mined from their different rates of decay and subtracted, or, more efficiently, the same sample can be irradiated again and counted in the neutron counting assembly in which the DzO has been replaced by natural water. Delayed neutron emission, due to its short half life (< 1 minute) is of no practical concern, since a -5-minute delay will result in the complete decay of this source. If a delay in counting cannot be permitted the delayed neutron effect can be subtracted by counting with natural water, as mentioned above. The extent of mutual interference of calcium and sulfur in samples containing both elements is seen in Table IX. Xnother possible source of interference in the analysis of sulfur is chlorine, and to a much lesser degree, argon. Table XI shows the extent of formation of S3’ from these sources. Since chlorine might be more abundant than sulfur in many types of samples, its effect would mask that of sulfur. The use of a well thermalized neutron flux is then important. Xnother possibility is to irradiate the sample behind a thermal neutron absorber-e.g., cadmium-and the difference will represent the net thermal effect due to neutron capture in sulfur. A minor source of inter-
ference is the high energy gamma ray of 37.3-minute C138,of -10-2~oabundance, which may give rise to photoneutrons (9), but because of the large difference in half lives, it can be corrected for by following the decay. The interference of argon is small as can be seen from the fact that 10 cc. of air present in the sample contribute only -1 c.p.m., when irradiated for 10 minutes at a flux of lOI3 neutrons/cm. l-second. These problems do not exist for Ca49 which is formed exclusively from calcium. LITERATURE CITED
Table XI. Formation of S3’from Different Sources [Using Mixed Neutron Flux of 7.4 X 10l2Thermal and 2.4 X 1 O I 2 Fission Spectrum Neutrons per c m 2 per sec.]
Photoneutrons/min./mg. parent after 10-min. irradiation Reaction Calcd. Exptl. SYn,r)S37 11.1 11.2 f 0 . 3 ClYn,p)S37 10.2‘ 3.4 f0.3 Arao(n,a)S37 84.00 6.0 f 0.5 Based on estimated cross sections quoted in Table I1 according to (11).
(1) Amiel, S., ANAL. CHEM. 34, 1683
(1962). (2) Amiel, S., Peisach, M., Ibid., 35, 1072 (1963). (3) Amiel, S., Stuhl, Z., Zsrael At. Energy Comm. Rept. IA-932 (1964). (4) Bouten, P., Hoste, J., Anal. Chim. Acta 27, 315 (1962). (5) Bowen, H. J. hl., Cawse, P. A., U . K .
At. Energy Authority Rept. AERER4309 (1963). (6)‘Bowen, H. J. hl., Gibbons, D.,
Radioactivation Analysis,’ ’ Oxford University Press, London, 1963. (7) Chingala, B., Cinffolotti, L., Malvano, R., Energia Xucleare 10 (7) 389 (1963). (8) Handbuch de Physik, XLII, p. 325, Springer Verlag, Berlin, 1957. (9) Juliano, J. O., Amiel, S., Atomic Energy Commission, Yavne, Israel, unpublished data (1964).
(10) Koch, R. C., “Activation Analysis Handbook,” Academic Press, New York, 1960. (11) Roy, J. C., Hawton, J. J., At. Energy of
Canada Rept. AECL-1181
(1960). (12) Simpson, H., Gibbons, D., “Radioisotopes in the Physical Sciences and Industry, ’ I International Atomic Energy Agency, Vol. 11, p. 269, Vienna, 1960. (13) Way,, K., et al. “Nuclear Data Sheets, National Academy of Sciences, National Research Council, Washington, D. C. RECEIVEDfor review June 18, 1964. Accepted December 15, 1964. One of us (J. 0. J.) thanks the Israel Atomic Energy Commission for a fellowship.
Application of Galvanic Cell to Measurement of Oxygen-Consuming Enzyme Systems HARRY LIPNER, L. R. WITHERSPOON,l and ANNE WAHLBORG Departments o f 6iological Science and Chemistry, Florida State University, Tallahassee, Fla.
b The application of a galvanic cell oxygen analyzer to the study of oxidative enzyme systems is discussed. Comparisons are made of results obtained on three enzyme-substrate systems using the Warburg manometric technique, spectrophotometry, and the galvanic cell with a bucking potential circuit. Agreement of kinetic data, high sensitivity, and versatility suggest that the galvanic cell is applicable in the analysis of oxidative systems. Data are presented which demonstrate that the galvanic cell can b e used to measure changes in oxidative systems which consume less oxygen than can b e measured b y conventional manometry or which involve no change in the ultraviolet or visible spectrum of reactants or products.
