175

Biochimica et Biophysica Acta, 578 ( 1 9 7 9 ) 1 7 5 - - 1 8 7 © E l sev ie r /Nor th -Hol l and Biomedica l Press

BBA 3 8 1 6 4

THE DESTRUCTION OF SERINE AND THREONINE THIOHYDANTOINS DURING THE SEQUENCE DETERMINATION OF PEPTIDES BY 4-N,N-DIMETHYLAMINOAZOBENZENE 4'-ISOTHIOCYANATE

J.Y. C H A N G *

Protein Biochemistry Unit, Research School o f Biological Sciences, The Australian National University, Canberra, A.C. T. (Australia)

(Received Augus t 3 0 t h , 1978)

Key words: Thiohydantoin; Dimethylaminoazotrenzene isothiocyanate ; Serine destruction; Threonine destruction; Amino acid sequence

Summary

1. A mechanism for the destruction of serine and threonine thiohydantoins during protein sequence analysis by the Edman-type degradation is proposed. The mechanism begins with the dehydration of serine and threonine side chains (at the cyclization stage) which occurs mainly in anhydrous acid solution. The dehydrated derivatives finally polymerize by way of the reactive methylene group (enamine) to form polymers with various molecular weights. In aqueous acid solution, the dehydrated thiohydantoins of serine and threonine undergo hydration (according to the Markovnikov rule) and ring fission, which leads to the irreversible breakdown of thiohydantoin ring. The serine derivative shows a much greater tendency to undergo these side-reactions than the threonine derivative.

2. In the presence of oxygen, the alkaline hydrolysis of amino acid thiohy- dantoins goes through an oxidation-deamination reaction at the C-N bond of the thiohydantoin ring and leads to the formation of thiourea derivative and keto acids. This reaction mechanism accounts for the low recoveries of amino acid obtained from the alkaline hydrolysis of amino acid thiohydantoins.

* Present address: Max-Planck Ins t i tu t fiir Molekulare Genetik, Abt. Wittmann, Ihnestrasse 63/73, D-1000 Berlin (West) 33-Dahlern, Germany. Abbreviations: DABITC, 4-N,N-dimethylarnin0azobenzene 4'-isothiocyanate; DABTC, 4-N,N-dimethyl- aminoaz obenzene 4'-thiocarbamyl; DABTH, 4-N,N-dimethyiaminoazobenzene 4'-thiohydantoin; DAAB, 4-N,N-dimcthylarnino-4'aminobenzene or N,N-dimethyl-4,4'-azodianiline; DABTZ, 4-N,N- dimcthylaminoazobenzene 4'-thiazolinone; H+/H2 O, w a t e r / a c e t i c acid saturated w i t h HCI (1 : 2, v/v).

176

Introduct ion

The coloured reagent, 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate (DABITC) has been introduced for identifying the N-terminal amino acid [1,2] and for the stepwise degradation [3,4] of proteins in amino acid sequence anal- ysis. The application of this new reagent to both liquid [3,4] and solid phase [5] sequence determination requires only 2--10 nmol of peptides and proteins. The sensitive detect ion of the DABTH-amino acids and their satisfactory separation on the thin-layer chromatography has enabled the proposed sequencing method to be carried out in a simple and sensitive way. One of the major drawbacks of DABITC method is, however, the extremely low recovery of serine and threo- nine thiohydantoins (as DABTH derivatives). The reasons for the low recovery have not until now been elucidated. Also, the phenylisothiocyanate technique has occupied a central position in protein chemistry since its introduction by Edman [6--8]. Some of its reaction mechanisms, such as the instability of phe- nylthiohydantoin-amino acids in alkaline solution [9] and the destruction of phenylthiohydantoin-serine, must still be finally clarified.

This s tudy endeavours to elucidate the possible pathways of these side reac- tions.

