Chronometric Method of Determining Polyphenol Oxidase Activity Potentiometric Device for Automatic Determination of End Point LLOYD L. INGRAHAM and BENJAMIN MAKOWER Western Utilization Research Branch, Agricultural Research Service,
A potentiometric deFice has been developed to determine automatically the chronometric end point in the estimation of polyphenol oxidase activity. This is accomplished by measuring the time for aerobic oxidation of a known quantity of ascorbic acid in the presence of the substrate catechol. The equipment essential11 consists of a verj sensitive mechanical relay which turns offan electric clock when all the ascorbic acid has been oxidized. A n improved graphical method has been suggested for the calculation of the enzyme activity from the chronometric data at several ascorhic acid concentrations.
T””(4),
catalytic activity of the enzyme pol! phenolovidase is commonly determined by means of the “chronometric method” which involves a measurement of the time required for oxidation of a given quantity of ascorbic acid added to the reaction system. I n this system polyphenoloxidase catalvzes t h e aerobic oxidation of catechol to o-quinone, which in turn oxidizes ascorbic acid to dehydroascorbic acid and is reduced back t o catechol. The amount of ascorbic acid oxidized, &, or quinone formed, has been shown (4)to vary nith time as described by Equation
U. S. Department OF Agriculture, Albany, Calif. PRINCIPLE OF METHOD
Because the enzymatic reaction continually produces o-quinone which reacts n ith ascorbic acid, the course of the reaction niav be likened to a titration of the ascorbic acid by the o-quinone Thus it is possible to follox the “titration” reaction electrometrically and to determine the end point from the change in t!ie electromotive force. A simple electrical circuit has been devised which follows t’ie e.m.f. change and automatically registers on a clock the time required to reach a potential difference selected as the end point. The details of this circuit are described below. I n order to decide on the potential difference a t the end point, the e.m.f. of the reaction system was measured lyith a platinu n electrode as a function of time against a calomel electrode. The results are shown in Figure 1. The curve is not symmetrical and the relatively S ~ O Kdecrease of e.m.f. n i t h time immediatelv after the inflection point, P, may be attributed to inhibition of the enzyme ( 5 ) by the accumulating o-quinone or t o decoinpoqition or polymerization of the o-quinone. This effect does not appear until after the end point, because the o-quinone does not begin to accumulate until the supplv of ascorbic acid is exhanqted.
1.
Differentiation of Equation 1 gives the initial rate of reaction.
(g)t
=
=
~ O E=Oa b
I n order to find the rate constant, KO,a t a given value of Eo, one must therefore evaluate a / b . This is accomplished (4) from a graph of l / & US. l / t . Since b l 1 Q = a j + ; 1
i 70A
I
t
(3)
the reciprocal of the slope of this graph will give KoEO. T o obtain values of Q and t, a given amount of ascorbic acid, &, is added to the reaction mixture and the time, t , to oxidize this amount is measured. T o determine the time when the ascorbic acid is oxidized, the reaction mixture is siphoned dropwise through a capillary tube into an outside starch iodide indicator solution. When the ascorbic acid is all oxidized, the o-quinone oxidizes the iodide ion to iodine and produces a localized momentary violet flash in the outside indicator. (Permanent iodine-starch color is not obtained until after oxidation of the small amount of ascorbic acid siphoned into the test solution prior t o the end point.) T h e advantages of this method over manometric methods have been discussed (4). I n the present paper is described a potentiometric method for determination of the end point and a slightly different treatment of the data t o obtain the enzyme activity. This method eliminates the inconvenient outside indicator and does not depend upon the skill of the operator. I n addition, it can be used for smaller amounts of ascorbic acid than the visual end-point method. K i t h the new method it is possible to determine automatically and to register the elapsed time when the end point has been reached.
