the preparation and evaluation of tritiated polyalanyl insulin

The Preparation and Evaluation of Tritiated PolyalanylInsulin Derivatives1

ROBERT E. CANFIELD/- GORDON I. KAYE,~ AND SUSAN B. WESTFrom the Department of Medicine and the F.H. Cabot Laboratory of the Division of Surgical Pathol-ogy, Departments of Surgery and Pathology, College of Physicians and Surgeons, Columbia Univer-sity, Nexv York, N.Y. 10032

ABSTRACT. A series of polyalanyl insulin deriva-tives (PAI) have been synthesized and character-ized. Their content of added alanine ranged from0.49 to 16.6 moles per mole of insulin. PAI de-rivatives with an average of up to 3.75 moles ofadded alanine per mole of insulin retained fullbiological activity as measured by the epididymalfat pad assay. Data from phenylisothiocyanate deg-radation indicate that 87% of the molecules insuch a derivative probably possess at least oneadded alanine, so it is concluded that insulin withminimal addition of alanine is biologically active.

A preparation of high specific activity tritiatedpolyalanyl insulin has also been synthesizedand shown to be fully biologically active by theepididymal fat pad assay. Preliminary autoradio-graphic studies using this insulin derivative demon-strate that the hormone is bound to the sarcolem-mal membranes of both striated and cardiacmuscle. 3H-polyalanyl insulin was also concen-trated in the proximal tubules of the kidney, butbarely detectable in the liver at seven minutesafter intravenous injection. (Endocrinology 90:112, 1972)

THE present study was undertaken to de-termine whether polyalanyl derivatives

of protein hormones might be prepared withpreservation of their biological function. Ifthis can be accomplished, protein hormoneswith added radioactive polyalanyl side chainscould be used in tracer studies to locate sitesof hormone binding at the target cell.

The reaction of proteins with N-carboxy-D,L-alanine anhydride has been used to syn-thesize derivatives with multiple polyalanylside chains attached to the alpha aminogroup and to the epsilon amino groups ofproteins (1,2). One notable feature of thesederivatives is that the addition of large num-bers of alanine residues is accompanied bylittle or no interference with the biologicalaction of a particular protein. For example,it has been demonstrated that up to an aver-age of 700 poly-D,L-alanine residues may beadded to gamma globulin without alteringthe capacity of the antibody to bind antigen(3). The addition to ribonuclease of up to SOalanine residues, in multiple polyalanyl side

Received May 17, 1971.1 Supported by USPHS grants AM 09579 and

AM 12396.2 Career Investigators of the Health Research

Council of the City of New York.

chains, actually appears to enhance enzymicfunction on certain substrates and does notinterfere with the normal reoxidation of thereduced protein (4). Polyalanyl derivativesof trypsin with more than 100 added alanineshave enhanced stability and no loss of en-zyme function. Similarly polyalanyl deriva-tives of chymotrypsin have been shown toretain their enzymic activity (5). Finally,a preparation of poly-D,L-alanyl rabbit my-osin has been shown to retain most of its CaATPase activity (6).

The present report describes the synthesisand characterization of a series of polyalanylinsulin derivatives as well as preliminary au-toradiographic studies with a biologically ac-.tive tritiated polyalanyl insulin. Studies withpolyalanyl human growth hormone have beendescribed elsewhere (7).

Materials and Methods

Crystalline bovine insulin was obtained fromthe Boots Pure Drug Co., Ltd. (Batch 2189,23 IU/mg). This particular lot of insulin wasprepared at the request of The Commission onProteins, IUPAC, and is accepted as standard-ized material. Phosphocellulose (P-70) was ob-tained from H. Reeve Angel. Leuch's anhydride(N-carboxy-D,L-alanine) was purchased fromthe Pilot Chemical Co. and Dowex (AG 50 X 4)

112

January 1972 SYNTHESIS OF 3H-POLYALANYL INSULIN 113

was purchased from Calbiochem. Tritiated D,Lalanine was prepared as a custom synthesis bythe method of Humphrey et al. (8) by the NewEngland Nuclear Corporation and D-glucose-1-14C was also purchased from the New EnglandNuclear Corporation.

