A FURTHER INVESTIGATION OF THE CHEMICAL PROPERTIES OF INSULIN.

BY D. A. SCOTT.

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

(Received for publication, July 15, 1925.)

In a recent paper (1) the writer showed that the primary action when trypsin was added to insulin was adsorption and that this was followed by proteoclastic destruction of the insulin. In the present study an attempt has been made to ascertain some of the chemical groups which insulin contains. With this object in mind, material of known potency has been subjected to the action of various reagents. The resultant material has been tested by the standard procedure to find out whether the chemical groups affected by each reagent are necessary for the physiological action of insulin. The nitrogen distribution of a purified insulin product has been determined and a combustion analysis has been carried out on a sample of the same material.

Since the discovery by Banting and Best (2) of the pancreatic hormone, insulin, much work has been done in attempting to ascertain its chemical nature, The original investigators first recognized its protein-like properties when they observed t.hat trypsin destroyed the activity of insulin. Best and Macleod (3) found that the insulin from the ox showed a distinct biuret test (protein) while the insulin from the skate showed no biuret reaction (non-protein). They also observed that insulin was readily absorbed by charcoal and kaolin. Dudley (4) found that both pepsin and trypsin destroyed insulin and concluded that it appeared to be a complex protein derivative. He also showed that his insulin which was purified by an entirely different method from the Toronto insulin, gave protein reactions and that its physiological activity was destroyed by alkali. Doisy, Somogyi, and Shaffer (5) found that insulin had an isoelectric point (pH 5.0) and the purified product gave characteristic reactions for proteins.

601

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602 Chemical Properties of Insulin

Independently the isoelectric point was discovered by Walden of Eli Lilly and Company (6). Widmark (7) attempted to purify insulin by separating the active material from inert matter by the use of various organic solvents. He was not successful in dialyzing insulin. He suggests that insulin is an albumose. Piper, Allen, and Murlin (8) state that insulin is not a protein. They did not obtain any of the protein reactions in a purified insulin product prepared from the ox pancreas. Kimball and Murlin (9) report the preparation of a product which gives no protein reaction and which has a nitrogen content of from 4 to 6 per cent of its dry weight. Shonle and Waldo (10) conclude that the pancreatic substance containing insulin appears to be a proteose which gives typical protein reactions, and that further research is necessary to determine whether the active principle is a proteose or is merely associated with the protein fraction.

The explanation of the failure of (1) Best and Macleod and (2) Murlin, to obtain positive reactions for protein may be due to the fact that these investigators were dealing with very dilute solutions of insulin and that the dilution was beyond the accuracy of the biuret test. This is also the view of Somogyi, Doisy, and Shaffer (11) who first suggested this possibility.

The chemical work on insulin appears to indicate clearly that the active substance is closely related to a protein. If insulin is a protein it would be expected to have amino groups. Amino groups react with certain chemicals, i.e. formaldehyde, nitrous acid, etc. Thus it was hoped that by subjecting insulin to the action of these various reagents its chemical nature would be more clearly demonstrated. Further it was hoped that it might be possible to form an insulin compound with some reagent which would aid in the purification or identification of the active substance. It also seemed of interest to find out whether or not the amino groups were essential for the physiological action of insulin.

The insulin used in the following experiments is lot No. 405 and was prepared according to the method of Scott and Best (12).

Action of Formaldehyde.

Dodds and Dickens (13) have reported that formaldehyde up to a concentration of 20 per cent at ordinary temperatures, has

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D. A. Scott 603

no effect on the physiological activity of insulin. Our experi- ments were carried out as follows :

5 cc. of formaldehyde solution were added to 20 cc. of 20 unit insulin (pH 2.5) and the temperature kept at 20°C. After 20 hours the formalde- hyde was removed in vucuo. The resultant product was diluted and tested for potency on a series of test rabbits according to the method adopted by the testing department of the Insulin Committee (14). Other experiments were made on insulin solutions which were made alkaline with excess sodium bicarbonate. Control experiments were made on insulin solutions without formaldehyde which were made alkaline with sodium bicarbonate.

TABLE I.

4cid- ity Experiment.

26 cc. insulin + 5 cc. HsO.. . . . . . . . . . . . . . . . . 20 “ “ + 5 “ HCOH.. . . . . . . . . . . . . . . .

