THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 239, No. 11, November 1964

Printed in U.S.A.

High Specific Activity Labeling of Insulin with 1311*

JOSEPH L. Izzo, WILLIAM F. BALE, MARY JANE Izzo, AND ANGELA RONCONE

From the Department of Medicine and the Department of Radiation Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14620

(Received for publication, March 19, 1964)

The immunoassay of endogenous insulin in plasma or other body fluids by means of radiochromatoelectrophoretic techniques which have been developed by Berson and Yalow requires as a tracer %I-insulin of high specific activity. However, the meth- ods which Berson, Yalow et al. (1, 2), as well as others (3), have employed to prepare highly radioiodinated insulin have usually resulted in low and unpredictable yields of labeled insulin. Fur- thermore, if large amounts of 1311 are used, damage to the insulin occurs which is roughly proportional to the amount of 1311 attached to the hormone. It is reported that the insulin which is damaged by the effects of 1311 radiation does not bind insulin antibody (2) and that purification procedures are usually neces- sary to remove the damaged components whenever they are present in appreciable quantities. Hence, a procedure for high level radioiodination of insulin which is efficient in the use of starting 1311 and which does not require repurification of the labeled hormone should be useful.

1%insulin of very high specific radioactivity should also be potentially very useful for study of the metabolism and action of insulin. The studies that have been reported thus far on the disposal and metabolic fate of injected 1311-insulin have been restricted to the use of radioiodinated insulin of very low specific activity, with the result that the total amount of injected insulin has been substantial compared to the amount of endogenous hormone generally present in the circulation (1,4-8). Although these studies have provided useful information on the metabolic fate of injected insulin, they do not necessarily reflect the disposal of endogenous insulin at physiological levels. Hence, a suitable preparation of highly radioiodinated insulin that would permit the use of truly tracer quantities of injected insulin could be a useful tool for studying the metabolism and action of endogenous insulin at physiological levels. Obviously, one of the prime req- uisites for such a radioiodinated insulin is that it possess full biological activity.

In the accompanying paper (9)) we have reported that the total amount of iodine (stable as well as radioactive) attached to insulin differentially affects the biological, electrophoretic, and immunochemical properties of insulin. The incorporation of an average of more than 1 atom of iodine per molecule of insulin of an assumed molecular weight of 6000 resulted in a progressive loss of biological activity which was roughly proportional to the number of iodine atoms incorporated. On the other hand, increasing iodination of insulin had little, if any, effect on the electrophoretic

* This investigation was supported by Research Grant A-3556 from the United States Public Health Service, by a grant from Eli Lilly and Company, and by the United States Atomic Energy Commission. Presented in part before the Federation of Ameri- can Societies for Experimental Biology, Atlantic City, April 14, 1962.

or immunochemical properties investigated. On the basis of these observations it is clear that the average incorporation of total iodine should not exceed 1 atom per molecule of insulin if the labeled insulin is to be used for physiological studies, whereas the same restrictions are not necessary if the labeled insulin is to be used solely for immunoassay purposes.

With these considerations in mind, we present a procedure for incorporating 1311 into insulin in the order of 100 to 400 mc of Is11 per mg of insulin, with good yield of product in terms of starting 1311 and with little or no damage to the insulin by radia- tion as determined by chromatoelectrophoresis. By precise control of the total amount of iodine attached to the insulin (i.e. stable as well as radioactive iodine), highly radioiodinated insulin can be prepared for use in immunoassay procedures or for use in physiological studies. The method is based on, although not identical with, a procedure developed by Helmkamp et al. (10) for protein labeling, which, in turn, is based on the use of iodine monochloride first suggested by McFarland (11).

EXPERIMENTAL PROCEDURE

1311 Xolutions-Samples of carrier-free Na?I solutions pro- duced by fission of uranium were obtained by air shipment from Oak Ridge National Laboratory soon after production to mini- mize relative increase in the content of 1271 and 1291 which are present in the original 1311 sample. Activity at the time of shipment ranged from 20 to 50 mc per ml of solution.

