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A micromethod for the estimation of oxaloacetic acid in tissues
596
SHORT COMMUNICATIONS
the product was dialyzed against several changes of distilled water and lyophilized. The poly-N4-(3-hydroxypropyl)-L-asparaginyl gliadin obtained sedimented with s20 = 2.6 (c = 1 % in dimethylformamide), while the polybenzyl-L-aspartyl gliadin from which it was derived sedimented with s20 = 3.1 (c = 1 % in the same solvent). The samples enriched with benzyl aspartate or N4-(3-hydroxypropyl)-L-asparagine were analyzed by the same procedure as used for polyalanyl gliadin. The analysis showed in both cases an enrichment with 6.3 mmoles of aspartic acid residues per gram gliadin (the ratio of Asp/Glu was 0.088 in gliadin and 2.5 in the aspartylated gliadin derivatives). Water-soluble poly-L-aspartyl gliadin was obtained upon the reaction of polybenzyl-L-aspartyl gliadin with anhydrous H B r in glacial acetic acid. Attachment of suitable peptides to gliadin m a y thus yield water-soluble derivatives. Enrichment with charged amino acids might help the study of individual components in the neutral and acid p H range 5. This work was supported by a grant, No. FG-Is-Io4-6o, from the U.S. Department of Agriculture. MICHAEL SELA
Department of Biophysics, The Weizmann Institute of Science, Rehovoth (Israel)
NOACH LuPu ARIEH YARON A R I E H BERGER
1 S. BROHULT AND E. SANDEGREN, in H. NEURATH AND K. BAILEY, The Proteins, Vol. 2, A c a d e m i c Press, Inc., N e w York, 1954, p. 487 • 2 j . H. WOYCHIK, R. J. DIMLER AND F. R. SENTI, Arch. Biochem. Biophys., 91 (196o) 235. 3 A. G. MCCALLA AND N. GRATEN, Can. J. Research, 20 (1942) I3O. 4 R. VERCOUTEREN AND R. LONTIE, Arch. Internat. Physiol., 62 (1954) 579. 5 j . H. WOYCHIK, J. A. BOUNDY AND R. J. DIMLER, Arch. Biochem. Biophys., 94 (1961) 477. 6 R. W. JONES, IN. W. TAYLOR AND F. R. SENTI, Arch. Biochem. Biophys., 84 (1959) 363. 7 R. R. BECKER AND M. A. STAHMANN, J. Biol. Chem., 204 (1953) 745. 8 E. KATCHALSKI AND M. SELA, Advances in Protein Chem., 13 (1958) 243. 9 C. B. ANFINSEN, M. SELA AND J. P. COOKE, J. Biol. Chem., in the press. 10 E. KATCHALSKI, M. GEHATIA AND M. SELA, J. Am. Chem. Soc., 77 (1955) 6175. 11 M. SELA AND E. KATCHALSKI, Advances in Protein Chem., 14 (1959) 391. 13 :N. LuPu, A. YARON, M. SELA AND A. BERGER, Bull. Research Council of Israel, IoA (1961) 47. 13 G. L. MILLS, Biochim. Biophys. Acta, 14 (1954) 274. 14 V. V. PFEIFER, C. VOJNOVICH AND R. A. ANDERSON, Cereal Chemistry, 35 (1958) 458.
