in favorable cases; the new instrument required highly sophisticated and highly purified chemicals; and manual dansyl-Edman chemistry had virtually superseded Edman chemistry for sequencing peptides because of its sensitivity and ease of obtaining results. Nonetheless, the direct Edman ap-

PROTEIN

CHEMICAL

C-TERMINAL

preach gradually took over as difficulties with it were overcome by ensuring that the protein did not become inaccessible to the reagents, by adding antioxidants to protect the relatively unstable derivatives, and by improving the instrumentation. Because of the similarities to the C-terminal procedure, the developments of the N-terminal procedure have already benefited the former (31,34) and they will continue to be a spur to it. Peptides

and Proteins

A number of physiologically active peptides such as hormones and neurotransmitters have amidated C-terminal amino acids, and these would not react in the thiohydantoin procedure. The amides are, of course, amenable to conversion to the free carboxylic acid. One of the alternative chemical procedures (4) in fact requires the amide as the activated derivative. The extent to which protein C-terminal carboxyl groups are modified is not known with certainty, but it is not expected to be great. On the other hand, often the N-terminal amino groups are either acetylated (or modified in some other way) after synthesis, reactive with solvents such as acetic acid and urea during purification, lost by cyclization (e.g., N-terminal glutamine in acidic solutions), or blocked during N-terminal sequencing, in the case of Asn-Gly sequences. A reliable C-terminal sequencing procedure should therefore help: protein characterization; assessment of a protein’s purity; primary structure determinations, especially when the N-terminus is blocked. Sequence Related Problems As expected from experiences with N-terminal amino acid sequencing, there are problems associated with certain amino acid residues in the C-terminal procedure. Kubo et al. (25) found that acetylproline forms a thiohydantoin, but in so doing, the acyl moiety is cleaved concomitantly. Conditions were gentle: 15 min at 30°C in acetyl chloride-trifluoroacetic acid, then addition of 3% thiocyanic acid in dioxane, and further reaction for 60 min at 30°C. Z-glycylproline also released the thiohydantoin of proline prior to the cleavage reaction. Therefore, during routine sequencing, direct evidence for a proline residue might require analysis of the coupling washings; possibly the preview of the residue next in the sequence would not be quantitative, thereby indicating that the cycle was abnormal. Albeit with a different reagent (trimethyl isothiocyanate), others have found that the proline residue is intractable at this stage (33). Hence, the problem with proline during C-terminal sequencing appears to be coupling related in contrast to N-terminal sequencing where it is cleavage related (some Pro-X bonds being particularly slow to cleave). As discussed above, the problems could stem from a slow cyclization reaction-in the former case, slow for-

189

SEQUENCING

mation of the thiohydantoin ring during coupling and, in the latter, a difficult cyclization to the thiazolinone derivative which is necessary to promote cleavage of the peptide bond with trifluoroacetic acid. The side chains of serine and threonine break down (38) but this does not affect subsequent degradations (34,38). Tryptophan is stable, for at least two degradation cycles (34). C-terminal asparagine apparently behaved normally using 0.5 M KOH in 33% methanol for cleavage (34) but hydrolysis of the peptidylthiohydantoin with 2% aqueous triethylamine gave unsatisfactory results for asparagine, some terminal amide being formed (33). The latter reagent was not overly successful in earlier work (29); it can give secondary products on reaction with acetylamino acid thiohydantoins (Inglis, unpublished work). Aspartic acid in a peptide was found to be amenable to the degradation procedure using HSCN at 80°C for the coupling reaction (31), only partly so for trimethyl isothiocyanate-pyridine reaction at 50°C (33). Liquid-Phase