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130 16
32
SECONDS
I 40
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64
Figure 1. E.m.f. of reaction cell as function of time Data obtained from a recording potentiometer. Reaction mixture. 10 r r d . of chronometric buffer*, 500 mg. of catechol, 1 mg. of ascorbic acid. and 1 nil. of mushroom juice enzyme2 in a total volume of 100 ml. Reaction initiated by adding enzyme
The equipment used in the potentiometric determination of the end point is shown in Figure 2. It consists essentially of a potentiometer, adjustable by R1 and R3,and a sensitive mechanical relay adjustable b y R4. The potentiometer is balanced against the reaction cell (shoTn in Figure 3) while the cell contains a solution consisting of the chronometric buffer (4), ascorbic acid, and dehydroascorbic acid (obtained from Bios Laboratories, Inc., to obtain an approximate initial e.m.f. setting for the potentiometer. The dehydroascorbic acid is added to give a better poised system. I n practice it has been found that day-old solutions of ascorbic acid or fresh so!utions of ascorbic acid with a little catechol added contain enough oxidized material
916
V O L U M E 2 7 , N O . 6, J U N E 1 9 5 5
917
- - -, - - - - - - -
equilibrium. The reaction is started by adding enzyme, and simultaneously switch 82 is closed. This allows current to flow through the normally closed contacts of relay RL1 to run the clock. When the voltage unbalance, as determined by resistance Rd between the reaction cell and potentiometer, reaches a large enough value to produce a 2-pa. current through relay RL1, the contacts of this relay close and this activates relay RL2, which stops the clock.
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RESULTS
A typical set of results is given in Table I with the corresponding curve in Figure 4. A correction called the mixing time, which applies to thevisual method as well ( I ) , has been subtracted from the experimental times. Relay RLI is slow acting, which ll0V. AC
Figure 2.
Circuit of equipment
SI. Double-throw double-pole switch RLi. Weston mechanical Sensitrol relay with sensitivity of 2 @a.and normally open contacts RL2. Electronic relay with normally closed contacts S2. Toggle switch K . Key GI. Galvanometer with sensitivity o f 0.02 Fa. per division (mm,) Ri. 200-ohm variable potentiometer Rz. 300-ohm fixed resistor Ri. IO-ohm variable potentiometer Rd. 50,000-ohm variable resistance Electric clock reads to 0.1 second.
I t o be sufficiently poised. ;ifter the initial e.m.f. is set approximately by means of R1and R3,the resistance R4is set to determine the end point time. The potentiometer need only be set approximately, because the final adjustment made with R4 determines the end point time. Resistance Rg is adjusted to make the end point time as measured b y the potentiometric method coincide with the time measured by the visual end point method. If actual catecholase units (4)are not required, either R4 is adjusted so that the end point occurs a t the time when the potential changes i-erv rapidly as observed with a milliviolet meter, or R4 is aajusted so that the relay acts when the potential is changed bv 30 to 50 mv. The exact e.m.f. a t the end point is not important, as long as the same value is used in all measurements, because the time nil1 always correspond to the same percentage of the time for the true end point, and the enzyme activity will be numerically different but proportional to the activity found by the visual method. This difference is of no consequence, as the enzyme activity is usually only a relative quantity. Since the curve in Figure 1 is steeper before the inflection point than after, it is better to select a potential difference less than 50 mv. for greater precision. METHOD
A diagram of the circuit for the automatic determination of the end point is shown in Figure 2 . Before any activity measurements are made a poised ascorbic acid solution is placed in the cell and switch 81 is placed in t h e b a l a n c e p o s i t i o n (Ba1.l The cell is then balanced against the 6-volt storage battery by means of resistanres R1 and Ra,using the key, K , and the galv a n o m e t e r . This adjustment may then be kept until the salt bridge or any other critical part of the cell is changed. When an actual run is made, switch 81 is placed in the run position. -4fter the solution is placed in the cell, 2 minutes are allowed for the cell to come t o
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!
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40
80
,
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I20
160
SECONDS
Figure 4 .
Ascorbic acid oxidized at various times
D a t a obtained by automatic chronometric method. Reaction mixtures, except for varying ascorbic acid, same a s in Figure 1. Intercept on time axis is mixing time, .M
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I 40
80
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I20
I 160
t (secsj
Figure 5 .