Borate Bu$ers-Borate buffer of pH 7.65, designated 2X borate buffer (lo), was prepared by adjusting a distilled water solution of 0.32 M NaCl and 0.40 M H3B02 to pH 7.65 by addition of 3.2 M NaOH to a final concentration of approximately 0.08 M

NaOH. This borate buffer was diluted with an equal volume of distilled water to produce a second buffer of pH 8.0, designated 1 X borate buffer.

Insulin-Bovine zinc insulin crystals, Lot 535664, assayed by Eli Lilly and Company at 25.6 units per mg, were generously supplied by Dr. William R. Kirtley of the Lilly Research Labora- tories. Prior to each iodination, a fresh solution of insulin was prepared as follows. Dried insulin cryst,als (2 mg) were dissolved in 1 ml of 0.01 N HCl, and 1 ml of 2X borate buffer, was then added. For iodination of 150 pg of insulin, 0.15 ml of the above solution was diluted with 0.85 mlof 1 x borate buffer.

Iodine Monochloride Reagent-As described more fully else- where (12), the iodine monochloride reagent was prepared ac- cording to the reaction

2 HI + KIOa + 6 HCl 3 3 ICI + 3 KC1 + 3 Hz0

A convenient stock solution is one that is 0.02 M in ICl, 2.0 M

in KCl, 1.0 M in HCI, and 2.0 M in NaCl and was prepared as

3743

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High Specific Activity Labeling of Insulin with 1311 Vol. 239, No. 11

follows. To a solution of 0.5550 g of KCl, 0.3567 g of KIO, and 29.23 g of NaCl were added 21 ml of concentrated hydro- chloric acid (sp. gr. 1.18) and enough distilled water to bring the final volume of the total mixture to 250 ml. The slight amount of free iodine which formed was removed by repeatedly extract- ing this solution with carbon tetrachloride. A current of air, saturated with water vapor, was then passed through the ICl solution to volatilize any suspended or dissolved carbon tetra- chloride. Such a solution has a molarity within 1% of that calculated on the basis of the amount of KIO, that was used, and is stable indefinitely at room temperature.

Just before use, a dilution of the stock solution of ICI in 2 M

NaCl was prepared so that 0.2 ml of this diluted stock solution contained the appropriate number of molecules of ICl for each insulin molecule of molecular weight 6000 that was to be iodi- nated.

Procedure for High Level I311 IO&nation-Depending upon the level of specific activity that was desired, from 50 to 100 mc of Oak Ridge 1311 in a volume of solution not exceeding 5 ml were transferred to a chemically clean test tube (A, 2 x 15 cm). To this solution was added 1.5 ml of 2~ borate buffer (2 ml if the volume exceeded 4 ml). I f the pH was not approximately 8 by wide range indicator paper, the solution was adjusted to this pH by the addition of a few drops of 1 N HCl. Hydrogen peroxide, which is produced by /3 radiation from 1311, was destroyed by the addition of 0.2 ml of freshly prepared 0.5 M Na2S03. If it is not removed, hydrogen peroxide can prevent iodination of insulin by reducing the iodine of ICl to iodide. The excess sulfite in the contents of tube A was, in turn, oxidized by aerating the un- stoppered tube in a shielded rotary Evapo Mix2 with a swirling action at 70” for 2 hours.