and Solid-Phase

Systems

With respect to the types of technique that are applicable, the chemistry would suggest that liquid phase methods probably pose problems for the C-terminal sequencing procedure because of the polarity of the amino acid thiohydantoins. Meuth et al. (29) found that ethyl acetate extraction of the thiohydantoin, following coupling with HSCN and cleavage with 0.2 M acetohydroxamate, gave excellent yields of C-terminal Leu-TH, Phe-TH, and Tyr-TH from di- and tripeptides. The method provided a rapid procedure for determining the C-terminal residue of a peptide, but was restricted to this. The authors carried out preliminary studies using triethylamine as a volatile cleavage reagent to overcome extraction problems, but with limited success (which may have been related partly to the coupling conditions employed). Their results using covalent attachment of peptides to porous glass activated with carbonyldiimidazole were promising, and this method of attachment was also used with success by later workers (31). The use of porous glass beads for high sensitivity work presents some problems although such supports should not be ruled out (41). Alternative solid-phase systems are also being developed (37). One uses activation of the amino groups of a peptide with disuccinimidoyl carbonate to form a urea link to a polyamide support. The other incorporates an acid-cleavable link which gives a convenient method of studying the fate of the peptide portion after the completion of the degradation procedure. Derivatized PVDF membranes (Sequelon-DITC, for example) also offer possibilities as alternative solid phase support materials, but losses of protein are probably greater when proteins are attached to these (36).

190

ADAM

However, it would be most convenient if the current practice of electroblotting proteins onto polyvinylidene difluoride (PVDF) membranes for subsequent characterization by amino acid analysis and N-terminal sequence analysis could also be utilized for a C-terminal sequence analysis. Further experimentation, possibly exploiting the approach using the SequeNet procedure (MilliGen), in which the immobilized protein is enveloped with a porous polymer, would therefore be worthwhile. AUTOMATIC

C-TERMINAL

SEQUENCING

Prototype automatic N-terminal sequencers can also be used as the basis for an automatic C-terminal sequencer (34,35). The converted instruments incorporated a feature that has been missing on N-terminal sequencers, namely a reaction module that can be heated and cooled rapidly, thereby facilitating coupling at an elevated temperature (e.g., 80°C) and cleavage at a lower temperature (e.g., 20°C). There is no need for the automatic converter for C-terminal sequencing. The amino acid derivatives cleaved from the peptide chain during N-terminal sequencing are anilinothiazolinones, which are unstable and must be converted to the more stable phenylthiohydantoins for identification, whereas thiohydantoins are released in the cleavage step of the C-terminal procedure and may be analyzed directly. The conversion flask was therefore used only for collection and mixing of the solvent extracts following cleavage, and a portion thereof was immediately injected into the on-line HPLC. Reagents The reagents used in the preliminary experiments (34) were generally high quality commercial products and, because of their nature and the polar nature of the thiohydantoins, polar solvents such as methanol, acetonitrile, and aqueous solutions of these were used for washing. Stark found that freshly distilled acetic anhydride gave the best results (21). Dithiothreitol was added to the solvents and the cleavage solutions (31). To overcome problems associated with admission of the volatile reagent, HSCN in acetone, to the reactor, it was premixed with acetic acid and acetic anhydride, and kept chilled. A better answer to the problem might be to prepare the HSCN in situ in the reactor, viz., by following the addition of a thiocyanate salt with addition of a solution of acetic acid, acetic anhydride and trifluoroacetic acid (36); or alternatively, isothiocyanate reagents offer additional possibilities (35,37). Preparation of Thiocyanic Acid The thiocyanic acid was prepared by ion exchange of a solution of ammonium thiocyanate in acetone, ini-

S. INGLIS

tially following method II of Dwulet and Gurd (28). The following modified procedure of Dwulet and Gurd was used subsequently (34): Dowex 50X8 (H+ form) was soaked in 2 M NaOH for several hours, washed with water and soaked in 0.1 M EDTA overnight. The resin was then washed with deionized water, soaked in 2 M HCl for several hours, washed again with water, and finally with acetone several times. The resin was then mixed in acetone, degassed under vacuum and poured into a column (2 X 30 cm). After the resin had settled, the column was washed through several times with acetone. With the flow adjusted to 1 drop/8 s, a solution of ammonium thiocyanate (6 g) in acetone (20 ml) was added. When the eluent produced a deep red color with ferric chloride solution (60% (w/v)), fractions were collected and the concentrations determined by titration. The concentration of HSCN obtained ranged from 0.2 to 2.5 M. HSCN solutions were stored in the freezer. To ensure high purity and high concentration, it was prepared under an inert atmosphere and the collection vessels were chilled. Preparations in dioxane (25) were not made. The fractions were not pooled and adjusted to 0.5 M because it was found manually that concentrations of 1.8-2.2 M gave the best yields.

