V O L U M E 26, NO. 6, J U N E 1 9 5 4
1049
high order of catalytic effect of fluoride on this example of electron transfer, discovered by Hornig and Libby (I), was confirmed. T h e same workers stated t h a t chloridc had a slight catalytic effect in the same direction as fluoride. However, in this work, chloride added as sodium chloride, in three trials over Tide ranges of concentration, produced a n apparent slight negative catalytic effect. Also, an inhibition in measured catalytic effect of fluoride was obNerved in the presence of chloride. A detectable inhibition \yap noted a t 0 . 5 m X chloride; at 1.Om.U chloride, the effect of 1 -1 of fluoride was reduced by i . 8 % , Calcium ion, whose slight cat,alytic effect decreases in increment with increasing amount, is (except for magnesium) the only cation for which definite evidencc of catalysis of the cerous-ceric electron transfer was obtained in this study. The effect of calcium and chloride on electron transfer could be controlled in practical fluoride analysis, by adjusting to a concentration above which their effects became nearly constant. The catalytic effect of 0.1 millimole of sulfate is of the order of 1/900th that of the same amount of fluoride. On this account, some materials would require removal of sulfate prior to fluoride determination b y electron transfer catalysis. The catalyt,ic effect of O.lmW phosphate is approximately 1/370th that of a n equal amount of fluoride (Figure 2), and the relative efficiency of phosphate increases with increasing amounts of the ion. The measured catalytic effect of phosphate on electron transfer cannot be attributed t o fluoride contaminating the dibasic sodium phosphate uscd to supply the phosphate ion, bccause the observed effect wap not altered by repeated treatments of the salt with concentrated nitric acid, followed by removal of the acid by evaporation at water bath temperature. Evidence was obtained to indicate that the catalytic effects of phosphate and fluoride, when both are present, are not additive. The results given in Figure 3 show that the effects of 1 and 2 y of fluoride are augmented in the presence of phosphate ion equivalent to 1.7 mg. of phosphorus. The high catalytic effect of phosphate introduces a serious interference in the application of electron transfer catalysis to fluoride analysis, since many materials, particularly those of biological origin, may contain upward of several hundred times as much phosphate as fluoride. While fluoride and phosphate may be separated by chemical manipulations and distillation ( d ) , it. would not be advantageous, on account of cost, to use electron transfer catalysis for fluoride determinations in those cases in which phosphate must be eliminated from the sample. This procedure w-ould have found a justifiable, routine use only in the
hoped for, but unrealized, circumstance that phosphate and other ions do not alter appreciably the rate of electron transfer. Fluoride determinations by electron transfer catalysis can be made without removing the interfering ions, if the quantities and, henre, the effects of these ions can be made constant. This can easily be attained with calcified tissues, since the composition of the mineral part of a given type of ralrified tissue-for example, bone-is virtually fixed by nature. Table I1 s h o w the results of fluoride determinations in bovine bone ash and gives the results of recovery of added fluoride. In this xyork, 5-nig, samples of bone ash were used. The fluoride contents of these samples were obtained by reference to standard curves of electron transfer, with known amounts of fluoride in the presence of the quantities of phosphorus and calcium found in 5 mg. of bone ash. These quantities of calcium and phosphorus were actual1)- obtained hy U R P of a solution of the same bone ash from which the fluoride had been removed by repeated treatment with nitric acid in a platinum dish, followed by removal of thtx excess acid by heat.
Table 11. Analysis of Bovine Bone by Electron Transfer Catalysis Bone S s h 10 2 20 t 0.17"
S o . of samples analyzed Fluoride found, y Fluoride recovered, y Fluoride. 7~ o . 0 ~ 0.003~ a Standard deviation of mean
*
+
Bone Ash 0.50 y Fluoride 8 2.7" 0.10" 0.52
ACKNOWLEDGMEKT
The authors thank I. 11.Kolthoff, who called their attention to the work of Hornig and Libby and who supplied helpful discussions LITER'ATURE CITED
Libby, W.F., J . Phys. C'hcm.. 56, 869 (1952). (2) Libby, W.F., Ibid.,56, 872 (1952). (3) Singer. L.. and Armstrong. W.D., Sucleonics. 11, 55 (1953) (4) Willard, H. H., and Winter, 0. B., IKD. EBG.CHEM.,. ~ - . A L , ,ED., 5 , 7 (1933). (1) Hornig, H. C., and
RECEIVED f o r review Xovember 22, 1953. Accepted lIarci1 3 , 1954, Investigation supported b y Medical Research and Development Board, Office of the Surgeon General, Department of the Army, under Contract KO, DA-49-007-AID-390.