After cooling to room temperature and measurement of the total radioactivity, the contents of tube A were transferred by jet to tube B, which contained 1 ml of a solution of the insulin to be iodinated.3 In these studies we used 150 to 250 pg of insulin. Immediately, 0.2 ml of the freshly diluted solution of stock ICI was added to 1.8 ml of 0.85% NaCl solution, and the mixture in turn was added by jet to tube B and thus rapidly mixed with the 13rI-insulin-mI-iodide mixture. Two reactions occur. The Ia11 which is present as iodide exchanges with the iodine mono- chloride to form mIC1. If the 1311 is present initially in a total amount of iodine that is small compared with the iodine in the ICl, the exchange will result essentially in a quantitative conver-

1 If the radioiodinated insulin is to be used solely as a labeled tracer for immunological studies, 4 molecules of ICI are used for every insulin molecule of molecular weight 6000. However, if the radioiodinated insulin is to be used for studies on the metabolism and action of insulin, not more than 1 molecule of ICl should be used for every insulin molecule of molecular weight 6000.

2 Buchler I*nstruments, New York, New York- 3 Tube B is fitted with a two-hole rubber stopper. U-shaped

capillary glass tubing with an internal diameter of 1 mm is used to connect tubes A and B. One end of the tubing, which is slightly constricted at its tip, is inserted for a distance of several centi- meters into tube B through one of the openings in the rubber stopper while the other end of the tubing is placed in unstoppered tube A so that its end just clears the bottom of the tube. By means of glass tubing (6 mm internal diameter) inserted through the other opening of the rubber stopper, tube B is connected to a glass stopcock which, in turn, is connected to a suction flask. By opening and closing the stopcock, solutions in tube A can be rap- idly transferred (jetted) and mixed with the contents of tube B. These operations are conducted behind a lead shield by remote handling appliances.

sion of the 1311 to the iodine monochloride form with great rapid- ity. The 1311C1 then reacts with the tyrosine residues of insulin to attach 1311 by stable covalent bonds. This reaction is also complete in a short time, probably in less than 1 minute. After 3 minutes, 1 ml of 6.25% human serum albumin in 0.85% NaCl solution was added to serve as a competitive sink or site of reac- tion for free radicals and peroxides, and thus acted as a protection against radiation damage to the insulin. The reaction mixture was then transferred to a cellophane dialysis sac* containing an additional 8 ml of 6.25% human serum albumin.

The reaction mixture was dialyzed overnight at 3” against two successive 2-liter portions of 0.850/, NaCl, each portion being adjusted to pH 3.0 with 6 ml of glacial acetic acid. At the end of the dialysis, the radioactivity in the sac containing 1311 was assayed. The contents of the sac were then transferred to a test tube containing 125 mg of 6.25% human serum albumin and centrifuged for 15 minutes at 10,000 r.p.m. to remove a very small precipitate of what is probably denatured albumin (with no appreciable loss of 1311). The supernatant solution was then removed, assayed for radioactivity, and divided into several portions; the frozen material was stored immediately at -18” until used. Such solutions may be kept in the frozen state for several weeks without damage to the insulin by radiation.

Radioactivity measurements were carried out by placing samples to be measured at a distance of approximately 55 cm in a lateral direction from a well shielded well-type sodium iodide scintillation counter, usually with 1.25 or 1.5 inches of lead filtration to reduce the y-ray sensitivity of the measuring ap- paratus to a usable level. Calculations of ?I content of samples were made in terms of the initial la11 activity corrected for radio- active decay. A 1311 standard measured under the same condi- tions was used to check counter stability. Care was taken to place all samples in containers of approximately the same diam- eter to make self-absorption corrections unnecessary.

Preliminary studies showed that after dialysis of 1311-labeled insulin, 1311 activity was quantitatively carried down in a tri- chloroacetic acid precipitate. Small corrections were made for losses of 1311-insulin which occurred while the material was being transferred from one container to another. This was of the order of 3% for loss on glassware and an additional 2 to 3% loss upon centrifugation after dialysis. This correction was applied to the calculation of the specific activity, which is expressed as mil- licuries of r3rI per mg of insulin.

The I311 attached to insulin is calculated as 13rI millicuries used, corrected for decay, multiplied by the ratio of the counts ob- tained in the final preparation of 1311-insulin after centrifugation to the counts of 1311 after aeration, corrected for decay.

