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840
C O E N Z Y M E SAND RELATED PHOSPHATE COMPOUNDS
[115]
pH of the effluent is not so low as with HC1. One difficulty in these procedures is that the substances emerge from the column in very dilute solution. Concentration has been effected by neutralizing, passing through a smaller column, and then displacing with more concentrated acid. In this manner the substance can be obtained in a small volume and may even crystallize in the column. Another method is to adsorb the substance on charcoal and then elute with aqueous ethanol or acetone-containing ammonia. A useful method for the separation of sugar esters and probably other compounds consists in using the anion exchange columns in the borate form and displacing with a borate solution. The polyhydroxy compounds form complexes with boric acid which have different acid strength and a different number of boric acid residues according to the number and position of the hydroxyl groups. This gives further possibilities of separation with the anion exchange columns3 4
Adsorbents Activated charcoal (Norit, Nuchar, etc.) adsorbs most of the nucleotide coenzymes. It is usually used batchwise, and elution is carried out with aqueous alcohol or acetone. If the substance is not eluted, ammonia may be added to these solvents. Aqueous pyridine is also a good eluant. Activated charcoal has also been used for analytical purposes in the separation of nucleotides from other compounds such as inorganic phosphate, sugar phosphates, and acetyl phosphate, which are not adsorbed3 5 ~4 j. X. Khym and W. E. Cohn, J. Am. Chem. Soc. 75, 1153 (1953). ~5 R. K. Crane and F. Lipmann, J. Biol. Chem. 201, 235 (1953).
[115] Characterization of Phosphorus Compounds by Acid Lability By L m s F. LELOIR and CARLOS E. CARDINI Measurements of the rate of hydrolysis of phosphoric esters have been carried out for analyzing mixtures, as a test of homogeneity, and as a criterion of identity. The application of this procedure became general after Lohmann 1used it for distinguishing the 6-phosphate of glucose from that of fructose. The rate of hydrolysis of phosphoric esters in acid solutions depends on several factors. The esterification of alcohol groups yields esters which are hydrolyzed with difficulty. This is the case with the a- and f~-glyceroi K. Lohmann, Biochem. Z. 194, 306 (1928).
[115]
ACID LABILITY OF PHOSPHORUS COMPOUNDS
841
phosphates, 3-phosphoglyceric acid, and phosphorylcholine. In some cases the phosphate group appears to be released not by simple hydrolysis but by decomposition of the rest of the molecule. Thus triose phosphates yield methylglyoxal instead of the corresponding triose. 2 Migration of the phosphate group under the influence of acids has been detected in several instances. Such is the case with the a- and B-glycerophosphoric acids 3 and with the 2- and 3-phosphates of purine or pyrimidine ribosides. Among the sugar phosphates the most stable are the 6-phosphoaldoses. The pentose phosphates are less stable and are affected by the nature of the substituents. Thus, although purine ribosides hydrolyze at the same rate as ribose phosphate, the pyrimidine phosphates are more stable. The phosphate groups at the hemiacetal OH group of sugars are all acid-labile, and in the case of ribose and deoxyribose they are still more labile. If the OH at position 2 is substituted by an amino group, the stability is increased. For instance, galactosamine-l-phosphate is much more stable than galactose-l-phosphate. 4 This is also the case for the corresponding glucose derivatives. 5 The stabilizing effect of the amino group occurs not only with the phosphates but is general for hexosaminides as compared with the glycosides, e When the phosphate is bound to a carboxyl or amino group, the compounds are very labile, as for instance acetyl phosphate and phosphocreatine. The influence of the concentration of acid has not been studied adequately. Probably the rate of hydrolysis is proportional to the H + concentration for most compounds. However, this is not the case for glucose6-phosphate, which hydrolyzes at about the same rate at pH 2 and in 1 N solution of acid. 7Another exception is ethanolamine phosphate, which has the maximum rate of hydrolysis at pH 4.5, 8 whereas the rate in 1 N acid is one-third as great. It has become usual to separate the organic phosphates into two main groups. Those which are hydrolyzed completely in 1 N acid at 100° during 7 minutes are usually called labile. Those which are not hydrolyzed under the same conditions are called stable. The conditions were selected originally for estimating the two terminal phosphate groups in ATP. Another group of compounds estimate like inorganic phosphate in the 2 O. Meyerhof and K. Lohmann, Biochem. Z. 271, 89 (1934). a E. Baer and M. Kates, J. Biol. Chem. 175, 79 (1940). 4 C. E. Cardini and L. F. Leloir, Arch. Biochem. and Biophys. 45, 55 (1953). 5D. H. Brown, J. Biol. Chem. 204, 877 (1953). 6 M. Viscontini and J. Meier, Helv. Chim. Aria 86, 807 (1952). R. Robison, Biochem. J. 26, 2191 (1932). s E. Cherbuliez and M. Bouvier, Helv. Chim. Acta $6, 1200 (1953).
