IV. THE ACTION OF INSOLUBLE ENZYMES. BY, Study notes of Physiology

Download IV. THE ACTION OF INSOLUBLE ENZYMES. BY and more Study notes Physiology in PDF only on Docsity! RESEARCHES ON THE NATURE OF ENZYME ACTION. IV. THE ACTION OF INSOLUBLE ENZYMES. BY W. M. BAYLISS. (From the Institute of Physiology, University College, London.) THERE are certain facts with regard to the solubility of enzymes which are of much significance in hypotheses as to the mode of action of these substances. I refer to the results of experiments in which the enzvme was found to be completely insoluble in the medium in which the reaction took place. This expression is used in the ordinary sense, meaning that the enzyme could be filtered off by the usual porous paper. It was found that, in many cases, although the suspension of the enzyme was active, the filtrate was inactive. In making this state- ment, it is to be remembered that, so far as true solution is concerned, all enzymes are insoluble, since they exist in the colloidal state. But when the theory is put forward that the activity of these substances is exercised by' their surfaces, a more convincing proof is afforded when they can be shown to be active, although present in particles of such a size as to be incapable of passing through ordinary filter paper. The chief difficulty in the investigation of various enzymes from this point of view is that of finding a solvent in which a particular enzyme is insoluble, while some substrate suitable for it is soluble. In the case of several different enzymes, this has been found possible. These cases will be taken in order and any results obtained by previous workers will be referred to in connection with each enzyme. Urease. Although Armstrong, Benjamin and Horton (1913, p. 343), stated that the results obtained by them could best be explained on the hypothesis that the enzyme acted at its surface, direct proof of the fact has not yet been afforded. The proof is possible owing to the fact that the substrate, urea, is soluble in strong alcohol, while the enzyme is not. The following experiments serve to show the correctness of the view. 86 W. M. BA YLISS. Exp. 1. A 20/0 filtered watery extract of soy beans was added in such amount to a 4% solution of urea in absolute alcohol that the resultant mixture consisted of 800/0 alcohol. The precipitate produced was filtered off through paper and the filtrate evaporated to dryness at 370 under reduced pressure. The residue left in the distillation flask was redissolved in water and found to give off no ammonia even after several days action at room temperature. That the alcohol does not destroy the enzyme under the conditions of the evaporation was shown by subjecting a similar unfiltered preparation to the same. treatment. This was very active, giving rise to the formation of ammonia in a few minutes. The object of this experiment was to test the solubility of urease in 800/0 alcohol. The fact that prolonged contact with alcohol does not destroy the enzyme was confirmed by the next experiment. Exp. 2. Finely ground soy beans were extracted with 760/0 alcohol for 24 hours. The extract was filtered off and concentrated as in Exp. 1. The residue in the flask was inactive. The enzyme is therefore insoluble in this strength of alcohol. The extracted beans were still very active on a watery solution of urea. The activity of the enzyme in strong alcohol was then tested as follows: Exp. 3. A 40/0 solution of urea in absolute alcohol was taken and to four samples different amounts of a 2 /0 watery extract of soy beans were added, so that the strength of the alcohol in each was, respectively, 89.3, 85-8, 83*3 and 800%. As control, a 40/0 solution of urea in 80 0/0 alcohol was taken. The enzyme was precipitated in all the first four cases. All the preparations, without filtration, were left at room temperature for three days. The ammonia produced was then estimated, after addition of sodium carbonate, by the aeration method of Folin, as used by Van $lyke and Cullen (1914.2), caprylic alcohol being used to prevent frothing. The ammonia found corresponded to a percentage hydrolysis in 8930/ alcohol of 1'8 0/ 85-8P0 4-3% 833% " 6.1° 800 °0/ ,, 7*8 %/ Control, without urease, of 0'06% A greater effect is obtained if a larger proportion of enzyme be taken, or the reaction allowed to take place at a higher temperature. Exp. 4. 2 grams of finely ground beans were soaked in 5 c.c. of water for an hour. 20 c.c. of 3 /0 urea in absolute alcohol were added to this paste, so that the strength of the alcohol would be about 800/. This, after shaking, was left at room temperature for 16 hours and then at 400 for an hour and a half. The ammonia produced showed 37 0/0 hydrolysis of the urea present. Evidence, of a confirmatory nature only, is afforded by comparing the effect of large particles of ground beans with that of fine particles. Exp. 5. Soy beans, coarsely ground, and free from husk, were sifted by means of a series of sieves with holes decreasing in size. Grains of 2 mm. diameter were compared with particles which passed through holes of 1 mm., and these again with those which ENZYME ACTION. 89 be - 1047 for the mercury green line. This filtered part was returned to the unfiltered part and placed in the incubator at 380 for five days. The rotation was then reduced to - 00.93. It was not thought necessary to make any further experiments with this enzyme. Invertase. An acetone precipitate from autolyzed yeast was tested first, but the action in 90 0/0 alcohol was very slow, owing to the sticking together of the enzyme preparation in gummy lumps. Exp. 10. A concentrated solution in water of the acetone precipitate was added to an alcoholic solution of saccharose in such quantity as to make the alcoholic content 900/0. A part was filtered off and found to have a rotation of 00.91. This part was placed along with an unfiltered portion at room temperature. No change could be detected in four days, but after 13 days the following rotations were obtained: Filtered ... 00 87 Unfiltered ... 