T
most commonly used in the study of oxidative reactions is the Warburg manometric technique (8). The spectrophotometer has also HE METHOD
been used in studies of oxidative reactions when the solutions are translucent and there are changes in either the visible or ultraviolet spectra (6). Polarographic techniques in which the reduction of oxygen is measured when proper potentials are applied are also used to measure oxidative reactions. The dropping mercury electrode (2) is the system usually associated with the term polarogaphy, however, the oxygen electrode (1) and the galvanic cell oxygen analyzer (3-5) are variants of the polarographic technique. Mancy (4) described a galvanic cell which utilized a membrane enclosed chamber containing electrodes and supporting electrolyte. Lipner ( 3 ) described a bucking potential circuit which resulted in a marked increase in sensitivity when used with the Mancy galvanic cell. The purpose of this study was to investigate the applicability of the galvanic cell to oxidative enzyme systems and to establish the limits of
accuracy, sensitivity, and the reproducibility of this apparatus. (The galvanic cells were constructed by E. J. Highsmith and the bucking potential circuits by F. Jordan of the Department of Biological Science, Florida State University, Tallahassee, Fla.) Oxidative systems chosen for study were ones which allowed direct comparison of data obtained with the galvanic cell, Warburg, and spectrophotometer. The tyrosinase oxidation of tyrosine to dopachrome was applicable for study with all three instruments. The tyrosinase oxidation of 3,4-dihydroxyphenylalanine (DOPA) to dopachrome was too rapid for accurate manometry and was measured only with the galvanic cell and spectrophotometer. The reaction catalyzed by snake venom L-leucine oxidase to the corresponding alpha keto acid was measured with the galvanic cell and the Warburg but 1 Present address, University of Wisconsin Medical School, AIadison, Wis
VOL. 37. NO. 3, MARCH 1965
a
347
a Table I.
Subatrate- T y r o a l n r
Calculated Apparent K,s
X Spectrophotometer 0 Galvanlc c r l l 0 Warburg
Reaction Snake venom bleucine oxidase
Instrument Galvanic cell Warburg Beckman DK Literature values
0.95 x 10-3 1.07 X 1x
10-3
(Singer and Kearney,
Tyrosinase-tyrosine
x 10-4 x 10-4 1 . 7 7 x 10-4 1 . 9 x 10-4 + 0 . 4
2.03 2.29
Tyrosinase-DOPA 6.61
x
5 . s g ' x ' 10-4
(Osaki,
1963)
1950)
Table II.
Cell Constants
Membrane 1.0-mil polyethylene 1.0-mil Teflon 0 . &mil Teflon
pl. Oz/mv./ml. 0.107 0.092 0.044
could not be measured directly with the spectrophotometer because no significant change in the ultraviolet or visible spectrum of the reactant or product occurs. EXPERIMENTAL
Instrumentation. A galvanic cell equipped with a bucking potential circuit and with a Beckman linear recorder was used to measure initial rates of oxidation in all systems studied (3). Polyethylene membranes, 1.0-mil (obtained from 0. L. Dube, Precision Scientific Co., Chicago, Ill.) were employed for all studies except as otherwise noted. The cells had a diameter of 1 cm. and when in use the membranes had an effective area of approximately 16 mm.2 The microm'inkler method was used to determine the equivalence of oxygen concentration to voltage generated (cell constant) (3). Three identical galvanic cells were used to facilitate the operation. -4switching device allowed the recorder to monitor any of the three cells. A rate could be recorded from one cell while the other two chambers were being prepared for subsequent runs. Care must be taken that oxygen does not become a limiting factor. When cold solutions, or solutions that had been tightly stoppered, were introduced into the cell chamber, it was necessary to aerate the solutions. This was done by aspiration through a syringe when the solution was introduced into the cell chamber. Because there is very little surface exposed to the air, there is insufficient diffusion of oxygen into the solution to influence the rate observed during the first several minutes. The choice of a cell membrane is important since the rate of diffusion of oxygen through the membrane determines the voltage output of the cell circuit. A comparison of three membranes, 1.O-mil polyethylene, 1 .O-mil Teflon (Du Pont), and 0.5-mil Teflon was made to determine the membrane best suited for the cell.
