THE HEAT PRECIPITATION OF INSULIN*

BY VINCENT DU VIGNEAUD, ROBERT H. SIFFERD, AND ROBERT RIDGELY SEALOCK

(From the Department of Biochemistry, School of Medicine, George Washing- ton University, Washington, and the Laboratory of Physiological

Chemistry, University of Illinois, Urbana)

(Received for publication, July 26, 1933)

While attempting to hydrolyze crystalline insulin under very mild conditions du Vigneaud, Geiling, and Eddy (1) observed that a solution of insulin in 0.1 N HCl when heated in a boiling water bath yielded a flocculent precipitate which came down rather slowly, reaching complete precipitation in 14 to 2 hours. Al- though this acid-insoluble material exerted no physiological action when injected in suspension, reactivation to an active acid-soluble form could be brought about by dissolving the precipitate in very dilute alkali followed by immediate acidification. In a later in- vestigation it was found that this regeneration of activity could be effected by dissolving the dilute acid-insoluble material in cold 20 per cent HCl (2). Either method of regeneration gave a product similar in general properties to the original insulin with only a slight loss of potency. The regenerated material could be precipi- tated again by heating under the same conditions as the first and again reactivated by acid or alkali.

Blatherwick et al. (3) had earlier noted a similar precipitation with their amorphous insulin and, although the conditions that they had laid down for the precipitation have since been found to be of much wider limits, there seems to be no doubt that they were dealing with the same type of heat precipitation that we had encountered later with crystalline insulin.

The most obvious change which accompanies heat precipitation is the splitting out of ammonia from the insulin molecule as noted

* The insulin used in the present investigation was kindly supplied by E. R. Squibb and Sons through the courtesy of Dr. John A. Anderson. The authors take this opportunity to express their sincere appreciation.

521

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522 Heat Precipitation of Insulin

by Freudenberg and coworkers (4) and by Jensen and Evans (5). This fact suggested to Jensen, after his isolation of glutamic acid from the hydrolysate of insulin, that the ammonia originated possibly in the amide group of glutamine. Jensen further sug- gested the interesting hypothesis that the formation of the heat precipitate might be due to some sort of ring closure between the amide grouping of glutamine and some adjacent amino or car- boxy1 group with the splitting out of ammonia. That the am- monia liberation might take place simultaneously with the forma- tion of the heat precipitate without being directly connected with it is easily conceivable; for example, the liberation of ammonia during the heat coagulation of albumin has been shown by &&en- sen (6) to be merely coincidental to the coagulation. It occurred to us that if the ammonia formation were intimately connected wit.h the heat precipitation the rate of formation of ammonia might be expected to parallel that of heat precipitation, and, further, it. seemed reasonable to expect that the formation of the precipitate should be conditioned by the liberation of a definite amount of ammonia and conversely that after a certain amount of ammonia had been split out the heat precipitate should make its appearance. We have therefore studied the liberation of ammonia from insulin in some detail.

This peculiar behavior of insulin to which we shall refer as heat precipitation appears at present to be the chief characteristic, aside from its physiological activity, which sets insulin apart from the host of animal and vegetable proteins. We have been unable so far to find any other substance not derived from insulin which will yield a heat precipitate under the conditions mentioned above for crystalline insulin. Furthermore, the inactivation of insulin by such mild reducing agents as cysteine and glutathione is accom- panied by a loss of its ability to yield a heat precipitate (7). In view of all these suggestive facts, this heat precipitation reaction appeared to us to merit a much closer study.

EXPERJMENTAL

Heat Precipitation with Various Acids and Temperature Coe& cient of Reaction-The observations that we had originally made on the heat precipitation of insulin with 0.1 N HCI were at 100”. In the present investigation we have extended these observations

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du Vigneaud, Sifferd, and Sealock 523

to lower temperatures and to other acids. The time required for the appearance of definite, flocculated particles with the formation of a thin gel at various temperatures with 0.1 N HCl was deter- mined. There is a considerable increase in the length of time re- quired for the precipitate to form at lower temperatures; in fact, no precipitate was obtained at 50” even after heating for 4 days, only a few small flakes having separated out. The temperature coefficient calculated from the data, however, showed consider- able variation particularly at the higher temperatures.