PH hrs.

2.5 20 2.5 20

20 (‘ “ + 5 “ HzO.. . . . . . . . . . . . . . . . . . . . 2.5 20 “ “ + 5 “ HCOH.. . . . . . . . . . . . . . . . 2.5

20 “ “ + 5 “ HzO.. . . . . . . . . . . . . . . . . . . . 2.5 20 “ ‘( +5“ HCOH . . . . . . . . . . . . . . . . . . . 2.5

20 “ “ + 5 “ H1O + NaHC08(excess). 20 “ “ + 5 “ HCOH + NaHCOa

(excess) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

-i-

-

Ihe.

20 20

20 20

1

1

-

1 1

_ -

-

rem- pern- ture.

“C.

20 20

40 40

60 60

20

20

-

2 1

-

P

-

IAX3S

In po-

X?llCY.

er cent

0.0

30.0

0.0 40.0

0.0 60.0

0.0

90.0

From Table I it will be seen that formaldehyde in acid solution decreases the physiological action of insulin. Increasing the temperature hastens the inactivation. In a solution made alka- line with sodium bicarbonate the insulin is inactivated almost immediately. If formaldehyde combines with an amino group, then there should be a decrease in the amount of amino nitrogen after the treatment of insulin with formaldehyde. Attempts to estimate any change in amino nitrogen by the Van Slyke micro amino nitrogen apparatus, however, were unsuccessful. This was probably due to the small amount of amino nitrogen in the insulin solution .

Attempts were made to activate the insulin made inactive with formaldehyde by (1) acidifying the solution (pH 1.5) and heating

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604 Chemical Properties of Insulin

at 80°C. for 30 minutes, (2) making the solution alkaline (pH 8.5). No increase in activity was obtained by either procedure.

Since aldehydes are good reducing agents, it was thought that the action of formaldehyde on insulin might be one of reduction. To test this hypothesis samples of insulin were subjected to the action of other reducing agents such as nascent hydrogen, sulfur dioxide, and sodium bisulfide. These substances all destroy the activity of insulin and attempts to recover the potency either by acid hydrolysis or oxidation of the reduced insulin were unsuccessful.

Action of Benzoyl Chloride.

Primary and secondary amines, alcohols, and phenols yield benzoyl derivatives by the replacement of the hydrogen of the NH2 NH and OH groups by the benzoyl radicle. The formation of such derivatives not only indicates the presence of XI& NH or OH groups, but also is used to identify the individual substances under examination, as the benzoyl derivatives crystal- lize well and have very definite melting points.

Experiments to demonstrate the action of benzoyl chloride on insulin were carried out as follows:

20 cc. (400 units) in insulin were made alkaline with sodium carbonate. To this were added 2 cc. of benzoyl chloride. The mixture was shaken for 3 hour, acidified with hydrochloric acid, and the benzoic acid ethered out. The aqueous fraction after the removal of traces of ether in vacua was tested for potency. No activity was present. Attempts were made to recover the active principle by acid hydrolysis, e.g. heating the solution for 30 minutes at 80°C. at pH 1.5. These attempts were unsuccessful (see Table II).

Action of Carbon Bisulphide.

Primary amines condense with carbon bisulfide with the for- mation of dialkylated thioureas. The latter are decomposed on boiling with concentrated hydrochloric acid yielding isothiocy- anates (mustard oils). Kodama (15) has shown that certain amino acids condense with carbon bisulfide in the presence of sodium bicarbonate.

Experiments were carried on as follows:

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D. A. Scott 605

100 cc. of insulin (1000 units) were made alkaline with sodium hydroxide (pH 9.5). To this were added 250 cc. of ethyl alcohol and 20 cc. of neutral carbon bisulfide. The mixture was shaken for 1 hour and was then acidi- fied with hydrochloric acid. The carbon bisulfide and alcohol were re- moved in vacua. .A precipitate settled out during the concentration. This precipitate was soluble in a dilute alkaline aqueous solution. Samples of this solution were tested on standard test rabbits. A control experiment was carried out in which no carbon bisulfide was added. The above frac- tions were also tested for potency after an attempt had been made to regain the activitv bv acid hvdrolvsis. These tests showed that no potency was obtainable”(see Table”II1) .”

