ANALYTICAL CHEMISTRY
1184 potential was -0.520 volt VS. S.C.E.; this is very near to the potential of the electrocapillary maximum of mercury in 1 F potassium chloride, so that dt/Ed e is neaily zero and the effect of small fluctuations in the applied potential is negligible. The maximum current during the drop life was about 0.3 Ha. These data show that the mean drop life is 7.0097 seconds, with a standard deviation of 1.2 milliseconds. Twenty determinations of ten drop lives by the conventional manual technique gave a mean drop time of 7.007 seconds, with a standard deviation of 4 milliseconds. The slightly lower mean value secured by the manual method reflects the vibrations set up by the manual manipulation of the clock. A more fundamental comparison was secured by timing 150 individual drops, under exactly the same conditions, Jvith the millisecond timer recently described by Sturtevant ( 7 ) and used in measurements of the instantaneous diffusion current by Grant, Mcites, and Sturtevant (6). With this instrument, the position of the sharp drop in current at the end of the drop life clan emily be determined u i t h a precision of better than a millisecond. However, because the action of the timer is not initiated until an indeterminate instant within the first niillisecond after the fall of the preceding drop, the measured time n i l 1 always be smaller than the true drop time. The average difference between the true and measured drop times in a large number of measurements would be expected to be about 0.5 millisecond. I n good agreement with this prediction, the mean drop time found from these data (B, Figure 2) was 7.0090 seconds with a standard deviation of 2.5 milliseconds. That this standard deviation is smaller than that which would be predicted from the data secured with the drop timer is probably due to fluctuations in the power line frequency. This would cause the indications of the electric stop clock to be in error, but would not affect the frequency of the tuning fork which serves as the standard of time in the millisecond timer. .4s a further test of the accuracy of the drop timer, the temperature coefficient of the drop time was determined. Five measurements of ten drop lives were made a t each of ten temperature.; between 10" and 40" in deaerated 0.1 F potassium chloride a t -0.520 volt vs. S.C.E. The data, shown in Table I, give the mean temperature coefficient of the drop time as - 2.16 ( f0.11) x 10-3 per degree. This mean deviation corresponds to an error of only a few tenths of a millisecond in the average drop time at each temperature.
Table I. Temperature Coefficient of Drop T i m e Temperature, C. t , See. ( - ~ t / t ax ~ )103 9.9
3.7526
12.9
3.7266
16.3
3.6976
19.5
3.6706
22.85
2 6-164
26. Oj
3.6202
29 3
3 5941
33,l
3,5646
2.30 2.32 2.24 2 00 2 25 2 20 2.17
The room in which these measurements were made has two solid brick walls about 15 inches thick and a very heavy steelreinforced concrete floor. It ie therefore extraordinarily free from vibration, so that these data probably provide a close approximation to the inherent precision of the drop time a t a dropping electrode of conventional form. It is evident that, under ideal conditions, the drop time is far more reproducible than has hitherto been believed ( 2 ) . ACKNOWLEDGMENT
The authors are indebted t o Stephen Boyan for his assistance in the design and construction of the circuit described in this paper. LITERATURE C l T E D
Colichman, E. L., J . Am. Chem. Soc., 73, 1795 (1951). English, F. L., Ax.4~.CHEM.,20, 889 (1948). Goldsmith, K., J. Sci. Instrumenls, 25, 385 (1948). Grahame, D. C., Larsen, R. P., and Poth, M.A., J . Am. Chenz. Soc., 71, 2978 (1949). Grant, D. IT.,hleites, L.. and Sturtevant, J. M.,Division of Analytical Chemistry, Symposium on Polarographic Methods, 120th Meeting, ~ E R I C . I N CHEMICALSOCIETY,Piew York,
x. I-.
hleites, L., J . Am. Chem. Soc., 73, 177 (1951). Rev. Sei. Instrumenlu, 22, 359 (1951). Sturtevant, J. M., RECEIVED for review December 6, l%l. Accepted March 1, 1952. Conmibution No. 1087 from t h e Department of Chemistry of Yale University.
Determination of Inorganic Phosphate Produced by Arterial Enzymatic Action FREDERICK K. BELL, C. JELLEFF CARR, AND JOHN C. KRANTZ, J R . School of Medicine, Unicersity of Maryland, Baltimore, ;Md.