TIME /N MIN. 4

IO 20 40 60 80 100 I20 I40 160

CHART I

0.1 N HzS04, however, showed more promise as a precipitant for determining the temperature coefficient, because the heat precipi- tate formed in definite particles without gel formation. The obser- vation of the formation of the first trace of precipitate and the time required for its formation were easy to duplicate. This superiority of H&SO4 is in accord with the finding of Gerlough and Bates (8) as is our observation that the speed of precipitation with H&O4 is 3 to 5 times that with HCl. From our data the tempera- ture coefficient of the heat precipitation with H&J04 was approxi- mately 4 for a 10” rise in temperature which agrees with that cal- culated from the data of Gerlough and Bates. The time required

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524 Heat Precipitation of Insulin

for the first appearance of the heat precipitate at different tempera- tures is shown in Chart I.

In studying the action of more dilute acid we found that no heat precipitate was obtained with 0.01 N HCl even after heating for 8 hours at 100’. This was surprising in that at a slightly higher pH a precipitate resulted after only a few minutes heating. We found however that when a little salt was added, enough to make the solution about 0.17 M, the precipitation was complete in 5 to 10 minutes. This speeding up of the precipitation by a little salt was found to be true of the other precipitations as well.

The precipitation velocity with HaPOd was found to be much slower than that with HCl or HzS04. A heat precipitate was produced with 0.1 N HsP04 at 100’ in approximately 160 minutes and with 0.5 N in 60 minutes.

Acetic acid in concentrations from 0.01 N to 1.6 N failed to yield a heat precipitate with insulin when the solutions were heated at 100” for 8 hours.

The difference between HCl and HzS04 as heat precipitants becomes more obvious in the more acid solutions. In solutions of acidity up to 1.5 N with respect to HCl the characteristic heat precipitate was formed, but with 2.0 N HCI no precipitate was given. With HCl from 0.5 N to 1.5 N insulin hydrochloride pre- cipitated at room temperature, but as the temperature was in- creased insulin hydrochloride dissolved and the insulin then pre- cipitated as the heat precipitate. That the failure to precipitate in the stronger acid was not a solubility phenomenon, but that the insulin had actually lost its ability to form the heat precipitate was shown by the fact that the insulin was not precipitated when the solution was subsequently made 1.0 N and again heated. The activity of the insulin was also lost.

In contrast to the behavior with HCI, heat precipitation is possible in HzS04 with as high a concentration as 6 N. The so called insulin sulfate forms at room temperature but dissolves on heating and the insulin then comes out as heat precipitate.

Liberation of Ammonia in Relation to Heat Precipitation-The NH3 liberated on heating the insulin with acid was determined by a method essentially the same as that described by Parnas and Heller (9) with certain modifications. In determining the amount of NH3 split off from 10 mg. of insulin dissolved in 2 cc. of 0.1 N HCI

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du Vigneaud, Sifferd, and Sealock 525

after heating for 1 hour at loo”, for example, the entire solution was transferred quantitatively with NHa-free water to the distilling flask, neutralized with NaOH, and 2 cc. of paraffin oil were added, followed by 8 cc. of a borax buffer solution which contained 8.5 volumes of 0.05 M borax to 1.5 volumes of 0.1 N HCl. The NH, was then distilled over in eracuo at a pressure of 100 mm. of mer- cury into 1 N H&304 and determined calorimetrically by Nessler- ization and comparison with (NH&SO4 standards. With care- ful attention to the details of manipulation and by rigid exclusion of laboratory air from the apparatus during the distillation, by using heavy walled tubing for all connections, and by covering all joints with collodion, we were able to obtain almost theoretical recoveries of known amounts of NH3. Repeated control deter- minations gave us definite proof that by our technique it was possible to estimate, with an error of less than 5 per cent, amounts of NH3 ranging upwards from 0.005 mg., with much smaller error in amounts from 0.02 mg. to 0.05 mg. Control experiments on our insulin samples showed the absence of ammonium salts and further that NH, was not split from the insulin under the conditions of the distillation.

