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The photodecomposition of dinitrophenyl-amino acids
364
SHORT COMMUNICATIONS
The photodecomposition of dinitrophenyl-amino acids* It is recogmzed that a principle shortcoming in the estimation of protein N-terminal amino acids as their dinitrophenyl derivatives is the difficulty encountered in accurately evaluating DNP-amino acid losses occurring during hydrolysis and the subsequent operations required for quantitation t In attempting to evaluate such losses employing DNP-EztC]amino acids as internal standards, we observed a variable and often an extensive loss of radioactivity when the derivatives were not shielded from light. The necessity for avoiding photodecompositlon of DNP-ammo acids has been mentioned by a number of authors 2-4. AKABORI et al. 5 have reported on the rate of the photolytic decomposition and recently have shown that certain DNP-peptides ~ are also light sensitive Similar observations have been reported for trmitrophenylated peptides 7 However, the nature or the extent of the decomposition has received little attention. The DNP derivatives of [2-14C]glycme, ~2-14C]tyrosme, I3-z4C]serine, E I - ~ 4 C ] valine (Nuclear Chicago Corporation), [i-14C]alanine and [I-t4C]glycine (Isotope Specialties) were prepared following the method of RAO AND SOBERs. The DNP-amino acids were not recrystallized but were purified by siliclc acid chromatography 9 thus removing dinitrophenol and dinitroaniline. Aliquots of the DNP derivatives in acetone solution were plated onto stainlesssteel planchets and exposed to two different lighting conditions occurring in the laboratory. Fig. IA demonstrates the rate of activity loss when the samples were exposed to light in the usual working area approximately 12 feet from outside windows. The loss was greatly accelerated when a parallel group of samples was exposed to daylight adjacent to an outside window but protected from direct sunlight DNP AMINO ACkDS
•
[~ "c] )
A
•
,
'"C]GLY
04
; 21
3-P'C] SEP
8 55
Lf %] ~L&
o T,
a
L2-"C] T~R
O,B26
•
[2 E~C]~L~
4,B2
I00 90
SPECiFiCACTIVITY uc/um
.L
[~
80
mm eo S
5e
~
40 3o
IO i
0
2
4
6
8
i
IO 12 14 16 18 20 22
0
2
4
6
8
IO 12 14 16 18 20 22
HOURS
Fig z Effect of light on the decomposltzon of
DI~Tp-[14C]&ITIlnO&clds
plated on st&m]ess steel.
A, c o m p o u n d s e x p o s e d to h g h t i2 feet from w i n d o w IB, c o m p o u n d s e x p o s e d to light i foot f r o m w i n d o w (See text.) Abbreviations: DNP-, dmltrophenyl. * P r e s e n t e d to t h e F e d e r a t i o n of A m e r i c a n Soclettes for E x p e r z m e n t a t Biology. Atlantxc Q t y , N e w Jersey, April, 1959
B~och~m B~opha,s 4cla, 39 (t90o) 364-367
365
SHORT COMMUNICATIONS
(Fig. IB). Other experiments showed that the decomposition rate varied with the nature and intensity of illumination, the area of sample distribution and the nature of the supporting medium. A group of samples kept in total darkness failed to lose activity over the same period. A decline in the rate of loss, more prominent in Fig. IB, is interpreted as the effect of decomposition products accumulating on the sample surface reducing the effective light intensity. In other experiments, aliquots of the radioactive DNP derivatives were subjected to photodecomposition in Conway-Kirk diffusion flasks. The center wells of the flasks contained saturated Ba(OH)~ or I N NaOH. Following exposure to daylight for periods of 48-72 h, the alkali trap was checked for radioactavity. The carboxyllabeled compounds yielded radioactive CO S which was recovered directly as BaCO v On the other hand, DNP-E3-14C]serine gave rise to volatile radioactive compounds which were trapped in NaOH and could be recovered as BaCO a only after persulfate oxidation. These results suggested that volatile acidic compounds are formed Although DNP-[2-14C]tyrosme did not lose radioactivity, chromatography of a sample which had been exposed to light demonstrated that DNP-[2-14C]tyrosine decarboxylates to form radioactive DNP-tyramine (vide mfra). Table I summarizes these experiments. Decarboxylation appears to be a principle but not the sole photolytic reaction. TABLE I RECOVI~RY OF RADIOACTIVITYFROM DNp-[14C]AMINO ACIDS EXPOSED TO LIGHT IN CONWAY-KIRK DIFFUSION FLASKS Added rachoactlvlty was determined vclth unexposed demvatives plated on stainless steel at essentially zero thxckness. Volatile radloactlvxty was c o u n t e d as BaCOs, n o t corrected for self absorption. All c o u n t s were corrected for coinmdence loss. R admactw;ly
DPNcompoundexposed
[I-14C] a.lanme [I-14C]glycme [i-l~C]valme [2-tlCJglycme [3-1~CJserine [2-x4C]tyrosine
Acttv*ty added xo a counts/m~n
64 o I26 28.8 17 ° 64.8 3o.o
CO s recovered as CO 2 recovered as BaCO, from r N No,OH after BaCO, from BaOH2 persulfate ox~aSton z o s counts/mzn zo s counts/ram
29.4 48.5 I9 8 0. 4 0.2 o.o
a* a* a* 40.0 17.2 o. 5
* a n o t determined.