Experimental

Materials DABITC was synthesizecl by the method described [1,3]. The L-amino acids

and serine peptides (Ser-Ala and Ser-Ser-Ser) were purchased from Sigma St. Louis, MO, U.S.A. Polyamide sheets were obtained from Chen-Chin Co. Taipei, Taiwan and the silica gel plates were from Merck (G-60, wi thout fluorescent indicator, 0.25 mm). All the other chemicals and solvents used in this report were commercial analytical grade and the solvents were redistilled before use.

DABTC-amino acids and peptides were prepared by reaction of DABITC with an excess of amino acids or peptides in the volatile buffer (pH 10) [10]. The DABTC-amino acid could be separated from the excess amino acid by extraction into ethyl acetate from aqueous solution and cyclized to form DABTH-amino acid in H÷/H20 [11]. The DABTC derivatives of Ser-Ala and Set-Set-Set were used in the presence of the excess peptides.

Stabilities o f DABTH-amino acids and DABTC-amino acids in alkaline solu- tion

DABTH-amino acid (or DABTC-amino acid) was dissolved (40 nmole in 100 ~1) in 1 N NaOH and incubated at 55°C for 1 h. No precautions were taken to exclude oxygen. After the reaction, the mixture was acidified with 1 N HC1 and the products were extracted into ethyl acetate (200 ~1). The converted products were identified using both polyamide and silica gel thin-layer chromatography.

Stabilities o f DABTH-Ser and DABTH-Thr in anhydrous trifluoroacetic acid 2 ~mol of DABTH-Ser or DABTH-Thr was dissolved in 4 ml of anhydrous

trifluoroacetic acid and incubated at 55°C. Portions of equal volume (50 pl) were removed after different time intervals and dried. The chemical composi-

177

tions (examined on both silica gel and polyamide TLC) and the spectrophotom- etric character in ethanol (on a Unicam SP 1800 Ultraviolet Spectrophotometer) of the aliquots were examined by comparing them with the standard DABTH- Set a~d DABTH-Thr.

Destruction o f serine and threonine residues during the formation o f their DABTH derivatives

DABTH-Ser, DABTH-Thr, DABTC-Ser-Ala and DABTC-Ser-Ser-Ser were first cyclized to give the corresponding DABTZ derivatives in the anhydrous trifluoroacetic acid for different time intervals. Equal samples were removed and dried and then isomerized to their DABTH derivatives in H÷/H20 solution (55°C, 50 min). The acid was evaporated to dryness after the reaction period. The nature and purities of the DABTH products obtained (as a function of anhydrous trifluoroacetic acid t reatment duration) were examined on TLC or spectrophotometr ic recordings (dissolved in ethanol).

Quantitative analysis o f the DABTH and DABTC derivatives recovered from thin-layer chromatography

Two methods were generally used for the quantitative determination of the DABTH and DABTC derivatives recovered from TLC.

(1) The derivatives recovered from the silica gel plates were extracted into absolute ethanol and the absorbances were measured at 420 nm [12].

(2) The derivatives recovered from the polyamide sheet (both polyamide and derivatives) were dissolved in 6 N HC1/ethanol (1 : 2, v/v) and the absorb- ances were measured at 520 nm.

The molar extinction coefficient (emax) of DABTH-amino acids at 420 nm (in ethanol) is about 34 000. When DABTH-amino acids were dissolved in the

p ,, ',, # - I

: i " ~ / \ i',

• i i I i i i i i

260 340 420 500 580 nm

F i g . 1 . Spectra o f D A B T H - V a l in e thano l ( ) , i n 1 N H C I ( . . . . . . ) a n d i n e t h a n o l / 6 N H C I (2 : 1 , v / v ) (-- --) in the ultraviolet and visible regions. Concentrat ion: 0 . 0 2 3 m M . ~.

178

acidic aqueous ethanol, the emax values increased with increasing acidity and the kmax was also shifted to longer wavelength (520 nm). The emax (in etha- nol)/emax (in ethanol/6 N HC1, 2 : 1, v/v) ranged from 0.72--0.78 for all the DABTH derivatives of common amino acids studied [11]. Fig. 1 gives the typi- cal absorption spectra of DABTH-Val in the solvents indicated.

