APRIL, 1938
INDUSTRIAL AND ENGINEERING CHEMISTRY
Combining Equations 2 and 17 gives the relation between increase in dielectric constant and increase in water,
2 (3 0.50
=
where and c1 are experimentally determined. This relation is illustrated in Figure 12, the data of which were obtained by combining the two curves in Figure 11. It is obvious that, if the relation between dielectric constant and water content is known, a measurement of dielectric constant alone will suffice to describe the water-sorbing characteristics of dielectric materials. Different rubber compositions differ in the amounts of water they sorb and also in the effect of this water on the dielectric constant. I n either case the curves, as in Figure 11, will be shifted along the ordinate but will maintain their respective slopes. I n Figure 12 a larger increase in dielectric constant per unit quantity of water will shift the curve upward on the graph in the same manner.
Dielectric Constant of Insulated Wire As with sheets, the greatest concentration of water in rubber-covered wire occurs in the outer layer of the insulation, decreasing rapidly toward the center. During the sorption process the insulation expands in volume, usually by an amount approximating the volume of the water taken up. Furthermore, owing to alteration in the structure of rubber by wetting and drying, the time required to reach a given
415
water content will be less during the second immersion of the redried specimen than during the fist. These factors must be taken into account if the calculated behavior of insulated wire is to coincide with the experimental. Consider, for instance, the mutual capacitance of a two-parallel-conductor circularly insulated wire immersed in water. As sorption continues, the diameter of the insulation increases, the spacing between the conductors is altered, and the dielectric content of the insulation varies with the water content. When these factors are properly accounted for and the wire is regarded insulated with graded rather than homogeneous insulation, the calculated and experimental values will check closely over extended periods of immersion.
Literature Cited Andrews, D. H., and Johnston, J., J. Am. Chenz. SOC.,46, 640 (1924). Boggs, C. R., and Blake, J. T., IND.ENQ.CHEM.,18, 224 (1926). Kemp, A. R.,Ibid., 29,643 (1937). Lowry, H.H., and Kohman, G. T., J.P h y s . Chem., 31,23 (1927). Messenger, T.H., and Scott, J. R., J. Research Assoc. B r i t . Rubber Mfrs., 5,No. 11, 121 (1936). Satake, S., J.SOC.Rubber Ind. Japan, 8 , 15 (1935). Soule, K.J., IND.ENQ.CHEM.,23, 654 (1931). Taylor, R.L., Herrmann, D. B., and Kemp, A. R., Ibid., 28, 1255 (1936). Wosnessensky, S., and Dubnikow, L. M., Kolloid-Z., 74, 183. (1 936). RECEIVEDDecember 10, 1937. Presented before the Division of Rubber Chemistry a t the 94th Meeting of the American Chemical Society, Rochester, N. Y . , September 6 t o 10, 1937.
Activity of Lipase a t Low Temperatures
A. K. BALLS A N D I. W. TUCKER
Food Research Division, Bureau of Chemistry and Soils, Washington, D. C.
T
HE practical experience of industries dealing with the
cold storage of foods points to the conclusion that enzyme action occurs a t low temperatures. The researches of Pennington and Hepburn (2) confirmed this view many years ago, as far as the enzymic hydrolysis of fats and similar esters is concerned. Because of the comparatively low temperatures used in food storage today, interest in enzyme action a t low temperature now centers in the demonstration of its occurrence in firmly frozen materials. Experience in this laboratory with proteinases a t low temperatures has shown that protein hydrolysis also occurs in the cold. The rate decreases with the temperature to about the extent that might be expected of any chemical reaction, until a point is reached where the system freezes. Below this point, proteolysis is slowed down very much more and becomes practically undetectable by the methods in use today. The change from liquid to solid must severely limit the sphere of activity of the enzyme by preventing diffusion and thus also preventing new contacts between enzyme and substrate. If, instead of being a solid solution, the substrate consisted of comparatively large discrete particles, the attack of the enzyme might be more successful. The distance between one substrate molecule and the next would be a t a minimum, and the enzyme could perhaps “eat its way through.” Such a system would still be hampered by the accumulation of end products, but it might conceivably show
comparative activity in the frozen state. To test out such an idea on the proteins is a t present not practicable. The lipases, however, are enzymes that attack large discrete particles of their substrates, for even in the finest emulsion t h e siae of the fat particles is enormous compared with any reasonable conception of a lipase molecule. A further investigation of lipase activity in frozen systems therefore seemed worth while, and the results obtained indicate that hydrolysis takes place, with surprising rapidity in some cases. Previous work on the lipase from pig pancreas showed that even under ordinary circumstances the action of the enzyme is greatly dependent on temperature ( I ) . Glycerides of t h e higher saturated fatty acids are not appreciably attacked at. +20° C. Glycerides of the lower saturated fatty acids, on the other hand, are split rapidly. Most rapidly are split the glycerides of the fatty acids containing from 6 to 10 carbon atoms. The “specificity” of lipase appears to vary with the temperature. If the high-molecular fatty acid is unsaturated, however, as is the case in olive oil, the €at behaves as though the acid contained 8 or 9 carbon atoms. The double bond in the center of the oleic acid chain is conceivably the cause of this behavior. Olive oil and the lower fats, such as tributyrin,
INDUSTRIAL AND ENGINEERING CHEMISTRY