Detection of Radiation Damage to 1311-InsuZin-Damage to each radioiodinated insulin preparation was determined by means of radiochromatoelectrophoresis according to a modification of the technique of Berson and Yalow (1, 2). 1311-Insulin (0.1 ml) was diluted to 5 ml with Verona1 buffer containing 0.25% human serum albumin. Of this diluted mixture, containing 8.9 pg of %I-insulin, 50 ~1 were added to each of two test tubes containing either 0.5 ml of 0.25% human serum albumin in Verona1 buffer of pH 8.6 and ionic strength 0.1 (Solution A) or 0.5 ml of human plasma (Solution B). Samples of Solution A (10 ~1) were then applied to each of two sets of duplicate strips of Whatman No. 3MM paper along a line 4 cm from the cathode end of the cell.

* Available from the Visking Corporation, New York.

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November 1964 J. L. Izxo, W. F. Bale, M. J. Ixxo, and A. Roncone

TABLE I

3745

Results of high level radioiodination of insulin with different ratios of starting ‘31I-Insulin and iodine monochloride (ICI)

Preparation

-

Al A2 A3

Bl B2 B3 B4 B5 B6

Cl c2 c3 c4 c5 C6

Moles of ICl per mole of

insulin*

4 4 4

4 4 4 4 4 4

1 1 1 1 1 1

Insulin used ‘3’1 usedt

Pg

250 250 250

44.8 29.5 49.7 28.7 43.7 25.7

150 150 150 150 150 150

150 150 150 150 150 150

92.2 42.9 46.5 305 49.6 0.111 104.0 58.9 56.6 406 58.6 0.148

90.4 33.9 37.5 237 39.4 0.087 137.9 56.9 40.8 409 43.9 0.149

88.0 41.9 47.6 306 50.2 0.112 99.1 47.5 47.9 336 50.8 0.123

98.1 15.4 15.8 112 17.2 0.041 82.3 12.2 14.8 90 16.4 0.033

112.0 15.6 13.9 106 14.2 0.039 136.5 11.4 8.4 88 9.2 0.030

66.6 14.4 20.7 107 23.0 0.039 84.2 14.1 16.7 93 19.1 0.034

la’1 recoveries attached to

insulin

I

mc

‘3’1 recovered attached to insulin at completion of

experiment

% mclmg %

65.8 121 67.8 57.8 120 60.6 58.8 105 60.0

Specific activity of insulin .adioiodinatior

Estimated average No. of atoms of 1311

per molecule of insulin*t

0.044 0.044 0.038

* Molecular weight = 6000. t The millicuries of 1311 were always corrected for decay up to the day on which the experiment was completed. $ Estimations were calculated on the basis that 100 mc of 1311 contains 0.8 pg of lZII.

One set of strips previously had been saturated with native insulin by immersing the strips for 1 hour in a 0.3% solution of unlabeled insulin, and then removing the strips and rinsing them three times with Verona1 buffer to remove the excess insulin. The other set of strips was previously soaked in Verona1 buffer prior to electrophoresis. A similar procedure was carried out for Solution B. The cell containing the four sets of duplicate strips was sealed except for the opening along the top.5 A constant voltage (300 volts) was applied, and the electrophoresis was conducted for 1 hour at room temperature in Verona1 buffer (pH 8.6 and ionic strength 0.1). The slit at the top of the cell was then sealed, and the electrophoresis was continued for 16 hours at 100 volts.

The paper strips were dried at 120” for 30 minutes. One strip from each of the four sets of duplicate strips was stained with Spinco B-l dye (bromphenol blue and hydrated zinc sulfate), while the other was not stained but was cut into 0.5-cm widths, and the radioactivity of each width was measured either in a gas flow counter (Nuclear-Chicago) or in a liquid scintillation system (Nuclear-Chicago), model 703.