In another series of experiments the derivatives were spotted on strips of No. I Whatman paper and exposed to daylight and artificial light for up to 240 h to obtain maximum decomposition. The strips were subsequently chromatographed in the ascending system of BISERTE AND OSTEUX1° along with unexposed control strips. These chromatographed strips were then scanned for radioactivity in an end-window counter adapted for manual strip counting using a slit width of 0.25 inch. Figs. 2A and 2B illustrate typical radiochromatograms. All exposed compounds yielded an increase in color intensity of the band at the solvent front as compared to unexposed samples. Only compounds containing radiocarbon at C-2 or C-3 exhibited a significant increase of radioactivity in this region. Unexposed samples of derivatives labeled at Bzoch~m. Bzophys
A c t a , 39 (196o) 364-367
366
SHORT COMMUNICATIONS
C-2 or C-3 yielded some radio-activity in the solvent-front band This band was shown to be due to decomposition occurring during normal handling operations as evidenced by the reappearance of a small radioactive peak at the solvent front when DNP[2-14C]glycine, purified by paper chromatography, was eluted and rechromatographed. 2o
,*. J
16
•
BEFORE
EXPOSURE
c
AFTER
EXPOSURE
'0
0 0
o
9
B
c_
E~
',i
~ 5
,,,
~ o U
+' r ,
I L
4
II ....
0 -~--~r: ~ 0
I0
15
if!
~ 20
25
DISTANCE
,2~_ 50
0
5
I
)
( I u n i t : ~- m
10
15
.:._m~_, 20
25
30
Fig 2. Typtcal radiochromatograms of DNP-[tiC]amino acids before and after exposure to light 2A, DNP-[2-1*C]glycme before and after exposure Arrows indicate solvent front The small peaks of low RF also appear in radiochromatograms of DNP-[3-x4C]serme. Bands corresponding to these peaks are colorless and remain colorless when treated with nmhydrm. 2B, [x-14C]glycme before and after exposure.
These observations and those cited above suggested that N-alkyl dimtroanilines result from photo-induced decarboxylation of DNP-amino acids. Hydrolysates of DNPproteins subjected to chromatography using the ascending system of BISERTE AND OSTEUX yield a yellow band at the solvent front said to be dinitroamline n. It appeared likely that N-alkyl dlnitroanilines, which are not resolved from dinitroanihne in this system, might also be present. Accordingly, the "dimtroaniline" bands were eluted with chloroform from chromatograms of exposed DNP-[2-14Cltyrosine, DNP-[2-t~C] glycine, DNP-[I-14C]glycine and DNP-[I-14Clalanine and subjected to reverse-phase chromatography 12 in parallel with authentic DNP-tyramine, N-methyldinitroaniline and N-ethyldinitroaniline. DNP-tyramine was Identified as the photodecomposition product of DNP-tyrosine and no dinitroaniline was evident with the reverse-phase system employed. While N-methyldmitroamline and N-ethyldinitroanihne have an RF identical with that of dinitroaniline, the compound obtained from exposed DNP[2-14C]glycine was radioactive whereas those obtained from DNP-[IA~C]glycine and DNP-[I-l*C]alanine were nonradioactive. If dinitroanlline were a product of the decarboxylation of DNP-[14Clamino acids, it should be nonradioactive. Since DNP[l*Cltyramlne was identified as a product formed from DNP-I2A4Cltyrosine while dinitroanihne was not formed, the experiments indicate that N-methyl and N-ethyldinitroaniline are products formed from DNP-glycine and DNP-alanine respectively. In summary the photodecomposition of DNP-amino acids yields COs, N-alkyl dinitroanilines and volatile acidic compounds of unknown structure. PhotodeBzo~h~m Bzoph>,s .Iota, 39 (196o) 364-367
367
SHORT COMMUNICATIONS
composition may cause large and variable losses of DNP-amino acids unless exposure to light is avoided. The technical assistance of Miss C. ROTHNEM is gratefully acknowledged. This work was supported by grants from the Life Insurance Medical Research Fund, the American Heart Association and Eh Lilly and Company.
