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The mechanism of the radiation chemical degradation of amino acids—II
Intem~tio~lJoum~lofAppliedRadiition
PcrgamcmPrenLtd.PrintcdinNmthmt
~d~topa,1962,Vol.13,pp.617622.
Irelatwl
The Mechanism of the Radiation Chemical Degradation of Amino Acids-II Degradation of a-Aminobutyric Acid in Aqueous Solution and Nitrogen Atmosphere and Comparison with the Degradation in Oxygen Atmosphere J. KOPOLDOVA, J. LIEBSTER and A. BABICKG Biological Institute of the Czechoslovak Academy of Sciences, Prague, Czechoslovakia
fm
(First received 3 January 1962 and in+1
18 June 1962)
In our previous paper(l) of this series the degradation of a-aminobutyric acid was studied in aqueous solution in oxygen atmosphere. A similar investigation has now been carried out in nitrogen atmosphere for the further elucidation of the reaction mechanism. Comparison of the products arising in oxygen and nitrogen atmosphere and their yields should make it possible to determine the effect of oxygen on the formation of the degradation products. A similar comparative study was made by WJZEKS and GARRISON(*) on glycine, who on the basis of various literature sources and their own experimental resultsproposed a mechanism for the action of radiation on aqueous solutions of glycine under different conditions. LE MECANISME
DE LA DEGRADATION CHIMIQUE DES AMINO-ACIDES PRODUITE PAR LE RAYONNEMENT-II
DEGRADATION DE L’ACIDE a-AMINOBUTYRIQUE EN SOLUTION AQUEUSE ET SOUS ATMOSPHERE D’AZOTE, ET COMPARISON AVEC LA DEGRADATION SOUS ATMOSPHERE D’OXYGENE Dans notre communication prkklente de cette strie nous avions Ctudit la degradation de l’acide a-aminobutyrique en solution aqueuse sous une atmosphere d’oxygene. Nous venons de faire une recherche pareille en une atmosphere d’aaote afin d’aider a dtcouvrir le mt5canismede la reaction. Une comparaison des produits rendus dans l’atmosphere d’oxygkne et dans celle d’azote devrait rendre possible la mesure de l’effet de l’oxygene sur la formation des produits de degradation. Wrmxs et GARRISON ont fait une pareille etude comparative sur la glycine; partant de diffkentes source publites et des rksultats de leun experiences ils proposent un mecanisme pour l’action du rayonnement sur des solutions aqueusesde glycine sous une varittt de conditions. MEXAHHSM
PA~HAHHOHHOI’G XHMHYECKOPO AMHHOKHCJIOT-II
PA3JI03KEHkH.I
PABJIOXEHHE a-AMHHOBYTHPOBOH KHCJIOTbI B ATMOCaEPE A30TA H CPABHEHHE C PABJIO7KEHBEM B ATMOCQEPE KWCJIOPOJJA B npeJ@IRymetiCTaTbe 11)gamofi
cepm paccraTpHsmocb pamomemie ~omoro pacmopa [email protected]~a. ~naBarKcBeHurr~exaa~Bvapea~ TaKoe~eEc~e~oa~Ee6~~0 IIpOBeAeHO BaTMOC@peaaOTa. CpaBHeHEeIIpO~yKTonpaCK~a H HX BHXOAOB B TOM B npyrOM CRJnlae, nOBWWIOMy, IIOaBOJIHTyCTaHOBHTbBJIHRaHe KEC-BMAHO6yTHpOBOti
nOpOAa
Ha
KHWIOT~IB
06paaOBaHm3
BTKX
~POAYKTOB.
YHKC
H
XE3ppECOH"'
IIpOBt?JIlr TaKOB
Hte
cpanKmenbEoe Bccne~oBaRHe Ea rrm.wie. Ha OCEOBBAEH paBJIimibIX JISiTepaTypKx AaHHbIX H co6cmeHHhtx BKC~epEMeETaJIbHbIX PeByJIbTaTOB, BTH aBTOpH ll~&lIOWiJflI urex~Hsu~egcTssrrobn~eKBR:KaBoZpiHe paoraopnrJrmuma~ paammnkrxycnoaaax.