Polarographic Determination of Alpha-Ketoglutaric Acid JAMES K. PALMER' and CLIFFORD 0. JENSEN D e p a r t m e n t o f Agricultural and Biological Chemistry, The Pennsylvania State University, State College, Pa.
I
S THE course of studies on the metabolism of plant tissues, a rapid, sensitive, and specific method for determining a-ketoglutaric acid was desired. The most commonly used method for determining a-ketoglutaric acid was developed by Friedemann and Haugen ( 4 ) . It is based on the formation of a red-brown color u hen the 2,4-dinitrophenylhydrazone of a-ketoglutai ic acid is treated with sodium hydroxide. This method is sensitive, but it is specific for a-ketoglutaric acid only v h e n carried out under carefully defined condition?. I t also suffers from the reintive lack of stability of the colored conipound and the rangc' is small. Several other procedures have been clmplored for determining keto acids. Clift and Cook ( 3 ) descrihcd a titration method 1 Piesent address f l a i e n Conn
Connecticut 4griculturai Experiment Station
Yew
based on thc formation of a bisulfite addition product. Keto acids also have been determined by measuring the carbon dioxide produced on oxidation with ceric sulfate or permanganate ( 7 , 11). These methods are not specific for a-ketoglutaric acid. Chi omatographic separations have been described employing silica gel ( 5 ) )ion exchange resins (I), and filter paper Celite (8), ( 2 , f0). Procedures using silica gel columns and paper chromatograms have given good recoveries of a-ketoglutaric acid, but these methods itre not convenient for the determination of a single acid. Previously, certain acids of the tricarboxylic acid cycle have been successfully determined polarographically ( 9 , 1 2 ) . The polarographic behavior of a-ketoglutaric acid is such that waves suitable for quantitative analysis are obtained in solutions buffered a t pH 1.8 and 8.2. The current increment observed under
ANALYTICAL CHEMISTRY
1050
T a b l e I.
Recovery of a - K e t o g l u t a r i c -4cid from Enzyme Reaction Mixtures
(At p H 1.8 in 0.7M potassium chloride-hydrochloric acid solution) a-Keto lutaric Acid I d d e d , Micromoles
--Keto lutaric Acid joound, Micromoles
Recovery, %
bubbling purified nitrogen through the solution for about 10 minutes. At p H 1.8, record the current produced over the applied voltage range of -0.35 t o -0.85 volt. At pH 8.2 record the current produced over the applied voltage range of - 1.05 to 1.60 volts. Calculate the a-ketoglutaric acid concentration from the equation :
-
where
Av. 9 8 . 6
=I= 1.5
c, c, i, i,
these conditions is proportional to the a-ketoglutaric acid concentration. WPARATUS AND MATERI4LS
Apparatus. The Fisher Elecdropode was employed in these studies. To facilitate determination of the limiting current, the galvanometer deflection was calibrated in terms of microamperes by substituting a 100,000-ohm Akra-ohm resistance for the polarographic cell. The galvanometer was calibrated a t each of the shunt settings used. All voltages were measured versus the saturated calomel electrode. The polarographic cell was of the H type described by Komyathp, Malloy, and Elving (6). The temperature of the cell was maintained a t 25’ f 0.2” C. by water flowing from a thermostatically controlled constant-temperature bath. The Beckman Model G pH meter was used in measuring pH. Materials. Alpha-ketoglutaric acid (General Biochemicals, melting point 110-112° C.) was dissolved in water and neutralized to bromothymol blue with 1 N potassium hydroxide. The supporting electrolyte solution of p H 1.8 consisted of a 0.7.V potassium chloride-hydrochloric acid solution. At p H 8.2 the electrolyte was a 1.0.11‘ ammonium chloride-ammonium hydroxide solution. EXPERIMENTAL
