ASSAY OF INSULIN IN VITRO BY FIBRIL ELONGATION AND PRECIPITATION

BY DAVID F. WAUGH

(From the Department of Biology, Massachusetts Znstitute of Technology, Cambridge)

AND ROBERT E. THOMPSON AND ROBERT J. WEIMER

(From the Chemical Research and Development Laboratory, Armour and Company, Chicago)

(Received for publication, December 2, 1949)

When heated, a solution of insulin in acid (pH about 2.0) will form a gel, a precipitate or coagulum, or a mixture of the two. The precipitate is composed of floes of spherites (1) whose subunits are radially oriented fibrils. Suspensions of dispersed fibrils (2) may also be prepared. In these, electron micrographs show that the fibrils have diameters of about 150 A and lengths of 15,000 to 40,000 A.

When one introduces fibrils formed at higher temperatures (80-100”) and cooled into solutions of native insulin at temperatures and pH values well within the stability range, the seeded fibrils will elongate at the lower temperature and quantitatively remove the native insulin from solution, thus transforming it into fibrous insulin (3). Elongation of insulin fibrils in the presence of native insulin has been found to take place in relatively impure solutions containing small amounts of insulin and in solutions which have been made acid by mineral acids, organic acids (for example, concen- trations of acetic acid up to 50 per cent have been used), or mixtures. A quantitative recovery of insulin is often achieved, indicating that the elongation reaction may afford the basis for an assay of insulin in vitro. This paper describes a method in vitro, based on fibril elongation and pre- cipitation.

Methods

Biological Assay-The sloped screen-mouse convulsion method devised by Thompson (4) was used with two 400 mouse assays. The results were calculated in terms of U. S. P. reference insulin run simultaneously with the samples. Experimental errors were determined by statistical analysis (5). The weighted mean values were obtained by weighting individual assays by the reciprocal of variance method.

Crude insulin solutions (still concentrates) were prepared for bioassay by adding 50 ml. of the sample to a mixture of 400 ml. of absolute alcohol and 400 ml. of commercial ethyl ether, and then stored at 5’ overnight.

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86 ASSAY OF INSULIN IN VITRO

The precipitate was dissolved in 100 ml. of hydrochloric. acid at pH 3.0. Appropriate dilutions of this solution were made for bioassay purposes.

Preparation of Initial Gel-A good quality crystalline pork insulin is dis- solved in 0.05 N hydrochloric acid in 2 per cent concentration. 2 ml. aliquots of this solution are pipetted into annealed Pyrex vials which are then sealed. The tubes are immersed in boiling water for a period of 1 to 2 minutes, withdrawn, cooled, and frozen rapidly by immersion in a mixture containing solid carbon dioxide and acetone. They are then rapidly thawed and subjected to a second 2 minute immersion in boiling water. The procedure of freezing and thawing, and heating is repeated, if necessary, until the contents of the tube show intense static double refraction between crossed Polaroid screens. This material will be referred to as the initial gel.

Preparation of Seeding Fibrils-The contents of a vial of initial gel are homogenized by repeated expulsion through a 10 inch, 18 gage hypodermic needle. The homogenate is then frozen in a solid carbon dioxide-acetone bath and rapidly thawed. The freezing-thawing cycle is repeated and the gel is then injected into 10 times its volume of 0.05 N hydrochloric acid. This material is added as rapidly as possible to the solution to be assayed. The freezing and thawing treatment breaks the long fib& into segments, shortening the reaction time by increasing the number of elongating ends.

Preliminary Experiments

Consideration of Variables-A more detailed consideration of the vari- ables involved in fibril elongation will be given later by one of the authors (D. F. W.). The following were considered to be of importance here: pH, temperature, ionic strength, and the nature of the inorganic anions. These factors will affect the rate at which insulin molecules associate with an individual fibril, and, in conjunction with the total aggregating area (see below) and concentration of free protein, will describe the course of a reac- tion involving dispersed fibrils. In the assay procedure it is desirable to terminate with the fibrils physically associated in a way which will allow mild centrifugation or filtration to separate them from the suspension medium. To keep the number of available ends maximum they should not form spherites as compact, for example, as those obtained by heating a 1 per cent solution of native insulin in 0.1 N HzS04 (1). Floccules of in- creasing compactness may be induced by an increase in ionic strength. Some salt also increases the rate of elongation to an extent which justi- fies an addition of salt before or just after seeding. Chlorides or sulfates of monovalent cations have been added to make the final salt concentra- tion 1 to 3 per cent, the sulfate ion (1) being more effective than the chlo- ride ion. Fibril elongation has been found to have a maximum at pH 1.5 to 2.0. 20 per cent sulfuric acid is therefore added to solutions to be