TABLE II.

Experiment.

100 cc. solution from benzoyl chloride experiment.

100 “ “ “ “ ‘I hydrolysis.

after acid

Control experiment (benzoyl chloride and water).

-

1 i

.-

-

Bload sugar. hmlnt - njected.

Normal -~

cc. per cent

1 0.110 5 0.118

1 0.115 5 0.104

1 0.118 5 0.110

After 2 hrs.

per cent

0.104 0.110

0.118 0.120

0.110 0.114

Action of Nitrous Acid.

Nitrous acid reacts with warm solutions of primary amines yielding alcohols, with secondary amines yielding nitrosamines. Nitrous acid was first used as a deaminizing agent for proteins in 1885 by Loew (16). This investigator found that one-third of the nitrogen of peptones was liberated by the action of nitrous acid.

Several procedures were used in an attempt to deaminize insulin. The first was the method of Levites (17). 50 cc. of 10 unit insulin were acidified with 5 cc. of glacial acetic acid. This solution was warmed to 40°C. in a water bath. To the solution were then added 10 cc. of a 10 per cent solution of sodium nitrite. This mixture was retained at a temperature of 40°C. and shaken at intervals over a period of 1 hour. A precipitate settled out. At the end of 1 hour the solution was cooled to 20°C. and made

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606 Chemical Properties of Insulin

alkaline (pH 8.5). At this pH the precipitate was completely soluble. Samples of the resultant product were tested on standard rabbits. Approximately 75 per cent of the activity was de- stroyed. Part of the solution was acidified (pH 1.5) and hydro- lyzed. It was then made alkaline and tested for activity. No potency was recovered. It is possible that the heat at 40” C. may cause a slight hydrolysis or other changes in the insulin protein structure, although this is scarcely likely, as it has been shown that insulin is quite stable in acid solution at much higher temperatures. However, in order to test this possibility the method of Dunn and Lewis (18) was tried. 50 cc. of 10 unit

TABLE III

Amount Blood sugar

iniectd. Before. After 2 hrs.

-- cc. pm cm1 per cent

100 cc. insulin (pH 9.5) treated with CL% and 0.3 0.110 0.118 alcohol as described. 2.0 0.110 0.098

4.0 0.118 0.098

50 cc. of above sample hydrolyzed at pH 2.0 0.114 0.104 1.5. 4.0 0.120 0.098

100 cc. insulin (pH 9.5) treated with alcohol 0.3 0.118 0.040 (control experiment). 0.4 0.110 ,Convulsions.

insulin were added to 5 cc. of glacial acetic acid. To this solution 10 cc. of a 10 per cent solution of sodium nitrite were added. The sodium nitrite was added dropwise and the solution was kept well shaken. A yellow precipitate gradually settled out. After standing 18 hours the solution was made alkaline (pH 8.5) and tested for activity. Attempts to recover the potency by acid hydrolysis were also made. Negative results were obtained. To determine whether or not the concentration of the reagents used in these experiments had any effect on blood sugar, experi- ments were carried out exactly the same as above with the ex- ception that water was used in the place of insulin. Amounts of this solution equivalent to the amounts used in the first two experiments were injected into standard test rabbits. The results were negative (see Table IV).

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607 D. A. Scott

Chemical Assay of Insulin.

This investigation is an attempt to obtain a better idea of the complexity of the insulin protein molecule. The first step in such an undertaking is the preparation of a pure product. After many efforts in which many different methods of separating proteins were tried, the following procedure was adopted for the prepara- tion and purification of insulin. The procedure of preparation is similar to that previously reported by Scott and Best (12).

TABLE IV.

Experiment.

Method of Levites as de- scribed, volume = 100 cc.

Method of Levites as de- scribed after acid hydrolysis

Method of Dunn and Lewis as described, volume = 100 cc.

Method of Dunn and Lewis as described after acid hy- drolysis.

Control experiment in which water replaced the insulin solution.

T

--

-

\cid- ity.

Pfi

8.5

2.5 8.5

8.5 2.5 8.5

8.5

nm”” in-

ectd.

-

t

?

-

Blood sugar.