Preparation of Polyalanyl Insulin Derivatives(PAI). Crystalline insulin (3.0 g) was dissolvedin 200 ml of 0.02N HC1. Following the addi-tion of 800 ml of 0.1M NaH2PO4, the mixturewas adjusted to pH 7.0 with concentratedNaOH. The solution, which was quite turbidfollowing addition of the phosphate, becameclear after adjustment of the pH. Nine 100 mlaliquots of this solution were chilled on ice.Two grams of N-carboxy-D,L-alanine were dis-solved in 50 ml of anhydrous dioxane and aslight amount of residual insoluble material waseliminated by centrifugation. Eight differentvolumes of the dioxane solution (0.25 ml, 0.5ml, 0.75 ml, 1.0 ml, 2 ml, 5 ml, 10 ml, and 25ml) were added with vigorous stirring to thechilled 100 ml insulin-containing aliquots. Thepolyalanyl insulin products obtained from theseeight mixtures will be referred to as PAI 1through 8, sequentially. A ninth sample (PAI9) received no N-carboxyl-D,L-alanine, but wascarried through all the other procedures as acontrol. Following one hour of stirring, suffi-cient IN HC1 was added to each sample to adjustthe pH to 3.0. The material was then frozen andstored at —20° C.

Chromatography of Polyalanyl Insulin (PAI)Derivatives. The frozen insulin samples werewarmed to room temperature and applied to a2.5 X 10 cm column of phosphocellulose thathad been equilibrated with IN acetic acid ad-justed to pH 3.5 with NH40H. The columnwas eluted at room temperature with ammoniumacetate buffers mixed in a three chamber vari-grad. The first chamber contained IN aceticacid adjusted to a pH of 3.5 with NH40H, thesecond chamber contained 2N acetic acid ad-justed to a pH of 3.8 with NH40H, and thethird contained 4N acetic acid adjusted to apH of 4.5 with NH4OH. The volume of eachchamber was 250 ml; the fraction size was 5 ml.These conditions were chosen to permit elutionof insulin without precipitation of the hormone.The fractions containing the insulin were pooledand passed through a 5 X 80 cm column of

Sephadex G-25, which previously had beenequilibrated with 0 .1N acetic acid. The samesolution was used to elute the column after thepassage of the PAI fractions. The insulin-con-taining eluate, free of ammonium ions, was ly-ophilized and stored at —20° C.

Chemical Characterization of PAI Derivatives.Portions of each PAI sample (1 mg) were hy-drolyzed in 6N HC1 in evacuated tubes for 24hours at 110° C and, following evaporation ofHC1, subjected to amino acid analysis on aSpinco Model 120 B Amino Acid Analyzer. Thenumber of added alanines per mole of insulinis tabulated in the second column of Table 1.

An additional portion of each sample (30 mg)was subjected to performic acid oxidation bythe method of Hirs (9). The resulting mixtureof oxidized A and B insulin chains was dis-solved in 15% aqueous formic acid at 70° Cand applied to a jacketed, 0.9 X 47 cm columnof Dowex AG 50 X 4 maintained at 70° C.The column was developed with 15% formicacid until a sharp peak of optical density ab-sorbing at 280 nm emerged. The eluting solu-tion was then changed to 10% NH40H and asecond UV absorbing peak emerged from thecolumn coincident with the change in effluentpH. The columns were operated at elevatedtemperatures to avoid precipitation of the oxi-dized insulin chains. The composition of mate-rial from the first peak was identical to thecomposition of the insulin A chain; the compo-sition of material from the second peak wasidentical to that of the insulin B chain. Thissame technique was then applied to the isola-tion of the oxidized A and B chains from eachof the PAI derivatives. The four cysteic acidresidues in the oxidized A chain made it suffi-ciently acid to prevent adsorption on Dowexunder the conditions of application, whereasthe histidine and the lysine residues in theoxidized B chain apparently provided sufficientpositive charges to permit binding. The numberof alanines in each insulin chain could then bedetermined by amino acid analysis. These re-sults are listed in the third and fourth columnsof Table 1. The calculated sum of these valuesfor the individual oxidized chains (fifth column)is in excellent agreement with the results of theamino acid analyses of the unoxidized PAIsamples (second column).