RESULTS AND DISCUSSION

Percentage of m1 Attached to Insulin-Table I summarizes the results of three series of radioiodinations of insulin with different ratios of 1311, insulin, and ICl. Note that the amount of Is11 incorporated was determined not only by the initial ratio of 1311 to insulin, but also by the amount of ICl used. The highest radioiodinating efficiencies, i.e. 60 to 70% of starting 1311 firmly attached to protein, were achieved with 50-mc lots of 1311, 250 pg of insulin, and 4 atom equivalents of ICl. Specific radio- activities of 100 to 125 mc of 1311 per mg of insulin were obtained

5 If the slit at the top of the cell cover is left open, hydrody- namic flow occurs. The resulting evaporation provides the flow necessary to move globulins away from the origin.

under these conditions (Series A). Higher specific activities (237 to 409 me of 1311 per mg of insulin) were achieved with only a slight loss in iodinating efficiencies (40 to 50%) by the use of lOO- mc lots of 1311, 150 pg of insulin, and 4 atom equivalents of ICl (Series B). In these preparations an average of 47 mc of 1311 attached to insulin was recovered. On the other hand, iodina- tions of 150 vg of insulin with lOO-mc lots of 1311 and only 1 atom equivalent of ICl resulted in specific activities in the range of 88 to 112 mc of I31 I per mg of insulin with efficiencies of 10 to 23 % in terms of starting 1311 (Series C). An average of 14 mc of 1311 attached to insulin was recovered in Series C. In terms of insulin used, the yields of radioiodinated insulin in Series A and B are higher than those that have been reported in other published procedures (2, 3). Studies are currently in progress to improve efficiencies of iodinations of the Series C type of preparation.

An additional and very important factor that limits the amount of radioactive iodine that can be attached to insulin or other protein is the presence of appreciable amounts of 1271 and l291 in the so-called “carrier-free” 1311 preparations. As was described more fully elsewhere (12), the production of 1311 with a half-life of 8.05 days either by the fission of uranium or by the bombardment of tellurium with thermal neutrons also results in the production of stable 1271 and a long lived radioactive isotope, lZ91 (half-life, 1.6 x 107 years). Stable 1271 may also be present as an impurity in the irradiated material or in reagents that are used in the extraction and purification of 1311. Hence, as presently manu- factured, so-called “carrier-free ” 1311 is never in a strict sense free of stable or carrier iodine. Furthermore, 1311 decays on storage, while the other two isotopes do not, with the result that the ratio of stable to radioactive iodine increases progressively and can reach very substantial amounts. With the ICl chemical ex- change method of preparing high level radioiodinated insulin, the presence of increased quantities of I271 and 12gI interferes with the quantitative conversion of 1311 to 131ICl and results in a reduc-

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3746 High Specific Activity Labeling of Insulin with I311 Vol. 239, No. 11

tion in the efficiency of the coupling of 1311 to protein. Difficulty of this kind has been reduced to a minimum in the present studies by the use of freshly processed 1311.

Percentage of Total Iodine Attached to Insulin-Although the percentage of 1311 coupled to insulin is influenced not only by the ratio of starting %I to insulin, but also by the ratio of ICl to insulin and by the ratios of 12’1 and 1291 to KI, the percentage of total iodine attached to insulin is largely a function of the ratio of ICl to insulin. On the basis of data we have obtained from iodination of 150 pg of insulin with 1 and 4 atom equivalents of ICl and trace quantities of 1311, it is estimated that the Series B preparations contained an average of approximately 3.5 atoms of total iodine per molecule of insulin. On the other hand, the total iodine content of the Series C preparations did not exceed an average of approximately 0.65 atom of total iodine per mole- cule of insulin (mol. wt. 6000). Approximately 3 to 4 y0 of the total iodine incorporated into insulin is 1311 in the Series B preparations or, in other words, about 1 out of 8 insulin molecules is labeled with 1311. In the Series C preparations, approximately 1 out of 28 insulin molecules contains 1311.