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D. F. WAUGH, R. E. THOMPSON, AND R. J. WEIMER 87

assayed to bring the pH within this region. Seeded solutions are stirred vigorously, but without frothing, not only to keep the floes distributed throughout the solution, but to break up the larger floes, thus reducing the time of diffusion of free insulin to the fibril end. Floe size in a stirred sus- pension has been found to be 2 to 5 I.C.

Quantity of Seeding Material-The over-all growth of a population of fibrils is given by

dC -z

= k (total aggregating area) C (1)

where lc is the growth constant for unit aggregating area and C is the con- centration of free insulin. The aggregating area of a single fibril is a func- tion of its size and shape and is therefore related to the surface area. Although applicable to dispersed systems, Equation 1 cannot be applied easily to the growth of fibrils in spherites and floes. The effect of varying the number of seeding fibrils may be approximated as follows. If N1 fibrils, each of constant aggregating area, A, are used in seeding, the total aggregating area is A .N. Using Equation 1 and integrating, we obtain

where 0 is the fraction of the original insulin present at time t. If Nz fibrils had been used, the time necessary to achieve a chosen value of 8 would have been changed by a factor NJN2; thus, doubling the number of fibrils is expected to halve the time axis. The approximation represented by Equation 2 has been examined briefly by assaying (biologically) the native insulin present during the course of the reaction (Fig. 1). The linearity of the plots of Fig. 1 indicates that Equation 2 may be applied to seeded solutions of the type described here. Equation 2 therefore suggests that the most effective seeding suspension is one containing the maximum number of fibril segments.

Temperature-Impure solutions impose limitations on the maximum allowable temperature, which will be considered below.

In the preliminary experiments beef still concentrates, clarified by filtra- tion, were acidified by the addition of 20 per cent sulfuric acid to pH 1.5 to 1.6. The concentrates contained sufficient salt. After seeding with a solution prepared as described from beef insulin assaying 23 to 25 units per mg., the concentrates were stirred by a glass rod at 2400 r.p.m. in en- closed vessels at 37.5” (no precipitate in unseeded solutions after several days). The reaction precipitates, obtained by centrifuging at 3200 X g, were suspended in 10 to 15 times their volume of 1 per cent ammonium chloride at pH 2.0 and centrifuged. This washing cycle was repeated twice additionally. The solutions were then dialyzed against distilled water,

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88 ASSAY OF INSULIN IN VITRO

dried at 110” for 12 to 18 hours, and weighed. 2 liter volumes were used and seeded at 20 mg. of fibrils per liter. The solutions were stirred for 28 to 72 hours. It was found that duplicate samples gave recoveries within 3 per cent, that only the seeding fibrils could be recovered on reseeding spent concentrate, and that crystalline insulin added to spent concentrate could be recovered quantitatively on reseeding. When the quantity of seeding material was varied from 20 to 4 mg. per liter of concentrate, the amount of insulin removed was independent of the amount of seed (within 3 per cent), provided sufficient time was allowed for completion of the elonga-

0 IO 40

FIG. 1. Fibril precipitation reaction. Line A, beef concentrate (3.09 unite per ml.) acidified to pH 1.55. Line B, pork concentrate (2.6 units per ml.) acidified to pH 1.6. Temperature 38”. 2 mg. of seeding fibrils per 100 ml. of concentrate.

tion reaction. The concentrates seeded with 4 mg. per liter were stirred for 75 hours.

On the basis of the above it seemed reasonable to devise an assay pro- cedure to be used with an unselected group of still concentrates. The volumes used in the preliminary assays were considered to be too large for routine use. Choice of a gravimetric analysis suggested still concentrate volumes of 100 ml. (about 20 mg. of insulin). Seeding material, not being critical, was placed at 2 mg. per 100 ml. of concentrate.