Results

Stabilities of DABTH-Ser and DABTH-Thr The recoveries of serine and threonine (as their thiohydantoin derivatives),

by either phenylisothiocyanate or DABITC method, has been found to be much poorer than those of other amino acid residues in the protein sequence analysis. There are two possible stages when serine and threonine can be destroyed during the formation of their thiohydantoins (see Scheme 1); (1) destruction of DABTC-Der- and DABTC-Thr- in the alkaline solution during the coupling stage, (2) destruction of DABTH-Ser and DABTH-Thr (or DABTZ-Ser and DABTZ-Thr) in the acid solution during the cyclization stage. Since there was no detectable change of DABTC-Ser and DABTC-Thr in the

AA-

DABITC (Purple)

I

c ~ ~ ~ .N'(" "*)" N : N "-(I "*).NH-C-NH-CH-C-

CH3 ~ J ~ II i II S R O

DABTC-A A- (Blne)

i Via DABTZ-and DABTC-AA

TFA (INDIRECT)

L CH..~ / ; ' - &

OAAB (Red)

I H÷/H20 (DIRECT)

j~'~NoCH3 ! I I

6 H~'H2 0 H

DABTZ-AA (Blue) DABTH-AA (Red)

S c h e m e 1. T he p a t h w a y of f o r m a t i o n of D A B T H - a m i n o acids b y r eac t i on of D A B I T C w i t h a m i n o acids or N- te rmina ls of pept ides . T h e colours of the der iva t ives a p p e a r e d on the p o l y a m i d e TLC aftez exposu re to HCI vapou r . T F A , a n h y d r o u s t r i f luozoacet ic acid.

179

moderately strong alkaline solution (1 N NaOH, 55°C, 1 h) (see following sec- tion), it is reasonable to rule out the possibility that DABTC-Ser and DABTC- Thr ~ e destroyed in the mild coupling buffer. In fact, bases do no t cause ready elimination of water from simple alcohols, unlike the elimination of hydrogen halide from alkyl halides. On the other hand, acids catalyse the dehydrat ion of alcohols to olefins. The acidic medium is the most likely case for these destruc- tions.

It is also interesting to point out that the direct formation of DABTH-Ser in H+/H20 (see Scheme 1) resulted in a much more satisfactory recovery than the indirect method (i.e. first treating with anhydrous trifluoroacetic acid and then H+/H20; DABTC to DABTZ to DABTH) [2,3]. Apparently, the destruction of serine and threonine were initiated during the cyclization (or cleavage from N-terminals of peptides) to form the thiazolinone derivatives in anhydrous trifluoroacetic acid.

The possible routes of serine and threonine destruction during the formation of their thiohydantoins were approached in two different ways:

(1) The stability of the standard DABTH-Ser and DABTH-Thr in the anhy- drous trifluoroacetic acid was first examined. The direct conversion of DABTC- amino acid to DABTH-amino acid in H*/H20 is mos t satisfactory in terms of yields. Most DABTC-amino acids were converted into their corresponding DABTH-amino acids quantitatively in this manner. Even the labile DABTC-Ser and DABTC-Thr gave 81% and 97% recoveries respectively [11]. Treatment of DABTH-Ser and DABTH-Thr with anhydrous trifluoroacetic acid, on the other hand, converted DABTH-Ser into DABTH-Ser ~ and DABTH-Ser ~ (a discontinu- ous tailing strip along the direction of solvent 2, see Fig. 2) and converted DABTH-Thr into DABTH-Thr ~. These new products have a characteristic absorption at 320 nm in addition to 269 nm. The rates of conversion were fol- lowed from the spectrophotometr ic changes {Fig. 3). The dec reaseof DABTH- Set and DABTH-Thr could also be quantitatively followed by TLC. DABTH-Ser shows a faster conversion rate than DABTH-Thr. Over 90% of DABTH-Ser was

DABITC DABITC c c )