We intended at first to find the velocity constant of NH, removal but the impracticability of such a determination became apparent. We found repeatedly that there was liberated, although at a slower rate, considerable NH, even after the mother liquor became biuret-free. In other words, during the course of the precipitation, part of the NH3 would be coming from the insulin in solution and part from the insulin already precipitated; hence, the constant would be meaningless. In the results that were obtained, how- ever, poor correlation was shown between the NH, liberation and the heat precipitation, the rate of the former being much slower.

The rate of NH3 liberation from insulin by 0.1 N H&304 was determined at 70°, 80”, 90”, and 100’. The concentration in all cases was 5 mg. per cc. For each point on the curves, either 10 mg. or 20 mg. samples were heated for the times and at the tem- peratures designated, and in every case the NH, determination was made on the entire sample. The data, checked by duplicate or trip- licate samples, are shown on Chart II.

The disparity between the amount of NH, liberated and the amount of heat precipitate formed is illustrated in Chart I, a com-

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526 Heat Precipitation of Insulin

parison of the time required for the cleavage of 0.1 per cent NH3 at 10” temperature intervals with the time required for the first appearance of the heat precipitate at the same temperatures. It is apparent that the temperature coefficients of the two reactions differ to the extent that at the higher temperatures precipitation precedes the liberation of 0.1 per cent NH,, while below 60” the removal of 0.1 per cent NH3 far precedes any trace of precipitation.

This lack of correlation is strikingly brought out by comparing the NH3 liberated at 60” and at 90”. At 60” for example it took

0. I8 L

LfBERATIOh’ 0 F

TIHE IN MIN.

20 40 60 80 100 120 /YO 160 /a0200 220

CHART II

160 minutes for the heat precipitate to begin to form in 0.1 N

H&04 and at this point 0.11 per cent of NH, had been liberated, while at 90” the heat precipitate began to form within 3 minutes but the amount of NH3 liberated was less than 0.03 per cent. Al- though the rate of heat precipitation, as we have already shown, is much greater in H&SO4 than in HCl, the rate of NH1 liberation by these two acids is the reverse. For example, at 100” 0.1 N HCl liberates 0.50 per cent NH3 in 90 minutes whereas 0.1 N H&S04 liberates 0.31 per cent under the same conditions. A more strik-

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du Vigneaud, Sifferd, and Sealock 527

ing contrast is the fact that 0.01 N HCl liberates 0.45 per cent of NH, in 5 hours at 100” without a trace of heat precipitate while HzS04 under the same conditions liberates only 0.35 per cent with an almost complete precipitation.

Inactivation and Loss of Heat Precipitability by Reducing Agents- As we have already reported (7), inactivation of insulin with cysteine or glutathione is attended by loss of heat precipitability. We have now extended these studies to reduction with H2S and with NaCN.

The inactivation with H2S was accomplished by allowing HzS to bubble for 45 minutes through a M/15 NazHP04 solution of insulin containing 0.5 mg. of insulin per cc. Nitrogen was then bubbled through the mixture to free it from HzS and the resulting solution was tested for potency. The material so inactivated was dissolved in 0.1 N HCl and placed in a boiling water bath. No heat precipi- tate was obtained.

After many preliminary experiments we found the minimum con- centration of NaCN and the minimum length of time necessary to bring about inactivation of crystalline insulin. We wished to obtain the inactive product with as little change as possible in the molecule for it became quite evident that long continued action of the cyanide caused a more deep seated change resulting in an acid- insoluble product. This was also true of HZS.

It was found that a solution containing 1 mg. of insulin and 2 mg. of NaCN became completely inactive within 3 hours. After 5 minutes of contact there was enough activity left so that when 0.1 mg. of insulin per kilo was injected into rabbits convulsions resulted. Within 30 minutes 0.1 mg. showed only a trace of activity but 0.5 mg. caused large blood sugar decreases. At 90 minutes the latter dosage produced only a slight fall and at 3 hours the insulin was completely inactivated. The insulin-cyanide solutions were injected directly, the amount of cyanide alone hav- ing been shown by control experiments to produce only a very slight rise if any in blood sugar. On heating the inactive material with acid no heat precipitate was obtained.