Received April 25th, 1962 Biochim. Biophys. Acta, 62 (1962) 594-596
SC 2 1 2 0
A micromethod for the estimation of oxaloacetic acid in tissues In the course of studies on respiratory metabolism of plants, we were faced with the problem of the estimation of oxaloacetic acid in plant tissues. The manometric method based on catalytic decarboxylation of oxaloacetate a and manometric measurement of C02 evolved proved to be inadequate, owing to the very small amount of oxaloacetate present. We tried to measure oxaloacetate in pea-stem extracts enzymically, as the amount of D P N H oxidized in the presence of malate dehydrogenase (EC 1.1.1.37) and D P N H in excess. We were unable to obtain by this method reproducible results, owing to lack of sufficient sensitivity. Indeed, only 20-50 mt~moles of oxaloacetate are present per gram of tissue, and, on the other hand, the extract Biochim. Biophys. Acta, 62 (1962) 596-598
597
SHORT COMMUNICATIONS
from I g of tissue has a very high absorbancy at 340 my,, the wavelength at which D P N H oxidation is measured. A method was worked out in which the DPN + produced from D P N H on addition of malate dehydrogenase was measured b y enzymic cycling. The tissue (I-cm long internodes of etiolated pea seedlings) is ground in a mortar with I vol. of chilled 7 To HCI04 and quartz sand.The mortar is washedwith i % HCI04, and the extract plus the mortar washings is centrifuged at 0-4 ° for 30 min at 20o00 × g. The precipitate is washed twice with 1 % HCI04, and the extract plus the washings are combined to give a volume of 5-6 ml/g of original tissue. The acid extraction insures total destruction of the D P N H present in the tissue, whilst DPN + is stable in acidic solution. The HC104 extract is neutralized to p H 7.0 with KOH, and the KCI04 formed is removed b y centrifugation after 30 min standing at 0% I t is important to avoid alkalinization beyond p H 7-7.2, because of the instability of oxaloacetate at alkaline pH's. As the estimation of oxaloacetate is based on the determination of DPN + formed from D P N H b y malate dehydrogenase, it is essential to remove any DPN originally present in the tissue. The neutralized extract is therefore incubated for IO rain at room temperature with an excess of alcohol dehydrogenase (EC I . I . I . I ) in the presence of I M ethanol. All the DPN + is completely reduced to D P N H , and this latter is destroyed upon acidification with H2SO ~. The extract is again neutralized (pH 7.o), and incubated with D P N H and malate dehydrogenase in excess for IO rain at room temperature. Starting with I g of tissue, the volume of the extract at this point of the procedure is about 6 ml. The addition of 1.5/~moles of D P N H is adequate to obtain complete reduction of oxaloacetate to malate in a few minutes. I mole of DPN per mole of oxaloacetate present is produced and the excess of D P N H is destroyed b y acidification. The extract is then brought to p H 7.5, and made up to a volume (usually 6.5-7 ml/g of tissue). DPN + is then measured on an aliquot of the
0.580-I'
o
-
o
~
. ]y
40
30 -
o
x
20-
10-
////
/
0"5501
or
i.500 0.450-
~ m ~0"~i ]I
I 0
0.400 DPN (m~moles)
Time (Min)
Fig. I. P r o p o r t i o n a l i t y of r e a c t i o n r a t e t o D P N + concentration.
F i g . 2. T y p i c a l d e t e r m i n a t i o n of D P N + i n p e a s t e m . C u r v e I, d e t e r m i n a t i o n of D P N + i n e x t r a c t e q u i v a l e n t t o 4 ° m g of t i s s u e ; C u r v e I I , n o ext r a c t , 0.57 r e # m o l e of D P N + ; C u r v e I I I , n o e x t r a c t , 0.285 m y m o l e of D P N + ; C u r v e IV, e x t r a c t e q u i v a l e n t t o 80 m g of t i s s u e n o t t r e a t e d w i t h m a l a t e d e h y d r o g e n a s e .