Standard Assay

Procedure-The sample to be assayed is filtered with the use of filter aid’ to a crystal clarity. To 100 ml. of solution in a conical centrifuge tube is

1 Hytlo Super-Cel, Johns-Manville, New York.

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D. F. WAUGH, R. E. THOMPSON, AND R. J. WEIMER 89

added 1.0 ml. of 20 per cent sulfuric acid (pH 1.5 to 2.0). It is then placed on a stirring apparatus. Seeding fibrils equivalent to 2 mg. of insulin are injected into the sample which is then stirred at 2500 r.p.m. with a straight glass rod for 40 hours at 38” or for 16 to 18 hours at 48”. Care must be taken not to stir air into the solution. The reaction is terminated by cen- trifuging at 2100 to 2300 X g until the supernatant is crystal-clear (45 minutes). The fibrous precipitate is transferred to 15 ml. centrifuge tubes with the aid of the first wash solution. Two different procedures for wash- ing the fibrils have been used. In Procedure 1, the precipitate is washed by centrifuging (15 ml. volume) once with 1 per cent ammonium chloride in 0.01 N hydrochloric acid, once with 0.01 N hydrochloric acid, twice with absolute methyl alcohol, and once with absolute ether. In Procedure 2, the precipitate is washed by centrifuging once with 1 per cent ammonium chloride and 0.01 N hydrochloric acid in 65 per cent alcohol, twice with 0.01 N hydrochloric acid in 65 per cent alcohol, and once with reagent grade acetone.

After washing, the wet precipitates are spread over large areas inside the tubes which are wiped with a towel wet with alcohol and dried under a vacuum to constant weight (2 hours or overnight) in the presence of phosphorus pentoxide. The tubes are then washed (inside) with 0.1 N

sodium hydroxide, water, alcohol, and ether, dried, and reweighed. The weight of the dried precipitate (less the weight of seeding fibrils) represents the weight of insulin contained in the solution assayed. Transferring and washing the precipitate are facilitated by means of a 5 ml. hypodermic syringe fitted with an 18 gage, 10 inch needle.

Materials-Comparative assays were conducted on the following types of insulin solutions: (a) Still concentrates derived from the original acid- aqueous alcoholic extracts of pancreas upon removal of the alcohol by low temperature vacuum distillation. Precipitated lipides were removed by filtration and the resultant product contained 2 to 5 units of insulin per ml., together with some salts and considerable protein material other than insulin. (b) Preliminary experiments have been done on solutions of crystal- line insulin, 18 to 25 units per mg.; one set of assays was conducted on a crystalline insulin solution subjected to a heat test for stability determina- tion.

Seeding fibrils were prepared from crystalline insulin assaying 25 f 2 units per mg. by the mouse method.

Results

Table I gives the results of comparative assays on thirty insulin still con- centrates. For the fibril assays the precipitation reaction proceeded at 38” for 40 hours.

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90 ASSAY OF INSULIN IN VITRO

TABLE I

Comparison of Bioassays and Fibril Assays

Fibril assays were run at 38” for 40 hours.

Sample’

B-l B-2 c-3 B-4 B-5 B-6 P-7 P-8 P-9 P-10 P-11 P-12 P-13 P-14 P-15 P-16 P-17 P-18 P-19 P-29 P-21 P-22 P-23 P-24 P-25 P-26 P-27 P-28 P-29 P-30

Mean . . . .

T- / / I- Ul

--

.I

Bioassay

nits per ml

3.6 3.0 9.7 3.9 3.7 2.9 2.6 3.6 3.9 3.7 3.9 3.8 3.6 3.7 2.7 3.4 2.6 3.0 2.6 2.6 3.4 2.6 3.7 2.9 3.4 4.2 4.0 3.7 3.5 3.5

- . P .-

L-

er cent se. Tube 1 Tube 2

6.1 3.9

9.2 3.5 9.2 11.7 7.2 3.7 6.4 3.6 9.2 2.9 9.4 3.0

10.3 5.0 8.0 4.1 9.7 4.1 6.9 4.2 9.2 4.0

10.5 4.4 7.2 3.5 8.0 2.3 6.4 3.0 8.3 2.6 9.4 3.0 9.2 3.0

10.7 3.0 9.7 3.1 9.7 2.9 9.0 4.4 8.5 3.5

11.5 3.5 8.5 4.2

11.0 4.2 9.2 3.6 9.2 4.1 9.2 3.3

3.9 3.5

11.7 3.5 3.7 2.9 2.9 4.8 4.1 4.1 4.2 4.0 4.6 3.4 2.4 3.1 2.5 2.8 2.9 3.0 3.1 2.9

3.3 3.5 4.1 4.2

4.3 3.2

r Fibril assay, units per m1.t Bioassay >ibx

x loo

3.9 92 3.5 86

11.7 83 3.6 108 3.6 103 2.9 100 2.9 90 4.9 74 4.1 95 4.1 90 4.2 93 4.0 95 4.5 80 3.4 109 2.3 117 3.0 113 2.5 104 2.9 103 2.9 90 3.0 87 3.1 110 2.9 90 4.4 84 3.4 a5 3.5 97 4.1 102 4.2 95 3.6 103 4.2 83 3.2 109

