Conductometric Determination of Enzyme Activity B. N. SASTRI
AND
M. SREENIVASAYA, Indian Institute of Science, Bangalore, India
T
H E possibility of utilizing Changes in electrical conductivity acThe third group is represented by changes in electrical conduccompanying the enzyme hydrolysis of urea, carbohydrases and the majority of glucosidases which, acting tivity for measuring enzyme acarginine, and peptone have been measured. on their respective substrates, tivity has been shown by the The changes are shown to be strictly prorelease sugars. The reactions workof Sjoquist ( I I ) , Oker-Blom portional to the release of ammonium carbelonging to the first group are (IO),Henri and des Bancels ( 6 ) , bonate in the first two systems and of obviously best suited for cona n d B a y l i s s (1, 2). More recently, Northrop (7, 8, 9) in amino groups in the third. The conductod u c t o m e t r i c studies* The second group reactions offer certhe course of his studies has utimetric method finds application in the detain difficultiesand impose cerlized the method for (1) estimattermination of enzyme and substrate contain limitations but can nevering pepsin, (2) determining the centrations in various physiological fluids. theless be followed by employing ionization of egg albumin-hydroa sensitive device. The third chloric acid with a view to elucigroup reactions which, for the present, lie beyond the scope of dating the dependence of hydrolysis on substrate ionization, these studies, could be brought within their scoDe by taking and (3) studying the kinetics of tryptic digestion in relation advantage of the fact that, ?n presence of borates, &senates, to its mechanism. Euler (5) has employed the method for and molybdates, polyhydric alcohols like sugars exhibit an studying the hydrolysis of glycylglycine. Bayliss (3) has sugincrease in their electrical conductivity. gested the possibility of employing electrical conductivity for The strongest objection put forward against the employinvestigating the action of urease, lipase, and the glucosidase, ment of this method is that the reactions cannot be studied in myrosin, but does not report any investigation relating to buffered systems. For determining enzymic activities, we these systems. are concerned essentially with the course of action in the For purposes of conductometric estimation, enzyme reacinitial stages when the disturbing factors, due not only to the tions can be classed as (1) those that release strong elecchanges of reaction but also to the accumulation of products trolytes, (2) those that release feebly ionizing ampholytes, of hydrolysis, are a t a minimum. During this period the and (3) those that give rise to what are conventionally known conductivity method is perhaps the only one which could be as nonelectrolytes, like sugars. Urea-urease, sinigrin-myrosin, applied with any advantage, since it is capable of giving a and methylglyoxal-glyoxalase are among those comprising the large number of values for the early stages of the reaction. first group, while the protein-proteolytic systems, resulting Further, there are reactions where very little change occurs in the production of amino acids, belong to the second group. in the p H of the medium; thus, SO r Northrop (9) found that a t pH 6.2 to 6.4 the h y d r o l y s i s of 5gelatin by trypsin is not accompanied by any a l t e r a t i o n in pH. The presence of e l e c t r o l y t e s in d 4rn small concentrations as impurities in the r e a c t i n g mixture does not interfere with the measurements, since appropriate conductivity cells can be chosen to give the desired -'2. accuracy. In common with other physicalmethods, conductivity measurements have 1 the advantage of being c a r r i e d out without disturbing the course of action and can be made FIGURE1. CONDUCTOMETRIC METHOD 0 1 0 2 0 3 0 0 applicable to very A . Urea-urease small quantities of B. Arginine-arginase-urease C. Peptone-trypsin-kinase substrates. FIGURE2. CHEMICAL METHOD D . Salioin-emulsin 458
NOVEMBER 15,1936
ANALYTICAL EDITION
VR FIGURE 3. CORRELATION GRAPHS Experimental The present communication deals with a conductometric study of urea-urease, arginine-arginase-urease, peptonetrypsin-kinase, and salicin-emulsin. For the measurement of conductivity the usual Kohlrausch bridge method was employed. A calibrated Kohlrausch slide wire, a 4-dial resistance box, and an Arrhenius-Ostwald cell formed the components of the circuit. A 5-cc. aliquot of the substrate solution was all that was necessary for the work. Platinized electrodes were employed and the cell was immersed in a water thermostat electrically maintained at 30.0’ * 0.1’ C. The audio oscillator (General Radio Co.) giving an alternating current with a frequency of 1000 was employed as the source of highfrequency current and a telephone receiver was used for detecting the null point. The capacitance of the conducting cell was balanced by means of an air-condenser which was connected in parallel with the resistance box. Readings were taken at short intervals a t the commencement of the reaction and later at longer intervals. Experiments with larger quantities were simultaneously carried out in a separate reaction vessel, the same relative enzyme-substrate concentration being employed. Aliquots of the reaction mixture, withdrawn from the reaction vessel at