Two to three milligram samples of each iso-

114 CANFIELD, KAYE, AND WEST Volume 90

lated A and B chain preparation were subjectedto the Edman degradation by a procedure de-scribed previously (10). The residues removedwere calculated by difference from amino acidanalyses of samples taken before and after onecomplete Edman cycle. The degree of alanyla-tion of epsilon amino groups of the lysine resi-due in position 29 of the B chain was determinedby deamination of the unreacted lysine sidechains with nitrous acid followed by acid hy-drolysis and amino acid analysis (4). Theseresults are expressed as the fraction of substi-tuted lysines (Table 2).

Epididymal Fat Pad Assay. Samples of each ofthe polyalanyl insulin derivatives were assayedfor biological activity by the epididymal fatpad assay procedure (11,12). As noted by theseauthors, the metabolic activity of portions ofan individual fat pad may be variable. There-fore, we rotated the tissue sections in the ex-perimental procedure to compensate for thesedifferences. In the assays reported here, eachfat pad was sectioned into three approximatelyequal pieces and these were placed in separateflasks. One flask contained 500 uU/ml of thestandard insulin, the second contained an equalamount of the PAI insulin derivative to betested, and the third flask contained no insulinand served as a blank. Each unknown was testedwith at least 20 sets of triple flasks, and theinner, middle and outer sections of the fat padswere rotated to minimize the effect of the ana-tomical differences. The biological activity listed

in Table 1 is expressed as the ratio of countsliberated from D-glucose-l-]4C by the poly-alanyl insulin derivative to the counts liberatedby an identical quantity of standard insulinafter each set of counts had been corrected forthe counts in the blank. A ratio of 1.0 wastaken to indicate full biological activity in thederivative being tested. The level of insulinassayed was chosen because it produced a ten-fold increase in radioactivity over the blankvalue. When the standard Boots insulin wascompared with the control sample (PAI 9) inthis assay system, the ratio of activity was 1.01with a standard error of 0.06. I t should benoted that each polyalanyl insulin test solutionwas prepared on a weight basis assuming 23IU/mg of insulin. Therefore, samples PAI 6, 7,and 8, with heavy alanine enrichment, wouldhave had higher biological activity ratios hadthe comparisons been made on a molar basis.No assay was performed on samples PAI 1and 3.

Preparation of Tritiated D,L Alanyl Insulin.N-acetyldehydroalanine was subjected to cata-lytic reduction in tritium and a portion of theproduct (7 Ci, 20 Ci/mM) was converted toLeuch's anhydride according to a modificationof the procedure of Humphrey et at. (8). Thelabeled alanine was suspended in dioxane at50° C and phosgene was continuously bubbledthrough the suspension with continuous stirringfor two hours. The dioxane was then evapo-rated by passing anhydrous nitrogen (Mathe-

TABLE 1. Relationship between alanine addition and biological activity ofpolyalanyl-insulin derivatives (PAI)*

Insulinderivative

PAH234567S9

(control)

Insulin

0.490.9S1.541.963.757.99

12.2516.600.01

Moles of added alanine/mole protein

A-chain

0.290.560.821.112.174.586.218.81

-0 .01

B-chain

0.210.380.600.791.673.465.767.63

-0.02

Calc.total

0.500.941.421.903.848.04

11.9716.44

-0 .03

Biological activity

Ratio

**1.03**

1.090.980.810.710.461.01

Standarderror

0.06—

0.060.050.050.050.060.06

* Biological activity of the PAI derivatives was measured by epididymal fat pad assay and a tabulationof data for PAI 4, illustrating the details of one particular assay, is given in Table 3. All of the other assaysemployed the same procedure.