T - I

-

95.5

l B, C, and P indicate samples from beef, calf, or pork pancreas. t The values are derived by multiplying mg. of recovered fibrils by 25/K@. This

assumes that the fibril precipitate is comparable in purity to insulin containing 25 units per mg.

Table II summarizes the results of similar assays on the thirty-four still concentrates in which the fibril precipitation WM obtained by heating at 48” for 16 hours.

Table III gives the results of an experiment on the recovery of a U. S. P.

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D. F. WAUGH, R. E. THOMPSON, AND R. J. WEIMER

TABLE II

Biological and Fibril Assays on Still Concentrates

Fibril assays were run at 48” for 16 hours.

91

hbril assay, units per m1.t Bioassay -ma-

x loo SXIlple*

T- nits per ml ‘er cent se. Tube 1 Tube 2 i Mean

_- _- P-31 3.5 7.6 3.5 3.6 97 P-32

3.6 I 3.4 8.0 4.4 4.4 4.4 77

P-33 2.8 6.7 2.8 2.8 109 P-34 3.2 9.0 3.3 3.4 3.3 97 P-35 3.9 9.2 3.4 3.5 3.5 111 P-36 4.2 10.3 4.8 4.8 88 P-37 4.6 9.2 4.2 4.2 4.2 109 P-38 3.4 9.4 3.5 3.5 3.5 97 P-39 4.2 8.5 4.1 4.2 4.1 102 P-40 4.i 8.0 4.7 5.0 4.9 96 P-41 3.0 7.4 2.9 2.9 2.9 103 P-42 4.6 10.6 4.3 4.4 4.3 107 P-43 3.2 9.0 4.1 4.2 4.2 76 P-44 4.2 9.4 4.2 4.2 4.2 100 P-45 2.1 11.7 2.3 2.3 2.3 91 B-46 3.3 9.0 4.3 3.7 4.0 83 B-47 2.6 10.5 2.5 2.5 104 B-48 1.5 9.2 1.5 1.6 1.5 100 B-491 3.0 10.3 2.9 2.9 2.9 103 B-50 2.7 6.9 2.4 2.4 2.4 112 B-51 3.1 8.3 3.1 3.0 3.1 100 B-51 2.9 3.0 2.9 B-51 3.0 3.1 3.1 B-52 3.4 12.9 4.5 4.2 4.3 79 B-53 4.4 12.9 3.5 3.5 126 B-54 2.1 9.0 2.2 2.2 2.2 96 B-55 4.0 7.6 4.0 4.1 4.1 98 B-56 5.2 9.7 4.7 5.0 4.9 106 c-57 9.9 10.9 11.9 11.9 11.9 83 B-58 2.3 7.4 2.7 2.7 85 B-59 3.4 7.1 2.8 2.8 121 B-60 3.0 11.3 3.1 3.2 3.1 97 B-61 2.7 6.3 2.8 2.9 2.9 93 B-62 5.4 12.0 5.4 5.2 5.3 102 B-63 4.0 11.5 3.7 3.7 3.7 108 B-64 3.0 7.8 3.2 3.2 I 3.2 94 -

Mean . . . 98.2

* The letters B, Cl, and P indicate samples from beef, calf, or pork pancreas. t The values are derived by multiplying mg. of recovered fibril by 25/100. $65 per cent alcohol washing procedure used in fibril assays on Samples B-49 to

B-64. Procedure 1 used in all others.

-

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92 ASSAY OF INSULIN IN VITRO

zinc insulin reference standard from aqueous acid solution, from still con- centrate with and without the original insulin removed by fibril precipita- tion, and from a duplicate assay on the original still concentrate.