842
COENZYMESAND RELATED PHOSPHATE COMPOUNDS
[115]
usual Fiske and SubbaRow procedure and may be called the extralabile compounds. The separation in these three groups is arbitrary, and the properties of many compounds are intermediate. However, the classification is useful. Therefore, organic phosphates may be grouped as follows. Extra-labile. Phosphocreatine, 1,3-diphosphoglyceric acid, ribose-1phosphate, deoxyribose-l-phosphate, acyl phosphates. Labile. 9 Adenosinetriphosphate (67% hydrolyzed), adenosinediphosphate (50%), uridine diphosphate (35%), aldose-l-phosphates (100%), fructose-l-phosphate (70%), fructose-l,6-diphosphate (32%), glucuronic acid 1-phosphate (100 %), inorganic pyrophosphate (100 %). Stable. Phosphopyruvic acid (40%), hexose-6-phosphate, pentose-3and 5-phosphates and the corresponding mono- and dinucleotides, 6-phosphogluconic acid, glycerol and glyceric acid phosphates, inositol phosphates, phosphorylcholine, and phosphorylethanolamine. More information on the hydrolysis of different compounds may be obtained by an examination of the table. Some of the compounds listed as labile are hydrolyzed completely under milder conditions. For instance, for aldose-l-phosphates it is sufficient to heat for a few minutes at 100° in 0.1 N acid. Other compounds such as uridine diphosphate need 30 minutes in 1 N acid at 100 ° to reach complete hydrolysis of the labile phosphate. Phosphopyruvic acid is intermediate between stable and labile. Liberation of the Phosphate Group by Methods Other Than Acid Hydrolysis Methods which are more or less specific for removing the phosphate group in certain compounds are as follows: Phosphopyruvate may be estimated by a method based on the liberation of phosphate by hypoiodite ~° or by mercuric ions.~° Dihydroxyacetone phosphate and glyceraldehyde phosphate lose their phosphate on standing at room temperature in 1 N alkali during 20 minutes. 11Fructose-l-phosphate, fructose-l,6-diphosphate, and glucose-2-phosphate may be estimated by a method based on the liberation of the phosphate by heating with phenylhydrazine. 12,~3 Alkaline hydrolysis leads to the liberation of phosphate from the sugar esters with a free reducing group; the 1-phosphates remain unaffected. 9The numbers in parentheses represent the amount of phosphate hydrolyzed in 7 minutes at 100° in 1 N acid. 10K. Lohmann and O. Meyerhof, Biochem. Z. 278, 60 (1934). ~ K. Lohmann and O. Meyerhof, Biochem. Z. 278, 413 (1934). 12H. J. Deutieke and S. Hollmann, Z. physiol. Chem. 258, 160 (1939). ~3A. C. Paladini and L. F. Leloir, Biochem. J. §1~ 426 (1952).