Since the total amount of iodine incorporated into insulin, as can be seen from the foregoing discussion, is a function of the ratio of ICl to insulin used and cannot exceed the iodine content of the added ICl, it is apparent that with the iodine monochloride method the presence of unknown amounts of nonradioactive iodine isotopes in a given sample of 1311 would influence merely the percentage of 1311 coupled to protein, the total amount of iodine incorporated into insulin remaining unaltered. This constitutes a built in safety device to prevent overiodination of insulin. On the other hand, with iodination methods which depend upon the conversion of iodide to free iodine (2), the total as well as radioactive iodine incorporated into insulin would be influenced by the presence of unknown amounts of non- a dioactive iodide in the 1311 sample. Hence, at high 1311 levels,

PREPARATION B-5 NON-SATURATED PAPER

6000 -UNDAMAGED FREE INSULIN

LABELED WITH i3’ k 5000

2 s 4000

5 3000

2 $ 2000

8 1000

? I 2 3 4 5 6 7 8 9 IO II I2 I3 14 15 Cms

1 ORtGlN ALGIN

FIG. 1 (Zeft). Distribution of radioactivity of ‘311-insulin Prepa- ration B 5 (specific activity, 306 mc of Ia11 per mg of insulin) by the naner chromatoelectronhoretic techniaue of Yalow and Berson (2) on-untreated paper. According to their criteria, undamaged free

overiodination of insulin can easily occur with oxidation methods unless the total iodine content of a given 1311 sample is accurately known. This factor assumes especial importance in the prepara- tion of 1311-insulins of high specific activity which are intended for physiological use, because the total average iodine incorpora- tion of such preparations should not exceed 1 atom per molecule of insulin (9).

Damage to 1311-Insulin Produced by Radiation-None of the 1311-insulin preparations listed in Table I exhibited damage by radiation in excess of 5 y0 as determined by chromatoelectropho- resis in the presence of added human serum albumin or normal plasma. In several preparations no damage at all could be detected, even though the specific activity was of the order of 300 or more mc of 1311 per mg of insulin. This is illustrated by the chromatoelectrophoretic behavior of Preparation B 5 with added normal plasma which had an initial specific activity of 306 mc of 1311 per mg of insulin (Fig. 1). According to Berson and Yalow (1, 2), “undamaged” free insulin is adsorbed at site of application to the paper strip and remains at the origin when an electrical voltage is applied, whereas the “damaged” com- ponents are said to migrate away from the origin with the serum proteins. On this basis the preparation was essentially un- damaged, since virtually all of the radioactivity was detected at the origin and no perceptible increase in radioactivity could be detected in the region of the serum proteins. Furthermore, on paper that was presaturated with stable insulin, the radioac- tivity of the same preparation moved as one peak just behind the serum albumin fraction.

The movement of the radioactivity of these 1311-insulin prepa- rations as one peak (Fig. 2) suggests that the preparations were probably homogeneous. This, however, does not preclude the presence of altered insulin molecules having an electrophoretic mobility similar to those of the unaltered molecule. Neverthe- less, since the migration of the radioactivity of these 1311-insulin

6000

1 PREPARATION B-5

SATURATED PAPER

Cms

2 ORkIN &&IN

insulin remains at the origin, while damaged insulin migrates with the protein components of admixed nonimmune plasma.

FIG. 2 (right). Distribution of radioactivity of 1311~insulin Prep- aration B 5 (specific activity, 306 mc of 1311 per mg of insulin) on paper presaturated with nonlabeled insulin.

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November 1964 J. L. Izzo, W. F. Bale, M. J. Izzo, and A. Roncone 3747

preparat,ions of high specific activity was similar to the migration patterns of radioactivity obtained by Berson et al. (1) and in our laboratory (9) with the use of insulin from the same manu- facturer and lot and labeled with only trace quantities of r311, it is reasonable to assume that our radioiodinating procedure did not produce gross changes in the insulin molecule that would affect its electrophoretic mobility.