Determination of initial rate
Relation between t / Q and time t ; t is measured time corrected for mixing time, and Q is amount of ascorbic acid oxidized. D a t a from Table I
Table I. Typical Results Lsing Potentiometric End Point" Ascorbic Acid, 1Ig.
Q
Figure 3.
Reaction cell
Removable electrode fitted with platinum gauze C. Tubular openings tangential t o jacket D for circulation of constant temperature water E . Sintered-glass bubbler F . Saturated KC1-agar plug for calomel cell 13. Drain Total capacity of t h e cell is a b o u t 160 ml. Large salt bridge between cell a n d calomel electrode is required t o obtain t h a t of a low resistance unit. A calomel electrode of type ordinarily used for determination of p H is too high in resistance
T
Corrected Timeh, Seconds t
l/Q
26 2 1 7 17 0 1 26.8 2 3 23 1 0 4.5 2 20 9 20 9 1 0 47.4 22 9 22 9 10 46.5 22 0 22.0 2 0 75 1 50 6 25.3 2 0 74.5 50 0 25 0 2 0 24.4 73.2 48 7 3 0 113.2 29.6 88 7 29.7 89 2 3 0 113.7 116 8 4 0 171 3 36.7 148 2 37.1 4 0 172.7 36.7 4 0 171.4 146 9 From plot in Figure 5 , h / a = 18.8 seconds/ing. of ascorbic acid. Enzyme activity = 381/18.8 = 20.2 catecholase units ( 4 ) or 0.018 millimole per minute per ml. The factor 381 converts results from sec./ma. t o catecholase units ( 4 ) . a Reaction mixtures as described in legend for Figure 1 , except for varying amounts of ascorbic acid. b4 !. = mixing time = 24.5 seconds = intercept on time axis in Figure 4. t = T - hl = corrected reaction time. 0.1
A.
Observed Time Seconds
ANALYTICAL CHEMISTRY
918 gives a large "mixing time" (1). This is of no disadvantage, because the mixing time map be measured accurately by using small amounts of ascorbic acid. Results in Table I show data for evperiments a t one enzyme concentration, with different amounts of ascorbic acid. The reproducibility of replicates is seen to be very good, showing the adequacy of the automatic end-point method.
method is advantageous, as a is large, the slope is small, and the intercept is easy t o determine. Figure 5 shows the expected straight line relationship. The intercept b/a is 18.8 seconds per mg., which corresponds to an enzyme activity of 20.2 catecholase units or 0.018 millimole (of ascorbic acid oxidized) per minute per milliliter as shown in Table I. REFERENCES
TREATMENT OF DATA
The treatment of the data in Table I is presented here to show a modified method of calculating the enzyme activity. The initial rate, KO& = a / b , is evaluated from the data by means of a plot of t / Q us. t according to Equation 4 (which is another form of Equation 3) rather than from a plot of 1/Q us. l / t ( 4 ) .
t
b
t
(1) Asimov, I., and Dawson, C. R., J . A m . Chern SOC.,72, 820 (1950). (2) Gould, B. S., Enzymologia, 7, 292 (1939). (3) Ludwig, B. J., and Xelson, J. AI., J . -4m. Chem. SOC.,61, 2601 (1939). (4) Miller, W. H., Alallette, 11.F., Roth, L.J., and Dswson, C. R., Ibid., 66,514 (1944). (5) Ponting, J. D., Ibid., 76, 662 (1954). RECEIVED for review November 26, 1954. Accepted February
Q=a+u
It is evident that t / Q is equal to b/a when t = 0. This plotting
14, 1935.
Mention of specific products does not imply t h a t they are endorsed or recornmended by the Department of Agriculture over others of a slmllar nature not mentioned.