NIX- After Loss in potency

mal. 2 hrs. -- xr cent per rent per cent

0.110 0.048 Approximately 0.118 0.040 75 0.120 0.054

0.115 0.051 Approximately 0.118 0.054 70 0.118 0.048

0.104 0.110 Normal.

Fresh pancreatic glands from the ox were obtained from abattoirs. As much fat and connective tissue as possible were separated from the glands. They were then collected hourly and placed in a refrigerator at 4°C. The glands representing the morning’s kill were collected and taken to the laboratory at 12 o’clock noon.

The glands were weighed. They were then run through a power meat chopper in which they were finely minced. The minced material dropped into large 50 gallon earthenware crocks. For every 100 lbs. of glands there were added 25 gallons of 95 per cent alcohol and 5 gallons of water containing 1200 cc. of concentrated hydrochloric acid. (The alcohol was ethyl dena- tured with 10 per cent methyl alcohol.) The mixture was slowly agitated in order to facilitate the extraction. At the end of 3 hours the acid-alcohol mixture was poured into a large rotary centrifuge which separated the

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Chemical Properties of Insulin

alcoholic extract from the solid materials. The solid materials remaining in the centrifuge were rcextracted with a volume of 60 per cent alcohol equal to that of the liquid obtained during the centrifuging. After this mixture had stood overnight, it was centrifuged as in the case of the first extraction The extracts from the first and second extractions were mixed and made alkaline with ammonium hydroxide. The mixture was filtered through large glass funnels which had been fitted with fluted filter papers. The filtrate was thenacidified withsulfuricacid. 125 cc. of concentrated sul- furic acid solution were added for each 50 gallons of filtrate. The nlco- holic extract was then concentrated to about one-tenth of its original volume in an efficient vacuum still. During the distillation, the tempera- ture of the distillate was not allowed to rise above 30°C.

After the completion of the distillation, the concentrate was quickly heated to 50°C. At this temperature, lipoid and other materials were readily removed by filtration. The concentrate had an acidity of pH 2. It is important that the acidity be within this range (pH 2 to 2.5) in order to ensure rapid filtration and to avoid loss of potency. After the concen- trate had cooled, ammonium sulfate was added to half saturation (37 gm. per 100 cc.). This mixture was stirred, and almost iimnediately protein material separated out and readily rose to the top of the liquid. After standing overnight, the protein precipitate was skimmed off and as much liquid as possible was pressed out. The weight of the precipitate from 300 Ibs. of glands was approximately 200 gm. This precipitate was dissolved in hot acid alcohol. 1 liter of 50 per cent alcohol containing 5 cc. of 5 N

HCl was sufficient. The temperature was kept between 30” and 40°C. When the precipitate had completely dissolved ten volumes of warm alco- hol were added. The solution was then neutralized by adding 5 cc. of 5 N

NaOH. After the solution had cooled to room temperature it was placed in a refrigerator at 5” C. for2days. At theendof that time the darkcolored supernatant alcohol was filtered through a hardened filter paper on a Wchner funnel. The alcohol contained practically no potency. The precipitate was washed with ether and dried. It was then dissolved in acid water in which it was readily soluble. The solution was made alkaline with 5 N NaOH to pH 7.5. At this alkalinity a dark colored precipitate settled out (see Shaffer et al.). This was immediately centrifuged off. This precip- itate was washed once or twice with alkaline water, pH 9.0, and the washings added to the main liquid. It is very important that this process be carried out fairly quickly as insulin is destroyed in alkaline solution. The acidity was adjusted to pH 5 and a white precipitate readily separated out. After standing overnight in an ice chest the supernatant liquid was decanted off, and the resultant liquid removed by centrifuging. The precipitate was then dissolved in a small quantity of acid water.

A concentrated solution of trichloroacetic acid was added to the acid insulin solution until a concentration of 3 per cent was reached. The insulin was completely precipitated by this con- centration of trichloroacetic acid. Hiller and Van Slyke (19)

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D. A. Scott 609

have shown that trichloroacetic acid up to a concentration of 5 per cent is practically specific for precipitating protein. The more recent work of Wasteneys and Borsook (20) confirms the findings of these investigators. The protein precipitate was centrifuged off. The precipitate was dissolved in acid water and the pH adjusted to 6.2. This was allowed to stand overnight. During this time a small precipitate settled out. This precipitate was removed and tpc supernatant liquid adjusted to pH 3.7. A precipitate readily separated out and was centrifuged off. The supernatant liquid was t.hen adjusted to pH 5.0. The precipitate which formed was centrifuged off. This precipitate was dis-

TABLE V.