THE authors have been interested in micromethods for thc 1 determination of inorganic phosphate in connection with studies of the adenosine triphosphatase activity of arterial tissue ( 3 ) . Experience with available methods was not satisfactory. The major disadvantages of these methods are: ( 1 ) the hydrolysis of adenosine triphosphate (ATP) by trichloroacetic acid; (2) the uncontrolled color development even in the absence of inorganic phosphate; and (3) the capricious color development in the final steps by the excess adenosine triphosphate substratc ( 2 ) . These difficulties thwarted the precise and absolute determination of the adenosine triphosphate activity of tissues of the vascular system, where the total amount of enzyme is small and the quantity of tissue is limited. A new method published by Griswold, Humoller, and McIntyre ( 2 ) for the estimation of inorganic phosphate in biological material involves several radical departures from earlier methods and appeared t o solve some of the difficulties. The inorganic phos-
phate is precipitated and isolated as magnesium ammonium phosphate, which is then converted, under dcfinite conditions, into the heteropoly molybdenum blue compound. The solution of this substance is very stable, displays a sharp absorption maximum, and is therefore suitable for accurate spectrophotometric analysis. By this method the excess adenosine triphosphate substrate can be removed from the reaction mixture and its subPequent interference with color development eliminated. Lowry and Lopez ( 4 ) used a buffer mixture of sodium acetate and ammonium sulfate for the precipitation of tissue protein. This reduces the capricious hydrolysis of adenosine triphosphate resulting from acid precipitation. The authors considered the probability that this step in their procedure could be carried out in this medium. This communication describes a method incorporating the advantages of the reaction mixture of DuBois and Potter ( I ) , the protein Precipitation procedure of Lowry and Lopez, and the
V O L U M E 24, NO. 7, J U L Y 1 9 5 2
1185
phosphatc precipitation, color development, and estimation of Griwold et al. This method has been used successfully t o nwasure thr adenosine triphosphatase activity of tidsues of the ular system and t o det,erniine the effect of drugs upon this enzyme systc,m. I t also appears applicable t o the determination of the :itic,nosinc t viphosphatase activity of othcxr tissues-e.g., brain : ~ n dheart, EXPERIMENT:\ L
To ev;ilu te t h e method, the dorsal aorta from several species of animals \vas used. This t,issue is relatively rich in adenosine triphosphatase enzyme. The aort,a is removed, washed in distilled water (4' C.), cleaned and split longitudinally, dried quickly on filter paper, and weighed to the second decimal place. The tissue (0.3 to 0.5 grain) is then transferred to a previouslj- chilled mortar, cut into fragments, arid ground \r.it,h approximately 0.5 gram of cold sand added in miall portions. K a t e r is added to make the final mixture of a 1% suspension of tissue. The suspension iti centrifuged for exact,ly 3 minutes a t 1200 r.p.m. This provides a uniform suspension. Aliquots arc removed from the distinctly turbid supernatant fluid. I
?v\IETHOI)
strate (adenosine triphosphatc tetrasodium salt, Rohm 8: Haas Co., Philadelphia, Pa.). To accumulat'e data for statistical analysis, multiple determinations were carried out for the adcnosine triphosphat,e blank and for the tissue plus adenosine triphosphate. The first experiments wrrc restricted t o the rabbit's aorta. .Isample experiment is shown in detail in Table I and is typical, exwpt for the fact that t h r tinsue phosphate was also determined. The values given for the tissue phosphate include the phosphate of the reagent blank. Both correct,ions are very small and are of no significance.
Table I.
Typical Experiment on Rabbit's Aorta Spectrophotometer Reading, Absorbancy -~2 3 4 5 6 7 8 A v .
1 ATPblank 0.50 ATP tissue 0 . 9 8 Tissue, no ATP 0.09
+
3%.
0 . 5 0 0.51 0 . 5 0 1.00 0 . 9 7 0 . 9 2 0.09
0.07
0 . 5 4 0.53 0 . 5 2 1.04 1.00 0.98
0.54 0.97
0.08 0 . 0 9
0.09
0.08 0.07
.4verage ATPase activity is 6.0, f standard deviation 0.18 unit;
0.05 .If sodium barbital huffwed wirh HCI to p H 7.4. nil. 0.032 .M calciiim chloride. inl. 0.0144 M hTP sodium salt, nil. H20, nrl.
