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[94A] Carbamyl phosphate
[94A]
CARBAMYL PHOSPHATE
653
vessel is V, and the volume of liquid which has passed through the mixing vessel is L. The values of x and V are fixed by the experimental conditions, and L may be determined by measuring the volume of eluate which has emerged from the column. Tables of (1 - e-s) are to be found in many reference books, and hence the calculation of X is a simple matter. The theoretical and practical aspects of gradient elution chromatography are discussed in papers by Lakshmanan and Lieberman 4 and Busch et al. 5 Elution patterns of mixtures of compounds are shown in Figs. 2 and 3. The results are highly reproducible both with artificial and tissue reaction mixtures. The volume at which a particular compound is eluted can be reproduced to within + 3 %. At the conclusion of each run the column is washed as described above and is then ready for further use. 4 T. K. Lakshmanan and S. Lieberman, Arch. Biochem. and Biophys. 45, 235 (1953). 5 H. Busch, R. B. Hurlbert, and V. R. Potter, J. Biol. Chem. 196, 717 (1952).
[94A] Carbamyl Phosphate B y L.
SPECTOR, M. E. JONES, and F. LIPMANN
Carbamyl phosphate is formed through the reaction of KH~P04 with KCNO. When the reaction is complete, potassium and most of the inorganic phosphate are removed simultaneously as the perchlorate and as the lithium salt, respectively, by the addition of lithium perchlorate. Lithium carbamyl phosphate is obtained from the filtrate by alcohol fractionation. Procedure. In 100 ml. of water are dissolved 13.6 g. (0.10 mole) of KH2P04 and 8.1 g. (0.10 mole) of KCNO. The solution is warmed at 30 ° for 30 minutes and then chilled. Subsequent operations (until the drying step) are best performed in the cold room. An ice-cold solution of 7.2 g. (0.30 mole) of lithium hydroxide and 16.7 ml. of 12 N perchloric acid (0.20 mole) in 83 ml. of water is added to the solution of crude potassium carbamyl phosphate. The mixture is thoroughly agitated and allowed to stand for 1 to 2 hours before removal of the precipitate 1 on a Biichner funnel. To the magnetically stirred filtrate cold absolute ethanol is added, dropwise, from a buret until 70 ml. has been added. The precipitate which 1 This precipitate is a mixture of potassium perchlorate and lithium phosphate. It is discarded.
654
PROTEINS AND DERIVATIVES
[94A]
forms is r e m o v e d b y filtration and discarded. ~,3 The filtrate is treated in the same m a n n e r with an additional 50 to 55 ml. of absolute ethanol. The precipitate is collected and dried over phosphorus pentoxide in a v a c u u m desiccator at r o o m t e m p e r a t u r e , the desiccant being changed once or twice a d a y until the evolution of volatile material f r o m the p r o d u c t is complete (1 to 4 days). Yield, 4 g.4.5 Stability. Solutions of lithium c a r b a m y l p h o s p h a t e in water h a v e been found to h a v e the following half-lives: 16 hours at 0°; 2 hours at 30°; 40 to 50 minutes at 37 ° . I n the frozen state the solutions a p p e a r to be stable indefinitely. The dried solid salt has stood at room t e m p e r a t u r e for over 3 weeks with no detectable change.
