Carbonic Anhydrase in the Titration of Carbon Dioxide Solutions A. L. UNDERWOOD Department of Chemistry, Emory University, Atlanta 22, Ga.
b Because most of the carbon dioxide in a solution of this gas exists as COZ and not as carbonic acid, and because the hydration of dissolved carbon dioxide i s slow, the titration of carbon dioxide solutions i s tedious and the end point i s uncertain. The addition of the enzyme carbonic anhydrase, which is known to accelerate the hydration of carbon dioxide, permits rapid titration to a permanent endpoint. This shortens the process and renders the end point less subjective, thereby improving the precision of the titration. Quantities of enzyme too small to contribute a noticeable titration blank are completely effective.
T
HE ALKALIMETRIC TITRATION of carbon dioxide solutions is important in certain fields such as water analysis. The chemistry of carbon dioxide solutions has recently been reviewed by Kern ( 2 ) , and the titration has been discussed thoroughly by Kolthoff and Stenger ( 3 ) . For the reaction CO, HzO = H&03, the equilibrium constant, [HzC03]/ [COe][H20], is rather small Thus most of the carbon dioxide in a solution of this gas exists as COP, not as carbonic acid. Further, the hydration reaction of carbon dioxide is slow. For these reasons, the titration of carbon dioxide solutions is tedious and the end point uncertain. As base is added t o the solution, carbonic acid is consumed but its replacement through hydration of more carbon dioxide is slow. This gives rise to a series of premature phenolphthalein end points which fade more and more slowly as the true end point is approached. It is recommended (3) that one accept as the end point a pink color that persists for 5 minutes. I n our experience, it may take 15 to 20 minutes t o complete a single titration if one is careful in this regard. Various substances catalyze the reaction COP HPO = H,C03 (6); for example, fairly large concentrations of phosphate are effective. However, such materials will obviously interfere h-ith the titration itself, and the enzyme carbonic anhydrase, which is a much more active catalyst, appeared more promising. This enzyme occurs in
+
+
relatively large amounts in mammalian erythrocytes, where it plays a n important role in the transport of carbon dioxide by the blood, and in the parietal cells of the gastric mucosa, nhere it is probably important in the secretion of hydrochloric acid by the stomach. Carbonic anhydrase is a zinc-containing protein whose properties have been rather thoroughly reviem ed in the biological literature ( I , 6, 8, 9). It is available in a purified form n i t h high enzymatic activity from suppliers of biological chemicals; alternatively, the preparation of a n active fraction from bovine red cells is not difficult ( I , 9). Actually, Roughton and Meldrum stated in 1933 that carbonic anhydrase was beneficial in carbonic acid titrations ( 7 ) . But this observation apparently escaped the attention of analysts generally, probably because it appeared only in the patent literature, although it was briefly mentioned again in a review by Roughton and Clark in 1951 (6). Thus it appears useful t o describe our recent experience with carbonic anhydrase. EXPERIMENTAL
The carbonic anhydrase was obtained from Worthington Biochemical Corp., Freehold, K. J. This product is prepared from beef blood by the procedure of Keilin and hlann ( I ) and assays about 700 enzyme units per mg. by the method of Wilbur and Anderson ( I O ) . A similar preparation from Pentex Inc., Kankakee, Ill., was also satisfactory. An aqueous solution containing 1 mg. per ml. was used in this nork. The dry material appears to keep indefinitely in the refrigerator, but because solutions may be unstable in varying degree depending upon their purity and other factors, fresh solutions nere prepared as needed. The enzyme is readily soluble in water. To study the reproducibility of titrations with and without the enzjme present, a carbon dioxide solution mas required whose concentration would remain reasonably constant for a n hour or two during which ahquots nould be withdrawn and titrated. X satisfactory method was to maintain a stock solution in equilibrium with carbon dioxide a t 1 atm. at room temperature by the continuous bubbling of carbon dioxide through a previously saturated solution, The carbon dioxide was led from a dry
