THE ACTION OF TRYPSIN ON INSULIN.

BY D. A. SCOTT.

(From the Connaught Laboratories and the Department of Biochemistry, University of Toronto, Toronto, Canada.)

(Received for publication, October 22, 1024.)

Various investigators have obtained very different yields of insu- lin from the pancreas. Reference to t.he important contributions in this field may be found in a recent article from this laboratory (1). The great variation in the yields of insulin may be partly due to different methods of assay, nevertheless, it is obvious that there are factors in Ohe preparation as yet not understood. It was thought that a more complete study of the action of trypsin on insulin might help to explain t,he discrepancies in the yields.

Ranting and Rest (2) were the first to observe that trypsin dest’roys insulin. Dudley (3) and later Witzemann and Livshis (4) showed that both pepsin and trypsin destroy insulin. Shonle and Waldo (5) confirmed t’hc findings of the previous investiga- tors. Epstein and Rosenthal (6) in more recent work state that the action of trypsin on insulin is immediate and that the insulin may be recovered by increasing the acidity of the solution. They also state that if trypsin is injected directly after the administration of insulin the action of the latter is inhibited. They conclude that t’he action of the trypsin is not proteoclastic.

EXPERIJIESTAL.

It was first necessary to determine approximately the least amount of trypsin necessary to inactivate completely a known amount of insulin. This was done by adding various amounts of a solution of trypsin to 10 units of insulin (1 cc., pH 2.5), and imme- diately injecting the mixture into a series of standard test rabbits. In Table I, I have indicated the manner in which the amount of trypsin was roughly determined.

Table I shows that 1 mg. of trypsin per 10 units of insulin is not 641

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642 Action of Trypsin on Insulin

sufficient to inactivat,e immediately the insulin. Therefore, in all subsequent work 10 mg. of trypsin per 10 units of insulin were used. The trypsinl was made up in water so that 1 cc. contained 10 mg.

Attempts to liecover Latent Potency.

Attempts to recover the potency from the insulin-trypsin complex were made by: (1) varying the acidity, (2) varying the temperature, (3) varying the solvent, and (I) the use of detergents (sodium taurocholate, sodium glycocholate, saponin, and ben- zoates).

The first series of experiments was attempts to recover by acid hydrolysis the potency from insulin inactivated by trypsin. Trypsin was added to the insulin solution in such amounts that

TABLI? I.

Insulin. Trypsin.

uniis mu.

10 0.1 10 1 10 10 10 100

Remarks

Convulsions in all rabbits. 3 units (average figure). No significant change in blood sugar. Hyperglycemia in all rabbits.

1 mg. of trypsin was present for every unit of insulin. The mix- ture was then hydrolyzed with IICl, added in sufficient quantities to give an acidity in the first experiments of pH 2.5. In these experiments the temperature and time wcrc variables, while the acidity was constant (pH 2.5). After hydrolysis, the equivalent, of 10 units was administered at each injection into standard test rabbits and the amount of reclaimed activity determined. The results arc expressed in Charts 1 to 3. Some of the numerical values obtained are indicated on the charts.

Chart 1 shows that little potency is recovered by heating under 60” C. and that the recovered activity is rapidly lost at or above 110” C. (pH 2.5). It also indicates that the rate of disappearance of the recovered potency is roughly proportional to the amount reclaimed at any time. This is interesting in the light of the rapid

1 Digestive Ferments Company trypsin was used.

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D. A. Scott 643

Time

CHART 1.

Units

n

r

Time CHART 2.

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644 Action of Trypsin on Insulin

disappearance of potency when very high yields of insulin are obtained from the pancreas.

In Chart 2 units of regained potency are plotted against time of heating at different temperatures. This chart shows that the initial rate of recovery increases directly with the temperature. It would also appear that the time of heating at any temperature to secure maximum activation is roughly proportional to the tem- perature. The best procedure for the recovery of potency at this acidity is heating the solution for 3 minutes at a temperature of approximately 110” C. In practice, however, it is inconvenient to heat for such a short period. For this reason, in a second series of

20

0

32 / 1" 0

0.5 1.0 1.5 2.0 2.5 3.0 PH

CHART 3.

experiments, the time of heating was set at 30 minutes and the temperature and acidity varied. The results are illustrated in Chart 3.