Biochim. Biophys. Acta, 62 (1962) 5 9 6 - 5 9 8
598
SHORT COMMUNICATIONS
extract by means of its coenzymic activity in a system containing o.2 M ethanol, a mixture of ethanol dehydrogenase and lipoamide dehydrogenase (diaphorase, ECI.6.4.3), 2/,moles of ferricyanide and 0.06 M Tris buffer (pH 7.5) in a final volume of 3 ml. The rate of ferricyanide reduction is measured as the decrease of absorbancy at 420 m/z, and it is proportional to the amount of DPN+ (see Fig. i). The reaction rate with known amounts of DPN+ has to be measured with each analysis, and will of course depend on the activity of the enzymes used. It is essential to destroy any substance present in the tissue (such as ascorbate) which reduces ferricyanide non-enzymically, before running the DPN + analysis This is easily obtained b y addition of ferricyanide to the neutralized extract, until all reducing substances are titrated. TABLE
I
OXALOACETATE CONTENT OF PICA-STEM INTERNODES
Fresh weight of tissue (g)
Oxaloacelate added (m~moles)
Oxaloacetate found (ml~moles)
% recovery of added oxaloacetate
2
-I6
33.6 49.5
99.5
2
-16
28.9 44.0
94-5
I
-28. 5
13.4 41.6
99
Fig. 2 shows a typical analysis. Curves I I and I I I illustrate the reaction rates in the presence of 0.57 ° and 0.285 m/~mole of DPN, respectively; Curve I is obtained with an extract equivalent to 4 ° mg of tissue. Curve IV is obtained with an aliquot of same extract equivalent to 8o mg of tissue, but not treated with malate dehydrogenase. It shows that the DPN + originally present is completely destroyed by the procedure outlined above. Table I shows that when small amounts of oxaloacetate are added during the disintegration of tissue in the mortar, over 95 % of the added oxaloacetate is recovered.
National Research Council Unit for Redox studies in Plants, Laboratory of Plant Physiology, Institute of Plant Sciences, University of Milano, Milano (Italy)
MARIA LUISA BERTOLE
GIORGIO FORTI
1 W . %,"v'.UMBREIT, 1:{. H . BURRIS AND J. F. STAUFFER, M a n o m e t r i c T e c h n i q u e s , B u r g e s s P u b h , Co., M i n n e a p o l i s , 1957, P. 211.
Received May Ist, 1962 B i o c h i m . B i o p h ) , s . A c t a , 62 (1962) 5 9 6 - 5 9 8
SHORT COMMUNICATIONS
the product was dialyzed against several changes of distilled water and lyophilized. The poly-N4-(3-hydroxypropyl)-L-asparaginyl gliadin obtained sedimented with s20 = 2.6 (c = 1 % in dimethylformamide), while the polybenzyl-L-aspartyl gliadin from which it was derived sedimented with s20 = 3.1 (c = 1 % in the same solvent). The samples enriched with benzyl aspartate or N4-(3-hydroxypropyl)-L-asparagine were analyzed by the same procedure as used for polyalanyl gliadin. The analysis showed in both cases an enrichment with 6.3 mmoles of aspartic acid residues per gram gliadin (the ratio of Asp/Glu was 0.088 in gliadin and 2.5 in the aspartylated gliadin derivatives). Water-soluble poly-L-aspartyl gliadin was obtained upon the reaction of polybenzyl-L-aspartyl gliadin with anhydrous H B r in glacial acetic acid. Attachment of suitable peptides to gliadin m a y thus yield water-soluble derivatives. Enrichment with charged amino acids might help the study of individual components in the neutral and acid p H range 5. This work was supported by a grant, No. FG-Is-Io4-6o, from the U.S. Department of Agriculture. MICHAEL SELA
Department of Biophysics, The Weizmann Institute of Science, Rehovoth (Israel)
NOACH LuPu ARIEH YARON A R I E H BERGER