Departments of Pediatrics and Physiological Chemistry, Research Laboratories of the Variety Club Heart Hospital, University of Minnesota, Minneapolis, M, nn. (U.S.A.)
B. POLLARA
R. W. VON KORFF*
1 H S. RHINESMITH,W .A SCHROEDER AND L PAULING,J . . 4 m . Chem. Soc, 79 (1957) 6o9. 2 G L MILLS, B~ochem J., 5o (1952 ) 707 3 F. SANGER, B,ochem J., 45 (1949) 563. 4 S. BLACKBURN, B~ochem. J., 45 (1949) 579. s S AKABORI, T [KENAKA,Y. OKADA AND K KOHNO,Proc Jap .4cad, 29 (1953) 5o9 6 S AKABORI, S SAKAKIBARAAND K SAKAKIBARA, Bull Chem Soc Japan, 32 (1959) 312 ? K SATAKE AND T. OKAUAMA, Bull. Chem. Soc. Japan, 32 (I959) 520. s K. R. RAO AND H. A. SOBER, J. A m Chem. Soc., 76 (1953) 1328. 9 C. H LI AND L. ASH, J. B,ol. Chem , 230 (1953) 419 10 G. BISERTE AND R. OSTEUX,Bull. soc, ch~m. b*ol, 33 (1951) 5 °. n H. FRAENKEL-CONRAT, J. I. HARRIS AND A L. LEvY in D. CLICK, Methods of BzochemzcM Analys~s, Vol. II, Intersclence P u b h s h e r s , N e w York, 1955, P. 539 12 I. M. LOCKHART, Nature, 177 (1956) 393.
Received October I9th, 1959 * Established Investigator, Minnesota H e a r t Association.
B~ochzm. B~ophys Acta, 39 (196o) 364-367
Synthesis of serine from glycine in mitochondrial fragments The conversion of two molecules of glycine to serine has been observed in the intact rat 1, In homogenates of turkey live# and in a particulate fraction of rat live#. Some of the intermediate steps in the synthesis have been studied in partially purified systems (see ref.4). Incidental to a search for an enzyme system capable of synthesizing ~-aminole~allinic acid, we observed that the entire complement of enzymes and cofactors required for the net synthesis of serine from glycine was present in a particulate fraction obtained by osmotic disruption of mitochondria. The mitochondrial preparations were obtained from chicken livers by the conventional technique involving differential centnfugation in 0.3 M sucrose 5. The preparation gave P/O ratios of 1.7-I. 9 succinate and 2.3-2.6 with glutamate. The sub-mitochondrial particles were obtained by suspending the mltochondria in i . io-SM tris(hydroxymethyl)amlnomethane at pH 7.0 for 30 rain at o ° and centrifuging at ioo,ooo × g for 15 min The residue was suspended in an amount of 0.3 M sucrose equal to the original mitochondrial volume. The treatment results in disintegration of mitochondria and loss of approximately one-third of the protein in the supematant. For the assay, an aliquot of the enzyme preparation was incubated in a volume of 3.0 ml with o.8-1.o- IO-z M [2-1~C]glycine (I8,65o counts//,mole), 8. IO-4 M pyridoxal phosphate, 3" Io-8 M glutamate, 2. Io 4 M tetrahydrofohc acid, I. IO-~ M B~och~m. Bzophys. Acta, 39 (196o) 367-369
SHORT COMMUNICATIONS
The photodecomposition of dinitrophenyl-amino acids* It is recogmzed that a principle shortcoming in the estimation of protein N-terminal amino acids as their dinitrophenyl derivatives is the difficulty encountered in accurately evaluating DNP-amino acid losses occurring during hydrolysis and the subsequent operations required for quantitation t In attempting to evaluate such losses employing DNP-EztC]amino acids as internal standards, we observed a variable and often an extensive loss of radioactivity when the derivatives were not shielded from light. The necessity for avoiding photodecompositlon of DNP-ammo acids has been mentioned by a number of authors 2-4. AKABORI et al. 5 have reported on the rate of the photolytic decomposition and recently have shown that certain DNP-peptides ~ are also light sensitive Similar observations have been reported for trmitrophenylated peptides 7 However, the nature or the extent of the decomposition has received little attention. The DNP derivatives of [2-14C]glycme, ~2-14C]tyrosme, I3-z4C]serine, E