[116]
843
ACID LABILITY OF PHOSPHORUS COMPOUNDS s.,
Method for the Estimation of Phosphate
Of the many methods available for the estimation of phosphate, that of Fiske and SubbaRow is one of the simplest and most widely used. In certain cases, however, it is convenient to use procedures in which the phosphomolybdate complex is extracted with an organic solvent such as isobutanol, 14,15 thus avoiding the interference due to colored substances, citrates, oxalates, buffers, etc. In the Fiske and SubbaRow procedure the extra-labile compounds are estimated as inorganic phosphate because of the relatively high acid concentration (pH 0.65) and because molybdate accelerates the hydrolysis of some organic phosphates. 15-17 By measuring the color immediately after adding the reagents, however, Fiske and SubbaRow 18 were able to estimate phosphocreatine. Better results are obtained by estimating the 'true' inorganic phosphate by precipitating it with magnesium mixture or with calcium salts and ethanol. In the Lowry and Lbpez method 19 the acid and molybdate concentrations are lower than in Fiske and SubbaRow's, so that some extra-labile compounds are hydrolyzed more slowly. However, certain compounds such as deoxyribose-l-phosphate are hydrolyzed even under these conditions. Method of Fiske and SubbaRow 2°
Reagents 5 N sulfuric acid. 2.5 % ammonium molybdate. 2 N nitric acid. Reducing reagent. This may be prepared in the powdered form and dissolved before use. The solution deteriorates slowly and should not be used after more than a week. The powdered reagent is prepared by mixing thoroughly 0.2 g. of 1-amino-2-naphthol-4sulfonic acid with 1.2 g. of sodium bisulfite and 1.2 g. of sodium sulfite. For use 0.25 g. is measured with a small spoon and dissolved in 10 ml. of water. Standard solution. 1.3613 g. of analytically pure KH~P04 is dissolved in 1000 ml. of water, a few drops of chloroform are added, 14 I. Berenblum and E. Chain, Biochem. J. 32, 295 (1938). 15 H. Weil-Malherbe and R. H. Green, Biochem. J. 49, 286 (1951). le F. Lipmann, J. Biol. Chem. 153, 571 (1944). 17 H. M. Kalckar, J. Biol. Chem. 167, 477 (1947). 18 C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 81, 629 (1929). 19 O. H. Lowry and J. A. LSpez, J. Biol. Chem. 162, 421 (1946). ~0 C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925).
844
COENZYMES AND RELATED PHOSPHATE COMPOUNDS
[115]
and the solution is stored in the refrigerator. For use it is diluted 1:10, so that 1 ml. corresponds to 1 micromole of phosphorus.
Deproteinization. The usual procedure is to use trichloroacetic acid (final concentration, 5 to 10%) or perchloric acid (final concentration, 8%). :~ Some extra-labile compounds such as acetyl phosphate are most stable at pH 5 to 6 and are not appreciably hydrolyzed if the trichloroacetic acid solution is added cold and if the procedure is carried out rapidly. Procedure. The standard and unknowns should contain from 0.1 to 1 micromole of phosphate. One milliliter of sulfuric acid is added, followed by 1 ml. of molybdate. After mixing, 0.1 ml. of reducing solution is added. The volume is made up to 10 ml. After mixing again, the absorbency at 660 mu is measured after 10 minutes. When phosphocreatine is determined, the sample is left standing with the acid and molybdate for 20 minutes before the reducing reagent is added. All the phosphocreatine is hydrolyzed under these conditions. It is important to avoid contamination with silicates which, like phosphate, give a blue color in the Fiske and SubbaRow procedure. Silicates are usually present in alkaline reagents stored in soft glass containers and are released by glass homogenizers. Arsenate also estimates like phosphate, and procedures have been devised in order to avoid its interference. 22 Estimation of Labile Phosphate. After the sulfuric acid is added in the Fiske and SubbaRow procedure, water is added to complete the 5 ml., that is, to make the concentration 1 N. The tubes are heated in a boiling water bath, usually for 7 minutes, cooled, and then the procedure is continued as described previously by adding the molybdate, etc. Estimation of Total Phosphate. One milliliter of 5 N sulfuric acid is added to the sample as described in the Fiske and SubbaRow procedure. The mixture is evaporated in the test tube over a free flame. When the contents become brown and have cooled, 1 drop of 2 N nitric acid is added and the heating is continued until white fumes appear. If the liquid does not become colorless, the addition of nitric acid is repeated. Excess nitric acid interferes with the subsequent color development. After cooling, about 1 ml. of water is added and the tube is placed in a boiling water bath for 5 minutes. After cooling, molybdate is added, etc., as described in the inorganic phosphorus procedure. ~ C. Neuberg, E. Strauss a n d L. E. Lipkin, Arch. Biochem. 4, 101 (1944). 23 L. B. Pett, Biochem. J. 27, 1672 (1933).