On-Line Detection Because the thiohydantoin derivatives of the amino acids are polar compounds, the acidic amino acid derivatives are eluted very close to the front using reversedphase columns for HPLC, hence it is critical that the sample solution is appropriate for maximum retention of the thiohydantoin by the column, that the polar byproducts are removed and on-line injection peaks do not interfere. The evidence obtained from both manual and automatic operations indicates that these conditions are attainable (34-36). The extinction coefficients of the thiohydantoins at 266 nm are similar to those of the phenylthiohydantoins (22) so the detectors of the HPLC equipment used for on-line identification are suitable also for analysis of the amino acid thiohydantoins. One can envisage the application of electrophoretie methods and mass spectrometric techniques to the identification of picomole and subpicomole quantities in the future.

HPLC IDENTIFICATION THIOHYDANTOINS

OF AMINO

ACID

In the past, amino acid thiohydantoins have been identified by thin-layer chromatography on silica gel plates (22), two-dimensional thin-layer chromatography on polyamide sheets (42), gas-liquid chromatography (43,44), mass spectrometry (30,45,46), and amino acid analysis after hydrolysis of the thiohydantoins

PROTEIN

0

FIG.

2

2.

Isocratic

4

6

and gradient

e TIME IMINS)

elution

ID

traces

CHEMICAL

12

for amino

14

C-TERMINAL

191

SEQUENCING

16

acid thiohydantoin

(38). During sequencing one can, of course, also analyze the residual peptide for amino acids after each degradation cycle. This indirect procedure is satisfactory providing the peptide is relatively small. HPLC procedures have also been developed (29,30, 32,34,36,47), although the methods described generally have shortcomings in that all of the amino acid thiohydantoins were not available. Since the use of analytical instruments is widespread, this technique offers a good alternative choice for analysis after either manual or automatic degradations (34). The extinction coefhcients are around 18,000 in ethanol at 266 nm, hence they may be detected readily at the low picomole level with microbore columns. The lysine side chain is acetylated and so does not have the additional absorption that one seeswith the phenylthiohydantoin of lysine. The phenyl group of phenylthiohydantoins, however, confers greater hydrophobicity on the thiohydantoin molecule and so the acidic amino acid derivatives can be retained on the columns relatively easily using low amounts of organic modifier in the buffer. However, the drawback to the current HPLC technology at this stage of development is that the commonly used C,, packings are not ideal for separation of the polar amino acid thiohydantoins. The thiohydantoins of the amino acids Asp, Asn, Glu, Gin, Gly, and Ser are not retained strongly on the normal C,, columns in the presence of organic solvents. This aspect of the methodology has undoubtedly been hampered because standard amino acid thiohydantoins have not been commercially available. The recent report that one company has been able to prepare them in the laboratory could foreshadow an end to this situation. Nonetheless, Fig. 2 shows two useful systems that have been developed for analysis of the common derivatives that would be obtained on degradation (34).

mixtures

(details

in text).

From

(34), reproduced

with permission.

Isocratic El&ion To achieve elution of the nonpolar thiohydantoins in reasonable times, it was necessary to use a C, column and to add a small amount of a less polar alcohol to the buffer solution, which then consisted of 3.3 mM ammonium acetate, 10.5% methanol, and 1.5% 2-propanol, 0.05 mM dithiothreitol. While simple equipment will suffice, the isocratic system was found to have drawbacks during sequencing, particularly for the automatic method. This is primarily because one needs a compromise solution which restricts removing polar impurities prior to elution of the polar amino acids yet leads to a broadening of the peaks and loss of peak height sensitivity as the chromatography proceeds. In the example shown, the peaks were improved by using a precolumn of Si60 and increasing the flow rate during the run. The proline derivative elutes in a region near Met-TH and Val-TH as expected; the elution of this group is sensitive to the temperature of the column. Two isomers of isoleucine are clearly evident on the isocratic elution trace.