I - ~ 4 C ] valine (Nuclear Chicago Corporation), [i-14C]alanine and [I-t4C]glycine (Isotope Specialties) were prepared following the method of RAO AND SOBERs. The DNP-amino acids were not recrystallized but were purified by siliclc acid chromatography 9 thus removing dinitrophenol and dinitroaniline. Aliquots of the DNP derivatives in acetone solution were plated onto stainlesssteel planchets and exposed to two different lighting conditions occurring in the laboratory. Fig. IA demonstrates the rate of activity loss when the samples were exposed to light in the usual working area approximately 12 feet from outside windows. The loss was greatly accelerated when a parallel group of samples was exposed to daylight adjacent to an outside window but protected from direct sunlight DNP AMINO ACkDS
•
[~ "c] )
A
•
,
'"C]GLY
04
; 21
3-P'C] SEP
8 55
Lf %] ~L&
o T,
a
L2-"C] T~R
O,B26
•
[2 E~C]~L~
4,B2
I00 90
SPECiFiCACTIVITY uc/um
.L
[~
80
mm eo S
5e
~
40 3o
IO i
0
2
4
6
8
i
IO 12 14 16 18 20 22
0
2
4
6
8
IO 12 14 16 18 20 22
HOURS
Fig z Effect of light on the decomposltzon of
DI~Tp-[14C]&ITIlnO&clds
plated on st&m]ess steel.
A, c o m p o u n d s e x p o s e d to h g h t i2 feet from w i n d o w IB, c o m p o u n d s e x p o s e d to light i foot f r o m w i n d o w (See text.) Abbreviations: DNP-, dmltrophenyl. * P r e s e n t e d to t h e F e d e r a t i o n of A m e r i c a n Soclettes for E x p e r z m e n t a t Biology. Atlantxc Q t y , N e w Jersey, April, 1959
B~och~m B~opha,s 4cla, 39 (t90o) 364-367
365
SHORT COMMUNICATIONS
(Fig. IB). Other experiments showed that the decomposition rate varied with the nature and intensity of illumination, the area of sample distribution and the nature of the supporting medium. A group of samples kept in total darkness failed to lose activity over the same period. A decline in the rate of loss, more prominent in Fig. IB, is interpreted as the effect of decomposition products accumulating on the sample surface reducing the effective light intensity. In other experiments, aliquots of the radioactive DNP derivatives were subjected to photodecomposition in Conway-Kirk diffusion flasks. The center wells of the flasks contained saturated Ba(OH)~ or I N NaOH. Following exposure to daylight for periods of 48-72 h, the alkali trap was checked for radioactavity. The carboxyllabeled compounds yielded radioactive CO S which was recovered directly as BaCO v On the other hand, DNP-E3-14C]serine gave rise to volatile radioactive compounds which were trapped in NaOH and could be recovered as BaCO a only after persulfate oxidation. These results suggested that volatile acidic compounds are formed Although DNP-[2-14C]tyrosme did not lose radioactivity, chromatography of a sample which had been exposed to light demonstrated that DNP-[2-14C]tyrosine decarboxylates to form radioactive DNP-tyramine (vide mfra). Table I summarizes these experiments. Decarboxylation appears to be a principle but not the sole photolytic reaction. TABLE I RECOVI~RY OF RADIOACTIVITYFROM DNp-[14C]AMINO ACIDS EXPOSED TO LIGHT IN CONWAY-KIRK DIFFUSION FLASKS Added rachoactlvlty was determined vclth unexposed demvatives plated on stainless steel at essentially zero thxckness. Volatile radloactlvxty was c o u n t e d as BaCOs, n o t corrected for self absorption. All c o u n t s were corrected for coinmdence loss. R admactw;ly
DPNcompoundexposed
[I-14C] a.lanme [I-14C]glycme [i-l~C]valme [2-tlCJglycme [3-1~CJserine [2-x4C]tyrosine
Acttv*ty added xo a counts/m~n
64 o I26 28.8 17 ° 64.8 3o.o
CO s recovered as CO 2 recovered as BaCO, from r N No,OH after BaCO, from BaOH2 persulfate ox~aSton z o s counts/mzn zo s counts/ram
29.4 48.5 I9 8 0. 4 0.2 o.o
a* a* a* 40.0 17.2 o. 5
* a n o t determined.