617
618
J. Kopoldo~, J. Liebstcr and A. Babickj MECHANISMUS
DES RADIOCHEMISCIIEN
ABBAUS VON AMINOSAUREN-II
DEGRADATION VON a-AMINOSAURE IN WASSERlGER L&SUNG UND VERGLEICH MIT DER DEGRADATION IN EINER SAUERSTOFFATMOSPHhiRE In unserem fri.iheren Beitragtl) dieser Serie hehandelten wir die Degradation von a-Aminobuttersiiure in wasseriger Losung in’einer Sauerstoffatmosphlre. Eine %hnliche Untersuchung in einer Stickstoffatmosphare ist nun fertiggestellt worden zur zukiinftigen KiPrung des Degradationsmechanismus. Ein Vergleich der Produkte, die in Sauerstoff und Stickstoffatmospharen erfolgen, sowie deren relative Ausbeute mtisste es miiglich machen, den Einfluss des Saueistoffes auf die Bildung der Degradationsprodukte festzustellen. Ein PhnlichesVergleichsstudium wurde von WEEKSund GARIU~ON(~) fur Glyzin unternommen. Gesttitzt auf verschiedene Ver6ffentlichungen, sowie auf ihre eigenin Versuchsergebnisse, schlagen diese Autoren einen Mechanismus der Stahlungswirkung auf wiisserige Losungen von Glyzin unter verschiedenen VerhUtnissen vor. EXPERIMENTAL THE conditions of irradiation and analysis were identical with those given in our previous paper. The a-aminobutyric acid solutions in this case were saturated with nitrogen before irradiation in the glass ampullae.
In the experiments concerning the carboxylation reaction of a-aminobutyric acid leading to glutamic acid 0.88.mg CY40, (NaHCP40,, 20 ,UC) per 1 ml of the irradiated solution was added, (giving a 0.02 M solution of CO,). This amount is four times the amount of CO, formed by irradiation of a 0.05 M aminobutyric acid solution with a dose of 1.25 x 1020 eV/ml. The position of Cl4 in the carboxylation product, i.e. glutamic acid, was determined by enzymatic decarboxylation with glutamic decarboxylase of the isolated product in the Warburg apparatus and measurement of the CO, evolved and its radioactivity. RESULTS
AND
DISCUSSIONS
The radiolysis of a-aminobutyric the formation of these compounds:
acid led to
The concentration and amounts of the main products of radiolysis were determined in dependence on the radiation dose and plotted in Figs. 1 and 2. The Gvalues of the principal products of radiolysis for the highest dose are given in Table 1. Good agreement was found between the sum of Gvalues of the products formed and the degradation of a-aminobutyric acid (G 5.7 against 5-l). On the other hand a discrepancy was found
between G NH, and the sum of the deamination compounds (2-l against l-1) and between G CO, plus G of the carboxylation products and the sum of the decarboxylation products(2.5 against 1.6). These differences could be due to complete radiation chemical destruction of the original compound occurring to a small extent. On the basis of these results it is possible to explain the mechanism of the formation of the principal products of the radiolysis of a-aminobutyric acid in nitrogen. According to DEWHURST and BURTON(~),
Amino acids. a,q-diaminosuberic acid, glutamic acid, a-aminoadipic acid, and traces of homoserine, threonine, alanine, and 3 higher a-aminomonocarboxylic acids (probably a-aminopimelic and a-aminosuberic acid and the methyl derivates of the first two). One diaminodicarboxylic acid chromatographically corresponding to 2.6 diamino-3-methylpimelic acid. Amines. Propylamine. Ktto a&.&. Traces of a-ketobutyric acid. Ala%h~&s. Traces of propionaldehyde, acet-
HsO + H, OH, H,, HsO,. (1) The most important reaction again is the formation of an a-aminobutyric acid radical according to
aldehyde, formaldehyde. A&3. Acetic, propionic,
x being an OH or H radical, y being either HsO or H,.
butyric
acid.
the basic chemical reaction induced by the absorption of radiation in water in the absence of oxygen is
HOOCCH
(NH,) CH&H,
+ x+
HOOCCH(NHJCH,CH,
+ y
(2)
The me&akm oj the radiationc-d
9
c
619
&gm&ationof amino ad&--II
TABLE 1. c-values of the products of radiolysis of a-aminobutyric acid in nitrogen atmosphere for an irradiation dose of 1.25 x IOr0eV/ml (0.05 M aqueous solution) Compound
-
Decrease of a-aminobutyric acid
0
0.25
050
0.75
hJ% Amines
I.25
IGO
5.7 2.1 0.65 2.1 1.4 0.2 0.4 0.15 0.2 0.1 0.2 0.2 0.2
CC, Diaminosuberic acid lDiaminomethylpimelic acid Glutamic acid Aminoadipic acid Acetic acid Propionic acid Butyric acid *a-aminosuberic acid +a-methylaminopimelic acid
IO20 eV/ml FIG. 1. Decrease of a-aminobutyric acid, function of ammonia, amine, CO, and diaminosuberic acid.
x-&radiation dose.in 10sseV/ml y-axis-W molecules/mlformed x - decrease of a-aminobutyric acid O-formation of ammonia O-formation of diaminosuberic acid
A-formation of amines A-formation of CO,.
l These compounds were identified by their chromatographic behaviour as compared to analogue.
or
HOOCCH
(NE&)CH,CH,
+ COOH.CH( NH,) CH&H, + (HOOC.CH(NH,)CH,CHJ,
0
0.25
0.50
0.75
1.00
1.25
HOOCCH(NHB)CH,CH,
Fro. 2. Formation of radiolysis products. x-axis-radiation dose in 10seeV/ml y-axis 101’ molecules/mlformed X -formation of total acids A-formation of glutamic acid O-formation of a-ketobutyric acid O-formation of propionaldehyde
From the a-aminobutyric acid radical, diaminosuberic acid is being formed by recombination according to
+
(HOOC~CHWHWWHA
+ CO,- +
HOOCCH(NHJCH,CH,COO-.