A |

J Thr ~ f

T 2 ki

c~ r

| ~Solvent 1

O

~oo.E Q I O

O Thr

B

Fig. 2. Th in - l aye r c h r o m a t o g r a p h y on the D A B T H derivatives ( o n p o l y a m i d e ) f r o m the anhydrous tri- f l uo roace t i c acid treatment o f D A B T H - S e z (A) and D A B T H - T h r (B). The dehydrated products (A) and the presumed p o l y m e r i z e d product s (o) are d e n o t e d . E is a s y n t h e t i c ma rke r , D A B T C - d i e t h y l a m i n e . Solvent 1: water /acet ic acid (2 : 1. v/v) and so lvent 2: toluene/n-hexane/acetic acid (2 : 1 : 1, by vol.) .

o

o

0.4

(18

!

260

/

~i:~:

! I 260 320 nm

A

320 n m

"8 l 0.4~

180

F i g . 3. The changes o f the ultraviolet absorpt ion spectra o f DABTH-Ser (A) and DABTH-Thr (B) after treatment wi th anhydrous tri f luoroacetic acid for 3 0 r a i n ( ~ - - ) , 6 0 m i n ( . . . . . . ) and 120 m i n ( . . . . )

at 55°C.

converted into DABTH-Ser ~ and DABTH-Ser ° after exposure to anhydrous trifluoroacetic acid for 60 min (at 55°C) while only 55% of DABTH-Thr was converted into DABTH-Thr ~.

These products possess a thiohydantoin structure since they all appear as red spots on TLC. DABTH-Ser ~ and DABTH-Thr ~ are probably the dehydrated products and DABTH-Ser ~ probably corresponds to polymerized products of DABTH-Ser ~ with varying molecular weights because of their behaviour on TLC. This will be discussed later in this report.

(2) DABTH-Ser and DABTH-Thr were prepared by ~he indirect route (Scheme 1) with different times of anhydrous trifluoroacetic acid incubation. The purities of DABTH-Ser and DABTH-Thr obtained were then examined as a function of the duration of the anhydrous trifluoroacetic acid incubation. This is the normal process for the formation of DABTH-Ser and DABTH-Thr during the protein sequence analysis when anhydrous trifluoroacetic acid is employed as the cleavage reagent. Since DABTZ derivatives are unstable, the anhydrous trifluoroacetic acid aliquots removed at different time interval were dried and isomerized (in H÷/H20) to the more stable DABTH derivatives for identifica- tion.

For threonine, again, the decrease of DABTH-Thr and subsequently the increase of DABTH-Thr ~ is a function of the time of incubating DABTC-Thr in anhydrous trifluoroacetic acid (Fig. 4B). About 40% of DABTH-Thr was dehydrated to DABTH-Thr ~ if exposure of DABTC-Thr in anhydrous trifluoro- acetic acid is 60 min (55°C). This result shows a very close similarity to that obtained from the direct t reatment of DABTH-Thr with anhydrous trifluoro- acetic acid (Fig. 3B).

For serine, the mechanism is more complicated. Although the destruction

1 8 1

C, H2OH DAB-NH-C-NH-CH-CO-

CH3, ~',',',',',',',',',-~ ,-... S DAB= CH3"N "-'f~'N'N t-'/~- DABTC-Ser-

I TFA

+HIN---CI_NH.DA B +HI~-C-NH-DAB , +HN NH-DAB, / , '~ / Ik o c . . s f .c . .c .