348
ANALYTICAL CHEMISTRY
14
10-4
A limitation imposed on the use of the galvanic cell is noise which may be introduced by erratic stirring and temperature fluctuation. The former can be reduced by rapid stirring at the membrane surface and the latter by using a large volume reservoir as a source of constant temperature water for the jacketed reaction chamber. These factors have been more extensively treated by Mancy ( 5 ) . A Beckman D K spectrophotometer was used to measure the oxidative activity of tyrosinase. The formation of dopachrome was observed spectrally a t 475 mp. The Warburg was used to measure the oxygen consumed in the tyrosinase-tyrosine and snake venomL-leucine oxidations. .4 paper accordion carbon dioxide trap was used for the snake venom catalized oxidations to trap carbon dioxide formed because of instability of the alpha keto acid product. Materials. L-Leucine was used as the substrate for snake venom-iamino acid oxidase. Lyophilized Agkistrodon p . piscivorous (moccasin) venom was used without purification (obtained from Ross Allen's Reptile Institute, Inc., Silver Springs, Fla.). Enzyme solutions contained 62.5 mg. venom/5 ml. buffer. L-tyrosine and L-DOPA were used as substrates for tyrosinase. A crude mushroom preparation was the source of the tyrosinase artivity (obtained from Earl Frieden, Department of Chemistry, FSU, Tallahassee, Fla.). All substrate and enzyme solutions were prepared in 0.1M phosphate buffer, pH 7.1. Measurements. Velocities (v) were determined when both the enzyme ( E ) and the substrate ( 8 ) concentrations varied. T h e rates plotted represent the mean of the observations. Values obtained for replicate runs differed by no more than *lo% from the mean and all slopes were determined using t h e method of least squares. Apparent Michaelis-Menten constants (K,) were calculated from the data collected with each instrument. The K,s were compared because they are independent of the units of the rate constants. The enzyme was added to fixed volumes and the assumption was made that small changes in volume did not appreciably alter concentrations. Rates measured by the galvanic cell as spectrophotometer were initial rates, observed within seconds after the reaction began. Since the Warburg technique does not lend itself to determina-
.-
99% of the PI (518keV) cesium was adsorbed. The column was washed with 2 ml. of lh‘ HC1 and 13, ( l l B 0 ke\ a period was allowed for growth of the Ba13Trndaughter. Then a Ba137m milking consisted of flowing 1N HC1 through the column and collecting individual drops of the effluent. Ba13’lrn Figure Decay scheme for C S * ~ ~ was eluted promptly and sharply and a separation factor from Cs137 of >lo5 was achieved. and more precise value for the 662 Ba137rnsources suitable for 4nB counting were prepared by evaporation of k.e.v. y-ray abundance and a value for small samples of the eluate onto gold the @-branching ratio to the 662 k.e.v. coated collodion films. These films level in Ba137. These new values have could be placed very close to an infrared been incorporated in the decay scheme lamp without damage. The time reshown in Figure 1. milking and source quired for a 13a137rn preparation usually was 1 mc. (3). Response curves were obtained for ampoules in three different conper mg. cesium. Part of a y-ray spectrum of this solution taken with a tainers: 0.002-inch AI, 0.016-inch Cd, lithium-drifted germanium 7-ray specand 0.048-inch Cd lined with 0.005-inch trometer (88) is shown in Figure 2 Ta. The latter two source holders were together with a spectrum of the same designed for use when it is necessary to material with 2.0y0 C S ’ added. ~~ From absorb out x-rays accompanying electhese spectra it was concluded that tron capture or internal conversion, but they are also useful in that they there was less than 0.02y0 Cs134in the Cs’37. provide three partially independent A method was developed for the rapid response curves for checking the preseparation of carrier-free samples of cision of the method. Ba137m. This method made use of the Ba137m Counting. The 1 3 a 1 3 7 m sources adsorption of Cs137 on Co&’e(cK)~ were counted in a system consisting of a 4np-proportional counter sandwiched (10). C O ~ F ~ ( Cadsorption N)~ columns were between two 3 inch X 3 inch ?;a1 y-ray detectors ( 3 ) . Essentially all prepared as follows: about 2 ml. each of K,Fe(CN), solution, water, Cos04 ( >99.9yo) of the conversion electrons solution, and water were passed in were detected in the 4xp counter (fy). turn through small (0.03 cm.2 X 6 The y-ray emission rates of the sources were determined by direct comparison cm.) columns of Dowex-1. I n this way
c 5‘3’ (30yl
n .
VOL. 37, NO. 3, MARCH 1965
351