Assay Since c a r b a m y l p h o s p h a t e is for practical purposes stable on short exposure to the conditions of the F i s k e - S u b b a R o w colorimetric test, 6 it is most conveniently measured b y differential p h o s p h a t e analysis. T w o tubes are required for each a n a l y s i s - - o n e for the assay of o r t h o p h o s p h a t e and another for the determination of the s u m of o r t h o p h o s p h a t e and carb a m y l p h o s p h a t e (after hydrolytic decomposition of the latter). Orthophosphate Assay. T h e Fiske and S u b b a R o w reagents are used, and the minor modifications m a d e in the conventional procedure are designed to avoid hydrolysis of c a r b a m y l p h o s p h a t e during the determination. For protein precipitation, ice-cold T C A (final concentration, 3 % ) is used and the protein precipitate is centrifuged down in chilled m e t a l cups (2 to 3 minutes) or in a refrigerated centrifuge. Samples are k e p t ice-cold until r e a d y for analysis. A suitable aliquot is transferred to an assay tube, diluted to 8 0 % of its final volume, the m o l y b d a t e and reducing reagents 7 are added to each t u b e separately at timed intervals, Ordinarily the solid begins to separate at 65 to 70 ml. of ethanol, but if the addition has been too rapid the precipitate will sometimes fail to appear. However, continued stirring has invariably resulted in the eventual appearance of precipitate. 3 This precipitate is a mixture of residual potassium perchlorate and lithium phosphate along with a forerun of lithium earbamyl phosphate. No attempt has been made to recover the latter from the mixture. 4 The product prepared in this way generally gave the following analysis: 75% (by weight) lithium carbamyl phosphate; 10% lithium phosphate; 1 to 2% urea; and traces of unidentified carbon- and nitrogen-containing substances. The remainder appears to be firmly bound water. 6 If desirable, the lithium salt can be converted to potassium carbamyl phosphate with potassium Dowex 50. All operations must be carried out at 0° to a:coid hydrolysis of carbamyl phosphate. 6 C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925); see Vol. I I I [114]. 7 The aminonaphtholsulfonic acid reagent, when mixed with standard orthophosphate solution and the molybdate reagent, should produce, within 5 minutes at room temperature, a color intensity not less than 95% of that obtained in 20 minutes. This reagent is stable when stored well-stoppered and in the refrigerator.
[94A]
CARBAMYL PHOSPHATE
655
and the t u b e is t h e n m a d e to volume and mixed. T h e color is read 5 to 7 minutes after the addition of the reagents. 8,9 Carbamyl Phosphate Hydrolysis. C a r b a m y l p h o s p h a t e can be hydrolyzed to yield o r t h o p h o s p h a t e b y heating in a boiling w a t e r b a t h for 2 minutes or b y exposure to 0.1 N base for 10 minutes at room t e m p e r a ture. 10 T h e heat t r e a t m e n t is more rapid and m a y be preferred when there are no other heat-unstable phosphates in the sample. Alkaline hydrolysis is in general a milder, more specific procedure and is preferable in the presence of other heat- and acid-labile phosphates, such as the adenosine polyphosphates. 1~ After hydrolysis of the c a r b a m y l phosphate, the total o r t h o p h o s p h a t e is determined b y the Fiske and S u b b a R o w method. The difference between this and the o r t h o p h o s p h a t e value represents the carb a m y l p h o s p h a t e content. T h e t e r m " h y d r o l y s i s " has been used in an inclusive sense to indicate inorganic p h o s p h a t e release from c a r b a m y l p h o s p h a t e regardless of the detailed mechanism. I t should be noted, however, t h a t at least two t y p e s of reaction are included here. I n acid solutions the reaction a p p e a r s to be a true hydrolysis while in neutral or basic solution, the reaction is pred o m i n a n t l y a splitting of c a r b a m y l p h o s p h a t e to inorganic p h o s p h a t e and c y a n a t e and not a hydrolysis. T h e f o r m a t i o n of c y a n a t e has been confirmed b y a differential a m m o n i a analysis and b y the specific blue color which e y a n a t e gives with cobalt. s The use of the timed interval limits the number of tubes which may be run as a set to approximately ten. If samples are kept well chilled, however, several sets may be run in sequence. 9 The reagents of O. H. Lowry and J. A. Lopez [J. Biol. Chem. 162, 421 (1946)] may be used in place of those of Fiske and SubbaRow, but the substitution offers no particular advantage; see also Vol. I I I [114]. lo The rate of hydrolysis of carbamyl phosphate is approximately the same from pH 1.5 to 9.0. In this range heating is necessary for rapid decomposition, whereas at strongly alkaline reaction decomposition occurs in a few minutes at room temperature. Therefore, the pH used for the hydrolysis is mainly dictated by the stability of any other phosphate compounds present in the sample. ~1Although carbamyl phosphate may be determined in the presence of the adenosine polyphosphates by hydrolysis with heat, the alkaline hydrolysis is more suitable. If heat is used, however, the TCA filtrate should be diluted to bring the molarity to 0.05, for under these conditions the adenosine polyphosphates are hydrolyzed only to the extent of 3 % in the l-minute heating.