Nitroprusside tests were carried out as quantitatively as possible for comparative purposes on the above solutions at various time intervals. At 5 minutes t,he test was very weak and increased with length of time until a maximum was reached in a little over

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Heat Precipitation of Insulin

an hour. The increase in strength of the nitroprusside test was roughly proportional to the degree of inactivation.

Inactivation and Loss of Heat Precipitability by Acid and Alkali and Formation of Inactive Heat-Precipitable Material-As men- tioned previously, heating insulin for a few minutes in 2 N HCI resulted in loss of both heat precipitability and activity. Some experiments were therefore carried out at a lower temperature with 20 per cent HCI, in which the so called insulin hydrochloride is soluble, to see how readily activity and heat precipitability disappeared by this mild hydrolytic procedure. A sample of in- sulin was allowed to stand in 20 per cent HCl at room temperature and samples were removed at 1 minute, 30 minutes, 5,24, and 48 hours. No activity and no heat precipitation were obtained with the material from the 24 and 48 hour samples. Slight positive tests were obtained at the 5 hour point.

The behavior of insulin with dilute NaOH particularly brings out the extreme lability of the groupings necessary for heat pre- cipitation as well as those necessary for activity. Jensen and Evans (5) have already found that material treated with 0.03 N

NaOH for 3 hours at 34” or 0.1 N NaOH at 50” for 1 hour does not yield a heat precipitate. We have found that contact with 0.04 N NaOH for 12 hours at 25” would cause inactivation of the insulin as would be expected from previous reports (4, 5) and that this very mild treatment also caused disappearance of the heat pre- cipitation reaction.

From the acid inactivations that have just been described, it is quite apparent that the groupings necessary for the formation of the heat precipitate can be modified readily by the action of acid so that the insulin loses its ability to yield a heat precipitate as well as its activity. We have found though that, once the heat pre- cipitate is formed, these groups involved in the formation of the heat precipitate are quite resistant to acid. They are probably closed in a ring structure and, if the acid does not regenerate the material, the activity can be irreversibly destroyed before these groups are modified. It was found that heating the heat precipi- tate with 6 N HzS04 for 5 hours destroys all the activity but leaves an insoluble residue which can be regenerated by our standard procedure to an acid-soluble product. This regenerated material although physiologically inactive can again be heat-precipitated.

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du Vigneaud, Sifferd, and Sealock 529

Various attempts have been made to reactivate the material in order to see if it were potentially active. For example, the ma- terial after being dissolved at pH 11.4 was precipitated again by heating jn 0.1 N HCl and again regenerated but no activity was demonstrable. Furthermore regeneration of solubility of the original modified heat precipitate by 20 per cent HCI led to no activity, and alkaline treatment according to Charles and Scott’s (10) regeneration of acid-alcohol-inactivated insulin (11) was likewise negative.

This represents the first time that we have been able to isolate an inactive substance which would yield a heat precipitate under the conditions for heat precipitation of insdin in 0.1 N HCI. We are carrying out a more detailed study of this inactive heat-pre- cipitable material, as a comparison of its composition and be- havior with that of the original insulin may prove to be significant.

Regeneration of Heat Precipitate-The possibility of partial de- struction of insulin in the regeneration of the heat precipitate by alkali led us to try to ascertain the lowest pH at which regenera- tion would occur. We also wished to establish definite standard conditions for regeneration that would be consistently duplicable. We therefore tried regeneration by various glycine-buffer mix- tures. The lowest pH at which we could obtain practically com- plete solution of the heat precipitate was 11.4, and we therefore selected this pH in our standard conditions for regeneration. The procedure is as follows: 50 to 100 mg. of heat precipitate are sus- pended in 15 cc. of glycine-NaCl solution (12) at room temperature and 15.65 cc. of 0.1 N NaOH added. After allowing the mixture to remain at this pH for 5 minutes, it is then acidified. We have usually found a very slight trace of insoluble material.

