Inorganic Phosphates and Phosphate Esters in Tissue Extracts BARBAKA L. GRISW-OLD, F. L. HUMOLLER, AND A. R. McINTYRE University of Nebraska, College of ,Fledicine, Omaha, .Veb. A method for the estimation of inorganic phosphate, phosphocreatine, and adenosine triphosphate in biological material is based in part upon the heteropoly blue method of Boltz and Mellon. It has greater sensitivity, convenience, and specificity than methods previously used for the estimation of inorganic phosphates and phosphate esters in biological material. Satisfactory determinations of inorganic phosphates, phosphocreatine, and adenosine triphosphate can be carried out in duplicate on as little as 300 mg. of tissue.
N
GMEROUS methods for the estimation of phosphates in
tissues and tissue fluids have been reported; the Fiske and Subbaron method ( 2 ) has gained perhaps the widest acceptance. This mtlthod, as well as modification8 thereof, depcuds upon the development of the blue color a t rrlatively low acidity and a t room temperature. .411 methods suffer from the disadvantages of a spontaneous development of color, w e n in the absence of phosphate, and of a progressive intensification of the blue color complex (4) In cases 1%here many determinations of inorganic phosphates and phosphate esirrs in the presence of each other are carried out routinely, these methods become impractical and it is highly desirable tu have available a method that is not subject to the limitatione of the Fiske and Subbarow method and its modifications. Such a method, if possible, should be more sensitive than prcvious methods-that is, i t should require smaller amounts of tissue or tissue fluids. Furthermore, the color produced should br proportional to the phosphate concentration over a wide range and once formed it should be stable for many hours, so that it could be estimated a t a convenient time. The method should be capable of determining phosphate esters in the presence of each other and of inorganic phosphates without the necessity of extrapolating the results to zero time of hydrolysis. This report describes a method that fulfills these requirements. I t has been used in this laboratory for hundreds of determinations of inorganic phosphates, creatine phosphate, and adenosine triphosphate in rat muscle. Obviously it can be employed for other tissues and tissue fluids with only minor changes in procedure.
The method used for obtaining tissue from rats under conditions that preclude more than an insignificant amount of hydrolysis of the phosphate esters during the process of removal has been described (3). The frozen and crushed tissue was treated with 20 ml. of ice-cold 5% trichloroacetic acid per gram of tissue, extracted for 15 minutes, and then filtered. PROCEDURE FOR INORGANIC PHOSPHATE DETERMINATION
For this estimation 1-ml. aliquots of the above filtrate are transferred to centrifuge tubes graduated a t the 10-ml. mark. The filtrate is neutralized with a drop of concentrated ammonium hydroxide and 1 ml. of magnesia mixture is added to each tube. After thorough mixing the tubes are allowed to stand overnight. The tubes are then centrifuged and the supernatant solution is carefully aspirated. The crystals of magnesium ammonium phosphate in the tubes are washed once with 2 ml. of dilute ammonia water and centrifuged, and the supernatant solution is aspirated and discarded. The crystalline residue in each tube is dissolved in 1 ml. of 0.2 N sulfuric acid and the contents are then diluted to 10 ml. with water.
IO0
APPARATUS AND REAGENTS
All transmittancy studies were made with a Beckman Model DU quartz spectrophotometer, using 1.000-cm. quartz cells. Water was used in the reference cell. Aminonaphtholsulfonic Acid. Prepare a sulfite-bisulfite solution by dissolving 59.5 grams of sodium bisulfite and 2.0 grams of anhydrous sodium sulfite in water and diluting to 250 ml. In this solution dissolve 1.0 gram of l-amino-2-naphthol-4-sulfonic :)rid (Eastman technical grade), purified as suggested b Fiske and Subbaroa (f2) and dilute to 1 liter. This solution will ieep for s t least 2 weeks if protected from light and air. Stock Sulfuric Acid Solution, 10 N. Dilute 280 ml. of concentrated sulfuric acid to 1 liter. Standardization is not necessary. >lake dilutions from this as required. Aqueous 2.5% Molybdate. Dissolve 25 grams of reagent grade ammonium molybdate tetrahydrate in water and dilute to 1 liter. Magnesia Mixture. Dissolve 17.5 grams of magnesium sulfate and 35.0 grams of ammonium chloride in 140 ml. of water and add i 8 ml. of concentrated ammonium hydroxide. Wash Ammonia Water. Dilute 50 ml. of reagent grade ammonium hydroxide to 1 liter. Acid Molybdate Reagent. Dissolve 50 grams of reagent grade ammonium molybdate in 10 N sulfuric acid and dilute to 500 ml. with 10 N acid.