[115]
ACID L A B I L I T Y OF PHOSPHORUS
COMPOUNDS
845
Method of Lowry and LSpez 19
Reagents Ascorbic acid, 1 g. in 100 ml. of water. Ammonium molybdate, 1 g. in 100 ml. of 0.05 N sulfuric acid. Acetate buffer, pH 4. 0.1 N in acetic acid and 0.025 N in sodium acetate. 0.1 N sodium acetate. Standard solution. The same as that of the Fiske and SubbaRow method may be used. Deproteinizing agent. (1) 5% trichloroacetie acid (0.3 N), (2) 3% perchloric acid (0.3 N) (3) saturated ammonium sulfate which is 0.1 N in acetic acid and 0.025 N in sodium acetate (pH 4).
Procedure. The sample to be analyzed is deproteinized under conditions which will not hydrolyze the particular ester; e.g., ice-cold 0.3 N trichloroacetic acid or 0.3 N perchloric acid, or, particularly with very labile esters, saturated ammonium sulfate with acetic acid and sodium acetate (pH 4). If either of the acid precipitants is used, the extracts are rapidly brought to pH 4 to 4.2 by adding 4 vol. of 0.1 N sodium acetate. Most of the labile esters are reasonably stable at this pH. The extracts are diluted with acetate buffer of pH 4 until the inorganic phosphorus is 0.015 to 0.1 raM. (0.015 to 0.10 # moles/ml.). Ammonium sulfate extracts should be diluted at least fivefold. To each volume of extract is added 0.1 vol. of 1% ascorbic acid and 0.1 vol. of 1% ammonium molybdate in 0.05 N sulfuric acid. Readings are made at 5 minutes and again at 10 minutes after the molybdate addition at a wavelength of 700 m~. (Any wavelength between 650 and 950 m~ is satisfactory.) Simultaneous readings are made on a standard (0.05 ~ moles of phosphorus per milliliter) and a blank, both of the same composition, as far as possible, as the unknown. If a difference is observed in the readings of the unknown at 5 and at 10 minutes compared to the standard, the values are extrapolated to zero time. The ascorbic acid and molybdate may be combined before addition but must then be used within 15 minutes. In the presence of certain tissue extracts, the reaction is delayed, in which case an internal standard must be used. A standard amount of inorganic phosphate is added to a duplicate tissue aliquot, and values of the unknown are calculated from the difference between the readings of the unknown and of the unknown with added phosphate. The inhibitory effect may be partially overcome by dilution. For example, in order to avoid undue inhibition, brain and muscle extracts should be diluted to a
846
COENZYMES AND RELATED PHOSPHATE COMPOUNDS
[115]
volume 150 to 250 times that of the tissue, and liver extracts to a volume 300 to 500 times that of the original liver. In addition, the molybdate concentration may be increased to 0.15%; i.e., 0.1 vol. of 1.5% ammonium molybdate in 0.05 N sulfuric acid is added in place of the 1% solution. This results in an acceleration of color development. In studying isolated enzyme systems, this problem of inhibition is ordinarily not encountered. The ascorbic acid concentration may be increased, if necessary, to accelerate the reaction, but with final concentrations of greater than 0.2 to 0.3% the readings of the standard increase unduly with time. The final pH may be varied, if desired, between 3.5 and 4.2. Removal of Inorganic Phosphate. 1. CALCIUM HYDROXIDE.TM To the neutralized sample add 0.2 vol. of 10% CaC12 saturated with Ca(OH)~. Let stand for 10 minutes at room temperature, centrifuge, and wash the precipitate with a small volume of water containing the CaCl~ reagent. The washed precipitate may be dissolved in dilute HC1 for the estimation of inorganic phosphate. 2. CALCIUM CHLORIDE AND ALCOHOL. This method has been used by Lipmann and Turtle 23 for the separation of inorganic phosphate from acetyl phosphate.
Reagents 3.3 % solution of anhydrous CaCl~ in 33 % ethanol. Neutralization solution. A mixture of 100 ml. of concentrated ammonia and 40 ml. of glacial acetic acid is made up to 1 1., and to that 100 ml. of 0.4 M bicarbonate solution is added.