Behavior of Regenerated Heat Precipitate-Having a standard set of conditions with which we could carry out regeneration of the heat precipitate we took up the study of the rate of precipitation of the regenerated material in order to see if the regenerated heat precipitate was different from the original insulin. We have found that the rate of precipitation of the regenerated material is far greater than that of the original, which fact indicates that we are dealing with a changed insulin. At temperatures from 70-100’ the precipitation is almost immediate in 0.1 N HCl. Although the original insulin shows no precipitation at 50’ even after heating

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530 Heat Precipitation of Insulin

for days, the regenerated material gives a precipitate within a few minutes at 50”. Still more remarkable, it gives a heat precipitate within a few hours at room temperature, and at 0” a heat precipi- tate overnight. These precipitates behave like the original heat precipitate in solubility and regeneration. It also became appar- ent in these studies that the ease of precipitation of the regener- ated material depends on the conditions under which the original precipitation has been carried out. The stronger the acid and the greater the length of time required to bring about the original precipitation, the more readily is the second heat precipitate formed. This can be brought out clearly by quoting the following experiments. A sample of regenerated heat precipitate that had been formed by heating under the mild condition of 0.01 N acid and salt for 10 minutes would not yield a heat precipitate at room tem- perature although it would at 37” after being heated at this tem- perature overnight, whereas a sample of a regenerated heat pre- cipitate that had been formed by heating the insulin in 0.1 N HCl for 2 hours gave not only a heat precipitate at room temperature, but also one at 0”. That the alkaline regeneration was not the cause of the increased ease of heat precipitation of the regenerated material was demonstrated by many control experiments.

DISCUSSION

The difference between ordinary protein coagulation and the process of precipitation observed when insulin is heated in acid solution is quite obvious. No protein of which we are aware can be coagulated by heat in acid of the concentrations that can bring about so readily the precipitation of insulin. Denaturation of the common proteins without coagulation may be effected, of course, by either acid or alkali and, on subsequent adjustment of the pH to the acid concentration favoring agglutination, the protein coagulates very rapidly. Insulin, on the other hand, does not show such behavior and if allowed to remain in contact with dilute alkali loses its ability to form a heat precipitate.

The coagulable proteins, owing to their high temperature coeffi- cient of denaturation, exhibit an extremely large temperature coefficient of coagulation. Coefficients from 58 to 9540 per 10’ rise in temperature have been found (13). Insulin, on the other hand, has a temperature coefficient of heat precipitation of only 4

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du Vigneaud, SifTerd, and Sealock 531

per lo”, a coefficient well within the range observed for ordinary chemical reactions. It must be admitted however that there is no a priori reason why a coagulation should not have as low a tem- perature coefficient as 4.

Since there is as yet no definite evidence that we are dealing with a coagulation according to the generally accepted meaning of the term, we prefer to use the term heat precipitation. Freudenberg has used the term Salzsiiureinsulin, but we feel that this termin- ology might lead to confusion with the term insulin hydrochloride as used by Dudley (14) to designate the insulin precipitate ob- tained at a concentration of 3 to 4 per cent HCl, a product entirely different from the precipitate formed in acid solution by heat.

We have attempted in these studies to throw some light on the possible relationship between the heat precipitation and the elimi- nation of NH, which accompanies it. We have been unable to obtain any evidence favoring such a correlation. In fact our results are more in accord with the idea that the NH3 liberation is merely coincidental to heat precipitation just as NH3 liberation is incidental to protein coagulation. S@rensen (6) has shown that, for example, in the process of heat coagulation small amounts of NH3 are liberated but concludes that the NH3 so obtained has not arisen in the process of denaturation nor in the actual coagulation itself, but is rather the result of a very mild hydrolysis of the coagulated material.