In another series of experiments the derivatives were spotted on strips of No. I Whatman paper and exposed to daylight and artificial light for up to 240 h to obtain maximum decomposition. The strips were subsequently chromatographed in the ascending system of BISERTE AND OSTEUX1° along with unexposed control strips. These chromatographed strips were then scanned for radioactivity in an end-window counter adapted for manual strip counting using a slit width of 0.25 inch. Figs. 2A and 2B illustrate typical radiochromatograms. All exposed compounds yielded an increase in color intensity of the band at the solvent front as compared to unexposed samples. Only compounds containing radiocarbon at C-2 or C-3 exhibited a significant increase of radioactivity in this region. Unexposed samples of derivatives labeled at Bzoch~m. Bzophys
A c t a , 39 (196o) 364-367
366
SHORT COMMUNICATIONS
C-2 or C-3 yielded some radio-activity in the solvent-front band This band was shown to be due to decomposition occurring during normal handling operations as evidenced by the reappearance of a small radioactive peak at the solvent front when DNP[2-14C]glycine, purified by paper chromatography, was eluted and rechromatographed. 2o
,*. J
16
•
BEFORE
EXPOSURE
c
AFTER
EXPOSURE
'0
0 0
o
9
B
c_
E~
',i
~ 5
,,,
~ o U
+' r ,
I L
4
II ....
0 -~--~r: ~ 0
I0
15
if!
~ 20
25
DISTANCE
,2~_ 50
0
5
I
)
( I u n i t : ~- m
10
15
.:._m~_, 20
25
30
Fig 2. Typtcal radiochromatograms of DNP-[tiC]amino acids before and after exposure to light 2A, DNP-[2-1*C]glycme before and after exposure Arrows indicate solvent front The small peaks of low RF also appear in radiochromatograms of DNP-[3-x4C]serme. Bands corresponding to these peaks are colorless and remain colorless when treated with nmhydrm. 2B, [x-14C]glycme before and after exposure.
These observations and those cited above suggested that N-alkyl dimtroanilines result from photo-induced decarboxylation of DNP-amino acids. Hydrolysates of DNPproteins subjected to chromatography using the ascending system of BISERTE AND OSTEUX yield a yellow band at the solvent front said to be dinitroamline n. It appeared likely that N-alkyl dlnitroanilines, which are not resolved from dinitroanihne in this system, might also be present. Accordingly, the "dimtroaniline" bands were eluted with chloroform from chromatograms of exposed DNP-[2-14Cltyrosine, DNP-[2-t~C] glycine, DNP-[I-14C]glycine and DNP-[I-14Clalanine and subjected to reverse-phase chromatography 12 in parallel with authentic DNP-tyramine, N-methyldinitroaniline and N-ethyldinitroaniline. DNP-tyramine was Identified as the photodecomposition product of DNP-tyrosine and no dinitroaniline was evident with the reverse-phase system employed. While N-methyldmitroamline and N-ethyldinitroanihne have an RF identical with that of dinitroaniline, the compound obtained from exposed DNP[2-14C]glycine was radioactive whereas those obtained from DNP-[IA~C]glycine and DNP-[I-l*C]alanine were nonradioactive. If dinitroanlline were a product of the decarboxylation of DNP-[14Clamino acids, it should be nonradioactive. Since DNP[l*Cltyramlne was identified as a product formed from DNP-I2A4Cltyrosine while dinitroanihne was not formed, the experiments indicate that N-methyl and N-ethyldinitroaniline are products formed from DNP-glycine and DNP-alanine respectively. In summary the photodecomposition of DNP-amino acids yields COs, N-alkyl dinitroanilines and volatile acidic compounds of unknown structure. PhotodeBzo~h~m Bzoph>,s .Iota, 39 (196o) 364-367
367
SHORT COMMUNICATIONS
composition may cause large and variable losses of DNP-amino acids unless exposure to light is avoided. The technical assistance of Miss C. ROTHNEM is gratefully acknowledged. This work was supported by grants from the Life Insurance Medical Research Fund, the American Heart Association and Eh Lilly and Company.