definite intervals, were analyzed and the course of the reaction was thus followed by an entirely independent chemical method. UREA-UREASE.A 1 per cent solution of urea (Kahlbaum) and an aqueous solution of soya urease precipitated by acetone were employed. The release of the ammonium carbonate was followed by the method developed by Sastri in 1935 (4,which consists in titrating aliquots of the reaction mixture in the presence of acetone against standard alcoholic hydrochloric acid (0.1 N ) . ARQININE-ARQINABE-UREASE. Arginine carbonate was prepared from &arginine, a solution of 0.5 per cent being saturated with carbon dioxide. The excess of carbon dioxide was removed by bubbling electrolytic hydrogen through the solution. The solution of arginine carbonate thus obtained showed a steady value for conductivity. An aqueous extract of the acetoneprecipitated liver extract of a ram was employed as the source of arginase. By using an excess of urease an almost instantaneous hydrolysis of the urea, progressively released during the decomposition of arginine, was secured so that the reaction mixture at any one time contained only ornithine and ammonium carbonate. The quantity of ammonium carbonate in the reaction mixture was estimated at different intervals of time by the titrimetric method referred to above. PEP’rONE-TRYPSIN-KINASE. A 1 per Cent aqueous SOlUtiOn of Witte’s peptone (B. D. H.) and 1per cent solution of Pfanstiehl’s preparation of trypsin-kinase were employed. The proportion of the substrate to enzyme was 10 to 1. The amino groups released during the hydrolysis were estimated by Linderstrgm Lang’s titrimetric method. SALICIN-EMULSIN. A 1 er cent solution of salicin and an D. H.) were used. The sugar reaqueous extract of emulsin leased was estimated by Bertrand’s method.
&.
Discussion The measurements, both conductometric and chemical, were carried out for at least two enzyme concentrations. The results of one set of experiments are graphically represented in Figure 1 (conductometric) and Figure 2 (chemical).
459
The general shape of the two corresponding sets of curves indicates t h a t the kinetics of these enzyme reactions can be followed conductometrically. The close relation between the physical and chemical methods is clearly brought out by the correlation graphs (Figure 3) prepared according to the method previously described (1.2). In the two systems urea-urease and arginine-arginase-urease, the change in conductivity is essentially due to the release of ammonium carbonate and this is strikingly brought out in the graphs. The correlation graphs (Figure 3) for urea-urease and argininearginase-urease are practically identical. The change in electrical conductivity per millimole release of amino acid can be calculated in the peptone-trypsin system also; there is a correlation between the change in conductivity and the release of amino groups. For the study of the salicin-emulsin system, however, the chemical method is by far the more accurate, since the changes in conductivity accompanying the system, although easily measurable and reproducible, are very slight in magnitude, The possibility of increasing the conductivity of the sugar released by the addition of borates is being investigated. At the moment the conductometric method, so far as sugar-releasing systems are concerned, offers no special advantage over the chemical method.
Literature Cited (1) Bayliss, Arch. sci. biol. (U.S. S. R.),11, 261 (1904). (2) Bayliss, J. Physiol., 36, 221 (1907). (3) Bayliss, “Nature of Enzyme Action,” p. 51, London, Longmans, Green & Co., 1925. (4) Dastur et al., Proc. I n d i a n Acad. Sci., 1, 867 (1935). (5) Euler, Vet. A k a d . Arloiv. Kemi., 2, No. 31 (1906). (6) Henri, Victor, and Bancels, Larguier des, Compt. rend. soc. biol., 55, 563 (1903). (7) Northrop, J. Gen. Physiol., 2, 113 (1920). (8) Ibid., 3, 211 (1921). (9) Ibid.. 4. 227 (1922). (ioj OkerLBlom, Skand. Arch. PhUsiOl., 13, 359 (1902). (11) Sjoquiat, Ibid., 5, 277 (1895). (12) Sreenivasaya et al., Biochem. J.,28, 351 (1934). RECEIVED June 20, 1936.
Correction In an article entitled “The Sampling and Analysis of Eggs”
[IND.ENQ.CHEM.,Anal. Ed., 8, 377 (1936)l the authors submitted a glycerol and a salt method with the statement that “here no A. 0. A. C. method applies,” and it has been called to our attention that this would imply that no A. 0. A. C. method is available for these analyses. This is not the case. The A. 0. A. C. method for chlorine on page 249 of the third edition of the “Methods of Analysis,’ of the association may be revised for the determination of salt in quantities such as are present in salt yolk. However, the time required for a series of determinations by this method is over 12 hours as against 10 minutes for the method we published. Similarly a method for glycerol published [ J . Oficial Agr. Chem., 15, 334 (1932)l and tentatively adopted [Ibid., 16’74 (1933)l requires 6 hours for six determinations as contrasted with 1.5 hours for a similar number of determinations by our method. Since our purpose, as mentioned in the third paragraph of our paper, is to give rapid methods which yield reproducible results, we omitted reference to the above A. 0. A. C. methods which are not suitable for plant control and trust that no ambiguity will have arisen because of such an omission. W. S. GUTHMANN W. L. TERRE