We believe that the early addition of human serum albumin to the radioiodinated insulin is largely responsible for preventing the degree of radiation damage that has been encountered by others (2, 3) in high level iodination of insulin with 13rI. This early addit’ion of protective protein is possible because of the rapidit.y with which 1311C1 reacts with the tyrosine residues of the insulin molecule to attach 1311 (10). Since the 1311C1 that has not. coupled with protein is promptly converted to iodide, serum albumin can be added within a few minutes of iodination of insulin with no appreciable labeling of albumin detectable within the limits of accuracy and sensitivity of our methods. As shown in Fig. 2, no discrete peak of radioactivity is detectable in the albumin region of the 16-hour chromatoelectrophoreto- gram of Preparation B 5, suggesting that perceptible labeling of albumin with 1311 did not occur under the conditions employed. This possibility was further investigated as follows. Two *311- insulin preparations were made with 0.25 mg of insulin, 1.5 mc of 1311, and 1 atom equivalent of ICl. To one of the preparations 32 atom equivalents of Na$03 were added just prior to the addition of the protective albumin. This was done to reduce any remaining ionic I311 to iodide. The percentage of iodination of both preparations was similar, 66.4 and 65.4$& respectively. Low level rather than high level radioiodination was carried out to eliminate or minimize the variable presence of small amounts of damaged components less than (5%) which have been ob- served to migrate with the serum proteins in some high level preparations and which, if present, might interfere with evalua- tion of the results. Chromatoelectrophoresis of both prepara- tions with hydrodynamic flow was carried out for 24 hours instead of 16 hours, with 0.25 y0 human serum albumin instead of normal plasma in order to achieve maximal sensitivity. Both prepara- tions gave identical results. No radioactivity was perceptible in the albumin region (Fig. 3).

High Level Radioiodination of Other Proteins--Preliminary studies indicate that with slight modifications in the method presented, high level radioiodination of glucagon can also be achieved. In fact, the relative simplicity of the method, coupled with its flexibility in permitting variable but controlled total as well as radioactive iodination of a protein of known composition, should make the procedure well suited for the labeling of other protein hormones of known chemical structure.

Greenwood, Hunter, and Glover (13) have recently reported a method for preparing small quantities of highly radioactive growth hormone based on 1311 labeling with r311 which has been oxidized from the initial iodide form with chloramine-T. They maintain that the degree of chemical substitution is minimized (0.5 to 1.0 atom of iodine per molecule of protein) by the use of carrier-free (m1) iodide. For reaspns that we have outlined earlier, the 1271 and I291 content of these starting preparations must be far higher than is consistent with the assumption of Greenwood et al. that 90% of the total iodine content of their starting preparation is 1311. Therefore, the number of iodine atoms attached to each growth hormone molecule must be greater than their calculations indicate. Bale et al. (12) have shown by cal-

-UNDAMAGED FREE INSULIN

LABELED WITH 113’

0 I2 34 5 6 789101112131415 Cms.

Ii c 1 I

OF&IN GiiGlN

FIG. 3. Chromatoelectrophoresis of ‘311-insulin of low specific activity in 0.25% human serum albumin and Verona1 buffer, pH 8.6, ionic strength 0.1, for 2f hours. Note the absence of per- ceptible radioactivity in the albumin region, indicating absence of any appreciable radioiodoalbumin in the preparation.

culations that are based on the physics of 1311 production by various methods, and also by chemical total iodine determina- tions of highly iodinated 1311 preparations, that all presently available 1311 preparations must contain much more total non- radioactive iodine than assumed by Greenwood et al. (13).