Departments of Pediatrics and Physiological Chemistry, Research Laboratories of the Variety Club Heart Hospital, University of Minnesota, Minneapolis, M, nn. (U.S.A.)
B. POLLARA
R. W. VON KORFF*
1 H S. RHINESMITH,W .A SCHROEDER AND L PAULING,J . . 4 m . Chem. Soc, 79 (1957) 6o9. 2 G L MILLS, B~ochem J., 5o (1952 ) 707 3 F. SANGER, B,ochem J., 45 (1949) 563. 4 S. BLACKBURN, B~ochem. J., 45 (1949) 579. s S AKABORI, T [KENAKA,Y. OKADA AND K KOHNO,Proc Jap .4cad, 29 (1953) 5o9 6 S AKABORI, S SAKAKIBARAAND K SAKAKIBARA, Bull Chem Soc Japan, 32 (1959) 312 ? K SATAKE AND T. OKAUAMA, Bull. Chem. Soc. Japan, 32 (I959) 520. s K. R. RAO AND H. A. SOBER, J. A m Chem. Soc., 76 (1953) 1328. 9 C. H LI AND L. ASH, J. B,ol. Chem , 230 (1953) 419 10 G. BISERTE AND R. OSTEUX,Bull. soc, ch~m. b*ol, 33 (1951) 5 °. n H. FRAENKEL-CONRAT, J. I. HARRIS AND A L. LEvY in D. CLICK, Methods of BzochemzcM Analys~s, Vol. II, Intersclence P u b h s h e r s , N e w York, 1955, P. 539 12 I. M. LOCKHART, Nature, 177 (1956) 393.
Received October I9th, 1959 * Established Investigator, Minnesota H e a r t Association.
B~ochzm. B~ophys Acta, 39 (196o) 364-367
Synthesis of serine from glycine in mitochondrial fragments The conversion of two molecules of glycine to serine has been observed in the intact rat 1, In homogenates of turkey live# and in a particulate fraction of rat live#. Some of the intermediate steps in the synthesis have been studied in partially purified systems (see ref.4). Incidental to a search for an enzyme system capable of synthesizing ~-aminole~allinic acid, we observed that the entire complement of enzymes and cofactors required for the net synthesis of serine from glycine was present in a particulate fraction obtained by osmotic disruption of mitochondria. The mitochondrial preparations were obtained from chicken livers by the conventional technique involving differential centnfugation in 0.3 M sucrose 5. The preparation gave P/O ratios of 1.7-I. 9 succinate and 2.3-2.6 with glutamate. The sub-mitochondrial particles were obtained by suspending the mltochondria in i . io-SM tris(hydroxymethyl)amlnomethane at pH 7.0 for 30 rain at o ° and centrifuging at ioo,ooo × g for 15 min The residue was suspended in an amount of 0.3 M sucrose equal to the original mitochondrial volume. The treatment results in disintegration of mitochondria and loss of approximately one-third of the protein in the supematant. For the assay, an aliquot of the enzyme preparation was incubated in a volume of 3.0 ml with o.8-1.o- IO-z M [2-1~C]glycine (I8,65o counts//,mole), 8. IO-4 M pyridoxal phosphate, 3" Io-8 M glutamate, 2. Io 4 M tetrahydrofohc acid, I. IO-~ M B~och~m. Bzophys. Acta, 39 (196o) 367-369