Source of material. Nitrogen per unit of insulin.

Pancreas* ............................................... Alcohol filtrate .......................................... Concentrate ............................................. Ammonium sulfate precipitate.. ......................... First isoelectric precipitate.. ............................ Final product. ..........................................

T7.

12.0 0.90 0.70 0.52 0.022 0.006

* The beef pancreas from which the insulin was prepared yields a maxi- mum of 3300 units per kg. by acid alcohol extraction as described above. Based on these figures, the nitrogen of the pancreas per unit of insulin is 12 mg.

solved in a small quantity of 50 per cent alcohol. The insulin was precipitated out by adding twenty volumes of acetone. After standing overnight the solid material was filtered off on a hardened filter paper. This material was transferred to a vacuum desiccator and dried over phosphorus pentoxide. The resultant product was snow-white.

Table V demonstrates the decrease in total nitrogen at different stages in the purification of the insulin product.

A small portion of the purified insulin powder was weighed and dissolved in acid water. This solution was thoroughly tested for pot,ency by the standard procedure. The solution was then diluted to a concentration of 100 units per cc. Samples of tllis solution were tested with various reagents to determine the different chemical groups which it contained. The results of a series of tests are shown in Table VI.

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610 Chemical Properties of Insulin

Combustion Analysis.

Samples of the standard insulin powder were subjected to a combustion analysis. The nitrogen was determined by the ordi- nary Kjeldahl method. The carbon and hydrogen were determined by oxidizing a known weight of insulin powder to carbon dioxide and water. The carbon dioxide was absorbed in a Fleming tower and the water in a phosphorus pentoxide tube. The operation

TABLE VI.

Chemical tests. Results.

Biuret (C 0 N H) ................................... Ninhydrin (a amino and free carboxyl) ............... Hopkins-Cole (tryptophane) ......................... Ehrlich (indole) (tryptophane) ....................... Millons (tyrosine) ................................... Xanthoproteic (benzene radicle) ...................... Ehrlich (diazo) ...................................... Folin and Looney (tryptophane) .....................

“ “ ‘I (tyrosine). ........................ Pauly (iminazol) ..................................... Organic sulfur (cystine). ............................. Molisch (carbohydrate) .............................. Phosphorus .......................................... Orcein (pentose) ..................................... Iron .................................................

Positive. “

“ (faint). “ “ “ “ “ “ “ “

Negative. “ “ “

TABLE VII.

Sample No. Weight. C H N Ash.

om. per cent per cent per cent per cent 1 0.2492 50.76 6.57 14.0 0.4 2 0.2714 50.03 6.66 14.0 0.4

was carried out in a silica tube heated in an electric furnace. The ash was determined by the difference in weight of the platinum boat before and after combustion. The results of the two ex- periments are shown in Table VII.

Distribution of -bogen in Insulin.

The Van Slyke (31) method of protein analysis was used. By this method of analysis the distribution of nitrogen in the following

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D. A. Scott 611

groups was determined: amide, humin, arginine, histidine, lysine, cystine, and the amino and non-amino nitrogen in the filtrate from the phosphotungstic acid.

Trytophane and tyrosine were determined on 0.8 gm. of insulin powder according to the method of Folin and Looney (22). The average results of four estimations are recorded in Table VIII.

Amino nitrogen was determined by the Van Slyke micro amino nitrogen apparatus. The amount of insulin used in each deter-

TABLE VIII.

Sample No. Tyrosine.

per cent

1 9.7 2 10.1 3 10.3 4 9.9

Average.........................lO.O

-

Tryptophane.

per cent

0.82 0.78 0.79 0.85

0.81

TABLE IX.

Temperature = 22” C.; pressure = 750 mn.