In a short communication, Banerjee and Gibson (14) have recently reported the production of radioiodinated insulin with a specific activity of 1 curie per mg with the method of Greenwood et al. (13). However, considerable radiation damage was ob- served, and repurification procedures were necessary to render the preparations sufficiently pure for use as tracers in immuno- assay procedures of insulin.

SUMMARY

1. A procedure is described for preparing r311-labeled insulin of high specific activity with efficient use of 1311 and with less than 5% radiation damage to insulin as measured by chromato- electrophoresis.

2. %I produced by fission of uranium is used soon after production to keep at a minimum the relative concentrations of 12rI and 1291. After destruction of the hydrogen peroxide which is invariably present in 1311 samples, solutions of I311 as iodide and of nonradiaoctive iodine monochloride (ICI) are successively added to solutions of insulin in borate buffer at pH 8.0. Chemi- cal exchange produces 1311C1, which, in turn, reacts with insulin to label it. Radiation damage is maintained at a minimum by prompt dilution with a solution of human serum albumin. 1311 that is not bound to insulin is removed by dialysis.

3. Iodination of 150 pg of insulin with lOO-mc lots of 1311 and 4 molecules of ICI per molecule of insulin resulted in specific activities of 237 to 409 mc of 1311 per mg of insulin. An average of 47 mc of 13rI was recovered firmly attached to insulin. The total iodine (radioactive plus nonradioactive) incorporated into insulin was estimated at 3.5 atoms of iodine per molecule of insulin with an assumed molecular weight of 6000.

4. Iodination of 150 Kg of insulin with lOO-mc lots of I311 and only 1 molecule of ICl per molecule of insulin resulted in specific activities of 88 to 112 mc of 1311 per mg of insulin. An average of 14 mg of 1311 was recovered attached to insulin. The total

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3748 High SpeciJic Activity Labeling of Insulin with 1311 Vol. 239, No. 11

iodine coupled to insulin did not exceed an average of 0.65 atom of iodine per molecule of insulin.

5. For 1311-insulin preparations that are to be used as tracers in physiological studies, the average incorporation of total iodine per molecule of insulin (mol. wt. 6000) should not exceed 1 atom. Such restriction is not required if the labeled insulin is to be used for immunoassay purposes only.

REFERENCES

1. BERSON, S. A., YALOW, R. S., BAUMAN, A., ROTHSCHILD, M., AND NEWERLY, K., J. Clin. Invest., 36, 170 (1956).

2. YALOW, R. S., AND BERSON, S. A., J. Clin. Invest,, 39, 1157 (1960).

3. SAMOLS, E., AND WILLIAMS, H. S., Nature, 190, 1211 (1961). 4. ELGEE, N. J., WILLIAMS, R. H., AND LEE, N. D., J. Clin.

Invest., 33, 1252 (1954).

5. 6.

7. 8.

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11. 12.

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14.

LEE, N. D., Endocrinology, 65, 347 (1959). WICK, A. N., AND DRURY, D. R., Proc. Sot. Exptl. Biol. Med.,

97, 514 (1958). STEIN, O., AND GROSS, J., Endocrinology, 66, 707 (1959). PROUT, T. E., AND EVANS, I. E., Ann. N. Y. Acad. Sci., 74,

530 (1959). 1.~~0, J. L., RONCONE, A., Izzo, M. J., AND BALE, W. F., J.

Biol. Chem., 239, 3749 (1964). HELMHAMP, R. W., GOODLAND, R. L., BALE, W. F., SPAR, I. L.,

AND MUTSCHLER, L. E., Cancer Research, 20, 1495 (1960). MCFARLAND, A. S., Nature, 183, 53 (1958). BALE, W. F., HELMKAMP, R. W., DAVIS, T. P., Izzo, M. J.,

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Joseph L. Izzo, William F. Bale, Mary Jane Izzo and Angela RonconeI131High Specific Activity Labeling of Insulin with

1964, 239:3743-3748.J. Biol. Chem. 

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