Procedure. Trichloroacetie acid extract (0.5 ml.) containing about 1 to 4 micromoles is transferred to a chilled test tube containing a drop of thymol blue, and the neutralization mixture is added quickly, with local alkalinization carefully avoided, until pH 8 is reached (grayish blue color). To the neutralized sample 2.5 ml. of alcoholic CaC12 solution is added. When the material is mixed, the color of the indicator usually turns more yellowish but should retain a bluish tinge. In order to make the precipitate bulkier, especially if only small amounts of phosphorus are present, 0.15 ml. of 0.04 M bicarbonate solution is added dropwise. The calcium precipitate (phosphate plus carbonate) is quickly centrifuged off; 1 to 2 minutes is sufficient. The supernatant is decanted carefully, the adhering fluid is washed off with 2 ml. of the alcoholic calcium chloride solution, and the precipitate is recentrifuged without stirring. The calcium precipitate is dissolved in 0.5 ml. of 0.5 N hydrochloric acid and quantitatively brought into a volumetric flask of convenient size. Phosphorus is determined by the procedure of Fiske and SubbaRow. 23 F. L i p m a n n a n d L. C. Tuttle, J. Biol. Chem. 158, 571 (1944).
[115]
ACID LABILITY OF PHOSPHORUS COMPOUNDS
847
OBSERVED HYDROLYSIS CONSTANTS OF SOME PHOSPHATES~
Compound
Normality Temperature of acid °C.
t~, min.
K X 103
Reference
A ldose-l-phosphates Deoxyribose-l-phosphate (pH 4) Ribose-l-phosphate Xylose-l-phosphate a-D-Glucose-l-phosphate a-D-Glucose-l-phosphate a-D-Glucose-l-phosphate a-D-Glucose-l-phosphate a-Glucose-l-phosphate 3-D-Glucose-l-phosphate a-Galactose-l-phosphate a-Galactose-l-phosphate a-Galactose-l-phosphate ¢l-Galactose-l-phosphate a-Mannose-l-phosphate Galactosamine-l-phosphate Glucosamine-l-phosphate Maltose- 1-phosphate
0.5 0.1 0.1 0.25 1 0.95 1 1 0.25 0.25 0.1 0.25 0.95
25 25 36 36 37 33 30 100 33 25 37 100 37 30
I2 25 2.5 120 111 2.7 158 1.9 230 1.30 60 5.00 200 1.15 1.05 200 20 15 333 0.90 50 5.9 ~2.1 ~-~140 53 5.6 360 0.82
1 1 0.1
100 100 36
4.1 ~--4 214
73.7 ~75.0 1.4
b c d d e I g h g
i k g i d
Other Sugar Phosphates Triose phosphates Erythrulose-l-phosphate Deoxyribose-5-phosphate Ribose-3-phosphate Ribose-3-phosphate Ribose-5-phosphate Ribose-5-phosphate Xylose-5-phosphate Ribulose-5-phosphate D-Glucose-2-phosphate Glucose-6-phosphate Glucose-6-phosphate 6-Phosphogluconic acid Fructose- 1-phosphate Fructose- 1-phosphate Fructose-6-phosphate Mannose-6-phosphate Mannose-6-phosphate 6-Phosphomannonic acid 6 - P h o s p h o m a n n o n i c acid ~-lactone L-Sorbose-6-phosphate Ketoheptose monophosphate
1 1 1 0.01 0.25 0.01 0.25 1 0.1 0:1 1 1 1 0.1 1 0.1 1 1
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
37 ~-~9.7 56 ~1.7 ~'~4.5 ~0.3 ~'~0.5 3.3 ~5 2.18 0.13 0.22 0.26-0.15 2.8 70 33 9 7O 4.36 2300 0.13 1034 0.29 0.199-0.131
1 1
100 100
2500 62
1
100
75
1
8.1 30 6.2 180 66 1000 600 90 60 136 2300 1300
° q P q *

0.12 4.8
' Y
4
h
848
[116]
COENZYMES AND RELATED PHOSPHATE COMPOUNDS
(Continued)
OBSERVED HYDROLYSIS CONSTANTS OF SOME PHOSPHATESa
Compound a-Glucose- 1,6-diphosphate a-Glucose-l,6-diphosphate t~-Glucose-l,6-diphosphate a-Mannose-l,6-diphosphate Fructose-l,6-diphosphate
Normality Temperature, of acid °C.
t~4, rain.