0° 55 Acetone dried yeast ("Zymin") was found to be free from the agglutinating properties and thus afforded a greater active surface and a more rapid action, thus: Exp. 11. About 2 grams of the powder were added to 50 c.c. of saccharose in 800/0 alcohol. A filtered sample gave an initial rotation of 120.57. After six days at room temperature, the rotations were: Filtered ... + 120-077 Unfiltered ... - 60.76 Although the passage of rotation to the lnvo-side of zero showed that fructose had been formed, the production of reducing sugar was confirmed by evaporating down the filtered alcoholic solution and redissolving the residue in water. The solution thus obtained had a rotation of - 10-7 and 3 c.c. reduced 20 c.c. of Fehling's solution. This reduction is practically equivalent to that of invert sugar corresponding to the rotation. The cane sugar had thus been almost completely hydrolyzed. Lactase. Lactose was found to be so insoluble in strong alcohol that it was necessary to proceed in another way. By the addition of excess of lactose to the reacting mixture, as the small amount in solution is attacked, more is dissolved, since glucose and galactose are more soluble. Thus an increase of rotation and reducing power implies a production of hydrolytic products, both from the lactose originally present in solution and from the additional amount dissolved as the reaction proceeds. W. M. BA YLISS. Exp. 12. A saturated solution of lactose in 70 0/0 alcohol was shaken with emulsin. A part was filtered and excess of lactose added to both. After six days at room tempera- ture, the following data were obtained: Copper Rotation reduction Ratio Filtered ... ... ... 00°55 2-4 4.3 Unfiltered ... ...10.20 18-5 15-4 The reducing power was determined in 5 c.c. of each after driving off the alcohol. The change of the ratio of rotation to reducing power is additional evidence of hydrolysis of lactose, but in comparing the numerical values, the following fact must be considered. From the work of Bourquelot et Bridel, it seemed probable that the glucose and galactose set free would, to some degree, combine with alcohol to form the respective f-glucosides, in the presence of emulsin. These glucosides, being laevo-rotatory, would cause the hydrolysis in this experiment to appear less than had actually taken place. A portion of the solution was accordingly evaporated to dryness, extracted first with absolute alcohol, which dissolved a considerable amount of the sugars and the possible glucosides. This solution was again evaporated to dryness and extracted with ethyl acetate to dissolve the latter, as in the method of Bourquelot et Bridel. A small amount of a substance was obtained which was laevo-rotatory and had a specific rotation for the mercury green line of - 250. The amount was too small for further purification. Trypsin. The chief difficulty in this case is finding a suitable substrate. Gliadin, which is soluble in 700/0 alcohol, although not very readily attacked, seemed to be a possible one. The solubility of trypsin in alcohol of this strength was first tested, as follows: Exp. 13. 2 grams of Schuchardt's trypsin were dissolved in 15 c.c. of water and added to 35 c.c. of absolute alcohol. Filtered and the filtrate evaporated nearly to dryness in vacuo. The residue in the flask was extracted with dilute sodium carbonate, which did not completely neutralize the acidity of the preparation, so that it was made faintly alkaline with ammonia, and a small piece of fibrin added. The insoluble part of the trypsin was suspended in 70°/0 alcohol, evaporated as above and fibrin added. Next morning, the fibrin was found to have been dissolved in hoth. Mett's tubes with gelatin were then added to both. The preparation from unfiltered trypsin was very active, while that from the ifitered part only dissolved 4 mm. in three days. It would appear thus that trypsin is very slightly soluble in 70% alcohol. The fact that the action is considerably more rapid in the unfiltered mixture indicates, however, that the undissolved enzyme is 90 ENZYME ACTION. 91 taking part in the reaction, since both solutions were presumably saturated. There is a possibility that the enzyme had partially disappeared from the less active solution, since trypsin is somewhat unstable. The effect was therefore tested on gliadin in 800/0 alcohol, although this protein is only slightly soluble in it. Exp. 14. A saturated solution of gliadin in 0-013 normal sodium hydroxide in 80 /0 alcohol was made at 40. Cooled and filtered. The amount of 0 05 normal NaOH required to neutralize 20 c.c. after addition of formaldehyde, was 4 c.c.. A part of this solution was set aside as control. 3 grams of dry trypsin were added to 125 c.c. of the remainder and made faintly alkaline with sodium carbonate in order to neutralize the acidity of the enzyme preparation. A part of this solution was filtered and had a form- aldehyde value of 4-4 c.c. for 20 c.c. The three preparations were kept at 38° for seven days and then had the following formaldehyde values: Without trypsin ... 1.5 With trypsin, unfiltered ... 19.1 With trypsin, filtered ... 3-7 We see that trypsin is active on gliadin in 800% alcohol, although the filtrate was inactive, the formaldehyde value being a little less than the original one. The decrease of the value in the first sample is unexplained. Pepsin. Exp. 15. About 2 grams of Merck's scale preparation of pepsin were added to 50 c.c. of a 2 %/ solution of gliadin in 70 /0 alcohol, made acid by the addition of hydro- chloric acid. After seven days' action at room temperature, the solution gave a red biuret reaction. whereas a solution from which the enzyme had been filtered, after standing for a day, gave, after seven days' standing, the same blue-violet colour as the original. The pepsin appeared to be destroyed at the end of the experiment, since neither the undissolved scales nor the supernatant fluid had any effect on fibrin. On repeating the experiment with a very active preparation by Schuchardt, which was in the form of a white powder, it was noticed that the substance was almost entirely dissolved by acid 70 /0 alcohol. But the filtered solution had no action on fibrin. Further experiments were made to elucidate the question: Exp. 16. About 1 gram of this pepsin was dissolved in 15 c.c. of water. The addition of 15 c.c. of absolute alcohol did not cause a precipitate and a further 20 c.c. only produced a faint turbidity. A drop added to absolute alcohol was precipitated. This solution in 70 /0 alcohol was filtered and the filtrate evaporated nearly to dryness in vacuo at 400: The residue was extracted with 0 05 normal acid and fibrin added. At 400 the fibrin was dissolved very slowly, especially as compared with the effect of a small amount of the original powder added to the same solution.