- " \ 6H ~ TFA O TFA

DABTZ-Ser DABTZ-Ser ~ DABTZ-Ser °

l l ' l~ O IH~I20 ~1H~H20

O~'CI N-DABI H~H20 O~CL~N"DAB H~H [ /C ..DAB CH2°CI~I~"C"SoH * - CH2"*~"N"~:"SH ~ " L"'IC""N"~, H N ~ CH2"

DABTH-Ser DABI'I"I-Ser ~ DABTH-'Ser a

%¢ Dec ~N-DAB N-DAB

CH3"C"~N "CeS /.t+/H20 CH~ "N"Ces H DABTH-Ser °

DAB-NH-C,-NH 2 s

DABTC-NH2

Scheme 2. The proposed pathway of the destruction of DABTH-Ser during the protein sequence deter° mination by DABITC degradation. TFA, anhydrous trifluoroacetic acid.

of DABTH-Ser is also a function of the length of the incubation of DABTC- Set in anhydrous trifluoroacetic acid, the converted DABTH-Ser does no t appear quantitatively as DABTH-Ser ~ and DABTH-Ser ° because, in addition, there were slightly increasing intensities of DABTC-NH2 and DABTH-Ser ° (a new red coloured spot which partially overlaps with DABTH-Asp on poly- amide TLC) with prolonged anhydrous trifluoroacetic acid incubation (Fig. 5A).

The nature of DABTC-NH2 was confirmed by comparing its colour and chro- matographic behaviour with those of an authentic sample. DABTH-Ser ° is

182

_G

o

0.4

!

250

|:"°.,.

I

320 nm

O.8

G

C~4

I, °°°°,° ~. . . . . -

~ ,°°°° ! i

260 320 nm

Fig. 4. The u l t rav io le t a b s o r p t i o n charac te r i s t i c of the p r o d u c t s ob t a ined f r o m the d i rec t ( ) and ind i rec t ( . . . . . . and . . . . . . ) cyc l i za t ion of DABTC-Ser (A) and D A B T C - T h r (B) w i th 10 m i n ( - . . . . . ) and 60 rain ( . . . . ) o f a n h y d r o u s t r i f luo roace t i c acid incuba t ion .

probably a hydrated product of DABTH-Ser ~ lacking the OH group in the a-position of the amino acid moiety (see Discussion).

Both DABTC-Ser and DABTC-Ser-Ala show a similar tendency for these side reactions, and the by-products can be separated on silica gel plate (Fig. 5B) and determined quantitatively. The recoveries of DABTH-Ser and by-products of DABTC-Ser-Ala as a function of anhydrous trifluoroacetic acid incubation is presented on Fig. 6.

From the spectrophotometric changes, it was found by the indirect method that the destruction of DABTH-Ser was not accompanied by the large increase

i

• A

(N

1 . Solvent 1

B

Fig. 5. Th in- layer c h r o m a t o g r a p h y on p o l y a m i d e (A) a nd silica gel (B) of the D A B T H der ivat ives (DABTH-Ser , D A B T H - S e r A, D A B T H - S e r a a nd D A B T H - S e r ° ) an d D A B T C - N H 2 der ived f r o m the des t ruc- t ion of D A B T H - S e r dur ing the D A B I T C degrada t ion . So lvent 3 was c h l o r o f o r m / m e t h a n o l (9 : 1, v /v) . F o r

the s t ruc tu res of the individual p r o d u c t s , please re fe r t o S c h e m e 2.

183

0 10 30 60 Time (rain)

Fig . 6 . R e c o v e r i e s o f D A B T H - S e r (¢ --), D A B T H - S e r ~ (~ ~), D A B T H - S e r ° (o o) , D A B T H - S e r ~ (D o) a n d D A B T C - N H 2 ( a A) o b t a i n e d f r o m t h e c y c l i z a t i o n o f D A B T C - S e r - A I a b y t h e d i r e c t m e t h o d ( t i m e o f a n h y d r o u s t r i f l u o r o a c e t i c ac id i n c u b a t i o n is ze ro ) a n d i n d i r e c t m e t h o d w i t h 10 , 3 0 a n d 6 0 m i n o f a n h y d r o u s t r i f l u o r o a c e t i c ac id i n c u b a t i o n .

in absorption at 320 nm (Fig. 4A). This indicates that the intermediate DABTH-Ser ~ which absorbs at 320 nm is not a major component under these conditions.

