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Identification of carboxymethylthiocysteamine as cleavage product of cystaldimine
418
BIOCHIMICA ET BIOPHYSICA ACTA
Short Communications -
m
.
Identification of carboxymethylthiocysteamine as cleavage product of cystaldimine
s(: 23060
Cvsteamine is oxidatively deaminated by hog-kidney or pea-seedling diamine oxidase (diamine :O2 oxidoreductase (deaminating), EC 1.4.3.0)1,2 t o cystaldimine 3, the corresponding eyclized aminoaldehyde. Some previous data indicated that cvstaldimine undergoes further degradation 4-6 and the following reaction ,sequence was suggested~: SS ('H e S S (,;|t.,
CIte
CtI.,
.... ~ ('Hz Ntt.,()H 2 N]ta
CH., .S-SH ~
(?I1~
CIt
-}- (.'2-fragment CHu.NHe
N cystamine
cvstaldimine
thiocysteamint.
The two-carbon fragment was in part identified as glycolaldehyde, but for thiocvsteamine only indirect data had been obtainedL In the present communication we report the identification of thiocysteamine, obtained by trapping it with monoiodoacetate in the form of the mixed disulphide I-amino-3,4-dithiapentane-5-cart~xylie acid (NHe-CHa-CHz-S-S-.CH.,-COOH). In
,¢ o e,
I
Q
O U
.•.-
~
~.
I*~
:--;i~
Fig. I. C h r o m a t o g r a p h y of pure c o m p o u n d . 8o/~g of cystamine, 13o ttg of CMTC a.nd 80 [tg of CMC have been subjected to p a p e r c h r o m a t o g r a p h y on W h a t m a n No. 4 paper, using phenol in the tirst run and collidine-lutidine in the second run. S p r a y reagent : n i n h y d r i n (o. i %) in butanol. S p o t No. i, CMC; No. 2, ('M'I'C; No. 3, cystaminc. Abbreviations: CMTC, c a r b o x y m e t h y l t h i o c y s t e a m i n e ; CMC, c a r b o x y m e t h y l c y s t e a m i n e .
tliochim. Hiophys...Icta, 93 (1904) 4 tS -4-' o
SHORT COMMUNICATIONS
419
analogy with the derivative obtained from cysteamine, we will refer to this compound as carboxymethylthiocysteamine (CMTC). CMTC has been synthesized by SCH6BE~L AXl) G R A F J E 8 from thioglycolic acid and the thiosulphinate ester of cystamine. In a simpler way we have obtained a few mg of the compound by passing through a small column of Dowex-5o (H*-form) an alkaline solution of cystamine and thioglycolic acid. Thioglycolic acid is washed out with water, CMTC is eluted by o.5 N NH3, and cystamine is retained on the column. By taking to dryness the ninhydrin-positive alkaline fractions, a white powder has been obtained which gives a ninhydrin reaction as well as a nitrot)russide-positive reaction after the addition of KCN, thus indicating the presence of a disulphide group. When subjected to paper chromatography, the compound gives only a spot detectable by ninhydrin or by Folin-Marenzi's spray reagent 9, with an Rv vah w. of 0.85 in phenol (o.73 reportedS), and 0.55 in collidine-lutidine. In the first solvent CMTC is well separated from cystamine (RF - o.9-I) but overlaps carboxymethyleysteanfine (CMC). In collidine-lutidine CMTC is well separated from CMC (RF 0.25) but overlaps cvstamine. In two-dimensional chromatography CMTC, CMC and cvstamine are well separated from each other (Fig. I).
t
1
4
o
F i g 2. Diamine oxidase incubated with ".5 p m o l e s of c y s t a m i n e and 30 pmoles of monoiodoacetate. At the right: the c h r o m a t o g r a m s p r a y e d with n i n h y d r i n ; at the left: the corresponding a u t o r a d i o g r a m (8 days on a 3 ° cm x 40 cm F e r r a n i a .X-ray film). Spot No. x, CMC ; No. -, CY,ITC ; No. 3, thioglycolic acid.
o
F i g 3. The same sample of Fig. 3 c h r o m a t o g r a p h e d with the addition on the p a p e r of Ioo p g of pure CMTC. At the right: the e h r o m a t o g r a m s p r a y e d with n i n h y d r i n : at the left : the corresponding a u t o r a d i o g r a m . Spot No. t, CMC; No. 2, CMTC.