K X 10 s
Reference
0.25
37
967
(a) 0.31
1
30
900
(a) 0.33
~
1
3U
220
(a) I. 37
'
30 100
1420 5.7
0.21 (a) 52 (b) 4 . 2
bb
0.95 1
o
Other Phosphate Esters a-Glycerophosphate ~-Glycerophosphate ~-Glycerophosphate 3-Phosphoglyceric acid Phosphorylcholine Phosphorylcholine Aminoethyl phosphoric acid Aminoethyl phosphoric acid Propanediol p h o s p h a t e
1 2.14 2. 035 1 1 2
100 127 124 100 100 124
3300 300 300 2140 2300 1870
0.09 0.98 0.97 0.14 0.13 0.16
h cc c' c~ ¢'
1
1'00
~940
N0.32
dd
4.5 5
100 100
300 540
1.0 0.55
dd ~'
P h o s p h o p y r u v i c acid
1
Enol Phosphate 100
8.3
36
h
1.2 11
250 27.8
h II
350
Ir
Acid Anhydrides Pyrophosphoric acid Acetyl p h o s p h a t e Acetyl p h o s p h a t e -Imolybdate 1,3-Diphosphoglyeerie acid
1 0.5
100 40
0.5
25
Neuter
38
0.86 27
11
g~
2.1 75
ah °
N0.4 ~-~0.5 N5 N1.60 0.085 0.26 0.26 38 ~'~15 ~'0.38
~ ~i i~ ' ii ' ~i kk k* u
Amide Phosphates Phosphoereatine Phosphocreatine
1.0 0.5
Uridine-3'-phosphate Cytidine-3'-phosphate Adenosine-3'-phosphate Guanosine-3ophosphate Uridine-5'-phosphate Adenosine-5'-phosphate Guanosine-5'-phosphate ATP UDPG DPN
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
20 25
150 4
Nucleotides 100 100 100 100 100 100 100 100 100 100
720 600 60 180 3590 1050 1050 8 20 780
[116]
ACID LABILITY OF PHOSPHORUS COMPOUNDS
849
FOOTNOTES TO TABLE ON PP. 847-848 I a a The constants are calculated with the formula K = ~- log~0 a---~k-k' or more usually I t~ log10 aa --- x~ K 12 -x~ The time is in minutes, and a is the initial concentration of the substance. The time for 50% hydrolysis, t~i = 0.30/K; the time for 98% hydrolysis, t = 1.7/K. M. Friedkin, H. M. Kalckar, and E. Hoff-JCrgensen, J. Biol. Chem. 178, 527 (1949). ° H. M. Kalckar, J. Biol. Chem. 167, 477 (1947). d W. R. Meagher and W. Z. Hassid, J. Am. Chem. Soc. 68, 2135 (1946). ' C. F. Cori, S. P. Colowick, and G. T. Cori, J. Biol. Chem. 121, 465 (1937). / M. L. Wolfrom, C. S~ Smith, D. E. Pletcher, and A. E. Brown, J. Am. Chem. Soc. 64, 23 (1942). g T. Posternak and J. P. Rosselet, Helv. Chim. Acta 36, 1614 (1953). h R. Robison and M. G. MacFarlane, in ' M e t h o d e n der Fermenforschung' (Bamann and Myrb~ck, eds.), Vol. 1, p. 296. G. Thieme, Leipzig, 1941. i H. W. Kosterlitz, Biochem. J. 38, 1087 (1939). i C. E. Cardini and L. F. Leloir, Arch. Biochem. and Biophys. 45, 55 (1953). k F. J. Reithel, J. Am. Chem. Soc. 67, 1056 (1945). D. H. Brown, J. Biol. Chem. 204, 877 (1953). O. Meyerhof and K. Lohmann, Biochem. Z. 271, 79 (1934). n F. C. Charalampous and G. C. Mueller, J. Biol. Chem. 201, 161 (1953). ° E. Racker, J. Biol. Chem. 196, 347 (1952). p P. A. Levene and E. T. Stiller, J. Biol. Chem. 104, 299 (1934). q H. G. Albaum and W. W. Umbreit, J. Biol. Chem. 167, 369 (1947). r p. A. Levene and A. L. Raymond, J. Biol. Chem. 102, 347 (1933). 'B. L. Horecker, P. Z. Smyrniotis, and J. E. Seegmiller, J. Biol. Chem. 193, 353 (1951). t K. R. Farrar, J. Chem. Soc. 1949, 3131. R. Robison and E. J. King, Biochem. J. 25, 323 (1931). R. Robinson, Biochem. J. 26, 2191 (1932). V. R. Patwardhan, Biochem. J. 28, 1854 (1934). B. Tanko and R. Robison, Biochem. J. 29, 961 (1935). v K. M. Mann and H. A. Lardy, J. Biol. Chem. 187, 339 (1950). ' C. E. Cardini, A. C. Paladini, R. Caputto, L. F. Leloir, and R. E. Trucco, Arch. Biochem. 