Table I gives the yields of DABTH-Ser from DABTC-Ser, DABTC-Ser-Ala and DABTC-Ser-Ser-Ser and DABTH-Ser(OBz) from DABTC-Ser(OBz) by both direct and indirect formation. The indirect method gave, in general, lower yields than the direct method. The much better recovery of DABTH-Ser(OBz) suggests that blocking of the serine hydroxyl group is an efficient way of pre- venting dehydration and the reactions which follow it.

Stability of DABTC-amino acids and DABTH-amino acids in alkaline solution The destruction of DABTH-amino acids in strong alkaline solution is almost

instantaneous, as observed by the disappearance of the absorption at 269 nm. The arbitrary condition chosen (1 N NaOH, 55°C, 1 h) converted most of the DABTH-amino acids (Glu, Ile, Leu, Phe, Gln, Val, Ala, Met, Thr, Set, Trp, Tyr and Gly) into one major (a) and two minor (b and c) blue products as shown in Fig. 7. The major blue product (a) was identified as DABTC-NH2 by comparing its colour, shape and RF Values on both polyamide and silica gel plate with

T A B L E I

R e c o v e r i e s o f t h e D A B T H - S e r f r o m D A B T C - S e r , D A B T C - S e r - A I a a n d D A B T C - S e r - S e r - S e r a n d D A B T H - Se t ( O B z ) f r o m D A B T C - S e r (OBz) b y b o t h d i r e c t ( D A B T C de r iva t ive w a s t r e a t e d w i t h H+/H2 O a t 55°C f o r 5 0 r a in ) a n d i n d i r e c t ( D A B T C der iva t ives w a s t r e a t e d w i t h a n h y d r o u s t r i f l u o r o a c e t i c ac id a t 55°C f o r I 0 m i n , e v a p o r a t e d t o d r y n e s s , t h e n w a s t r e a t e d w i t h H+/H2 O a t 55°C f o r 5 0 m i n ) c y c l i z a t i o n m e t h o d s .

D A T B C der iva t ives D i r e c t m e t h o d I n d i r e c t m e t h o d (%) (%)

Ser ine 81 58 S e r y l a l a n i n e 56 4 4 S e r y l s e r y l s e r i n e 4 7 4 2 O - B e n z y l s e r i n e 9 9 7 6

184

CN

O u')

2

DMITC O

~ a

Oc

O

A a O O 0

b o o

cO + Q H 5 M

B C')

O t ~

T 1 = Solvent 1

Fig. 7. T h i n - l a y e r c h r o m a t o g r a p h y o n p o l y a m i d e (A) a n d si l ica gel (B) o f t he t h r e e m a j o r p r o d u c t s (a, b and c) r e s u l t e d f r o m the a lka l ine h y d r o l y s i s o f D A B T H - a m i n o acids under t h e p r e s e n c e o f o x y g e n . H is h y d r o l y s e d p r o d u c t s . S is a s t a n d a r d s a m p l e s o f D A B T C - N H 2. M is t he m i x t u r e o f H a n d S. So lven t 3

w a s c h l o r o f o r m / m e t h a n o l (9 : 1, v/v) .

those of an authentic sample. The other two products (b and c) were not identified. They are believed to be DABTC-type products, since they appeared on TLC as blue coloured spots. The same alkaline condition, however, con- verted DABTH-Pro and DABTH-Hyp to DABTC-Pro and DABTC-Hyp, respec- tively.

The addition of sodium dithionite (0.2 M) in 1 N NaOH solution altered the hydrolysis mechanism by converting most of the DABTH-amino acids into their corresponding DABTC-amino acids. However, part of the azo bond was also reduced by sodium dithionite. No significant chemical change of DABTC- amino acids, including serine and threonine, was observed by TLC after alkaline hydrolysis (1 N NaOH, 55°C, 1 h). These results indicate that the major destruction of DABTH-amino acids in alkaline solution takes place by way of oxidative-deamination (see Scheme 3). The resistance of DABTH-Pro and DABTH-Hyp toward oxidation is due to the fact that they are secondary cyclic amines.