The second and third columns in Tables I and II give the bioassay results and their per cent standard errors, while Columns 4 and 5 give the fibril assay results on duplicates in units per ml. of sample, on the assumption that the fibril precipitates obtained contained 25 units per mg. This unit conversion factor of 25 will be considered further in the discussion. Tubes

TABLE III Recovery of U. S. P. Reference Insulin (23 Units Per Mg.) from Aqueous Solutions

and Still Concentrates

Assays were run simultaneously at 48’ for 16 hours.

0.01 N HCl in water 1 2 3

Still concentrate, Sample B-51, 4 insulin removed by fibril pre- 5 cipitation

Still concentrate, Sample B-51, 6 original sample 7

8 Still concentrate, Sample B-51, 9

original sample 10 11

-

1

-

-

w. 10.0 10.0 10.0 10.0 10.0

0 0 0

10.0 10.0 10.0

-

’ 1

__

Fib& .wmered

2.3 2.3 2.3 2.3 2.3

w. 8.7 9.2 9.1 9.6 9.4

3.0t 11.9 3.0 11.6 3.0 11.9 5.3 20.0 5.3 20.0 5.3 19.2

-

1

I

I

-

4ssayed’ mits Ill., fi v r1I weight x .la I00

-

i c

-- Q

Re- :overy

2.2 2.3 2.3 2.4 2.4

cr Gem

96 109 100 104 104

3.0 100 2.9 97 3.0 100 5.0 94 5.0 94 4.8 91

-

(

-

! i

-

Mean re-

:overy

)C? cm;

99

104

99

93

* Procedure 2 (65 per cent alcohol washing) used. t Previous fibril assay, 3.02 units per ml .; bioassay, 3.1 units per ml. ~8.3 per

cent (Table II).

broken from centrifuging, obvious loss of fibrils in withdrawing super- natants, or stirring failure accounts for the lack of some duplicate values. Samples B-49 to B-64 were assayed by Procedure 2 (65 per cent alcohol washing) as described previously. All other results were obtained with Procedure 1.

Pork and Beef Insulin Seeding Fibrils-A number of cross-seeding ex- periments were performed with beef and pork insulin seeding fibrils on crystalline pork and beef insulin solutions and on pork and beef still con- centrates. It appeared from these experiments that pork seeding fibrils enabled the recovery of the full amount of pork or beef insulin from solu-

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D. F. WAUGH, R. E. THOMPSON, AND R. J. WEIMER 93

tion, while beef seeding fibrils gave complete recovery of beef insulin, but only about 50 to 60 per cent recovery of pork insulin. All except Samples 1 to 7 have, therefore, been assayed with pork insulin seeding fibrils. Fur- ther examination of these observed differences is in process.

Washing Fibril Precipitate-It is essential to wash the precipitate free of all substances which will interfere with the final measurement (gravimetric in these experiments). Washing by Procedure 1 works well with still con- centrates. The fibril precipitate from more purified samples tends to dis- perse when Procedure 1 is used and cannot be centrifuged easily. Pro- cedure 2, designed to overcome this difficulty, works quite well. Assuming 5 per cent impurity in the residual liquid after the first centrifugation, Pro- cedure 2 should reduce this to about 1 y. The final acetone wash which facilitates drying may also remove some impurity.

Temperature-With pure insulin solutions the rate of elongation of the fibrils increases by a factor of about 4 for each 10” rise in temperature over a wide range (2030”; 90-100”). Other factors being constant, a 10” rise in temperature should decrease the assay time by the same factor. When pure insulin is to be converted to fibrils, relatively high temperatures may be used (e.g., 100”). However, in assaying, no precipitate other than insulin should form in seeded samples or unseeded controls. For still con- centrates we have chosen temperatures (48’ or below) at which the un- seeded controls remain clear. A precipitate forms increasingly at temper- atures of 52” or above. The overnight reaction time at 48” is considered practical, for a minimum of working hours is involved.

It is possible that higher temperatures may be used, particularly for the assay of more purified samples. This matter is under investigation.

DISCUSSION

The precision of the fibril assay procedure, independent of the assumed conversion factor of 25 units per mg., appears to be within f5 per cent as indicated by the variance of duplicates. The third column of Tables I and II indicates standard errors of 6 to 13 per cent for the bioassay used. A measured deviation from actual insulin content of 2.6 standard errors could be expected once in 100 trials on the basis of statistical variation alone. In addition, the interference of impurities on absorption in mice and the possibility of incomplete precipitation in preparing extracts for bioassay constitute other sources of error.