Chart 3 shows that over a considerable range for each pH value there is an optimum temperature for the activation of the latent insulin. When the temperature is increased and the acidity decreased very little activity is recovered. However, when the acidity is increased and the temperature decreased, as far as we have investigated, the activity continues to be recovered in large amounts. Further research in this direction was prevented by the high salt content formed on neutralizing the acid before the

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Il. A. Scott GG

injection. -1 30 minute hydrolysis at pH 1.5 at SO” C. was used in all subsequent, work as it offered a fair compromise between efiici- ency in recovery of activity and ease of control. These conditions permitted a recovery of SO per cent of the initial potency.

Sulfuric acid was founcl to be as effective as hydrochloric acid for similar pII values. The det,ergents, sodium t,aurocholate, sotlilun gl~cocholatc, and asponin. were completely ineffective in recovering the latent potency.

-1 large part of the insulin could be recovered from t’he insulin- trypsin comples by scid alcohol as is shown in the following cxperi- ment. To 20 cc. of 10 unit insulin were added 20 cc. of trypsin (200 mg.). The mist,ure was sh:kcn and t.he acidity lowered to pII 1.5. Immediately -L vohnnes of 9.5 per cent alcohol were added. The mixture was left to eskact for 3 hours at room tem- perature. The precipitate which formed was filtered off and the alcohol removed in NKXO. The concentke was made up to its original volume and assayed for potency. Approximately GO per cent of the insulin wa,s recovered.

Treatment of the inact’ive insulin with sotlium benzoate was also effectiw in recovering part of the latent potency. 2 gm. of sodium bcnxoatc were added to ‘20 cc. of insulin, ma.de inactive with trypsin. Aft,er stranding 4 hours, t,he solution was acidified with hydrochloric acid. and the benzoic acid ethered out. Approsimntely half of the original potency was recovered.

Benzoic aci(I alcohol was very cffectivc in recovering the latent pot c11cy. 20 cc. of trypsin solution were added to 20 cc. of insulin. This mixture was shnltcn and immediately ,.I0 CC. of a snt~urat,etl solution of bcnzoic acid alcohol were added. The mixture was acidified with 1 cc. of 1 N HCl. After standing 15 minutes the mixture was heated in a boiling watclr bath until most of t,he alcohol distilled off. Ether was then added to remove the benzoic acid. The aqueous fraction, after the removal of traces of ether in z’uc~o, contained practically all of the original potency.

In the following experiments the action of inactivated trypsin on insulin was studied. 20 cc. of a solution of trypsin (200 mg., pH 2.5) were killed by heating at SOY’. for 30 minutes. After

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646 Action of Trypsin on Insulin

cooling, this solution was added to 20 cc. of 10 unit insulin. No loss in potency occurred. In a second experiment 50 cc. of the insulin were inactivated with trypsin in the usual manner. After

Amount injected.

cc.

0.6

0.8

0.8

TABLE II.

Experiment.

20 cc. insulin + 20 cc. trypsin in- activated by heat.

20 cc. insulin + 20 cc. trypsin heated together.

After standing 1 week.

Blood sugar:

Before.

0.104 0.045 loo

0.110 0.048 80 0.098 0.040 80

After (lt hn.).

I

Amount eclairned.

per cent

* All estimations of potency of insulin solutions were made by injecting graded doses into a series of standard rabbits. The blood sugar of each rabbit was determined 18, 3, and 5 hours after the injection. The potency was calculated as described in a recent article from the Insulin Committee’s laboratory by Macleod and Orr (7). As it would require too much space to report all the blood sugar values, only the l$ hour blood sugar in one typi- cal experiment is recorded.

AmCXllV injected

cc.

0.3 2 4

50 cc. supernatant liquid from charcoal. 0.104 0.110 50 “ “ “ “ “ 0.110 0.066 50 “ ‘< “ ‘< ‘I 0.118 0.040

0.3 50 cc. suspension of charcoal. 0.110 0.104 1 50 ‘I ‘I IL ‘I 0.098 0.110 4 50 “ “ ‘I “ 0.110 0.116

0.3

0.6

45 cc. product reclaimed from charcoal by benzoic acid alcohol.

“ I‘ 0.110 0.066 0.124 0.040

TABLE III.

Experiment.

Blood sugar.

-

A

Before. After ‘( I+ hrs.)

c

-

1

-

Lmount re-

lsimed.