Sample No. Weight of sample. Amino nitrogen. Amino nitrogen Free amino N.

per gm. insulin. Total N

w. cc. per cent 1 0.08 1.20 6.0 2 0.08 1.25 6.2

After the deaminiaation of the insulin solution, the resultant product was neutralized and tested for potency on a series of rabbits. More than 95 per cent of the potency disappeared.

mination was 0.08 gm. The insulin was dissolved in dilute acetic acid. This was sucked into the deaminizing chamber. The chamber was shaken for 30 minutes at about 400 revolutions per minute. Dunn and Lewis (18) found that such a procedure was necessary to completely deaminize casein. The total nitrogen was determined on 0.2 gm. of insulin by the Kjeldahl method. The results are shown in Table IX.

The estimation of the different groups of amino acids were carried out on 3 gm. of the purified insulin powder. This powder was dissolved in 50 cc. of 20 per cent hydrochloric acid. The

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612 Chemical Properties of Insulin

solution was refluxed in a 300 cc. flask for 30 hours. Van Slyke (23) has shown that 24 hours boiling at 100°C. is usually sufficient to obtain complete hydrolysis of a protein. The procedure followed in determining the different groups was that described by Van Slyke. Duplicate analyses were carried out. The phosphotungstic acid which was used to precipitate the hexone bases was purified according to the method of Winterstein (24). Before analyzing the insulin samples of gelatin were processed in order to become familiar with the technique and to serve as a check on the reagents used. The product after hydrolysis showed no potency when tested on rabbits.

TABLE X.

Distribution of Nitrogen in Insulin.

I Total nitrogen.

2

per cent

Ammonia ................................. Humin ................................... Argenine ................................. Histidine ................................. Lysine ................................... Cystine .................................. Totalbases ...............................

“ filtrate .............................. Amino nitrogen of filtrate ................. Non-amino nitrogen of filtrate ............

Total nitrogen recovered .................

9.6 9.8 0.4 0.4

10.0 10.0 5.2 4.7 4.8 5.4 0.5 0.6

20.5 20.5 67.0 68.4 64.2 66.2

2.8 2.2

97.5 99.1

DISCUSSION.

Insulin of known potency has been subjected to the action of various reagents. The resultant product has been tested on a series of standard rabbits and the change in the physiological action of the insulin determined. The experiments dealing with the action of benzoyl chloride and carbon bisulfide show that these reagents completely inactivate insulin in an alkaline solution. It was thought that the action might be merely one of absorption. Hence attempts have been made to free the insulin by acid hy- drolysis. These experiments were unsuccessful. Insulin is only slightly acted on by formaldehyde in acid solutions at room tem-

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D. A. Scott 613

perature. .4t higher temperatures a greater loss in potency occurs. Almost complete inactivation of the insulin occurs, if the reaction proceeds in an alkaline solution. Attempts to reactivate the insulin by acid hydrolysis were unsuccessful. Since aldehydes are good reducing agents it was thought that the action might be one of reduction. Accordingly attempts were made to activate the insulin by oxidation. No activation occurred. Insulin is de- stroyed by other reducing agents such as nascent hydrogen, sulfur dioxide, and calcium bisulflte. In these experiments at- tempts were made to follow the decrease in amino nitrogen before and after the action of each reagent on insulin by the Van Slyke micro amino nitrogen apparatus. These experiments were un- successful, due probably to the fact that the amount of amino nitrogen in the solutions under test was too small.

The action of nitrous acid on insulin was studied. Levites and Dunn and Lewis studied the action of nitrous acid on proteins. Their methods when applied to insulin showed that there was a decided decrease in the activity of the insulin after the treatment with nitrous acid. In later experiments when the amount of free amino nitrogen was determined by the Van Slyke nitrogen ap- paratus, the mixture which remained showed practically no potency. The greater action of the nitrous acid on the insulin here is probably due to the greater concentration of the reagents and to the vigorous agitation, Attempts to activate the insulin were unsuccessful.

A very pure insulin product has been prepared. The essential steps in this preparation are (1) extraction with hydrochloric acid alcohol, (2) precipitation of the concentrate with ammonium sulfate, (3) dissolving the precipitate and reprecipitating in warm alcohol, (4) fractional precipitation at different acidities, (5) pre- cipitation with trichloroacetic acid. The resultant product was practically snow-white and had a nitrogen content of 0.006 mg. per unit of activity. The physiological assay showed 25,OOOunits of insulin per gm. of powder. This assay was checked by the testing laboratory of the Insulin Committee. Many experiments have been made in an attempt to purify the insulin further. These results have been negative.