A-formation of acetaldehyde O-formation of diaminosubericacid.
3
(3b)
This reaction participates with 60 per cent in the total degradation of the originally irradiated compound. From the a-aminobutyric acid radical glutamic acid arises as well by the addition of CO, on C4.
IO= eV/m 1
BHOOCGH (NHJCiCH,
+ H.
(3a)
(4)
This equation was written according to WEISS et uL.(~) who found formation of malonic acid upon irradiation of aqueous solutions of acetic acid which represents an analogous reaction. Addition of CO, to an amino acid leading to the formation of the corresponding dicarboxylic acid was observed by DOSE and ETTRI@ who irradiated aqueous solutions of alanine and found aspartic acid as one of the irradiation products. In thii wnnexion it is instructive to consider the curves in Fii. 1 and 2. When the curves for the formation of diaminosuberic acid and glutamic acid are considered the flattening of the curve for diaminosuberic acid wincides with the
620
J. Kojocdovd, J. Licbslcr and A. Babickj
beginning of the formation of glutamic acid. This shows that as soon as the CO, concentration in the irradiated solution is sufficiently high, the carboxylation reaction replaces the recombination reaction. In both these reactions the identical radicals are involved. The CO, supply necessary for the carboxylation reaction can be formed by decarboxylation of aminobutyric acid to propylamine, by decarboxylation and deamination of aminobutyric acid to propionaldehyde and by decarboxylation of ketobutyric acid. The type of curve obtained for CO, (FIG. 1) shows its formation through secondary reactions, e.g. decarboxylation of a-ketobutyric acid its curve of formation showing a characteristic maximum at the third dose. The second major reaction is the formation of propylamine by decarboxylation of a-aminobutyric acid, for which the following mechanism is suggested HOOCCH(NH,)CH,CHs
+ x -+
CH(NH,)CH,CH,
+ CO, +r.
(5)
The propylamine radical under the given conditions can react in two ways CH(NH,)CH,CHs
+ H -+
CHs(NH,)CHsCHs (8) yielding propylamine and CH(NH,)CH,CH,
+ CO,- -+ -OOCCH(NH,)
CH,CHs
(7)
giving a-aminobutyric acid. This reaction is supported by our observation that irradiation of an aqueous solution of aminobutyric acid and C140s gives rise to the formation of a-aminobutyric acid-l-Cl4 besides glutamic acid-5-Ci4. * Butyric acid, in analogy to the suggestion for glycine, could be formed by reductive deamination of a-aminobutyric acid. On the chromatograms spots are observed corresponding to the positions of a-aminosuberic acid and a-amino+methylpimelic acid which obviously were found by reaction of the butyric acid on the a-aminobutyric acid radical. For the formation of a-ketobutyric acid, propionaldehyde and propionic acid an analogous * These resultswillbe the subject of a further paper.
mechanism as in our previous communication(l) seems acceptable. Acetaldehyde and acetic acid could be formed either from propionic acid or another scission of the C-C-chain of aminobutyric acid between C2 and C3 after previous formation of a radical on either of these carbon atoms. As proof for the formation of a C3 radical the observation of traces of threonine and a dimerisation product (see below) can be considered. The acetic acid radical could combine with an aminobutyric acid radical to a-aminoadipic acid which was identified. Comparison of the yields of the different products of radiolysis shows the products of the reactions of the a-aminobutyric acid radical at C4 to account for 70 per cent of the total of a-aminobutyric degradation acid, 2,7diaminosuberic acid being the main product. The reaction on C2 and C3 accounts for 30 per cent of the total degradation. It is not possible, however, to differentiate exactly between the reaction on C3 and C2 as e.g. acetic acid may arise from either radical. COMPARISON OF THE DEGRADATION OF a-AMINO BUTYRIC ACID IN 0, AND N, ATMOSPHERE The Gvalues for the decrease of the irradiated compound and the Gvalues for the products in nitrogen media are given in Table 1. The data show a considerable decrease of the degradation of the irradiated compound in N, atmosphere i.e. 27 per cent as compared with 0,. If the products formed in traces (below G 0.1) are neglected, a much smaller amount of products ( 12 against 18) is formed in nitrogen. Two additional products were formed in nitrogen atmosphere i.e. butyric acid and probably a-aminosuberic acid and diamino+ methylpimelic acid. The missing products or those formed only in traces are compounds arising through oxidative processes, which in nitrogen atmosphere are suppressed. In both media the identical two recombination products were observed i.e. 2,7-diaminosuberic acid and a-aminoadipic acid. For the formation of the other recombination products found in oxygen atmosphere, one radical of aminobutyric acid and one radical formed by an oxidative process
-1
Propionic acid
Acet-
Acetic acid
aldehydc
CH, t COOH
Propionaldehyde
fCO,
CH, CHO
Y
CH,
\
\
CH,
-NH,
\\
tz&adicaI
CHO t
_j
I__FOOH.