WAVE LENGTH IN Figure 1.
m p
Effect of Molybdate Concentration on Absorption Spectrum
All solutions 0.5 Nin H&O', no added phosphorus. A m m o n i u y molybdate concentrations: 1,0.05%; 2,0.10%; 3.0.1596; 4,0.20%; a, 0.259'0
192
V O L U M E 23, N O . 1, J A N U A R Y 1 9 5 1 For color development, from 1 to 5 ml. of the above solution, drpendirig upon the expected phosphate content, are transferred to borosilicate glass test tubes, graduated for 10 ml. Next, 2 ml. of 5 sulfuric acid are added, followed by 1.0 ml. of aminonaphtliolsulfonic acid and 1.0 ml. of 2.5% aqueous molybdate and wati,] to the 10-ml. mark. After thorough mixing the tubes are placed in a large, vigorously boiling water bath for 10 minutes and then cooled to room temperature in a large bath of tap nTater. \\lien cool the volume is readjusted and the color intensity is nirasured a t 820 m,u. The color is stable for a t least 24 hours. By plotting the ohserved density against the concentration (or more conveniently by plotting the per cent transmittaricy iigainst concentration on semilogarithm paper) a standard curve will be obtained. From this chu1.t the phosphate content of the uiiknown samples can be obtained. PROC.EL)UELE FOR CREATINE PHOSI’€IhTE DETERMINATION
For this estimation 1.0 ml. of the original trichloroacetic acid filtrate (1 to 20) is transfrrred to centrifuge tubes graduated for 10 ml. Then 0.2 nil. of acid molphdate reagent is added and the hydrolysis is allowed to proceed for 1 hour a t room temperature. ‘I’he contents of the tubes are then neutralized to phenolphthalein with micentrated ammoniuni hydroxide.
I h m this point on, the procedure is identical n-it,h that used i n the estimation of inorganic phosphates and the same standard (‘urve ciin be used. Because in this estimation inorganic phospliute a* !vel1 as phosphates formed in the hydrolysis of phosphocreatine are determined, sm:illcr amounts of tissue extract a r e used for the color development.
193 tion for color development from 50 nil. to 10 ml. and by substituting thin-walled test tubes for volumetric flasks, the procedure was made more convenient for routinely carr,ying out large series of determinations. More important is the fact that the small volume of the solution quickly assumes the temperature of the water bath and thus the danger of incomplete hydrolysis of pyrophosphates is minimized. The color produced in the proposed method by using aminonaphthol sulfonic acid as reducing agent is stable for a t least 24 hours as compared with 12 hours found by Boltz and Mellon. The added color stability may be due to the fact that in this method the protective agent is driven off in the form of sulfur dioxide and hence a t the end of the heating period the reducing properties of the solution are diminished. Because absorption spectra of the heteropoly blue color may 1~ aflected by changes in reducing agent or conditions of reduction, i t became necessary to determine the absorption spectrum of the color complex of the proposed method. Within the range of the method, changes in phosphate concentration do not alter the absorption maxima of the blue complex. I n all cases a sharp ptbak a t 820 to 830 mG was observed. The specific absorbancy I 1 grsm/liter , was found to be 860. Known intiex, log om
i)l
(-
:mounts of phosphate were carried through the proposed nirthod for inorganic phosphates. The results obtained show that the blue complex follows Beer‘s law through the entire useiul range of phosphate concentration
I oc
PROCEDURE FOR ADEKOSINE TRIPHOSPHATE DE1’ER.MINATION 0 1 1 0 volume of the original tricliloroncetic acid (1 to 20) fikratc. is diluted with 19 volumes of water to make a total dilution of 1 to 400. Next 1.0-ml. quantities of this dilute filtrat,e are placed ill tvst tubes graduated for 10 ml. and the reagents for the devrlopmi~ritof color are added as before. The 10 minutes in the boiling witisr bath serve the dual purpose of hydrolyzing the pyrophos111i:itearid developing the color. Subtracting t,he value obtained for the apparent creatine phosphate from the value obtained i n this determination gives two thirds of the adenosine triphosphate l,ilosphorUs ( 6 ) .
