Barlett - Quantidade de Fosfatos

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Grant R. Bartlett

Phosphorylated Glyceric AcidsColorimetric Assay Methods for Free andARTICLE:

1959, 234:469-471.J. Biol. Chem.

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Calorimetric Assay Methods for Free andPhosphorylated Glyceric Acids*

GRANT R. BARTLETT

From the Scripps Clinic and ResearchFoundation La Jolla California(Received for publication, August 4, 1958)

Eegriwe (1) introduced 1,3-dihydroxynaphthalene (naphtho-resorcinol) as a spot test reagent for glycerate, and Rapoport (2)adapted the reaction fo r quantitative assay. In the presentstudy it was found that, in addition to the naphthoresorcinol,2,7-dihydroxynaphthalene and 4,5-dihydroxy-2,7-naphthalene-disulfonic acid (chromotropic acid) when heated with free orphosphorylated glycerates in concentrated sulfuric acid, produceddistinct ive colors which could be used to advantage for theidentification of these compounds in column chromatographiceluates. The chromotropic acid reaction displayed the highestsensi tivi ty and selectiv ity and was preferentially used for thequantitative analysis of glycerates.

EXPERIMENTAL2,7-Dihydroxynaphthalene was recrystallized from water, and

naphthoresorcinol was purified according to Meyer and Bloch(3); both were Matheson, Coleman, and Bell chemicals. Chro-motropic acid (Eastman Organic Chemicals) was precipitatedfrom a saturated aqueous solution with 4 volumes of ethanol.MPG (Schwarz Laboratories), 3- and 2-PG (Biochemica Boeh-ringer), 2-PG (gif t of Dr. C. Ballou), and DPG from red cellswere purified by ion exchange chromatography on Dowex 1resin. Glyceric acid was prepared by the hydrolysis with in-testinal phosphatase (Armour) of a sample of purified DPGuniformly labeled with CY, which had been isolated after incu-bation of human red cells with uniformly labeled glucose-W.The glyceric acid-V, after purification on Dowex 1, was phos-phorus- and nitrogen-free and contained 3.3 pmoles per ml. byradioact ivity (based on the original DPG specific act ivi ty) and3.4 pmoles per ml. by periodate analysis.

Each of the three reactions of glycerates with the hydroxy-naphthalene derivatives was carried out under the same condi-tions except for the time o f heating. A volume of 0.2 ml. ofsolution containing glycerate was mixed with 5.8 ml. of freshlyprepared 0.01 per cent hydroxynaphthalene in concentrated sul-fur ic acid, and the mixture was heated at 100 in a water bath.In the naphthoresorcinol and 2,7-dihydroxynaphthalene reac-tions, glyceric acid, MPG, and DPG were heated for 30, 60, and120 minutes, respect ively. In the chromotropic acid test , both

* Supported in part by Grant No. H-1071, Nationa l Institute sof Health, United States Public Health Service.

1 On accoun t of the very large number of data presented in thispaper, the Editors have permitted the use of a set of highly con-densed abbreviations which are not ordinarily employed in theJournal, accordin g to its present rules.

The abbreviations used are: MPG, monoph osphoglyceric acid;2- and 3-PG, 2 and 3 monop hosphoglyceric acid; DPG, 2,3-diphos-phoglyceric acid.

glyceric acid and MPG were heated for 30 minutes, and DPGwas heated for 120 minutes. The spectra of the colors formedare shown in Figs. 1 to 3.

The phosphorylated glycerates reacted more slowly than didthe free acid, and, as shown in the figures, each produced a some-what different color, both quantitatively and qualita tively.2-PG and 3-PG gave identical spectra in the three hydroxy-naphthalene reactions.

Formate ion or an excess of water inhibited the color formation.Aliquots for analysis greater than 0.2 ml. were dried at 80-90in a mechanical convection oven. Samples containing ammo-

w .5I-2; .4;:5.3N

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in .I

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I I I I I I I I

500 600 700 800 m/iFIG. 1. Spectra of the reaction of glycerates with chromotropic

acid. @---a, glyceric acid; O--O, MPG; O---O, DPG

I I I I I I I I

w .5I-2: .4?i5 .3sA .2i0 .I

I I I 1 I I I I I 1500 600 700 000 m/ L

FIG. 2. Spectra of the reaction of glycerates with naphtho-resorcinol. O---O, glyceric acid; O--O, MPG; O---O.DPG.

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470 Free and Phosphorylated Gtyceric Acid Vol. 234, No. 3

500 600 700 800 m/LFIG. 3. Spectra of the reaction of glycerates with 2,7-dihydrox-ynaphthalene. a-----0, glyceric acid; O--O, MPG; O----O,DPG.

nium formate were evaporated first at 80-90 and then placedunder vacuum at the same temperature for more rapid sublima-tion o f the formate. After the drying procedure, 0.2 ml. of wa-ter was added and the analysis continued as above. Chlorideon did not interfere.