ice flask through a 10-foot coil of copper tubing and then into the solution through a gas dispersion tube. The copper tubing permitted sufficient heat exchange to bring the gas to room temperature and prevent cooling of the solution. A 100-ml. portion of the saturated carbon dioxide solution was placed in a n Erlenmeyer flask, 0.1 ml. of the enzyme solution and 0.3 ml. of a 0.5% solution of phenolphthalein in 50% ethyl alcohol were added, and the solution was titrated with 0.1M sodium hydroxide t o the first permanent pink. To minimize loss of carbon dioxide, the base was added as rapidly as possible and the solution was not stirred until the latter stage of the titration. I n the cases where the enzyme was not used, the titrations were performed according to Kolthoff and Stenger (3)-i.e., the end point was taken when the pink color persisted for 5 minutes. RESULTS
The outstanding fact is the speed with which the titration can be performed in the presence of carbonic anhydrase. When 0.1 mg. of the enzyme is added t o 100 ml. of carbon dioxide solution, the titration can be completed as rapidly as one can perform the manipulations. There is no fading of the pink color, and the titration takes no longer than a similar titration of, say, hydrochloric acid, perhaps 1 or 2 minutes. Quantities of less than 0.1 mg. of carbonic anhydrase shorten the titration appreciably , but with the enzyme preparation used here, 0.1 mg. was roughly the minimum required for a near-instantaneous reaction. There is no advantage in using larger quantities of the enzyme. Based upon quotations for gram quantities, purified carbonic anhydrase appears expensive, but the cost per titration is trivial (less than a penny). The titration of 100 ml. of the carbon dioxide consumed about 40 nil. of 0.1JP sodium hydroxide. Twelve titrations performed seriatim showed an average deviation of 0.26 ml. (7.0 parts per thousand) and a standard deviation of 0.32 ml. (8.6 parts per thousand). A corresponding series without the benefit of the enzyme showed a n average deviation of 0.45 ml. (11.4 parts per thousand) and a standard deviation of 0.58 ml. VOL. 33, NO. 7,JUNE 1961
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(14.7 parts per thousand). It appears, then, that the precision of the titration is improved by carbonic anhydrase. The enzyme naturally does not enhance the sharpness of the end point, only the rate a t which it is attained, but this faster rate is very helpful in titrating labile solutions. The quantity of protein introduced in these experiments did not add a detectable blank to the titration. The effect of carbonic anhydrase qpon the titration of carbonate with hydrochloric acid to both the phenolpbthalein and the methyl orange end points was also investigated. We were able to perceive no advantage gained by using the enzyme over titrations performed with reasonable care in its absence. Like other protein enzymes, carbonic anhydrase may be inactivated under
certain conditions. For example, it is destroy::: by 30 minutes’ heating a t 65” C., and it is unstable dt pH values above 13 and h e l o w 3 (4,6,8).The pH a t whicll carbonic anhydrase exhibits maximal catalytic activity is not clearly stated in the literature, but fortunately the activity is high in the pH region of importance in the carbon dioxide titration. Certain substances act as inhibitors of carbonic anhydrase; among these are copper, silver, gold, mercury, zinc, and vanadium salts, as well as sulfides, cyanides, thiocyanates, and azides (4,6,8). LITERATURE CITED
(1) Keilin, D., Mann, T., Biochem. J . 34,
1163 (1940).
(2) Kern, D. M., J . Chem. Educ. 37,
14 (1960). (3) Kolthoff, I. M., Stenger, V. A,, “Volumetric Analysis,” 2nd Ed., Vol.
11, p. 131, Interscience, New York,
1947. (4) Meldrum, N. U., Roughton, F. J. W., J . Physiol. 80, 113 (1933). (5) Roughton, F. J. W., Booth, V. H., Biochem. J. 32, 2049 (1938). (6) . . Roughton. F. J. W.. Clark. A. SI..
in Sumner, ‘J. B., Myrback, K.,Eds.; “The Enzymes: Chemistry and Mechanism of Action,” Vol. I, Part 2, p. 1250, Academic Press, New York, 1951. ( 7 ) Roughton, F. J. W., Meldrum, N. U., Brit. Patent 403,096,p. 6, clauses 60 to 100, (Dec. 1, 1933). (8) Vallee, B. L., in Anson, M. L., Bailey, K., Edsal, J. T., Eds., “Advances in Protein Chemistry,” Vol. X, p. 333, Academic Press, New York, 1955. (9) Waygood, E. R. in Collqwick, S. P., Kaplan, N. O., Eds., Methods of Enzymology,” Vol. 11, p. 836, Academic Press, New York, 1955. (10) W.ilbur, K. M., Anderson, N. G., J . Bzol. Chem. 176, 147 (1948).