75 >0 Z Q
t 50
DISCUSSION
Of considerable help in this investigation was the work of Holtz and >fellon ( I ) , who carried out a fundamental study of the reduction by hydrazine of molybdic acid and the heteromolybdic acids which are formed in the presence of phosphates, arsenates, silicates, and germanates. They made the important observation that in the presence of heat and 1 S sulfuric acid the heteropoly acids are readily reduced to a blue complex, whereas molybtiic acid is not affected in the least. When color development is c:trried out at a lower acidity (such as is specified in the Fiskr t u d Subbarow method) molybdic acid itself is readily reduced to a blue complex even in the absence of phosph:ttes, arsenates, etc. (Figure 1). The Boltz and Mellon method as published, however, 11:~s several disadvantages which make it, unsuitable for the estimation of inorganic phosphates and phosphate esters in biological material. The color is developed in a 50-mI. volumetric flask l)y heating in a boiling water bath. Aside from the inconvenience of handling a large number of such flasks, Roltz and Mellon found that a t the end of the 10-minute heating period, which they specify, the contents of the flasks had reached a temperature of only go”, which is inadequate for the hydrolysis of pyrophosphates. Fiske and Subbarow ( 2 ) have demonstrated the value of aminoriaphtholsulfonic acid as reducing agent in phosphate determinat ions involving biological material. It wis considered desirable, therefore, to replace hydrazine as a reducing agent by aminonnl)littiolsulfonic acid. By reducing the volume of the solu-
25
t ai 400 Figure 2.
600 800 WAVE LENGTH IN m
30 p
Effect of Molybdate Concentration on Absorption Spectrum
All solutions 1.0 N i n H2SO.r 0.3 p g. of phosphorus. Ammonium molybdate Concentrations: 1, O.d5%; 2, 0.10%; 3, 0.15%; 4, 0.20%; 5, 0.25%; 6, 0.30%; 7, 0.50%
Figure 1 shows the results obtained in a study in which the acid concentration was kept a t 0.5 A’ and the molybdate concentration was varied from 0.05 to 0.25’%, No unspecific color is formed a t this acid concentration, if the molybdate concentration is kept sufficiently low (curves 1 and 2). On the other hand, even a t 1 N acid concentration, nonspecific molybdenum blue is formed if the molybdate concentration is increased enough (Figure 2, curve 7). The concentrations of acid (1 Ar) and of
ANALYTICAL CHEMISTRY
194 molybdate (0.25%) of the proposed method are such that neither is critical. SUMMARY
A spectrophotometric method for the estimation of phosphate fractions has been developed which takes advantage of the desirable features of the Fiske and Subbarow method (use of aminonaphtholsulfonic acid as reducing agent) and of the Boltz and Mellon method (development of color by heat in 1 N sulfuric acid solution). The advantages of the proposed method are greater sensitivity, greater convenience, and greater specificity over previousmethods. The method is particularly suited for the routine estimation of inorganic phosphate, phosphocreatine, and adenosine triphosphate in biological material (3).
LITERATURE CITED
(1) Boltz, D. F., and Mellon, M. G., ANAL.CHEW,19, 873 (1947). (2) Fiske. C. H., and Subbarow,Y . ,J . Biol. Chem., 66, 375 (1925). (3) Humoller, F. L., Griswold, B., and McIntyre, A. R., Am. J . Phglsiol., 161, 406 (1950). (4) Lowry, 0. H., and Lopez, J. A., J . Biol. Chem., 162,421 (1946). ( 5 ) Potter, V. R., in Umbreit.