Determination of Small and large Amounts of Fluorine in Rocks F. S. GRIMALDI, BLANCH INGRAM, and FRANK CUTTITTA
U. S.
Geological Survey, Washington 25,
D. C.
Gelatinous silica and aluminum ions retard the distillation of fluorine in the Willard and Winter distillation method. A generally applicable, simple method for the determination of fluorine in rocks containing aluminum or silicon or both as major constituents was desired. In the procedure developed, the sample is fused with a mixture of sodium carbonate and zinc oxide, leached with water, and filtered. The residue is granular and retains nearly all of the silica. The fluorine in the filtrate is distilled directly from a perchloric acid-phosphoric acid mixture. Phosphoric acid permits the quantitative distillation of fluorine in the presence of much aluminum at the usual distillation temperature and without the collection of large volumes of distillate. The fluorine is determined either by microtitration with thorium nitrate or colorimetrically with thoron. The procedure is rapid and has yielded excellent results on silicate rocks and on samples from the aluminum phosphate (leached) zone of the Florida phosphate deposits.
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N T H E determination of fluorine it is common practice to isolate fluorine as fluosilicic acid by some modification or adaptation of the Willard and Winter distillation method ( 1 1 ) . It is known that gelatinous silica or large amounts of aluminum, or both, as well as other elements of the ammonium hydroxide group, retard the distillation of fluorine. For example, with the usual distillation temperature of 135" to 140" C. and with the collection of about 150 ml. of distillate, fluorine is not completely recovered in the presence of more than about 50 mg. of silicon dioxide in the gelatinous form ( 7 , 9, 11) and about 20 mg. of aluminum oxide. The interference from these sources is especially serious for silicate rocks, because silicon and aluminum are major constituents, and large samples are required for determination of small quantities of fluorine, about 0.0301,. The analyst is confronted with these same difficulties in the determination of fluorine in samples from the aluminum phosphate (leached) zone of the Florida phosphate deposits. It has been reported ( 2 , 11) that better recovery of fluorine is obtained by distilling a t higher temperatures (160" to 165' C.)
and by collecting larger volumes of distillate. It is the authorq' experience, however, that such procedures fail to give quantitntive recovery of fluorine on such samples as described above. Up t o the present time the best approach has been to separate fluorine from silica and from the elements of the R203group before the distillation step. Some adaptation of the Berzeliua method is usually used for this separation, silica and aluminum being precipitated by zinc oxide from a nearly neutral or slightli ammoniacal solution ( 4 , 11). This procedure is time-consuming. and there is danger of loss of fluorine by coprecipitation on the large gelatinous precipitates obtained. Recently Shell and Craig (9) described a procedure primarily designed for the simultaneous determination of fluorine and silica in JThich a flu.: mixture of sodium carbonate and zinc oxide is used for decomposing silicate samples. This feature represents an important advance, because a granular precipitate retaining most of the silica is obtained when the melt is leached with water. The behavior of aluminum in the fusion depends on the composition of the sample being analyzed, and frequently as much as 95% of the aluminum will be found accompanying fluorine in the IT ater leach. Where the aluminum cannot be tolerated, it must be separated. This is ordinarily taken care of in Shell and Craig's procedure, where ammoniacal zinc oxide is added directly and prior t o the filtration of the water leach of the melt, in order to recover most of the dissolved silica. This investigation was undertaken to develop a generally applicable, simple method for the determination of both small and large amounts of fluorine in rocks, especially those containing aluminum and silicon as major constituents. Independent of Shell and Craig, the authors arrived a t a similar flux mixture for the decomposition of silicate rocks, and this feature of the procedure is the same as that of Shell and Craig. After the sample is fused, leached with water, and filtered, the fluorine in the filtrate is distilled directly from a perchloric acid-phosphoric acid mi.:ture. Phosphoric acid permits the quantitative distillation of fluorine in the presence of much aluminum a t the usual distillation temperature and viithout the collection of large volumes of distillate. The fluorine in the distillate may be determined either by microtitration with thorium nitrate or colorimetrically by a modification of the thoron [2-(2-hydroxy-3,6-disulfo-l-naphthylazo)-