Butyric acid
CH, COOH
CH, tCOOH
CHa
CH,
I CHNH+-‘CNH,
CH,
I
t + 0’0 4 ’
a-aminobutyric acid- 1-W
CK
a&iamino-&methylpimclic acid MI
t
CHNH, COOH
Ketobutyric acid
CH, +NH, co COOH
CH,
a-aminobutyric acid FH, -1 jCHI j [C=NHi _ ! COOHJ\
t +
c=h
CH, CH,
;
ICH
CHNH 4 COOH 2 + a-aminobutyric acid
-j,
i%H,
Y -:
Diioaubcric acid
CH, CH NH, 1 COOH-1 -
I I
CH,
.-
CA-radical
CH NH,
CH NH, COOH
‘W
AV= Aldch. COOH CH, CH, CHNH, COON
pyruvic acid CHO CH, CH NH,+ COOH
Hydmxy-
COOH
CH,OH
acid
.&p-tic
COOH (=I CH NH,COOH
+co* =b
Alanine
+ CH NH, COOH
Aminoadipic acid
CH NH, COOH
CH,
COOH
Glutamic acid + acetic acid
Diaminopimelic acid
m* i c~NH,j+cOoCOOH /
__+
L4Crim
-1
Peroxyde itarmed.
Homo-
-a,
kH,o* --1 fcH* 1 / CH NH, ‘+ FOOH 1 -_
scrine
U&OH c=* CH NH, t COOH
Alanme
CH,OH CHNH,+CHNH,+CO COOH COOH
CHs
+HCHO rcsp. CH,CHO -t CdY
Diagram of the radiolyxia of a-aminobutyric acid in oxygen and nitrogen
COOH CH NH, ~--cH, CH;
f
=!
I
0
!! E.
h
i F
f 2
622
.I. Kojoiahi,
J. L.iebstcr and A. Babickj
is required. In nitrogen on the other hand the recombination products from the butyric acid radical with a-aminobutyric acid were found plus an a-aminobutyric acid C3-C4 recombination product. These results suggest the radiolysis of the originally irradiated amino acid to occur in oxygenated as well as in oxygen-free solution through the primary formation of the identical radicals. These radicals then react differently in 0, and Ns atmosphere, in Ns the recombination predominates whereas in 0, oxidation proceeds to a certain extent as well. For the reactions at C, leading to decarboxylation or deamination the existence of a common primary radical can be accepted e.g.
existence of e.g. the propylamine radical is supported by the observed carboxyl exchange reaction. The formation of a radical at C3,in oxygen is proved by the existence of threonine in the irradiated solution. In nitrogen only traces of threonine were observed, but a dimerization product most probably 3-methyl-2,6-diaminopimelic acid was observed. From the aminobutyric acid radicals at C3 by scission of the C-C chain acetic acid and acetaldehyde can be formed. Analogous studies on the higher aliphatic amino acids are being made. Their results will contribute to a better understanding of the radiolytic processes discussed here.
HOOCC(NHJCH,CH, which according to the conditions (N, or 0,) reacts differently. In oxygen-free solutions the predominant reaction is suggested to be the decarboxylation of propylamine and in oxygenated soluticns the formation of propionaldehyde. These reactions could account for the predominant formation of one type of.product under each of the two specific conditions. The
REFERENCES 1. KOPOLDOVAJ., LIRBSTER J. and BABICK+A. ht. J.
appl. Rad. [email protected] 11, 139 (1961).
2. WEEKSB. M. and GARRISONW. M. Radiat. Res. 9, 291 (1958).
3. DJrwIiuRsTH. A. and BURTON M. J. Amer. &em. sot. 77,578l (1955). 4. SCHOLE~G., SIMC M. and WEISSJ. J. Nature, Lmd. 188, 1019 (1960). 5. Dow. K. and Em~k K. Z. Natur- 13 B, 784 (1958). 6. STEING. and Wms J. J. them. Sot. 3256 (1949).