W.IT., Burris, R. H., and Stauffer, J. F., "Manometric Techniques and Related Methods for the Study of Tissue Metabolism." Minneapolis,Burgess Publishing
Co.. 1945. RECEIVED N a y 1, 1950. Presented before the Division of Biological Chemistry at the 117th Meeting of the AMERICAK CHEMICAL SOCIETY, Philadelphia, Pa. Aided by a grant from the National Foundation for Infantile Paralysis.
Separation and Identification of 2,4=Dinitrophenylhydrazones of Aldehydes and Ketones, and 3,5=Dinitrobenzoates of Alcohols by Filter-Paper Chromatography RANDALL G . RICE, GEORGE J. KELLER, AND JUSTUS G. KIRCHNER Fruit and Vegetable Chemistry Laboratory, U . S. Department of Agriculture, Pasadena, Calg. T H E course of a study on the chemistry of the volatile IandNconstituents in orange juice, it became necessary to separate identify less than milligram quantities of mixtures of 2,4dinitrophenylhydrazones of aldehydes and ketones, and mixtures of the 3,5-dinitrobenzoates of alcohols. The procedures herein outlined require only microgram quantities of the mixtures and involve very little time for an analysis. 2.4-DINITROPHENYLHYDRAZONES
The filtei-paper partition chromatography of 2,4-dinitrophenylhydrazones of keto acids has been published (3, 4),and subsequent investigation has demonstrated that filter-paper chromatography can be used to effect the separation and identification of a number of other 2,4-dinitrophenylhydrazones. Analytical Procedure. The capillary-ascent test tube method of filter-paper chromatography for separating microgram quantities of amino acids ( 7 ) has been adopted with a feK minor variations. The method of application of the 2,4-dinitrophenylhydrazones and the use of filter paper are the same as described for amino acids (8), except that the compounds are dissolved in a more suitable solvent such as chloroform, and no drying is necessary either before or after development of the chromatogram. Furthermore, it has been found necessary to place a second pencil dot approximately 10 mm. from the top of the paper strips before the chromatogram is developed, because the clear, colorless developing solvents volatilize so rapidly that the solvent boundary cannot be marked after the strip has been removed from the test tube. I n addition, it has been found necessary to place a light source behind the tubes to facilitate the observation of the solvent boundary ascent. Immediately after the solvent boundary has reached the second pencil dot, the paper strip is removed from the tube, suspended, and sprayed with a lOy0 solution of potassium hydroxide in water. While still wet, the strips are placed upon a white background and the colored areas, resulting from the reaction of potassium hydroxide with the respective 2,4-dihydrophenylhydrazones,can be outlined with a pencil.
The careful spraying of the paper strips is very important if resolution is to be observed between certain compounds. The use of a conventional atomizer as a spraying device for this purpose has proved to be unsatisfactory in some instances, owing to erratic, excessive, and uncontrollable amounts of heterogeneous spray. This condition causes otherwise carefully defined spots to lack definition, to diffuse, and to run together. An artist's airbrush produces a more consistent and homogeneous spray, which also can be regulated easily to produce any desired amount and shape of spray. By the use of an artist's airbrush, resolution between some compounds has been observed, whereas, on comparable paper strips which were sprayed conventionally, no resolution was indicated.
Table I. Typical Rj Values of Some 2,4-Dinitrophenylhydrazones Carbonyl Compound 2,4-Dinitrophenylhydrazine) alicylaldehyde Cinnamaldehyde Propionaldehyde Furfural Benzaldehyde Formaldehyde 2-Hexenal Acetone Isovaleraldehyde n-Butyraldehyde Decyl aldehyde Nonyl aldehyde Methyl ethyl ketone Methyl propyl ketone Methyl isopropyl ketone Acetaldehyde Glycol aldehyde Biacetyl Acetylmethylcarbinol
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