The chemical tests on the purified insulin solution containing 100 units per cc. show characteristic protein reactions. The

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614 Chemical Properties of Insulin

color reactions for tryptophane were faint. Phosphorus was not present. The Molisch and orcein tests were negative.

The results from the combustion analysis show that the values obtained for carbon and hydrogen are within the limits of those values obtained for a typical protein. The nitrogen figure is somewhat low. However, this nitrogen value was obtained by the Kjeldahl method of estimating nitrogen and may be somewhat lower than the true nitrogen value, since some substances do not give their total nitrogen by this method of estimation. Cruto (25) reports the results of his analysis of insulin to be C 47.73, H 7.27, N 14.53. While these results are in fair agreement with the author’s, yet we would seem to be dealing with quite a different product. He reports that 0.8 mg. is the amount of insulin neces- sary to lower the blood sugar of a 2 kilo rabbit from normal to 0.45 per cent in 2 hours. 0.12 mg. of our product is necessary to give the same results or our product is 6.7 times as potent as his. Shonle and Waldo’s (10) combustion analysis on a highly purified product shows a much lower hydrogen value and a slightly higher nitrogen value than the results reported here. The ash of their product was high.

The ratio of amino nitrogen to total nitrogen is similar to that obtained for a true protein.

The following points are of special significance in the distri- bution of nitrogen: (1) the high amide and low humin nitrogen, (2) the low cystine value, (3) the high amino nitrogen in the filtrate, (4) the low non-amino nitrogen in the filtrate, (5) the high t.yrosine and low tryptophane values. Certain values reported in the nitrogen distribution are in fair agreement with some values reported by Doisy and Weber (26) and by Shonle and Waldo (10). The cystine value of our insulin, however, is much lower. The cystine value for insulin was checked against the sulfur of the blood. This standard gave a value for cystine of 0.9 per cent of the total nitrogen. This value is in fair agreement with the figures obtained by the method of Van Slyke. Probably the high cystine values which other investigators have obtained are due to the fact that sulfuric acid was used in their method of preparing insulin, whereas only hydrochloric acid was used in our method of prepara- tion and purification.

From a consideration of the following points - (1) the act,ion

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D. A. Scott 615

of trypsin on insulin, (2) the action of specific reagents, (3) the preparation, (4) the chemical tests, (5) the combustion analysis, (6) the distribution of nitrogen-the similarity between insulin and a protein is apparent. Whether the purified product used in this study contains two or more proteins, having very similar physical properties, can only be determined by further research.

CONCLUSIONS.

1. Benzoyl chloride and carbon bisulfide completely inactivate insulin in an alkaline solution.

2. Formaldehyde and nitrous acid greatly decrease the activ- ity of insulin.

3. Carbon, hydrogen, nitrogen, and ash values have been de- termined on a purified insulin product.

4. The distribution of nitrogen and the tyrosine, tryptophane, and amino nitrogen have been determined in a purified sample of insulin.

I wish to thank Professor Rogers of the Department of Chem- istry for the carbon and hydrogen estimations and the Insulin Committee for checking the physiological assay. I am also in- debted to Dr. C. H. Best and Dr. A. Hunter for their helpful criticism.

BIBLIOGRAPHY.

1. Scott, D.A., J. Biol. Chem., 1925, lxiii, 641. 2. Banting, F. G., and Best, C. H., J. Lab. and Clin. Med., 1921-22, vii,

251. 3. Best, C. H., and Macleod, J. J. R., J. Biol. Chem., 1923, Iv, p. xxix. 4. Dudley, H. W., Biochem. J., 1923, xvii, 376. 5. Doisy, E. A., Somogyi, M., and Shaffer, P. A., J. Biol. Chem., 1923, Iv,

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Chemical Properties of Insulin

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D. A. ScottCHEMICAL PROPERTIES OF INSULINA FURTHER INVESTIGATION OF THE

1925, 65:601-616.J. Biol. Chem. 

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