Gradient E&ion All of the expected derivatives were resolved by gradient elution using a Waters PicoTag ODS column (34). The thiohydantoin peaks with asterisks added in Fig. 2 were prepared in situ and added to the mixture of pure thiohydantoins. It was found to be best to use very low salt concentrations (0.25 g ammonium acetate/liter) in buffer A, a pH of 4.76 and a column at 40°C for this resolution, which is complete in 16 min. Lowering the column temperature (33,36) may also help in some systems. Dithiothreitol was added to the buffers to ensure

192

ADAM

CYCLE

I

S. INGLIS

CYCLE

2

CYCLE

S

CYCLE

5

CYCLE

6

‘l_L.1.:1L AttN CYCLE

276

4 M

V

Y

JL,1.

C:YCLE

FIG. 3. reproduced

HPLC with

traces of products permission.

that the derivatives through the column.

Ij(_

7

CYCLE

from

nine

manual

_J

6

degradation

CYCLE

cyc cles on a synthetic

do not decompose on passage

Preparation of Amino Acid Thiohydantoins Several of the amino acid thiohydantoins have been difficult to prepare by classical methods. The varying solubilities, reactivities and stabilities of the amino acids, reactions of the side chains of the polar amino acids, and the relatively unstable nature of the thiohydantoin ring are all contributory factors to this situation. Yamashita (23) prepared all of them. Cromwell and Stark (22) prepared most of the common derivatives, some by slightly different routes, but believed that neither aspartic acid nor proline would form a thiohydantoin. Swan (17) could not prepare Arg-, Ser-, Asp-, and Glu-THs. However, Haurowitz et al. (20) had described the preparation of Asp-TH some years earlier. Also, Kubo et al. (25) reported success starting from the acylamino acids and using HSCN at low temperatures. From the analytical viewpoint this, of course, is not of much consequence unless the products were obtained with high yields, and it is this aspect that has caused

9

decapeptide

(Y-L-A-I-Y-V-M-A-F-V).

From

(31),

concerns as to the suitability of the thiohydantoin procedure. Since the properties and elution characteristics of the derivatives are now well known, there does not seem to be a great deal of point in using classical techniques and crystallization procedures for preparation. Methods akin to in situ preparations used for the HPLC work (see Fig. 2) should be applicable, possibly starting with amino acids linked to solid supports where they can be washed thoroughly after coupling, then cleaved with base, and immediately purified by HPLC. The ability to work quickly with such an approach is advantageous with substances such as amino acid thiohydantoins. ANALYTICAL

RESULTS

FOR PEPTIDES

AND

PROTEINS

Darbre (40) has reviewed his work and others with respect to solid-phase methodology. More recently, Meuth et al. (29) showed that controlled-pore glass (lo75 A, 200-400 mesh) could be derivatized so that amino groups of the peptide could be covalently attached by an amide link. This involves silanizing the glass with (&aminopropyl)triethoxysilane, succinylation of the amino group, then reaction of the carboxyl group with

PROTEIN

FIG. 4. Results of the first four C-terminal 10 nanomol of peptide was sequenced after

CHEMICAL

C-TERMINAL

degradations of Leu-enkephalin (Y-G-G-F-L) with benzoyl covalent attachment to a polyamide resin via a urea linkage.

N,N’-carbonyldiimidazole to form the activated product, an acyl imidazole. Protein attachment was found to be more efficient when wider pore glass (170 A) was used (34). Meuth et al. (29) obtained clean products after degradations of peptides attached to the glass. This approach was later taken up by others (31) who obtained improved repetitive yields with shorter reaction cycles. These were accomplished by heating for shorter periods at higher temperatures and replacing the acetohydroxamic acid reagent with 0.5 M KOH in 33% methanol containing DTT as antioxidant for cleavage. The procedure was also scaled down from the high nanomole level of Meuth et al. (29) to the low nanomole level by using 1.5ml polypropylene centrifuge tubes with screw caps containing an O-ring. A more rapid procedure for proteins based on coupling to a PVDF-DITC membrane illustrates the simplicity of the manual methodology for establishing the C-terminal amino acid sequence (36): Apply protein (1 nmol, 2.5 ~1) in 98% formic acid to each side of the Sequelon-DITC disc on an aluminum block at 52°C and allow to dry. Add N-ethylmorpholine (7% in 50% methanol, 3 ~1) at 5- to lo-min intervals over a period of 50 min, turning the disc over during this period. Cut the disc in half. Wash half the disc with acetonitrile, then

193

SEQUENCING

isothiocyanate

(37).