Biochim. Biophys. Acta, 93 (I964) 4r8-4 zo
420
SHORT COMMUNICATIONS
Having established the chromatographic behaviour of CMTC, we attempted to detect it in our cnzvlnic reaction mixtures. Partially purified hog-kidney diamine oxidase 6 (3o rag) was incubated with 2. 5 /,moles of cystamine and 8 ~C of [a~Sicystamine (Radiochemical Center, Amersham, England) with, or without, 3o ?moles of monoiodoacetic acid (Fluka, recrvstallized), in a final volume of 3 ml of o.I M phosphate buffer (pH 7.4). The reaction mixture was incubated for 2 h at 3 8o in a water-bath, with continuous shaking, and the reaction was then stopped by adding 0.3 ml of lOO% trichloroacetic acid. After centrifugation, the supernatant was extracted with ether to remove trichloroacetic acid and o.3-ml portions were subjected to paper chromatography in parallel with samples of pure CMTC. The chromatogram of the incubation mixture without added monoiodoacetate did not show any radioactive Sl:Ot in the corresponding autoradiogram. Fig. a shows the. cllromatogram and the corresponding autoradiogram of the incubation mixture made in the presence of nmnoiodoacetate. It is well evident that eystamine has completely disappeared, whereas some other radioactive spots are present. One of these (No. I), detectable also by ninhydrin on the chromatogranl, is C31(', which was already known to originate under these conditionsL Another (No. 2), which is in insufficient amount to give a ninhydrin-positive spot, was identified ;is (TMT(" hy the addition on the paper of the pure compound (Fig. 3). Spot No. 3 was identified an thioglycolic acid by chromatography of the pure acid, and possibly derives from ttle partial degradation of CMT('. The spot of thioglycolic acid disappears in the control chromatogram prepared t73" adding ('blTC to the palter (Fig. 3). This fact may be explained b.\' a sulphide--disulphide exchange reaction, as is indicated by the increased radioactivity of the CMTC spot. The present results confirm our previous suggestions as to the sequence of reactions which evstaldimine undergoe:, and explain some side-reactions which may be coupled to cvstaminc oxidation4,'~,L
Institute of Biolog,ical Chemistry, Universit3' of Rome, Rome (Italy)
1 1). 2 l). 3 1). 4 C. s C. s B.
('. DE M:x~(;o G. B()MI¢ARDIE I~,I
(_'AVALLINI, ('. L)E 3'IARCO AND I{. MONDOV|, Experientta, I2 (I05(~) 377. (.TAVALLINI, ('. ]-)1-2 3~'ARCO AND B. ~IONl)OVt, Boll. £'oc. ltal. Biol. Nper., 32 (195t')) t l.t. 5. ('AVALLIN1, (~. I)F .MARCO AND B. MONI)OV]', Biochim. Biophys. Acta, 24 (1957) 3.53. l)l-: MARC(), 3'I. COLE'rTA AND B. MONDOVt, Ital. J. Btochem., 9 (x90o) 77I_)E M a R c o , ltal. J. Biochem., lo ( I 9 0 0 I90. MONDOVt, C. I.)E MARCO, L. TENTORI AND I). (~AVALLINI, Boll..S'oc. llal. Biol. 5;per., 37 (r()01)
x(,95. 7 C. I ) E MARco, l;roc. Syrup. Chem. Biol. Aspects oJ l°3,rtdoxal Catalysis, Rome, 1962, l ' e r g ; . t m o n P r e s s , L o n d o n , 1O03, p. 403 . 8 A. SCIt(~BERI. AND H . (~,RAI.'JF, .q[~*ll. Chem., 017 (1958) 71 . t~ B. MONI)OV|, G. MOI)IANO Ar~'l) C. I_)E MARCO, Giorn. Biochim., 4 (1955) 324 •
Received May I9th, I964
Biochim B.~phys..4eta, 93 (tgb4) 4 IS 42o
BIOCHIMICA ET BIOPHYSICA ACTA
Short Communications -
m
.