CARBAMYL PHOSPHATE
653
vessel is V, and the volume of liquid which has passed through the mixing vessel is L. The values of x and V are fixed by the experimental conditions, and L may be determined by measuring the volume of eluate which has emerged from the column. Tables of (1 - e-s) are to be found in many reference books, and hence the calculation of X is a simple matter. The theoretical and practical aspects of gradient elution chromatography are discussed in papers by Lakshmanan and Lieberman 4 and Busch et al. 5 Elution patterns of mixtures of compounds are shown in Figs. 2 and 3. The results are highly reproducible both with artificial and tissue reaction mixtures. The volume at which a particular compound is eluted can be reproduced to within + 3 %. At the conclusion of each run the column is washed as described above and is then ready for further use. 4 T. K. Lakshmanan and S. Lieberman, Arch. Biochem. and Biophys. 45, 235 (1953). 5 H. Busch, R. B. Hurlbert, and V. R. Potter, J. Biol. Chem. 196, 717 (1952).
[94A] Carbamyl Phosphate B y L.
SPECTOR, M. E. JONES, and F. LIPMANN
Carbamyl phosphate is formed through the reaction of KH~P04 with KCNO. When the reaction is complete, potassium and most of the inorganic phosphate are removed simultaneously as the perchlorate and as the lithium salt, respectively, by the addition of lithium perchlorate. Lithium carbamyl phosphate is obtained from the filtrate by alcohol fractionation. Procedure. In 100 ml. of water are dissolved 13.6 g. (0.10 mole) of KH2P04 and 8.1 g. (0.10 mole) of KCNO. The solution is warmed at 30 ° for 30 minutes and then chilled. Subsequent operations (until the drying step) are best performed in the cold room. An ice-cold solution of 7.2 g. (0.30 mole) of lithium hydroxide and 16.7 ml. of 12 N perchloric acid (0.20 mole) in 83 ml. of water is added to the solution of crude potassium carbamyl phosphate. The mixture is thoroughly agitated and allowed to stand for 1 to 2 hours before removal of the precipitate 1 on a Biichner funnel. To the magnetically stirred filtrate cold absolute ethanol is added, dropwise, from a buret until 70 ml. has been added. The precipitate which 1 This precipitate is a mixture of potassium perchlorate and lithium phosphate. It is discarded.
654
PROTEINS AND DERIVATIVES
[94A]
forms is r e m o v e d b y filtration and discarded. ~,3 The filtrate is treated in the same m a n n e r with an additional 50 to 55 ml. of absolute ethanol. The precipitate is collected and dried over phosphorus pentoxide in a v a c u u m desiccator at r o o m t e m p e r a t u r e , the desiccant being changed once or twice a d a y until the evolution of volatile material f r o m the p r o d u c t is complete (1 to 4 days). Yield, 4 g.4.5 Stability. Solutions of lithium c a r b a m y l p h o s p h a t e in water h a v e been found to h a v e the following half-lives: 16 hours at 0°; 2 hours at 30°; 40 to 50 minutes at 37 ° . I n the frozen state the solutions a p p e a r to be stable indefinitely. The dried solid salt has stood at room t e m p e r a t u r e for over 3 weeks with no detectable change.