PcrgamcmPrenLtd.PrintcdinNmthmt
~d~topa,1962,Vol.13,pp.617622.
Irelatwl
The Mechanism of the Radiation Chemical Degradation of Amino Acids-II Degradation of a-Aminobutyric Acid in Aqueous Solution and Nitrogen Atmosphere and Comparison with the Degradation in Oxygen Atmosphere J. KOPOLDOVA, J. LIEBSTER and A. BABICKG Biological Institute of the Czechoslovak Academy of Sciences, Prague, Czechoslovakia
fm
(First received 3 January 1962 and in+1
18 June 1962)
In our previous paper(l) of this series the degradation of a-aminobutyric acid was studied in aqueous solution in oxygen atmosphere. A similar investigation has now been carried out in nitrogen atmosphere for the further elucidation of the reaction mechanism. Comparison of the products arising in oxygen and nitrogen atmosphere and their yields should make it possible to determine the effect of oxygen on the formation of the degradation products. A similar comparative study was made by WJZEKS and GARRISON(*) on glycine, who on the basis of various literature sources and their own experimental resultsproposed a mechanism for the action of radiation on aqueous solutions of glycine under different conditions. LE MECANISME
DE LA DEGRADATION CHIMIQUE DES AMINO-ACIDES PRODUITE PAR LE RAYONNEMENT-II
DEGRADATION DE L’ACIDE a-AMINOBUTYRIQUE EN SOLUTION AQUEUSE ET SOUS ATMOSPHERE D’AZOTE, ET COMPARISON AVEC LA DEGRADATION SOUS ATMOSPHERE D’OXYGENE Dans notre communication prkklente de cette strie nous avions Ctudit la degradation de l’acide a-aminobutyrique en solution aqueuse sous une atmosphere d’oxygene. Nous venons de faire une recherche pareille en une atmosphere d’aaote afin d’aider a dtcouvrir le mt5canismede la reaction. Une comparaison des produits rendus dans l’atmosphere d’oxygkne et dans celle d’azote devrait rendre possible la mesure de l’effet de l’oxygene sur la formation des produits de degradation. Wrmxs et GARRISON ont fait une pareille etude comparative sur la glycine; partant de diffkentes source publites et des rksultats de leun experiences ils proposent un mecanisme pour l’action du rayonnement sur des solutions aqueusesde glycine sous une varittt de conditions. MEXAHHSM
PA~HAHHOHHOI’G XHMHYECKOPO AMHHOKHCJIOT-II
PA3JI03KEHkH.I
PABJIOXEHHE a-AMHHOBYTHPOBOH KHCJIOTbI B ATMOCaEPE A30TA H CPABHEHHE C PABJIO7KEHBEM B ATMOCQEPE KWCJIOPOJJA B npeJ@IRymetiCTaTbe 11)gamofi
cepm paccraTpHsmocb pamomemie ~omoro pacmopa [email protected]~a. ~naBarKcBeHurr~exaa~Bvapea~ TaKoe~eEc~e~oa~Ee6~~0 IIpOBeAeHO BaTMOC@peaaOTa. CpaBHeHEeIIpO~yKTonpaCK~a H HX BHXOAOB B TOM B npyrOM CRJnlae, nOBWWIOMy, IIOaBOJIHTyCTaHOBHTbBJIHRaHe KEC-BMAHO6yTHpOBOti
nOpOAa
Ha
KHWIOT~IB
06paaOBaHm3
BTKX
~POAYKTOB.
YHKC
H
XE3ppECOH"'
IIpOBt?JIlr TaKOB
Hte
cpanKmenbEoe Bccne~oBaRHe Ea rrm.wie. Ha OCEOBBAEH paBJIimibIX JISiTepaTypKx AaHHbIX H co6cmeHHhtx BKC~epEMeETaJIbHbIX PeByJIbTaTOB, BTH aBTOpH ll~&lIOWiJflI urex~Hsu~egcTssrrobn~eKBR:KaBoZpiHe paoraopnrJrmuma~ paammnkrxycnoaaax.