1 S. BROHULT AND E. SANDEGREN, in H. NEURATH AND K. BAILEY, The Proteins, Vol. 2, A c a d e m i c Press, Inc., N e w York, 1954, p. 487 • 2 j . H. WOYCHIK, R. J. DIMLER AND F. R. SENTI, Arch. Biochem. Biophys., 91 (196o) 235. 3 A. G. MCCALLA AND N. GRATEN, Can. J. Research, 20 (1942) I3O. 4 R. VERCOUTEREN AND R. LONTIE, Arch. Internat. Physiol., 62 (1954) 579. 5 j . H. WOYCHIK, J. A. BOUNDY AND R. J. DIMLER, Arch. Biochem. Biophys., 94 (1961) 477. 6 R. W. JONES, IN. W. TAYLOR AND F. R. SENTI, Arch. Biochem. Biophys., 84 (1959) 363. 7 R. R. BECKER AND M. A. STAHMANN, J. Biol. Chem., 204 (1953) 745. 8 E. KATCHALSKI AND M. SELA, Advances in Protein Chem., 13 (1958) 243. 9 C. B. ANFINSEN, M. SELA AND J. P. COOKE, J. Biol. Chem., in the press. 10 E. KATCHALSKI, M. GEHATIA AND M. SELA, J. Am. Chem. Soc., 77 (1955) 6175. 11 M. SELA AND E. KATCHALSKI, Advances in Protein Chem., 14 (1959) 391. 13 :N. LuPu, A. YARON, M. SELA AND A. BERGER, Bull. Research Council of Israel, IoA (1961) 47. 13 G. L. MILLS, Biochim. Biophys. Acta, 14 (1954) 274. 14 V. V. PFEIFER, C. VOJNOVICH AND R. A. ANDERSON, Cereal Chemistry, 35 (1958) 458.
Received April 25th, 1962 Biochim. Biophys. Acta, 62 (1962) 594-596
SC 2 1 2 0
A micromethod for the estimation of oxaloacetic acid in tissues In the course of studies on respiratory metabolism of plants, we were faced with the problem of the estimation of oxaloacetic acid in plant tissues. The manometric method based on catalytic decarboxylation of oxaloacetate a and manometric measurement of C02 evolved proved to be inadequate, owing to the very small amount of oxaloacetate present. We tried to measure oxaloacetate in pea-stem extracts enzymically, as the amount of D P N H oxidized in the presence of malate dehydrogenase (EC 1.1.1.37) and D P N H in excess. We were unable to obtain by this method reproducible results, owing to lack of sufficient sensitivity. Indeed, only 20-50 mt~moles of oxaloacetate are present per gram of tissue, and, on the other hand, the extract Biochim. Biophys. Acta, 62 (1962) 596-598
597
SHORT COMMUNICATIONS
from I g of tissue has a very high absorbancy at 340 my,, the wavelength at which D P N H oxidation is measured. A method was worked out in which the DPN + produced from D P N H on addition of malate dehydrogenase was measured b y enzymic cycling. The tissue (I-cm long internodes of etiolated pea seedlings) is ground in a mortar with I vol. of chilled 7 To HCI04 and quartz sand.The mortar is washedwith i % HCI04, and the extract plus the mortar washings is centrifuged at 0-4 ° for 30 min at 20o00 × g. The precipitate is washed twice with 1 % HCI04, and the extract plus the washings are combined to give a volume of 5-6 ml/g of original tissue. The acid extraction insures total destruction of the D P N H present in the tissue, whilst DPN + is stable in acidic solution. The HC104 extract is neutralized to p H 7.0 with KOH, and the KCI04 formed is removed b y centrifugation after 30 min standing at 0% I t is important to avoid alkalinization beyond p H 7-7.2, because of the instability of oxaloacetate at alkaline pH's. As the estimation of oxaloacetate is based on the determination of DPN + formed from D P N H b y malate dehydrogenase, it is essential to remove any DPN originally present in the tissue. The neutralized extract is therefore incubated for IO rain at room temperature with an excess of alcohol dehydrogenase (EC I . I . I . I ) in the presence of I M ethanol. All the DPN + is completely reduced to D P N H , and this latter is destroyed upon acidification with H2SO ~. The extract is again neutralized (pH 7.o), and incubated with D P N H and malate dehydrogenase in excess for IO rain at room temperature. Starting with I g of tissue, the volume of the extract at this point of the procedure is about 6 ml. The addition of 1.5/~moles of D P N H is adequate to obtain complete reduction of oxaloacetate to malate in a few minutes. I mole of DPN per mole of oxaloacetate present is produced and the excess of D P N H is destroyed b y acidification. The extract is then brought to p H 7.5, and made up to a volume (usually 6.5-7 ml/g of tissue). DPN + is then measured on an aliquot of the
0.580-I'
o
-
o
~
. ]y
40
30 -
o
x
20-
10-
////
/
0"5501
or
i.500 0.450-
~ m ~0"~i ]I
I 0
0.400 DPN (m~moles)
Time (Min)
Fig. I. P r o p o r t i o n a l i t y of r e a c t i o n r a t e t o D P N + concentration.