O"c__ N_DA B O~'C __ N_DA B DAB-N H-~S- NI"I2 I I I I

R.CHN ..C..s (o) R.C,.N ,,C.. S ~ ÷ H R-I~-COOH

S c h e m e 3. T h e o x i d a t i o n - d e a m i n a t i o n r e a c t i o n w h i c h l eads t o t h e d e s t r u c t i o n o f a m i n o acid res idues d u r i n g t h e a lka l ine h y d r o l y s i s o f a m i n o ac id t h i o h y d a n t o i n s .

Discussion

The major pathway for formation of DABTH-amino acids by reaction of DABITC with N-terminal amino acid of a peptide is summarized in Scheme 1.

185

The isothiocyanate group reacts with nucleophiles such as water to form thio- carbamic acid which subsequently decomposes to give the respective amine, or with amino acids, amino alcohols [13] and amines to form thioureas. If a thiourea was formed between DABITC and a peptide N-terminus, it could be cyclized to give the relatively stable thiohydantoin by way of two different processes {see Scheme 1).

{1) By the direct route, the cyclization is carried out in strong aqueous acid solution such as water/acetic acid saturated with HC1. In this process, the cleavage {DABTC-AA-AA1 to DABTZ-AA) and its following conversion reac- tions (DABTZ-AA to DABTC-AA to DABTH-AA) proceed in the same aqueous acid solution [14].

(2) By the indirect route, the cyclization is carried out in anhydrous acid solution such as anhydrous trifluoroacetic acid. In this process, the released product is DABTZ-AA. The DABTZ-AA is then separated from the shortened peptide and converted (by way of DABTC-AA) into DABTH-AA in aqueous acid solution in a separate reaction.

In the consecutive N-terminal degradation of peptides, the cleavage is normally carried out by way of the indirect route {in anhydrous trifluoroacetic acid) to protect the amide bonds frorn hydrolysis. The Edman-type degrada- tion cycle for amino acid sequence determination is therefore composed of three separate reactions, namely coupling (in alkaline solution), cleavage (in anhydrous acid solution) and conversion (in aqueous acid solution).

In the single N-terminal amino acid determination using DABITC, a rather drastic coupling condit ion (75°C, 1.5 h) [3,11] and a direct cleavage (in water/ acetic acid saturated with HC1) is recommended [11]. The direct cleavage avoids the extra conversion reaction and the extensive destruction of serine and threonine thiohydantoins caused by exposure to anhydrous acid.

Some earlier observations on the phenylisothiocyanate method [15,16] have indicated that serine and threonine tend to undergo dehydrat ion through a fi-elimination of the side chain and the dehydrated products have a distinct absorption at 320 nm. The dehydrated serine thiohydantoin slowly undergoes a further reaction leading to a product with a low intensity absorption maximum at 272 nm. Ilse and Edman believe, although wi thout direct evidence, that this further reaction is the polymerizat ion of the ~-fi unsaturated dehydrated prod- uct. If those assumptions are correct, then in the DABITC method, one should be able to follow all those conversions by observing the positions of the coloured spots.

The results obtained in this s tudy are, in general, consistent with those pre- vious observations. It is shown that dehydrat ion occurred mainly at the anhydr- ous trifluoroacetic acid t reatment stage in which DABTH-Ser reacted at a faster rate than DABTH-Thr. The positions of DABTH-Ser ~ and DABTH-Thr ~ (overlap with DABTH-Phe on polyamide TLC) on TLC should be used to help the confirmation of DABTH-Ser and DABTH-Thr.

The dehydrated DABTH-Ser (i.e. DABTH-Ser~), however, exhibits a much higher tendency to undergo further reactions than DABTH-Thr ~. The destruc- t ion of DABTH-Ser in acid solution is obviously no t a simple dehydration- polymerization reaction. The dehydrat ion must be followed by further reac- tions which lead to the five major products as shown in Fig. 5 (Namely,

186

DABTH-Ser, DABTH-Ser ~, DABTH-Ser [], DABTH-Ser ° and DABTC-NH:; for their structures, see Scheme 2). The possible routes of these reactions are postulated in Scheme 2, and it could be concluded that: (1) all the side reac- tions are initiated by the dehydrat ion of serine side chain; (2) the dehydration and polymerization reactions occurred mainly at the anhydrous trifluoroacetic acid stage; and (3) in the aqueous acid solution, the dehydrated serine thiohy- dantoin tends to undergo hydrat ion and deamination, leading to irreversible breakdown of the thiohydantoin ring.