Identification of carboxymethylthiocysteamine as cleavage product of cystaldimine
s(: 23060
Cvsteamine is oxidatively deaminated by hog-kidney or pea-seedling diamine oxidase (diamine :O2 oxidoreductase (deaminating), EC 1.4.3.0)1,2 t o cystaldimine 3, the corresponding eyclized aminoaldehyde. Some previous data indicated that cvstaldimine undergoes further degradation 4-6 and the following reaction ,sequence was suggested~: SS ('H e S S (,;|t.,
CIte
CtI.,
.... ~ ('Hz Ntt.,()H 2 N]ta
CH., .S-SH ~
(?I1~
CIt
-}- (.'2-fragment CHu.NHe
N cystamine
cvstaldimine
thiocysteamint.
The two-carbon fragment was in part identified as glycolaldehyde, but for thiocvsteamine only indirect data had been obtainedL In the present communication we report the identification of thiocysteamine, obtained by trapping it with monoiodoacetate in the form of the mixed disulphide I-amino-3,4-dithiapentane-5-cart~xylie acid (NHe-CHa-CHz-S-S-.CH.,-COOH). In
,¢ o e,
I
Q
O U
.•.-
~
~.
I*~
:--;i~
Fig. I. C h r o m a t o g r a p h y of pure c o m p o u n d . 8o/~g of cystamine, 13o ttg of CMTC a.nd 80 [tg of CMC have been subjected to p a p e r c h r o m a t o g r a p h y on W h a t m a n No. 4 paper, using phenol in the tirst run and collidine-lutidine in the second run. S p r a y reagent : n i n h y d r i n (o. i %) in butanol. S p o t No. i, CMC; No. 2, ('M'I'C; No. 3, cystaminc. Abbreviations: CMTC, c a r b o x y m e t h y l t h i o c y s t e a m i n e ; CMC, c a r b o x y m e t h y l c y s t e a m i n e .
tliochim. Hiophys...Icta, 93 (1904) 4 tS -4-' o
SHORT COMMUNICATIONS
419
analogy with the derivative obtained from cysteamine, we will refer to this compound as carboxymethylthiocysteamine (CMTC). CMTC has been synthesized by SCH6BE~L AXl) G R A F J E 8 from thioglycolic acid and the thiosulphinate ester of cystamine. In a simpler way we have obtained a few mg of the compound by passing through a small column of Dowex-5o (H*-form) an alkaline solution of cystamine and thioglycolic acid. Thioglycolic acid is washed out with water, CMTC is eluted by o.5 N NH3, and cystamine is retained on the column. By taking to dryness the ninhydrin-positive alkaline fractions, a white powder has been obtained which gives a ninhydrin reaction as well as a nitrot)russide-positive reaction after the addition of KCN, thus indicating the presence of a disulphide group. When subjected to paper chromatography, the compound gives only a spot detectable by ninhydrin or by Folin-Marenzi's spray reagent 9, with an Rv vah w. of 0.85 in phenol (o.73 reportedS), and 0.55 in collidine-lutidine. In the first solvent CMTC is well separated from cystamine (RF - o.9-I) but overlaps carboxymethyleysteanfine (CMC). In collidine-lutidine CMTC is well separated from CMC (RF 0.25) but overlaps cvstamine. In two-dimensional chromatography CMTC, CMC and cvstamine are well separated from each other (Fig. I).
t
1
4
o
F i g 2. Diamine oxidase incubated with ".5 p m o l e s of c y s t a m i n e and 30 pmoles of monoiodoacetate. At the right: the c h r o m a t o g r a m s p r a y e d with n i n h y d r i n ; at the left: the corresponding a u t o r a d i o g r a m (8 days on a 3 ° cm x 40 cm F e r r a n i a .X-ray film). Spot No. x, CMC ; No. -, CY,ITC ; No. 3, thioglycolic acid.
o
F i g 3. The same sample of Fig. 3 c h r o m a t o g r a p h e d with the addition on the p a p e r of Ioo p g of pure CMTC. At the right: the e h r o m a t o g r a m s p r a y e d with n i n h y d r i n : at the left : the corresponding a u t o r a d i o g r a m . Spot No. t, CMC; No. 2, CMTC.