617
618
J. Kopoldo~, J. Liebstcr and A. Babickj MECHANISMUS
DES RADIOCHEMISCIIEN
ABBAUS VON AMINOSAUREN-II
DEGRADATION VON a-AMINOSAURE IN WASSERlGER L&SUNG UND VERGLEICH MIT DER DEGRADATION IN EINER SAUERSTOFFATMOSPHhiRE In unserem fri.iheren Beitragtl) dieser Serie hehandelten wir die Degradation von a-Aminobuttersiiure in wasseriger Losung in’einer Sauerstoffatmosphlre. Eine %hnliche Untersuchung in einer Stickstoffatmosphare ist nun fertiggestellt worden zur zukiinftigen KiPrung des Degradationsmechanismus. Ein Vergleich der Produkte, die in Sauerstoff und Stickstoffatmospharen erfolgen, sowie deren relative Ausbeute mtisste es miiglich machen, den Einfluss des Saueistoffes auf die Bildung der Degradationsprodukte festzustellen. Ein PhnlichesVergleichsstudium wurde von WEEKSund GARIU~ON(~) fur Glyzin unternommen. Gesttitzt auf verschiedene Ver6ffentlichungen, sowie auf ihre eigenin Versuchsergebnisse, schlagen diese Autoren einen Mechanismus der Stahlungswirkung auf wiisserige Losungen von Glyzin unter verschiedenen VerhUtnissen vor. EXPERIMENTAL THE conditions of irradiation and analysis were identical with those given in our previous paper. The a-aminobutyric acid solutions in this case were saturated with nitrogen before irradiation in the glass ampullae.
In the experiments concerning the carboxylation reaction of a-aminobutyric acid leading to glutamic acid 0.88.mg CY40, (NaHCP40,, 20 ,UC) per 1 ml of the irradiated solution was added, (giving a 0.02 M solution of CO,). This amount is four times the amount of CO, formed by irradiation of a 0.05 M aminobutyric acid solution with a dose of 1.25 x 1020 eV/ml. The position of Cl4 in the carboxylation product, i.e. glutamic acid, was determined by enzymatic decarboxylation with glutamic decarboxylase of the isolated product in the Warburg apparatus and measurement of the CO, evolved and its radioactivity. RESULTS
AND
DISCUSSIONS
The radiolysis of a-aminobutyric the formation of these compounds:
acid led to
The concentration and amounts of the main products of radiolysis were determined in dependence on the radiation dose and plotted in Figs. 1 and 2. The Gvalues of the principal products of radiolysis for the highest dose are given in Table 1. Good agreement was found between the sum of Gvalues of the products formed and the degradation of a-aminobutyric acid (G 5.7 against 5-l). On the other hand a discrepancy was found
between G NH, and the sum of the deamination compounds (2-l against l-1) and between G CO, plus G of the carboxylation products and the sum of the decarboxylation products(2.5 against 1.6). These differences could be due to complete radiation chemical destruction of the original compound occurring to a small extent. On the basis of these results it is possible to explain the mechanism of the formation of the principal products of the radiolysis of a-aminobutyric acid in nitrogen. According to DEWHURST and BURTON(~),
Amino acids. a,q-diaminosuberic acid, glutamic acid, a-aminoadipic acid, and traces of homoserine, threonine, alanine, and 3 higher a-aminomonocarboxylic acids (probably a-aminopimelic and a-aminosuberic acid and the methyl derivates of the first two). One diaminodicarboxylic acid chromatographically corresponding to 2.6 diamino-3-methylpimelic acid. Amines. Propylamine. Ktto a&.&. Traces of a-ketobutyric acid. Ala%h~&s. Traces of propionaldehyde, acet-
HsO + H, OH, H,, HsO,. (1) The most important reaction again is the formation of an a-aminobutyric acid radical according to
aldehyde, formaldehyde. A&3. Acetic, propionic,
x being an OH or H radical, y being either HsO or H,.
butyric
acid.
the basic chemical reaction induced by the absorption of radiation in water in the absence of oxygen is
HOOCCH
(NH,) CH&H,
+ x+
HOOCCH(NHJCH,CH,
+ y
(2)
The me&akm oj the radiationc-d
9
c
619
&gm&ationof amino ad&--II
TABLE 1. c-values of the products of radiolysis of a-aminobutyric acid in nitrogen atmosphere for an irradiation dose of 1.25 x IOr0eV/ml (0.05 M aqueous solution) Compound
-
Decrease of a-aminobutyric acid
0
0.25
050
0.75
hJ% Amines
I.25
IGO
5.7 2.1 0.65 2.1 1.4 0.2 0.4 0.15 0.2 0.1 0.2 0.2 0.2
CC, Diaminosuberic acid lDiaminomethylpimelic acid Glutamic acid Aminoadipic acid Acetic acid Propionic acid Butyric acid *a-aminosuberic acid +a-methylaminopimelic acid
IO20 eV/ml FIG. 1. Decrease of a-aminobutyric acid, function of ammonia, amine, CO, and diaminosuberic acid.
x-&radiation dose.in 10sseV/ml y-axis-W molecules/mlformed x - decrease of a-aminobutyric acid O-formation of ammonia O-formation of diaminosuberic acid
A-formation of amines A-formation of CO,.
l These compounds were identified by their chromatographic behaviour as compared to analogue.
or
HOOCCH
(NE&)CH,CH,
+ COOH.CH( NH,) CH&H, + (HOOC.CH(NH,)CH,CHJ,
0
0.25
0.50
0.75
1.00
1.25
HOOCCH(NHB)CH,CH,
Fro. 2. Formation of radiolysis products. x-axis-radiation dose in 10seeV/ml y-axis 101’ molecules/mlformed X -formation of total acids A-formation of glutamic acid O-formation of a-ketobutyric acid O-formation of propionaldehyde
From the a-aminobutyric acid radical, diaminosuberic acid is being formed by recombination according to
+
(HOOC~CHWHWWHA
+ CO,- +
HOOCCH(NHJCH,CH,COO-.