Approximately

with the acetylation mixture (AM), acetic acid-acetic anhydride (1:5) in a 1.5-ml capped centrifuge tube. Remove AM and add acetic anhydride (100 pl), ammonium thiocyanate (1.4 M in acetic acid, 20 /*l), TFA (3.8 pl), acetone (30 pl), seal, vortex, and heat at 80°C for 25 min. No precautions were taken to remove oxygen (DTT did not increase yields). Remove reagents, wash successively (taking 200 bl) with acetonitrile (x2), methanol/ DTT (x2), 70% acetonitrile/DTT (x3) and 30% methanol/DTT (x2), allowing time (approx 2 min) for solvents to extract (DTT approx 0.01% DTT). Add base (0.16 M NaOH in 30% methanol, approx 0.01% DTT, 100 ~1)~vortex and cleave for 4.5 min at 20°C. Neutralize with 5% acetic acid in 20% acetonitrile (50 pl), transfer to HPLC vial, wash membrane once with 20% acetonitrile (50 pl), add to vial, and stir. Analyze (20 ~1) by HPLC using isocratic elution with buffer A (3.3 mM ammonium acetate, pH 4.7, adjust for best positions of Asp and Glu derivatives) for 4 min and then a linear gradient to 75% acetonitrile with buffer B (CH,CN) over 20 min (flow, 0.8 ml/min; column temperature, 30°C). A HP 1090 HPLC was used with a Merck LichroCART 125-4 column containing Lichrospher RP-18, 5 pm. Figures 3 and 4 are illustrative of sequencing results obtained after solid phase attachment of hydrophobic peptides. Figure 3 was obtained with the HSCN proce-

194

FIG. 5. Y-G-G-F-L,

ADAM

Manual sequence data from the minor, . . . V-M-A-F-V.

C-terminal degradations (*) Overlap residue;

(+)

S. INGLIS

of a mixture of unrelated preview residue. From

dure described above for a peptide attached to glass activated with carbonydiimidazole (31). Some preview of the amino acid adjacent to the C-terminal residue is evident, as well as a gradual increase in the carry over of residues from previous cycles. It is clear that the residue in cycle 9 is Leu, in accord with the known sequence of the peptide. The same procedure was applied successfully to peptides containing polar amino acids (34). Figure 4 was obtained for a solid-phase development system (37) and was provided by courtesy of Dr. D. Hawke. The first four residues from the C-terminus of Leu-enkephalin were obtained with low lag and good efficiency using benzoyl isothiocyanate as reagent. It confirms the conclusions drawn earlier that the hydrophobic amino acids do not present problems with the methodology. The C-terminal method could also be applied to establish whether peptide mixtures are unrelated, are similar but have substitutions, or have “ragged” ends (34). Figure 5 shows the traces obtained for degradation cycles on a mixture of two unrelated peptides. The two sequences are clear, expect for the last cycle where the peptide in larger amount gives a smaller signal. The residue, Tyr, was the N-terminal residue linked to the solid support. Automatic sequencing of peptides and proteins is now being pursued actively (34,35). Figure 6 shows that there are improvements to be made yet, but despite the poor background near the front, and the loss of sensitivity for Leu and Phe because of the isocratic elution, the HPLC traces suggest that the degradation continued out to eight cycles. Loss of protein from the support (PVDFDITC) was a factor contributing to low repetitive yields (36).

peptides. The (34), reproduced

major with

component permission.

has the sequence

C-terminal sequencing data from an updated version of the sequencer used for the work in Fig. 6 is shown in Fig. 7. The peptide (Arg-Glu-Asp-Leu-Val-Ala-Glu, 3 nmol) was degraded from the C-terminus with guanidine isothiocyanate (35) and the amino acid thiohydantoins were identified using gradient elution. These data suggest that the time is near when C-terminal sequence analysis will be as useful as N-terminal and internal peptide sequences in identifying the sequence of cDNA clones corresponding to a particular protein and for designing DNA cloning strategies.