!mr cent 10

0.0

60

the acidity had been adjusted to pH 1.5 the solution was heated at 80°C. for 30 minutes. On cooling, the acidity was adjusted to pH 2.5 and no inactivation of insulin occurred even on standing 1 week at room temperature (Table II).

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D. A. Scott 647

Since charcoal readily adsorbs insulin from an aqueous solution, we were interested t.o determine the activity of the insulin-charcoal complex. c 0.0 gm. of powdered charcoal was shaken for 2 minutes with 50 cc. of 10 unit insulin at pH 2.5. The charcoal was removed by centrifuging, washed with water, and the washings were added to the supernatant liquid. An assay showed that t,here was a 90 per cent loss of potency. The charcoal was shaken with 50 cc. of water and portions of the suspension equivalent to 3, 10, and 40 units of insulin were injected. In each case the blood sugar remained normal. The remaining charcoal was centrifuged off and the insulin extracted lvith benzoic acid alcohol (8). Approximately 60 per cent of the original potency was recovered (Table III).

Proteoclastic Action.

The proteoclastic action of trypsin on insulin was next investi- gated. 25 cc. of 10 unit insulin, pII 2.5, were mixed with 25 cc. of trypsin solut.ion (250 mg.). After adjusting to pH 7.5 and adding a few drops of xylene t,he solution was incubated at 40°C. Satnples were withdrawn hourly and attempts were made to recover the latent potency by heating at 80%. for 30 minutes at pH 1.5. The latent pot.ency of the original solution fell progres- sively to half its initial value in 4 hours. After standing overnight no activity could be recovered by acid hydrolysis or by acid alco- hol extraction, even on injecting quantities equivalent t.o 3, 10, and 60 units of the original material. Control experiments on insulin alone under similar conditions showed little loss in activ- ity. However, when the insulin-trypsin solution (pH 7.5) was left in the ice chest overnight, most of the activity could be reclaimed (Table IV). In some experiments strict aseptic technique was employed.

Action in Viva.

The action of trypsin on insulin in viva was studied. Standard test rabbits were injected with 3 unit.s of insulin and immediately afterwards with 0.3 cc. of trypsin (3 mg.). The blood sugar deter- minations showed that there was no appreciable loss in potency. The same results were secured when 0.6 and 0.9 cc. of the trypsin solution were injected. Thus more than three times the quantity

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648 Action of Trypsin on Insulin

T.&BIX IV.

Amount injected.

cc.

1 4

0.5

1.5

0.3

1.0 6.0

0.6

0.8

0.6

0.8

I-

25 cc. insulin + 25 cc: trypsin (pH 7.5). 25 “ “ + 25 “ “ ( “ 7.5).

Portions of solution after 4 hrs. incubatior hydrolyzed at 80°C. (pH 1.5).

“ “

Tryptic digest after 12 hrs. hydrolyzed at 80°C. (1’~I 1.5).

“ ‘I “ “

20 cc. insulin + 20 cc. 1120, pH 7.5 at 4O’C. for 12 hrs.

“ ‘I

25 cc. insulin + 25 cc. trypsin, pH 7.5 in ice chest 12 hrs., activated by acid.

“ “

TABLE 1’.

cc.

0.3

0.3

0.3

0.3

Experiment.

10 unit insulin immediately followed by 0.3 cc. of trypsin (subcutaneous).

10 unit insulin iminedintely folloved by 0.G cc. of trypsin (subcutaneous).

10 unit insulin immediately followed by 0.0 cc. of trypsin (subcutsncous).

10 unit insulin immediately followed by 0.3 cc. of trypsin (intravenous).

-

I -

T

After !1 hrs.)

0.098 0.104

0.072 0.048

0.110 0.098 0.110

0.060 0.048

0.066 0.048

0.098

0.110

0.040

0.054

0.104 ’ 0.048

0.098 0.040

-

A

cl

T

-

mount re-

aimed.

,er cent

0.0

40

0.0

90

70

90

90

SO

160

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D. A. Scott

of trypsin necessary to inactivate completely insulin in vitro had no appreciable effect ilz vivo. Both subcutaneous and intravenous injections were made (Table V). Injections of larger amounts of trypsin produce hyperglycemia which counteracts the effect of insulin.

DISCUSSION.