A-formation of acetaldehyde O-formation of diaminosubericacid.
3
(3b)
This reaction participates with 60 per cent in the total degradation of the originally irradiated compound. From the a-aminobutyric acid radical glutamic acid arises as well by the addition of CO, on C4.
IO= eV/m 1
BHOOCGH (NHJCiCH,
+ H.
(3a)
(4)
This equation was written according to WEISS et uL.(~) who found formation of malonic acid upon irradiation of aqueous solutions of acetic acid which represents an analogous reaction. Addition of CO, to an amino acid leading to the formation of the corresponding dicarboxylic acid was observed by DOSE and ETTRI@ who irradiated aqueous solutions of alanine and found aspartic acid as one of the irradiation products. In thii wnnexion it is instructive to consider the curves in Fii. 1 and 2. When the curves for the formation of diaminosuberic acid and glutamic acid are considered the flattening of the curve for diaminosuberic acid wincides with the
620
J. Kojocdovd, J. Licbslcr and A. Babickj
beginning of the formation of glutamic acid. This shows that as soon as the CO, concentration in the irradiated solution is sufficiently high, the carboxylation reaction replaces the recombination reaction. In both these reactions the identical radicals are involved. The CO, supply necessary for the carboxylation reaction can be formed by decarboxylation of aminobutyric acid to propylamine, by decarboxylation and deamination of aminobutyric acid to propionaldehyde and by decarboxylation of ketobutyric acid. The type of curve obtained for CO, (FIG. 1) shows its formation through secondary reactions, e.g. decarboxylation of a-ketobutyric acid its curve of formation showing a characteristic maximum at the third dose. The second major reaction is the formation of propylamine by decarboxylation of a-aminobutyric acid, for which the following mechanism is suggested HOOCCH(NH,)CH,CHs
+ x -+
CH(NH,)CH,CH,
+ CO, +r.
(5)
The propylamine radical under the given conditions can react in two ways CH(NH,)CH,CHs
+ H -+
CHs(NH,)CHsCHs (8) yielding propylamine and CH(NH,)CH,CH,
+ CO,- -+ -OOCCH(NH,)
CH,CHs
(7)
giving a-aminobutyric acid. This reaction is supported by our observation that irradiation of an aqueous solution of aminobutyric acid and C140s gives rise to the formation of a-aminobutyric acid-l-Cl4 besides glutamic acid-5-Ci4. * Butyric acid, in analogy to the suggestion for glycine, could be formed by reductive deamination of a-aminobutyric acid. On the chromatograms spots are observed corresponding to the positions of a-aminosuberic acid and a-amino+methylpimelic acid which obviously were found by reaction of the butyric acid on the a-aminobutyric acid radical. For the formation of a-ketobutyric acid, propionaldehyde and propionic acid an analogous * These resultswillbe the subject of a further paper.
mechanism as in our previous communication(l) seems acceptable. Acetaldehyde and acetic acid could be formed either from propionic acid or another scission of the C-C-chain of aminobutyric acid between C2 and C3 after previous formation of a radical on either of these carbon atoms. As proof for the formation of a C3 radical the observation of traces of threonine and a dimerisation product (see below) can be considered. The acetic acid radical could combine with an aminobutyric acid radical to a-aminoadipic acid which was identified. Comparison of the yields of the different products of radiolysis shows the products of the reactions of the a-aminobutyric acid radical at C4 to account for 70 per cent of the total of a-aminobutyric degradation acid, 2,7diaminosuberic acid being the main product. The reaction on C2 and C3 accounts for 30 per cent of the total degradation. It is not possible, however, to differentiate exactly between the reaction on C3 and C2 as e.g. acetic acid may arise from either radical. COMPARISON OF THE DEGRADATION OF a-AMINO BUTYRIC ACID IN 0, AND N, ATMOSPHERE The Gvalues for the decrease of the irradiated compound and the Gvalues for the products in nitrogen media are given in Table 1. The data show a considerable decrease of the degradation of the irradiated compound in N, atmosphere i.e. 27 per cent as compared with 0,. If the products formed in traces (below G 0.1) are neglected, a much smaller amount of products ( 12 against 18) is formed in nitrogen. Two additional products were formed in nitrogen atmosphere i.e. butyric acid and probably a-aminosuberic acid and diamino+ methylpimelic acid. The missing products or those formed only in traces are compounds arising through oxidative processes, which in nitrogen atmosphere are suppressed. In both media the identical two recombination products were observed i.e. 2,7-diaminosuberic acid and a-aminoadipic acid. For the formation of the other recombination products found in oxygen atmosphere, one radical of aminobutyric acid and one radical formed by an oxidative process
-1
Propionic acid
Acet-
Acetic acid
aldehydc
CH, t COOH
Propionaldehyde
fCO,
CH, CHO
Y
CH,
\
\
CH,
-NH,
\\
tz&adicaI
CHO t
_j
I__FOOH.