The evidence for the above is based mainly on the following conversion reac- tions (TFA = anhydrous trifluoroacetic acid):

(A) (B)

DABTH-Ser , . . lh) (81%)

DABTH-Ser h (19%) TFA

• DABTH-Ser (11%) 2 h

DABTH-Ser A (45%) I~/H20

• DABTH-Ser ° (44%)

DABTH-Ser .(10%) DABTH-Ser a (23%) DABTH-Ser a (30%) DABTH-Ser ° (32%) DABTC-NH2 (trace)

Reaction A, as demonstrated in Fig. 3A, resulted in a large increase of absorp- tion at 320 nm, which is a characteristic absorption peak of dehydrated serine thiohydantoin. The nature of DABTH-Ser ~ is therefore confirmed with little doubt. The nature of DABTH-Ser ° remains to be finally clarified although it seems likely that these products were polymerized DABTH-Ser ~ with varying molecular weights, on account of their behaviour on TLC (see Fig. 5). The ten- dency to form DABTH-Ser: is, in fact, the major reason for the absence or very low yield of DABTH-Ser during the sequence determination.

i

08 f A , ; \

0.4

! | !

2 6 0 320 nrn

Fig. 8. The change o f u l t r av io le t absorp t ion when a m i x t u r e o f D A B T H - S e r (11%), D A B T H - S e r z~ (45%) and D A B T H - S e r D (44%) ( . . . . . . ) was treated with H+/H20 at 55°C for 2 h ( - ). The reaction r e s u l t e d in a p a r t i a l c o n v e r s i o n o f D A B T H - S e r ~ a n d D A B T H - S e r ~ t o D A B T H - S e r ° . T h e d e c r e a s e o f a b s o r p t i o n a t 3 2 0 n m a n d i n c r e a s e o f a b s o r p t i o n a t 2 7 0 n m i m p l y t h a t p a r t o f t h e c o n j u g a t e d t h i o h y d a n - t o i n r i n g ( d e h y d r a t e d D A B T H - S e r ) w a s c o n v e r t e d b a c k t o t h e n o n - c o n j u g a t e d f o r m .

187

Reaction B converted approx. 50% of DABTH-Ser ~ and 30% of DABTH- Set D to a major red coloured compound DABTH-Ser ° and a minor blue product DABTC-NH2. This reaction, which resulted in a decrease of absorption at 320 nm and an increase of absorption at 269 nm (Fig. 8), suggests that part of the dehydrated serine thiohydantoin was converted back to the nonconjugated (hydrated) th iohydantoin ring. Since the hydrated serine thiohydantoin is dif- ferent from the parent DABTH-Ser, the hydrat ion of DABTH-Ser ~ according to the Markovnikov rule to DABTH-Ser ° is the most likely explanation. The formation of DABTC-NH2 from DABTH-Ser ° could also be explained in this way (see Scheme 2). It should be noted that dehydrated threonine thiohydan- toin exhibits a smaller tendency to react further, as might be expected from the presence of the sterically inhibiting methyl group.

Although the final confirmation of this suggestion must await the isolation and characterization of the individual products, the foremost importance of the results obtained is to help in tracing of the destroyed DABTH-Ser during the protein sequence analysis by the DABITC degradation [4].

Acknowledgement

This work is extracted from the Chapter 6 of the Ph.D. thesis (1977) which the author submit ted to the Australian National University. The author thanks Dr. W.L.F. Armarego for discussion and Ms. W.F. Chu for help in preparation of this manuscript.

References

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