It has been shown, confirming Epstein and Rosenthal, that insulin is immediately inactivated by trypsin i?l vitro at certain acidities. Various methods of recovering the insulin from the trypsin-insulin complex have been tried. By immediately heat- ing the complex to 80°C. for 30 minutes (pH 1.5) SO per cent of the original potency is recovered. Acid alcohol or sodium benzoate is also effective in recovering a large part of the insulin made inactive by trypsin. Since acid alcohol has been found to be very effective in extracting insulin from the pancreas it is possible that much of the insulin exists there in an inactive form. Other results which indicate that insulin may exist in the body in an inactive form are: (1) A simple aqueous extraction of the pancreas thus far has resulted in low yields of insulin. (2) Insulin is present in appreciable amounts in diabetic tissues. (3) The best yields of insulin from urine have been obtained by the benzoic acid method of purification. These yields may be due to the liberation of the latent potency by the strong hydrochloric or benzoic acid. (4) On several occasions the potency of the final product has been increased several fold by the useof strong hydro- chloric acid.

The preliminary action of trypsin on insulin would seem to be similar to the action of charcoal on insulin, both since the complex is not active and since the activity may be recovered by suitable extraction.

The results of the experiments on the proteoclastic action of trypsin on insulin would suggest that the primary action is a mutual adsorption phenomenon and that this is followed by com- plete proteoclastic destruction. This is in accord with the theory of enzyme action (9).

The amount of trypsin necessary to inactivate completely III~~I:II 111 z~zfrn had no appreciable effect in vivo when injected immediateiy after the insulin. These results are contrary to

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650 Action of Trypsin on Insulin

those of Epstein and Rosenthal who found that trypsin inacti- vates insulin in vivo as well as in vitro. Probably their results may be attributed to an overdose of insulin, which may produce surprisingly little effect on the blood sugar, or to an overdose of trypsin which produces hyperglycemia.

The experiments on the recovery of the latent potency and the proteoclastic action of trypsin on insulin indicate the advisability of using fresh pancreas and high acidity for obtaining a high yield of insulin.

CONCLUSIONS.

1. Insulin is immediately inactivated by trypsin at certain acidities.

2. The latent potency may be partly reclaimed by suitably adjusting the temperature and acidity, or by the use of alcohol or benzoates.

3. When insulin and trypsin are incubated together (PH. 7.5, 40°C.) for about 12 hours no insulin can be recovered by the procedures referred to above.

4. Trypsin in amounts which inactivate insulin in vitro does not inhibit insulin when the substances are injected separately.

I wish to acknowledge by indebtedness to Mr. K. L. MacAlpine for his efficient assistance. I also wish to thank Dr. C. H. Best and Dr. A. Hunter for their helpful criticism.

Addendum.-A paper by Epstein and Rosenthal on the nature of the action of trypsin on insulin (10) has appeared since our communication was sent to press. l’hese writers were able to activate the insulin after a 42 hour digestion by trypsin. I have made numerous attempts to repeat these experiments. Purified trypsin was used. These attempts have been unsuccessful. Even benzoic acid alcohol, which in my experience is by far the most efficient agent for the recovery of latent potency has given negative results in these experiments.

BIBLIOGRAPHY.

1. Best, C. H., Endocrinology, 1924, viii, 617. 2. Banting, F. G., and Best, C. H., J. Lab. and Clin. Med., 1921-22, vii,251. 3. Dudley, H. W., Biochem. J., 1923, xvii, 376. 4. Witzemann, E. J., and Livshis, L., J. Biol. Chem., 1923, lvii, 425. 5. Shonle, H. A., and Waldo, J. H., J. Biol. Chem., 1923-24, lviii, 731

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D. A. Scott 651

6. Epstein, A. A., and Rosenthal, N. E., J. Am. Med. Assn., 1924, Ixxxii, 1990.

7. Macleod, J. J. R.? and Orr, M. D., J. Lah. and Clin. Med., 1923-24, ix, 591. 8. hloloney, I’. J., and Findlay, D. hl., J. Phys. Chem., 1924, xxviii, 402. 9. Bayliss, W. M., The nature of enzyme action, New York, 3rd edition,

1914. 10. Epstein, A. A., and Rosenthal, IT. E., Am. J. Physiol., 1924, lxx, 225.

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D. A. ScottTHE ACTION OF TRYPSIN ON INSULIN

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