Biochim. Biophys. Acta, 93 (I964) 4r8-4 zo
420
SHORT COMMUNICATIONS
Having established the chromatographic behaviour of CMTC, we attempted to detect it in our cnzvlnic reaction mixtures. Partially purified hog-kidney diamine oxidase 6 (3o rag) was incubated with 2. 5 /,moles of cystamine and 8 ~C of [a~Sicystamine (Radiochemical Center, Amersham, England) with, or without, 3o ?moles of monoiodoacetic acid (Fluka, recrvstallized), in a final volume of 3 ml of o.I M phosphate buffer (pH 7.4). The reaction mixture was incubated for 2 h at 3 8o in a water-bath, with continuous shaking, and the reaction was then stopped by adding 0.3 ml of lOO% trichloroacetic acid. After centrifugation, the supernatant was extracted with ether to remove trichloroacetic acid and o.3-ml portions were subjected to paper chromatography in parallel with samples of pure CMTC. The chromatogram of the incubation mixture without added monoiodoacetate did not show any radioactive Sl:Ot in the corresponding autoradiogram. Fig. a shows the. cllromatogram and the corresponding autoradiogram of the incubation mixture made in the presence of nmnoiodoacetate. It is well evident that eystamine has completely disappeared, whereas some other radioactive spots are present. One of these (No. I), detectable also by ninhydrin on the chromatogranl, is C31(', which was already known to originate under these conditionsL Another (No. 2), which is in insufficient amount to give a ninhydrin-positive spot, was identified ;is (TMT(" hy the addition on the paper of the pure compound (Fig. 3). Spot No. 3 was identified an thioglycolic acid by chromatography of the pure acid, and possibly derives from ttle partial degradation of CMT('. The spot of thioglycolic acid disappears in the control chromatogram prepared t73" adding ('blTC to the palter (Fig. 3). This fact may be explained b.\' a sulphide--disulphide exchange reaction, as is indicated by the increased radioactivity of the CMTC spot. The present results confirm our previous suggestions as to the sequence of reactions which evstaldimine undergoe:, and explain some side-reactions which may be coupled to cvstaminc oxidation4,'~,L
Institute of Biolog,ical Chemistry, Universit3' of Rome, Rome (Italy)
1 1). 2 l). 3 1). 4 C. s C. s B.
('. DE M:x~(;o G. B()MI¢ARDIE I~,I
(_'AVALLINI, ('. L)E 3'IARCO AND I{. MONDOV|, Experientta, I2 (I05(~) 377. (.TAVALLINI, ('. ]-)1-2 3~'ARCO AND B. ~IONl)OVt, Boll. £'oc. ltal. Biol. Nper., 32 (195t')) t l.t. 5. ('AVALLIN1, (~. I)F .MARCO AND B. MONI)OV]', Biochim. Biophys. Acta, 24 (1957) 3.53. l)l-: MARC(), 3'I. COLE'rTA AND B. MONDOVt, Ital. J. Btochem., 9 (x90o) 77I_)E M a R c o , ltal. J. Biochem., lo ( I 9 0 0 I90. MONDOVt, C. I.)E MARCO, L. TENTORI AND I). (~AVALLINI, Boll..S'oc. llal. Biol. 5;per., 37 (r()01)
x(,95. 7 C. I ) E MARco, l;roc. Syrup. Chem. Biol. Aspects oJ l°3,rtdoxal Catalysis, Rome, 1962, l ' e r g ; . t m o n P r e s s , L o n d o n , 1O03, p. 403 . 8 A. SCIt(~BERI. AND H . (~,RAI.'JF, .q[~*ll. Chem., 017 (1958) 71 . t~ B. MONI)OV|, G. MOI)IANO Ar~'l) C. I_)E MARCO, Giorn. Biochim., 4 (1955) 324 •
Received May I9th, I964
Biochim B.~phys..4eta, 93 (tgb4) 4 IS 42o