22, 87 (1949). ' T. Posternak, J. Biol. Chem. 180, 1269 (1949). bb M. MacLeod and R. Robison, Biochem. J. 27, 286 (1933). ~¢O. Meyerhof and W. Kiessling, Biochem. Z. 264, 40 (1933). d~ E. Cherbuliez and M. Bouvier, Helv. Chim. Acta 36, 1200 (1953). ' O. N. Miller, C. G. Huggins, and K. Arai, J. Biol. Chem. 202, 263 (1953). / / F . Lipmann and L. C. Tuttle, J. Biol. Chem. 153, 571 (1944). gg E. Negelein and H. BrSmel, Bioehem. Z. 303, 132 (1939). hh K. Lohmann, Biochem. Z. 194, 306 (1928). ii A. M. Michelson and A. R. Todd, J. Chem. Soc. 1949, 2476. ii p. A. Levene and R. S. Tipson, J. Biol. Chem. 106, 113 (1934). kk R. Caputto, L. F. Leloir, C. E. Cardini, and A. C. Paladini, J. Biol. Chem. 18~ 333 (1950). u F. Schlenk, J. Biol. Chem. 146~ 619 (1942).
850
COENZYMESAND RELATED PHOSPHATE COMPOUNDS
[116]
3. MAGNESIA MIXTURE
Reagent. Dissolve 5.5 g. of MgC12.6H20 and 10 g. of NH4C1 in about 50 ml. of water. Add 10 ml. of 15 M ammonia and make up to 100 ml. Filter if necessary. One milliliter of the solution corresponds theoretically to 270 micromoles of phosphoric acid. Procedure. The sample is neutralized with 10% ammonia and excess magnesia mixture is added. The solution should be about 1.5 M with respect to NH4OH. The solution is stored for 2 to 3 hours in the refrigerator. The filtrate can then be neutralized with HCI and used for the estimation of extra-labile esters.
[116] Determination and Preparation of N-Phosphates of Biological Origin By A. H. ENNOR I. Phosphocreafine
/
CH3 N'CH2.COOH
C= N H

NPOsH2 H
Determination Because of the ease with which the N-P bond may be broken in acid solution, the methods generally employed for the determination of phosphocreatine (PC) depend on the estimation of the P moiety. Thus a commonly employed technique is that described by LePage, 1 which depends on the determination of the P~ by the method of Fiske and SubbaRow 2 after precipitation as a Ca ++ salt from alkaline solution. This figure is then subtracted from that obtained by a direct estimation of the P~ in the extract, since this latter is assumed to represent the sum of P~ and PC-P because of the lability of the PC in the H~SO4-ammonium molybdate reagent. This method lacks the sensitivity desirable if low concentrations of PC are present and has the further disadvantage that its accuracy depends on the quantitative precipitation of P~. as the Ca salt i G. A. LePage in ' M a n o m e t r i c Techniques and Related Methods for the Study of Tissue Metabolism' (Umbreit, Burris, and Stauffer, eds.), p. 185. Burgess Publishing Co., Minneapolis, 1951. C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925).
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The Electric Kool Aid Acid

Molecular test, nucleic acid; blood test, serology test: amplification tests (NAAT), RT-PCR tests. Download the form or call 1-800-332-1088 to request a form, then complete and return to. 1% Acid alcohol until light pink and color stops running. Wash in running tap water for 5 minutes. Rinse in distilled water. Working methylene blue for 30 seconds. Dehydrate, clear, and coverslip.Conventional Method: 60°C oven for 1 hour. RESULTS: Acid-fast bacilli bright red Background blue. The mixture is evaporated in the test tube over a free flame. When the contents become brown and have cooled, 1 drop of 2 N nitric acid is added and the heating is continued until white fumes appear. If the liquid does not become colorless, the addition of nitric acid is repeated. Excess nitric acid interferes with the subsequent color development.