Formula A
roriiluln
1.2
1.2 0.: 1.0 1.1
0.3 1. O 2.1 1.8
-
B
-
3.8
The tubes are immersed in a water bath a t 37" for several minutes and then 1 nil. of 1% tissue extract is added to each of the B tubes, followed by mixing. At, the end of 15 minutes the tubes are removed from the bat'h. To each tube is added 0.2 ml. of 0.1 A I sodium acetate saturated with ammonium sulfate. The. contents of the tubes are mixed by inversion and then immersed in cold water for several minutes. The contents of the B tubes are filtered. The filtrates may be slightly cloudy. Two-milliliter portions of the contents of the A tubes and of the filtrates from t h r B tubes are transferred to 15-ml. graduated borosilicate glass centrifuge tubes. For the precipitation of the phosphate and the subsequent color development, the m5thod of Griswold (5') was employed. However, the magnesium ammonium phosphate IWS allowed to stand for 18 hours at, 25" and the final volume was adjusted to 5 ml. For the spect.rophot'ometric determination this was diluted 1t o 10. The average of the absorbancy values obtained from the solutions of thr, A tubes is subt'racted from a similar value obt,ained from the B tuhesj and this difference is a direct measure of t,he tissue activity, in that it represents the amount of phosphatc hydrolpzed from t,he adenosine triphosphate suhstrate tiy enzymatir action under the conditions of t,he experiment. This value can he readily converted into micrograms of phosphoms 1)y reference to a standard phosphorus curve. The value of the tissue artivity obtained hy t,hk procedure can be espresscd in ternis of milligrams of tissue. The method conipensatcls for the phosphate in the reagents :tnd that,fronithr:ulenosin(: triphosphate s u h t r a t e . T h r phoPph:tt(' l~lanlifor thc reagents \\-as found to be w r y m a l l . Efficiency of Method. Incubation mixtures were prepared as indicxtd, incbliidinp thc adenosine triphosphate and tissue, and known aiiiounts of inorpanic phosphate wcre added. The mixture. n-ere carried through t,he complete procedure. I n the phosphorus concentration range of from 40 t o 60 micrograms, corresponding t o the level in most of the tissue determinations, 90 t o 100% recaovery was obtained. EXPERIMENTAL R E S U L T S
All experiments were carried out, n.ith t,he same sample of sub-
0.09b C.V.
=
Readings obtained on solutions not diluted tenfold. Therefore this value becomes 0.008 for comparison with values for A T P blank a n d ATP tiwiw. h
Thc tictermination of the adenosine triphosphatase xctivity of the tissue \vas started n-it,hin 30 minutes after preparation. The incubation mixture is essentially that reported by I h n t z , Carr, and Bryant (S), which was based on the recommcndat~ions of DuBois and Potter ( 1 ) . Into two sets of t,ul)es (borosilicate glass 13 X 100 mni. culture tubes) ingredients iwrc introduced in accordance with formulas -1and B:
0.33 0.9Sa
+-
In ten clperiments conducted on different days the variation in adenosine triphosphatase activity of the aorta of different animals was determined. One rabbit was used for each experiment. The tissue activity, in terms of the adenosine triphosphate unit, is defined as the micrograins of phosphorus liberated by 1 ma. of tissuc in 15 minutes at 37" C. ( 1 ) . The following values \Terc found: 3.5, 5.5, 3.0, 6.8, 6.0, 5.8, 5.0, 6.3, 6.3, and 4.3. In other experiments the adenosine triphosphatase activity of aortic tissue of the dog, rat, turtle, chicken, and frog was investigated. This method yielded satisfactory results with tissue from all these qources. Experienres have indicated that the adenosine triphosphatase activity of tissue from the same species shows \vide variation from animal t o animal and there appears to be a marked difference between speries. DISCUSSION
.In important, consideration in t,he use of this method is t.ho degree of hydrolysis of t,he adenosine t,riphosphate during the rourse of the det,ermination. Gross variations in the length of time that elapses betn-een the addition of the magnesia mixture and the final color development produce significant changes in the adenosine triphosphate blank and also in the observed tissue activity. Therefore for a comparison of experiments from day to day this time should be controlled. Experience with differcsnt samples of adenosine triphosphate has indicated that the nature of the substrate is most important. Significant differenctis in activit?. have been observed in t,he use of adenosine triphosphate samples of different manufacturers, and even different lots of adenopine triphosphate from the same source show important, variations. .kCBNOWLEDGRIENT
The authors wish t o ac~knonledgethe technical a Robrrt C. Holcombe in this investigation. LITERATURE CITED
and Potter, V. R., J . B i d Chem., 150, 186 (1943). ( 2 ) Griswold, B. L., Humoiler, F. L., and McIntyre, A. R., ANAL. CHEM.,23, 192 (1951). ( 3 ) Krante, J. C., Jr., Carr, C. J., and Bryant, H. H., J . Pharmacol. Exptl. Therap., 102, 16 (1950). (4) Lowry, 0. H., and Lopez, J. A , J.Bid. Chem., 162, 421 (1946). i l j DuBois, K. P.,
RECEIVED for review January 18, 1952. Accepted March 14, 1952. T h e expense of this investigation was defrayed in part by a grant from t h e National Institutes of Health.