FIG. 6. Results of automatic C-terminal sequence analysis of a riboattached to a Sequesomal protein, L12 (. . . A-A-G-L-G-A-L-F-M) lon-DITC membrane. HSCN coupling and isocratic HPLC were employed (36). Reproduced with permission.

PROTEIN

FIG. 7. reagent

Analytical and on-line

CONCLUDING

CHEMICAL

data from automatic C-terminal degradations gradient elution HPLC (35). Reproduced with

C-TERMINAL

of a peptide, permission.

REMARKS

Development of a reliable C-terminal sequence analysis should have several useful applications. There is still a substantial need for amino acid sequence determinations of peptides and proteins. A chemical C-terminal sequencing procedure would provide an alternative to the well-proven Edman N-terminal degradation which has limitations with respect to efficiency and which may not be applicable because of N-terminal blocking. Often the C-terminal sequence is the prime interest of the molecular biologist. It would help to characterize a protein and provide a criterion of purity. The thiohydantoin procedure might also provide a route for the labeling of C-terminal amino acids for subsequent isolation of peptides. Because of the sequence data generated from it, or from some alternative procedure, it would presumably supersede methods such as amidation (48), tritiation (49), and hydrazinolysis (50) for C-terminal end group determinations. The rapid, sensitive identification by HPLC of the amino acid thiohydantoins raises expectations that the thiohydantoin procedure will match the sensitivity of the Edman procedure in the near future. If one accepts the positive results that have emerged over the years on chemical methods, there are several approaches that can be taken in modifying the carboxyl group with a view to weakening the adjacent peptide bond, and some of these are encouraging enough to warrant a new look. In this respect, the work of Le and Tatemoto (5) is very interesting, not only because it keeps alive a potentially useful method, but also because it converts it from an indirect to a direct one. Likewise, the new look (31) at the Schlack-Kumpf procedure built on the earlier work of Meuth et al. (29) with HSCN, and led to a method providing better coupling yields and an improved cleavage procedure. Recently also, the work of Waley and Watson (15) and Kenner et al. (19) on isothiocyanate reagents has been taken up again (30,33,35,37). Time will tell whether this approach will be as good, or better than that of using HSCN, or a thiocyanate salt in the presence of a strong acid, for all the common amino acids. It is most interesting that the organic isothiocyanates react best in a mildly basic solution, thereby opening

195

SEQUENCING

R-E-D-L-V-A-E,

with

guanidine

isothiocyanate

as coupling

questions as to what the reaction pathways are for the thiocyanate and the isothiocyanate procedures, and whether the widely accepted route is, in fact, the major one. This review has been framed to a large extent around the postulated reaction pathway for the thiohydantoin degradation as one way to try and bring together a number of pertinent, and often discordant, research findings. It was also to attempt to sow the seeds for a solution to the problems of the coupling reaction with amino acids such as proline, aspartic acid, asparagine, and glutamine. The naive observations, and comparisons with existing quantitative coupling and cyclization reactions of peptides, were made with a view to challenging dogma based on generalities and belief, rather than on hard scientific data. Indeed, experiments with potassium hydroxide in aqueous methanol (31) for cleavage were undertaken because it had not been established clearly (22) that the reagent was too severe for the reactions involved, and on the contrary, there was literature that suggested that sodium hydroxide (15), or an alcoholic solution of it (51), might well be suitable. In the relatively small number of degradation cycles that have been made on peptides with it so far, nonspecific cleavage reactions have not been obvious. The major hurdle to clear before full rewards can be obtained from application of the thiohydantoin procedure is the one designated proline. Although it has not recently been shown to be amenable to analysis by the thiohydantoin procedures, Pro-TH can be made, is as stable as other amino acid thiohydantoins, and was reported (25) to be released from a peptide as the thiohydantoin. Should the proline leap be successful, it is worth remembering that the thiohydantoin procedure is in principle simpler than the N-terminal degradation procedure in that the released amino acid derivatives require no further conversion and can be analyzed immediately. This represents a very significant advantage.

ACKNOWLEDGMENTS The author is indebted to Dr. L. Packman (Department of Biochemistry, University of Cambridge, UK) for readingand commenting on the manuscript, and to Ms. Natalie Wech for kindly typing it.

196

ADAM

S. INGLIS

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