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VOL. 30, NO. 4
are therefore very suitable substrates on which to test lipase action a t low temperatures. With a slight modification in technic, the method followed was the same as that previously described (1) except that a small amount of sodium oleate was added to the emulsified olive oil to give the emulsion greater stability:
molecular reaction a t the start and to calculate a velocity constant from titrations made as near the beginning of the digestion as is possible without an unreasonable sacrifice of accuracy. This method of presenting the results seems to be justified by the observation that the hydrolysis of olive oil by lipase follows rather closely a monomolecular course a t ordinary temperatures, when TABLEI. COURSEOF HYDROLYSIS OF OLIVE OIL= the solution is continuously buffered (Table IA). A . Buffered System, Pancreas Ext. 0.08 B. Unbuffered System, Pancreas Ext. 0.33 Cc. per Cc. per Titrationn Titrationb When the system contains olive oil -30’ C . 7 7 2 0 ° C.7 - 3 ’ C.7 - 1 8 ’ C.~ - 2 C.-5 ~ but is not buffered except at the start, k t x k t x t x k t x k t x k 2.8 0.006 6 3.2 0.002 the reaction is mono5 15.4 0.015 5 7.9 0.0071 2 29.0 0.07 2 10 30.2 0.016 10 17.7 0.0085 4 35.2 0.04 6.3 10.0 0.007 24 6.2 0.001 molecular, a t least a t first (Table IB). 20 42.7 0.016 15 28.8 0.0098 I n Figure 1 a n attempt was made 20 36.4 0.0099 t = time in minutes; x per cent hydrolysis, calculated from the total saponificationvalue of the to depict the relative initial rates of - log !loo - x). oil determined by long-continued lipase hydrolysis; k = digestion of olive oil a t different tem- log (loo- z ) . peiatures. The velocity constant, as 6 t = time in hours; z = per cent hydrolysis, k = calculated from the observed sdittinm. varies as shown in Table IB. By inter: polating between these variations, a Olive oil (0.500 gram) was dissolved in 5 cc. of a 10 per cent value of the “constant” may be obtained for the point solution of dried bile in glycerol. To this were added 100 mg. where the hydrolysis of the fat has reached 5 per cent. From of calcium chloride, 10 cc. of 0.05‘ M ammonium chloride, these values the time required to effect a splitting of 5 per and 5 cc. of 5 per cent sodium oleate. All solutions were held cent was calculated and is plotted against the temperature at 0” C. It is essential to maintain this emulsion at a temperature of 0’ C. or below to prevent calcium oleate from coagulating. (curve A ) . Five cubic centimeter portions of the resulting emulsion were It is obvious that the initial velocity approximated by curve placed in test tubes together with one drop of three per cent A has no connection in this case with the total amount of phenolphthalein and enough 5 N ammonia t o produce a definite lipolysis ultimately to be observed. The extent to which pink coloration. While the emulsion was still at a temperature of 0’ C. or below, 0.33 cc. of a glycerol extract of dried pig panolive oil and tributyrin a t low temperatures can be hydrolyzed creas was added. The test tube was stoppered and swirled for in unbuffered systems over longer periods is shown in Table 20 seconds, and it was immediately immersed in a bath below 11. If it is assumed that the reaction is still monomolecular, a “constant” may be calculated from the hydrolysis observed 5 in 3 or in 5 days, and expressed in terms of the time required for 5 per cent splitting. Curves B and C (Figure 1) were I 1 0 calculated in this way for tributyrin and olive oil, respectively. There is no thought of justifying the assumption, but it is felt that a rough comparison of these data with those for initial velocity may be afforded by thus bringing them all into Figure 1 in the same form. I
Q
-
TABLE11. PERCENTHYDROLYSIS OF OLIVEOIL AND TRIBUTYRIN AFTER LONQ EXPOSURE TO LIPASE Tpv., C. - 1
FIGURE 1. ESTIMATED TIME FOR 5 PER CENT SPLITTINQ OF TRIBUTYRIN AND OF OLIVEOIL -25’ C. to freeze quickly. The sample was then ready for incubation at any desired temperature. A blank was determined by dissolving the contents of a test tube in 75 cc. of alcoholether and titrating with 0.1 N alcoholic potassium hydroxide. Subsequent titrations at convenient times, following this same procedure, indicated the extent of the hydrolysis. When 0.171 gram of tributyrin was used as a substrate, the same procedure was followed, except that the sodium oleate was left out.
It was impossible to neutralize the fatty acid formed during the hydrolysis. This can be done in liquid systems and it accelerates the reaction. I n the solid state the reaction therefore slows down abnormally. The freezing point of the test mixtures was not sharp, but solidification was complete a t -15“ C. No method of comparing the rates of digestion a t different temperatures will prove highly accurate, but apparently the most logical procedure is to regard the hydrolysis as a mono-
- 7 - 12 - 18 - 19 - 20 - 25 -31
7 -
3 days 52.4 51.5
..
2s:o
..
6:4
Olive Oil 5 days 10 days
.. 52:5 40.0 36:5 30.0
..
.. .. 4618 .. 3316 ..
-Tributyrin3 days 5 days 50.9 46.4
..
35:9
..
10 days
..
.. ..
36:4
4210
..
..
It follows from the foregoing data that the splitting of olive oil and of tributyrin takes place with measurable velocity a t temperatures far below the point where the system is solid. No sudden change was observed in the rate of hydrolysis corresponding to the change in state. The enzymic hydrolysis of fat in frozen tissues presents, therefore, a definite and detectable change in chemical composition which probably has an important bearing on the quality of the stored product as a food.
Literature Cited (1) Balls, A. K., Matlack, M. B., and Tucker, I. W., J. Biol. Chem., 122, 126-37 (1937). (2) Pennington, M. E., and Hepburn, J. S., U. 8. Dept. Agr., Bur. Chem. Circ. 103 (1912). RECEIVED January 7,1938.
Food Research Division Contribution 356.