LITERS THROUGH COLUMNFIG-. 5. Chromotropic acid assay of a Dowex 1-formate chroma-tographic separation of the red cell MPG-ADP mixture (4).0~0, chromotropic acid, 1.0 ml. for assay with 30 minutes ofheating; O--O, total phosphorus, 0.5 ml. for assay; O-O,adenine (LIza ml*) . The ADP-MPG mixture from Fig. 4C wasrechromatographed on a 1 X 14-cm. column of Dowex l-X8 for-mate (100 to 325 wet mesh) at 0.7 ml. per minute with 11. of lineargradient 0 to 1 N, pH 6.4 ammonium formate. The first peak hasbeen identified as MPG by other tests (4).

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.2 .4 .6 .8 1.0 1.2 1.4 1.6 1.8 2.0LITERS THROUGH COLUMNFIG. 4. Chromotropic acid assay of a Dowex l-chloride chromatographic separation of red cell phosphates (4). O---O, chromo-tropic acid, 1.0 ml. for assay with 30 minutes of heating; a---@, total phosphorus, 0.5 ml. for assay; C-C, adenine (D, 260 mp).A trichloroacetic acid extract of 53 ml. of human red cells (P. W., see text) was chromatographed on a 1 X 14-cm. column of Dowex

l-X8 chloride (60 to 80 wet mesh) at 2.5 ml. per minute. Eluting agents: A, 0.003 N HCl; B, 0.01 N HCl; C, 0.02 N HCl; D, 0.1 N NH l;E, 0.2 N NH l; F, 0.5 N NH l; G, 1.0 N HCl. Posi tive chromotropic acid reactions in C (= MPG) and in E (= DPG). See (4) fora description of compounds. DPG peak values at eluate volumes: 1260, 1280, 1301, 1322, and 1342 ml. were 7.9, 6.9, 4.2, 2.7, and 1.2pmoles of total P per ml. and 3.9,3.6,2.0, 1.3, and 0.53 pmole of glycerate per ml. (chromotropic acid assay with 120 minutes of heat-ing).glyceraldehyde, dihydroxyacetone, lactic acid, pyruvic acid,glycolic acid, acetic acid, glycerol, formaldehyde, and the mono-,di-, and triphosphates of adenosine.

The chromotropic acid reaction gave the most color per equiv-alent of glycerate and was also the most speci fic of the threetests. Naphthoresorcinol and 2,7-dihydroxynaphthalene re-acted with most of the glycolyt ic and pentose shunt intermedi-ates to form colors which could interfere with the analysis ofglycerates. The following compounds gave less than 5 per centinterference in the chromotropic acid reaction (30 minutes ofheating, color read at 695 mp) when present in concentrationsequivalent to the glycerates: glucose and fructose and their l-and g-mono- and diphosphates, sedoheptulose, ribose, erythrose, with the use of IO-mm. light path cuvet tes.

The chromotropic acid reaction produced a stable color theintens ity of which was closely proportional to the concentrationof glycerate up to 1 pmole in the reaction mixture. The densityreadings for 0.2 pmole of glycerate were 0.48 for glyceric acid,0.49 for MPG, and 0.28 for DPG when measured at 695 rnpwith a Beckman model B photometer (red-sensitive photocell)

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March 1959 G. R. Bartlett 471A practical demonstration of the analytical use of the method

is given in Fig. 4 which presents a Dowex l-chloride chromato-graphic separation of the mixture of human red blood cell phos-phate compounds (4) in which the chromotropic acid reactionvalues, optical density at 260 rnp, and total phosphorus areplotted. Fig. 5 shows the curves obtained by the same analyseson a Dowex I-formate chromatographic resolution of the MPG-ADP mixture from human red cells (4). It can be seen that onlythe eluate fractions containing glycerate gave a positive reac-tion with the chromotropic acid.

DISCUSSIONAs an analytical method fo r glycerates, the chromotropic acid

reaction has the advantages of greater sensit ivity and specificityin comparison with the naphthoresorcinol method of Rapoport

(2, 5), the only previous ly available calorimetric procedure. Areaction of glycerate with 2,7-dihydroxynaphthalene is alsodescribed but, like that with naphthoresorcinol, it, too, lacksspecificity . The characteristic spectra of these reactions canprovide useful information for the identification of glycerates inpurified solutions.

SUMMARYSpectra are given for the reaction of glyceric acid, monophos-

phoglyceric acid, and 2,3-diphosphoglyceric acid with 1,3-dihydroxynaphthalene, 2,7-dihydroxynaphthalene, and 4,5-di-hydroxy - 2,7 - naphthalene - disulfonic acid. The last namedreagent is recommended for the identification and quantitativeassay of glycerates.

REFERENCES1. EEGRIWE, E., 2. anal. Chem., 96, 323 (1933). Organic Syntheses, Collective Vol. III, John Wiley and Sons,2. RAPOPORT, S., o em. Z., 289, 406 (1937). Inc., New York, 1955, p. 637.3. MEYER, K., AND BLOCH, H. S., in E. C. HORNING (Editor), 4. BARTLETT, G. R., J. Biol . em., 234, 449 (1959).5. NEUBERG, C., AND LUSTIG, H., Erptl. Med. Surg., 1,314 (1943).

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