Assay Since c a r b a m y l p h o s p h a t e is for practical purposes stable on short exposure to the conditions of the F i s k e - S u b b a R o w colorimetric test, 6 it is most conveniently measured b y differential p h o s p h a t e analysis. T w o tubes are required for each a n a l y s i s - - o n e for the assay of o r t h o p h o s p h a t e and another for the determination of the s u m of o r t h o p h o s p h a t e and carb a m y l p h o s p h a t e (after hydrolytic decomposition of the latter). Orthophosphate Assay. T h e Fiske and S u b b a R o w reagents are used, and the minor modifications m a d e in the conventional procedure are designed to avoid hydrolysis of c a r b a m y l p h o s p h a t e during the determination. For protein precipitation, ice-cold T C A (final concentration, 3 % ) is used and the protein precipitate is centrifuged down in chilled m e t a l cups (2 to 3 minutes) or in a refrigerated centrifuge. Samples are k e p t ice-cold until r e a d y for analysis. A suitable aliquot is transferred to an assay tube, diluted to 8 0 % of its final volume, the m o l y b d a t e and reducing reagents 7 are added to each t u b e separately at timed intervals, Ordinarily the solid begins to separate at 65 to 70 ml. of ethanol, but if the addition has been too rapid the precipitate will sometimes fail to appear. However, continued stirring has invariably resulted in the eventual appearance of precipitate. 3 This precipitate is a mixture of residual potassium perchlorate and lithium phosphate along with a forerun of lithium earbamyl phosphate. No attempt has been made to recover the latter from the mixture. 4 The product prepared in this way generally gave the following analysis: 75% (by weight) lithium carbamyl phosphate; 10% lithium phosphate; 1 to 2% urea; and traces of unidentified carbon- and nitrogen-containing substances. The remainder appears to be firmly bound water. 6 If desirable, the lithium salt can be converted to potassium carbamyl phosphate with potassium Dowex 50. All operations must be carried out at 0° to a:coid hydrolysis of carbamyl phosphate. 6 C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925); see Vol. I I I [114]. 7 The aminonaphtholsulfonic acid reagent, when mixed with standard orthophosphate solution and the molybdate reagent, should produce, within 5 minutes at room temperature, a color intensity not less than 95% of that obtained in 20 minutes. This reagent is stable when stored well-stoppered and in the refrigerator.
[94A]
CARBAMYL PHOSPHATE
655
and the t u b e is t h e n m a d e to volume and mixed. T h e color is read 5 to 7 minutes after the addition of the reagents. 8,9 Carbamyl Phosphate Hydrolysis. C a r b a m y l p h o s p h a t e can be hydrolyzed to yield o r t h o p h o s p h a t e b y heating in a boiling w a t e r b a t h for 2 minutes or b y exposure to 0.1 N base for 10 minutes at room t e m p e r a ture. 10 T h e heat t r e a t m e n t is more rapid and m a y be preferred when there are no other heat-unstable phosphates in the sample. Alkaline hydrolysis is in general a milder, more specific procedure and is preferable in the presence of other heat- and acid-labile phosphates, such as the adenosine polyphosphates. 1~ After hydrolysis of the c a r b a m y l phosphate, the total o r t h o p h o s p h a t e is determined b y the Fiske and S u b b a R o w method. The difference between this and the o r t h o p h o s p h a t e value represents the carb a m y l p h o s p h a t e content. T h e t e r m " h y d r o l y s i s " has been used in an inclusive sense to indicate inorganic p h o s p h a t e release from c a r b a m y l p h o s p h a t e regardless of the detailed mechanism. I t should be noted, however, t h a t at least two t y p e s of reaction are included here. I n acid solutions the reaction a p p e a r s to be a true hydrolysis while in neutral or basic solution, the reaction is pred o m i n a n t l y a splitting of c a r b a m y l p h o s p h a t e to inorganic p h o s p h a t e and c y a n a t e and not a hydrolysis. T h e f o r m a t i o n of c y a n a t e has been confirmed b y a differential a m m o n i a analysis and b y the specific blue color which e y a n a t e gives with cobalt. s The use of the timed interval limits the number of tubes which may be run as a set to approximately ten. If samples are kept well chilled, however, several sets may be run in sequence. 9 The reagents of O. H. Lowry and J. A. Lopez [J. Biol. Chem. 162, 421 (1946)] may be used in place of those of Fiske and SubbaRow, but the substitution offers no particular advantage; see also Vol. I I I [114]. lo The rate of hydrolysis of carbamyl phosphate is approximately the same from pH 1.5 to 9.0. In this range heating is necessary for rapid decomposition, whereas at strongly alkaline reaction decomposition occurs in a few minutes at room temperature. Therefore, the pH used for the hydrolysis is mainly dictated by the stability of any other phosphate compounds present in the sample. ~1Although carbamyl phosphate may be determined in the presence of the adenosine polyphosphates by hydrolysis with heat, the alkaline hydrolysis is more suitable. If heat is used, however, the TCA filtrate should be diluted to bring the molarity to 0.05, for under these conditions the adenosine polyphosphates are hydrolyzed only to the extent of 3 % in the l-minute heating.