RECEIVED for review Kovember 21, 1960. Accepted March 6, 1961.
Particle Size Measurements with an Improved Contin uo us1y- Recording Sed imenta t ion A pparatus EMlL S. PALIK Chemical Products Plant, lamp Metals and Components Department, General Nectric Co., Cleveland, Ohio
b An improved continuously-recording sedimentation apparatus has been developed and its performance characteristics compared with other methods of particle size analysis. A comparison of the apparatus with two sedimentation methods shows acceptable agreement for a fluorescent powder whereas comparison with a method utilizing changes in electrolytic resistivity shows a significant difference. The apparatus has been found useful in the particle size analyses of fluorescent powders and may be used to analyze other powders with a size distribution in the range of 2 to 60 microns.
S
SEDIMENTATION balances for the measurement of particle size have been reported in the literature. The earliest form of this a p paratus was described by Oden ( I S ) who employed an analytical balance for weighing particles settling out of suspension, The Oden balance was modified by Coutts and Crowther (6) into a continuously-recording instrument by incorporating electromagnetic weighing and control with the original method of adding small weights a t the requisite times. Later, Bostock (6) substituted a torsion wire for the conventional beam balance to detect the weight of particles settled, but his design did not provide for automatic continuous weighEVERAL
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ANALYTICAL CHEMISTRY
ing of the particles. More recently, emphasis has been placed on developing automatic recording balances utilizing a variety of transducers (1, 3, 8, 12) with which to convert weight changes into an electrical signal suitable for amplification and recording. The present paper describes a sedimentation apparatus, referred to hereafter as the sedimeter, in which the sensitive springoptical arrangement of Rabatin and Gale (1’7)has been modified by the use of a torsion balance. The convenient feature of the optical transducer has been retained in the present design. Similar applications of the torsion balance principle have been described by Rabatin and Card (16)and also by Avgustinik and Dzhansis (2) to detect weight changes in thermogravimetric analyses and surface area measurements. The above modification together with additional changes in design resulted in a recording sedimentation apparatus having improved performance over the original spring design. This paper reports the first known application of the torsion-optical principle to the measurement of the settling rate of fine powders. APPARATUS
Construction. A diagrammatic sketch of the sedimeter is illustrated in Figure 1. The light source, 1, consists of a 6.3-volt tungsten filamentlamp
operating from a constant voltage transformer mounted in the housing directly behind the instrument panel, 3. The light originating a t 1 passes through a fixed slit, 2. The collimating lens, 4, is a double convex lens with a 15-cm. focal length. The torsion balance, 5, is of standard design except that it has been modified by employing more sensitive torsion elements. These elements (Sandsteel Mainsprings No. 2125, Watch-Motor Mainspring Co., Inc., N. Y.) have a 1.10 111111. width and 0.17 mm. thickness. The balance is mounted on a platform, 6, and shielded from stray light and air currents. Attached to the left extremity of the balance is an adjustable counter - weight, 7, with which to counterbalance the pan. A stainless steel pan, 8, 6.50 em. in diameter and soldered to a stainless steel stem 20 cm. in length, is attached to the right arm of the balance through an opening in the platform. These dimensions provide for an initial particle settling distance of 11.7 cm. The pan is immersed in an unsilvered, flat-bottomed Dewar settling vessel, 9, having a Lucite cover into which is cemented a thermometer, 10. During sedimentation the thermometer is immersed to a depth of about 2 cm. in the suspension. Although no temperature control is used, the temperature during sedimentation remains a t 25” i 1” C. To the left arm of the balance there is attached a rigid opaque metal shield, 11, 2.5 cm. X 5.5 cm., which intercepts the collimated beam emerging from the fixed slit, 12. The transmitted