Solutions of a-ketoglutaric acid were electrolyzed at pH 1.8 and 8.2 at various ionic strengths of supporting electrolyte solution. The usual polarographic procedure was followed. The mercury column of the dropping electrode was maintained a t a constant height of 47.0 cm. Capillary Constants. The weight of mercury delivered by the capillary under the conditions employed was 1.062 mg. per second. .4t pH 1.8 the drop time a t an applied potential of -0.85 volt was 5.6 seconds and the value of m2’3t1’5 was 1.39 mg.2’3 sec.-112 At p H 8.2 the drop time a t -1.60 volts was 4.4 seconds and the value of m 2 W 5was 1.33 mg.2’3 sec.-112 Effect of Concentration, Ionic Strength, and pH. Solutions of a-ketoglutaric acid give well defined polarographic waves whose values of E,,z(-0.63 volt a t p H 1.8 and -1.34 volts a t p H 8.2) are independent of concentration over the range of 0.20 to 2.00 millimolar. The average diffusion current constant ( I = i / C m2’3t1’6) is 2.71 f 0.04 a t p H 1.8 (10 observations) and 3.32 i 0.07 a t p H 8.2 (18 observations). A p H variation of 0.1 unit or id. At p H 8.2 a small secondhad no significant effect on E1/2 ary wave appeared a t ionic strengths lower than 1.0; the combined wave heights were equal to that of the single wave a t higher ionic strength. Bt p H 1.8 ionic strengths lower than 0.7 produced slight irregularities in the limiting portion of the wave. ANALYTICAL PROCEDURE
Select an aliquot (1 t o 12 ml.) of the test solution so that the concentration of a-ketoglutaric acid in the solution electrolyzed to 2 X l O - 3 M . Transfer this aliquot t o is in the range 2 X a 50-ml. volumetric flask, with 35 ml. of supporting electrolyte solution, and dilute the contents t o volume. Place the solution in the polarographic cell, and remove the dissolved oxygen by
molar concentration of a-ketoglutaric acid in the sample = molar concentration of a-ketoglutaric acid in the standard = current produced by a-ketoglutaric acid in the sample = current produced by a-ketoglutaric acid in the standard
=
SPECIFICITY
The presence of other anions is immaterial as long as they are not reducible over the range of measurement. Relatively few of the organic acids encountered in metabolic studies involving the tricarboxylic acid cycle are reducible polarographically. Of the reducible acids, only maleic acid will interfere a t both p H 1.8 and 8.2. Pyruvic and cis-aconitic acids will not interfere a t p H 1.8, but may interfere a t p H 8.2. Fumaric acid will interfere a t p H 1.8, but not a t pH 8.2. In the presence of proteins at p H 8.2, the limiting portion of the wave may be obscured by the appearance of an interfering wave a t about - 1.60 volts. At pH 1.8 the current produced is measured readily in the presence of protein, although the half-wave potential may be shifted t o about -0.69 volt. APPLICATION
.4t p H 1.8 the polarographic method provides a rapid means for measuring the enzyme-catalyzed appearance or disappearance of a-ketoglutaric acid in the absence of maleic and fumaric acids. Addition of supporting electrolyte buffered a t acid p H will usually stop the enzyme reaction and the polarogram may be obtained at any convenient time. This procedure is useful especially in kinetic studies. Table I shows the recovery of added a-ketoglutaric acid from enzyme control mixtures in which the enzyme has been inactivated. Aliquots were withdrawn folloving incubation of the mixtures for 1 to 6 hours a t 37” C. From these data it appears that a-ketoglutaric acid may be determined within 3 t o 4% under such conditions. The sensitivity of the method is of the order of 30 y per ml. LITERATURE CITED
Busch, H., Hurlbert, R. B., and Potter, V. R.. J . Biol. Chem., 196, 717 (1952).
Cavallini, D., Frontali, Tu’., and Toschi, G., Yature, 163, 568 (1949).
Clift, F. P., and Cook, R. P., Biochem. J., 26, 1788 (1932). Friedemann, T. E., and Haugen, G. E., J . Biol. Chem., 1 4 7 , 4 1 5 (1943).
Frohman, C . E., Orten, J. M., and Smith, A. H., Ibid., 193, 277 (1951).
Komyathy, J. C., hlalloy, F., and Elving, P. J., ANAL.CHEM., 24, 431 (1952).
Krebs, H. d.,and Johnson, W.A, Biochem. J., 31, 645 (1937). Phares. E. F., llosbach. E. H., Denison, F. W., Jr., and Carson, S.F., ANAL.CHEM.,24, 660 (1952). Sacks, W., and Jensen, C. O., J . Biol. Chem., 192, 231 (1951). Seligson, D., and Shapiro, B., Axat. CHEM.,24, 754 (1952). Virtanen, A. I., Arhimo, A. A, Sundman, J., and Jonnes, L., J. pTakt. Chem., 162, 71 (1943). Warshowsky, B., Elving, P. J., and Mandel, J., ANAL.CHEM., 19, 161 (1947).
RECEIVEDfor review October 16, 1953. Accepted March 6 , 1954. Authorized for publication a s Paper 1824, journal series, Pennsylvania Agricultural Experiment Station.