Butyric acid
CH, COOH
CH, tCOOH
CHa
CH,
I CHNH+-‘CNH,
CH,
I
t + 0’0 4 ’
a-aminobutyric acid- 1-W
CK
a&iamino-&methylpimclic acid MI
t
CHNH, COOH
Ketobutyric acid
CH, +NH, co COOH
CH,
a-aminobutyric acid FH, -1 jCHI j [C=NHi _ ! COOHJ\
t +
c=h
CH, CH,
;
ICH
CHNH 4 COOH 2 + a-aminobutyric acid
-j,
i%H,
Y -:
Diioaubcric acid
CH, CH NH, 1 COOH-1 -
I I
CH,
.-
CA-radical
CH NH,
CH NH, COOH
‘W
AV= Aldch. COOH CH, CH, CHNH, COON
pyruvic acid CHO CH, CH NH,+ COOH
Hydmxy-
COOH
CH,OH
acid
.&p-tic
COOH (=I CH NH,COOH
+co* =b
Alanine
+ CH NH, COOH
Aminoadipic acid
CH NH, COOH
CH,
COOH
Glutamic acid + acetic acid
Diaminopimelic acid
m* i c~NH,j+cOoCOOH /
__+
L4Crim
-1
Peroxyde itarmed.
Homo-
-a,
kH,o* --1 fcH* 1 / CH NH, ‘+ FOOH 1 -_
scrine
U&OH c=* CH NH, t COOH
Alanme
CH,OH CHNH,+CHNH,+CO COOH COOH
CHs
+HCHO rcsp. CH,CHO -t CdY
Diagram of the radiolyxia of a-aminobutyric acid in oxygen and nitrogen
COOH CH NH, ~--cH, CH;
f
=!
I
0
!! E.
h
i F
f 2
622
.I. Kojoiahi,
J. L.iebstcr and A. Babickj
is required. In nitrogen on the other hand the recombination products from the butyric acid radical with a-aminobutyric acid were found plus an a-aminobutyric acid C3-C4 recombination product. These results suggest the radiolysis of the originally irradiated amino acid to occur in oxygenated as well as in oxygen-free solution through the primary formation of the identical radicals. These radicals then react differently in 0, and Ns atmosphere, in Ns the recombination predominates whereas in 0, oxidation proceeds to a certain extent as well. For the reactions at C, leading to decarboxylation or deamination the existence of a common primary radical can be accepted e.g.
existence of e.g. the propylamine radical is supported by the observed carboxyl exchange reaction. The formation of a radical at C3,in oxygen is proved by the existence of threonine in the irradiated solution. In nitrogen only traces of threonine were observed, but a dimerization product most probably 3-methyl-2,6-diaminopimelic acid was observed. From the aminobutyric acid radicals at C3 by scission of the C-C chain acetic acid and acetaldehyde can be formed. Analogous studies on the higher aliphatic amino acids are being made. Their results will contribute to a better understanding of the radiolytic processes discussed here.
HOOCC(NHJCH,CH, which according to the conditions (N, or 0,) reacts differently. In oxygen-free solutions the predominant reaction is suggested to be the decarboxylation of propylamine and in oxygenated soluticns the formation of propionaldehyde. These reactions could account for the predominant formation of one type of.product under each of the two specific conditions. The
REFERENCES 1. KOPOLDOVAJ., LIRBSTER J. and BABICK+A. ht. J.
appl. Rad. [email protected] 11, 139 (1961).
2. WEEKSB. M. and GARRISONW. M. Radiat. Res. 9, 291 (1958).
3. DJrwIiuRsTH. A. and BURTON M. J. Amer. &em. sot. 77,578l (1955). 4. SCHOLE~G., SIMC M. and WEISSJ. J. Nature, Lmd. 188, 1019 (1960). 5. Dow. K. and Em~k K. Z. Natur- 13 B, 784 (1958). 6. STEING. and Wms J. J. them. Sot. 3256 (1949).