INHIBITION OF CARBONIC ANHYDRASE BY THIOCYANATE A Demonstration ROBERT I. MCGEACHIN University of Louisville School of Medicine, Louisville, Kentucky
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of a student experiment in biochemistry TABLE 1 demonstrating the inhibition of carbonic anhydrase Ml. qf Reaction time activity, it was deemed of interest to show the inhibition blood (seed.?). by thiocyanate which was first reported by Davenport 0.10 1.7 (1) and later menhned by others (8, 9, 4). Since the 0.08 1.7 "boat" method of Meldrum and Roughton (5), for 0.06 1.7 carbonic anhydrase was too cumbersome and compli0.04 1.8 0.02 2.8 cated for general student use, a modification of the 0.01 3.7 0.005 8.4 methods of Brinkman (6) and of P h i l ~ o tand P h i l ~ o t (7) was adopted as a simbler procedure. This method 0.003 ma* 9.? """l 11.1 measured the rate of formatibn of carbonic acid from "Averages of duplicate or triplicate determinations. dioxide and wateras catalyzed by carbonic an. hvdrase. To 25 ml. of distilled water in a large test tube was added one ml. of five per centsodium bicarbonate, one cent sodium bicarb0nat.e instead of one ml. This variaml. of 0.02 per centbromthymol blue, and one ml. of the tion proved unsatisfactory since theend points were then inhibitor solutioll (omitted in controls). hi^ mixture much less sharp than with the lower bicarbonate concentrations. Phenol red was tried in place of bromwas cooled to 4" C. in an ice bath. ~h~ carbonic hydrase, in this case in the form of human blood, thymol blue but again end points were not sharp. The although blood from other animals would be suitable, Presence of blood interfered more with phenol-red end was added after the cooling. Carbon dioxide from points than with bromthymol-blue. However, reaction cylinder fitted with a reducing valve was bubbled times with phenol red seemed to fall in abont the same through the mixture and the time required to reach range as those with bromth~molblue. From the tables one can see that it was possible under a color change from blue to yellow-green was measured. hi^ reaction tirne is an inverse function of carbonic an. some conditions to have considerable inhibition of carhydrase activity. Since the reaction tirne will vary bonic anhydrase without influencing the rate of the with the rate of buhbliug, the carbon dioxide was ad- reaction it was catalyzing. In Table. 1 it is apparent mitted at the maximal rate that could be achieved that 0.1 ml. of blood was a lalge excess over that without blowing the reaction mixture out of the test amount necessary to achieve maximal reaction rates. tube. Blank reaction times (i. e., no blood added) The limiting factor with excess blood may have been the rate of diffusion of carbon dioxide from the bubbles under these conditions were almost invariably 11 1 seconds. A Kipp generator using marble chips and into the solution. Not until the amount of blood was reduced below 0.03-0.04 ml. was there any detectable hydrochloric acid can be used as a source of dioxide, but the reaction times are longer and not as change in the reaction time. If, as was done in this experiment when it was first tried by students, the reproducible as those using the cylinder. Varying amounts of blood were used to test the amount of blood used were 0.1 ml. and the data on the method and obtain a standard curve, typical results of which are shown in Table 1. TABLE 2 To test the effect of thiocyanate, one-ml. portions of R e d i o n Time in Seconds one per cent and five per cent solutions of potassium Added 0.1 ml. blood 0.01 ml. None thiocyanate were added under varying conditions. See typical results in Table 2. A trial with Dirnate, an No SCN 1.7 3.9 10.7 exceptionally potent carbonic anhydrase inhibitor (8), 1% SCN 2.6 8.5 .. .. 3.8 9.9 5% SCN was run for comparative purposes. 1%Dirnrttea 10.9 .. .. In an attempt to extend the reaction times and thus reduce the percentage error in measurements of reaction Bii:t,w~ m d Dohme trade mmefor pcarboxybenzenesdfontimes, trials were made using two-ml. portions of five per PART
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standard curve were not available, it would appear that thiocyanate had very little inhibitory action on carbonic anhydrase since the difference in reaction times was small compared t o the difference with the blank. However, comparing results in the two tables, the reaction time for 0.1 ml. of blood with one per cent thiocyanate added was the same as that for 0.02 ml. of blood, indicating about 80 per cent inhibition. The time for 0.1 ml. of blood with five per cent thiocyanate added matches that for 0.01 ml. of blood, showing about 90 per cent inhibition. Note that even with such inhibition the differences in reaction times were small compared to the differences with the control times. When the amount of blood used was reduced to 0.01 ml., the inhibiting effect of thiocyanate solutions was immediately apparent since the differences in reaction times were appreciable. Reaction times with inhibitor present approached that of the blank. It should be noted that Davenport ( I ) used chloro-
JOURNAL OF CHEMICAL EDUCATION
form extracts of red blood cells as a source of carbonic anhydrase, whereas this work used whole blood. He used a carbonic-anhydrase assay method which measured manometrically the evolution of carbon dioxide from carbonic acid (5). The method described here meamres the reverse reaction, the formation of carbonic acid from carbon dioxide and water. LITERATURE CITED (1) DAVENPORT, H. W., Am. J . Physiol., 129, 505 (1940). (2) FELDBERG, W., D. KEILIN,AND T. MANN,Nature, 146, 651 (1940). AND E. B. HART,J. Bid. Chem., (3) HOVE,E., C. A. ELVEHJEM, 136, 425 (1940). (4) DRIVER, R . L., Am. J. Physiol., 135, 330 (1941). (5) MELDRCM, N. U., AND F. 3. W. ROCGHTON, J. Phvsiol. (London), 80, 113 (1933). (6) BRINKMAN, R., ibid., 80, 171 (1933). F. J., AND J. ST. L. PHILPOT, Bwchem. J. (London), (7) PHILPOT, 30, 2191 (1936). (8) KREBS,H. A.. ibid., 43, 525 (1948).