F i g . 2. T y p i c a l d e t e r m i n a t i o n of D P N + i n p e a s t e m . C u r v e I, d e t e r m i n a t i o n of D P N + i n e x t r a c t e q u i v a l e n t t o 4 ° m g of t i s s u e ; C u r v e I I , n o ext r a c t , 0.57 r e # m o l e of D P N + ; C u r v e I I I , n o e x t r a c t , 0.285 m y m o l e of D P N + ; C u r v e IV, e x t r a c t e q u i v a l e n t t o 80 m g of t i s s u e n o t t r e a t e d w i t h m a l a t e d e h y d r o g e n a s e .
Biochim. Biophys. Acta, 62 (1962) 5 9 6 - 5 9 8
598
SHORT COMMUNICATIONS
extract by means of its coenzymic activity in a system containing o.2 M ethanol, a mixture of ethanol dehydrogenase and lipoamide dehydrogenase (diaphorase, ECI.6.4.3), 2/,moles of ferricyanide and 0.06 M Tris buffer (pH 7.5) in a final volume of 3 ml. The rate of ferricyanide reduction is measured as the decrease of absorbancy at 420 m/z, and it is proportional to the amount of DPN+ (see Fig. i). The reaction rate with known amounts of DPN+ has to be measured with each analysis, and will of course depend on the activity of the enzymes used. It is essential to destroy any substance present in the tissue (such as ascorbate) which reduces ferricyanide non-enzymically, before running the DPN + analysis This is easily obtained b y addition of ferricyanide to the neutralized extract, until all reducing substances are titrated. TABLE
I
OXALOACETATE CONTENT OF PICA-STEM INTERNODES
Fresh weight of tissue (g)
Oxaloacelate added (m~moles)
Oxaloacetate found (ml~moles)
% recovery of added oxaloacetate
2
-I6
33.6 49.5
99.5
2
-16
28.9 44.0
94-5
I
-28. 5
13.4 41.6
99
Fig. 2 shows a typical analysis. Curves I I and I I I illustrate the reaction rates in the presence of 0.57 ° and 0.285 m/~mole of DPN, respectively; Curve I is obtained with an extract equivalent to 4 ° mg of tissue. Curve IV is obtained with an aliquot of same extract equivalent to 8o mg of tissue, but not treated with malate dehydrogenase. It shows that the DPN + originally present is completely destroyed by the procedure outlined above. Table I shows that when small amounts of oxaloacetate are added during the disintegration of tissue in the mortar, over 95 % of the added oxaloacetate is recovered.
National Research Council Unit for Redox studies in Plants, Laboratory of Plant Physiology, Institute of Plant Sciences, University of Milano, Milano (Italy)
MARIA LUISA BERTOLE
GIORGIO FORTI
1 W . %,"v'.UMBREIT, 1:{. H . BURRIS AND J. F. STAUFFER, M a n o m e t r i c T e c h n i q u e s , B u r g e s s P u b h , Co., M i n n e a p o l i s , 1957, P. 211.
Received May Ist, 1962 B i o c h i m . B i o p h ) , s . A c t a , 62 (1962) 5 9 6 - 5 9 8