In the previous work on the inactivation of insulin by cysteine and glutathione (7) it became apparent that the groupings in- volved in the heat precipitate could be modified readily with loss of this characteristic reaction. We have now been able to show that inactivation by H2S or NaCN also results in loss of heat pre- cipitability. The study of this relationship between inactivation and loss of heat precipitability has also been extended to inactiva- tions by acid and alkali. Insulin inactivated in either of these two ways fails to give a heat precipitate. All of these studies indicate the lability of the molecule with respect to this heat precipitation reaction as well as to activity. The fact that these reactions seem to accompany each other led us to believe for a time that the groups involved in both behaviors were identical. The isolation however of an inactive heat-precipitable material from insulin decomposi- tion has demonstrated that this cannot be the case. This repre-

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532 Heat Precipitation of Insulin

sents the first time we have been able to isolate an inactive sub- stance which would yield a heat precipitate under the conditions for heat precipitation in 0.1 N HCl or H&Sod. It might be pointed out, however, that so far we have found no active insulin prepara- tion which would not yield a heat precipitate.

The fact that the active regenerated heat precipitate will yield again a heat precipitate more readily than the original insulin is definite evidence that the insulin has been modified by this pre- cipitation. This finding indicates the way in which the data that we have obtained on the rate of precipitation of insulin can be utilized as a tool to detect changes in the molecule that might not otherwise be observed. The fact that this change has been brought about with retention of activity is an encouraging indica- tion that we may be able to modify insulin still further without losing the hypoglycemic action. We are also hoping to study the action of enzymes on the active regenerated material since it may be possible that the behavior towards enzymes has also been modified.

SUMMARY

1. A study has been made of the heat precipitation of insulin with respect to .the effect of various acids and acid concentration with special reference to the temperature coefficient of precipita- tion.

2. The amount of ammonia liberated from insulin under vari- ous conditions has been determined and compared with the amount of heat precipitate formed. No evidence has been obtained that ammonia removal is directly connected with the heat precipita- tion reaction; the results rather point to the fact that ammonia liberation is merely coincidental.

3. The effect of various agents on the heat precipitability and activity of insulin has been determined.

4. An inactive heat-precipitable material has been obtained by the action of acid on the heat precipitate itself.

5. Conditions for regenerating the activity and solubility of the heat precipitate yielding duplicable results have been worked out.

6. It has been demonstrated that the regenerated heat precipi- tate yields a heat precipitate again in 6.1 N HCl or H2S04 more readily than does the original insulin.

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du Vigneaud, Sifferd, and Sealock

BIBLIOGRAPHY

1. du Vigneeud, V., Geiling, E. M. K., and Eddy, C. A., 6. Pharmacol. and Exp. Therap., 33,497 (1928).

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yun, M., J. Biol. Chem., ‘73, 57 (1927). 4. Dirscherl, W., 2. physiol. Chem., 139, 223 (1929). Freudenberg, K.,

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5. Jensen, H., and Evans, E. A., Jr., 2. physiol. Chem., 2OQ, 134 (1932). Jensen, H., Science, 76, 614 (1932).

6. Serensen, S. P. L., Proteins, New York (1925). 7. du Vigneaud, V., Fitch, A., Pekarek, E., and Lockwood, W. W., J. Biol.

Chem., 94, 233 (1931). 8. Gerlough, T. D., and Bates, R. W., J. Pharmacol. and Exp. Therap., 46,

19 (1932). 9. Parnas, J. K., and Heller, J., Biochem. Z., 162, 1 (1924).

10. Charles, A. F., and Scott, D. A., J. Biol. Chem., 92, 289 (1931). 11. Carr, F. H., Culhane, K., Fuller, A. T., and Underhill, S. W. F., Bio-

them. J., 23, 1010 (1929). 12. Clark, W. M., The determination of hydrogen ions, Baltimore, 3rd edi-

tion (1928). 13. Lepeschkin, W. W., Biochem. J., 16, 678 (1922). 14. Dudley, H. W., Biochem. J., 17, 376 (1923).

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Robert Ridgely SealockVincent du Vigneaud, Robert H. Sifferd and

INSULINTHE HEAT PRECIPITATION OF

1933, 102:521-533.J. Biol. Chem. 

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