In the last column of Tables I and II the bioassays are expressed as per cent of the fibril assays. Good agreement is obtained within the limits of the bioassay error. The greatest differences are for Samples P-32 and B-59 in which deviations of 2.9 and 3.0 standard errors are obtained. A further analysis of the limits of the fibril assay will require more accurate

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94 ASSAY OF INSULIN IN VITRO

bioassays and a thorough analysis of the regenerated insulin obtained from the assay precipitates. The reliability of the precision accuracy indicated above (5 per cent) is strengthened, however, by the recoveries of crystalline insulin from pure solution and after addition to normal and spent still con- centrates as sho1T-n in the preliminary experiments and in Table III.

We have assumed that the weight-potency conversion factor (25 units per mg.) of a good grade of crystalline insulin applies to t,he fibril precipi- tates. That this value is reasonable may be deduced from the following. First, the assays of Tables I and II have been compared by use of a value of 25 units per mg. The ratios of the assays have a relatively normal dis tribution about the mean. The fact that the fibril assay indicates about 2 to 5 per cent more insulin than the bioassay does not affect this inter- pretation. Second, the results shown in Table III for the assay procedure indicate a quantitative recovery of cryst.alline insulin from pure solution and after addition to normal and spent concentrates. Third, two samples of purified insulin containing 18.3 and 28.7 units per mg. (~t6.5 per cent) by bioassay gave 16.9 and 24.8 units per mg. by fibril assay. Fourth, the fibril precipitates obtained with the technique described may be made to revert to a product having an activity close to 25 units per mg. Some forms of biologically inactive insulin may be found which will interfere, in the sense that they contribute to the fibril precipitate (but not to activity). This possibility is being examined. All of the experiments thus far, how- ever, suggest that the technique reported here will be useful in research problems as well as in those associated with the production and purification of the hormone.

The fibril assay has also been used to follow the results of the standard heat test on one sample of insulin (40 units per ml., pH 3.0, 52”, and 10 days). Bioassay of the control and heat-tested solutions gave 38.4 units per ml. f14 per cent and 27.3 units per ml. ~7.1 per cent, respectively. Fibril assays resulted in values of 38.3 and 27.5 units per ml. with or with- out filtration of the original samples. The latter indicates that the biolog- ically inactive insulin produced during the course of the test is not measured in the fibril assay; therefore, the loss is probably not due to fibril formation in this experiment.

SUMMARY

1. A method for the assay of insulin in vitro, based on the specific elonga- tion reaction of the insulin fibril, was devised. The over-all process is termed fibril precipitation.

2. The method in vitro was compared with a biological mouse convulsion method on 64 unselected crude pancreas extracts (still concentrates) and several solutions of crystalline insulin. The fibril assay described has a

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D. F. WAUGH, R. E. THOMPSON, AND R. J. WEIMER 95

precision error within f5 per cent. Assuming the fibril assay to be correct, the bioassays had a distribution over a range of 74 to 126 per cent of the fibril assay values. This range is reasonable on the basis of random selec- tion of mice alone.

3. It wan found that the fibril precipitation assay could be conducted at 38” for 40 hours or at 48” for 16 hours (overnight). Two other washing procedures were examined. One, involving 65 per cent alcohol, was found most practical from the standpoint of separation of the precipitate by centrifugation.

4. An apparent difference between pork and beef insulin seeding fibrils was found, pork fibrils being the most effective for seeding purposes.

5. Two samples of insulin having 18.3 and 28.7 units per mg. by bioassay gave 16.9 and 24.8 units per mg. by fibril assay.

6. The biologically active insulin remaining after a standard heat t,est applied to one sample was measured accurately by the fibril assay.

BIBLIOGRAPHY

1. Waugh, D. F., J. Am. Chem. Sot., 99.247 (1946). 2. Waugh, D. F., J. Am. Chem. Sot., 70,185O (1948). 3. Waugh, D. F., Federation Proc., 6, 111 (1946). 4. Thompson, R. E., J. Endocrinol., 39, 1 (1946). 5. Gaddum, J. H., Med. Ree. Council, Special Rep. Series, No. 183, 31 (1933).

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Robert J. WeimerDavid F. Waugh, Robert E. Thompson and

PRECIPITATIONFIBRIL ELONGATION AND

ASSAY OF INSULIN IN VITRO BY

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