** Epididymal fat pad assays were not performed on these two derivatives.


son) through the reaction vessel. The oily residueof :!H-alanine Leuch's anhydride (N-carboxy-D,L-a1anine anhydride) remaining after evapo-ration of the dioxane was redissolved in 5 mlof anhydrous dioxane and immediately addedto a chilled aqueous solution of insulin (20 mg)prepared as described earlier. After 30 minutesthe product was acidified (pH 3.0) and purifiedby the techniques for the isolation of PAI de-rivatives described above using a 5 X 1 cmcolumn of Sephadex G-25. The purified 3H-PAIwas then lyophilized and redissolved in 0.00INHC1 and its concentration determined by mea-suring absorption at 280 nm. Within 24 hoursof the reaction of insulin with the 3H-N-carboxy-D,L-alanine anhydride, the derivative was puri-fied and assayed for biological activity with theepididymal fat pad system. At the same timethat the assay flasks were incubating, 100 u.gof the radioactive insulin preparation were in-jected into the tail vein of an anesthetized 300g male albino rat. Seven minutes after the in-jection of the labeled insulin, the rat was sacri-ficed and samples of skeletal and cardiac muscle,kidney and liver were removed, fixed in Bouin'ssolution for 24-48 hours, dehydrated in ethanoland embedded in paraffin.

Preparation of Autoradiographs. Sections cut at5 u. and mounted on glass slides were subse-quently deparaffinized in toluene and rehydratedthrough a series of ethanol solutions of decreas-ing concentration.

Kodak NTB-2 nuclear track emulsion wasmelted at 45° C and then maintained at 37° Cin a continuous flow water bath. In total dark-ness, racks of slides were dipped for 30 secondsin the emulsion, drained for five minutes, andplaced for two hours in a desiccator containinga saturated atmosphere of hydrogen peroxidein order to oxidize artifactually reduced silverin the emulsion. The slides were dried for threehours in a light-proof box through which astream of cool air was passed. The emulsion-coated slides were stored in lightproof boxes at— 10° C and allowed to expose for 11, 18 and 20days. Two dippings were made, one of threeslides of each tissue, and one of two slides ofeach tissue, using a fresh lot of emulsion for eachdipping. Only slides from the same dipping werecompared in evaluating uptake of 3H-insulin.

Following exposure, slides were warmed to20° C, developed for three minutes in Kodak

3.0 -

A 280 nryj

2.0 -

1.0 -

-

-

r

I \

I '

1 •

• I< I

1 •

* \1II

1I

«

-

PH

-

- 5

PH

- 4

- 3

50 100

FRACTION NUMBER

150

FIG. 1. Elution of a 300 mg sample of PAI-1 fromphosphocellulose P-70 under conditions described inthe text. The closed circles indicate absorbance at280 nm and the closed triangles indicate the pH ofselected effluent fractions.

D-19, rinsed in running water for 30-60 seconds,and fixed in Ansco Rapafix (1:3 dilution) forseven minutes, all in total darkness. The de-veloped slides were washed for 20-30 minutes,placed in 4% formalin for 20 minutes to hardenthe emulsion, and then washed for an additionalten minutes. The sections were then stainedeither with Harris' hematoxylin and eosin Y(H&E) or with hematoxylin, phloxine and safran(HPS) (13), dehydrated, cleared in tolueneand mounted.

Results

Fig. 1 depicts the results of chromatog-raphy of the insulin derivative, PAI 1? onphosphocellulose. Phosphocellulose is a use-ful ion-exchange resin for peptide chromatog-raphy (14) and was chosen in this instancebecause most of the monophosphate groupsare fully ionized at pH 3.5. At this pH in-sulin is bound to the phosphocellulose, butalanine and polyalanine, by-products of thereaction with N-carboxy-D,L-alanine anhy-dride, pass through the column. It was ob-served that when the column was developedwith high concentrations of ammonium ace-tate at room temperature, the insulin andpolyalanyl insulin derivatives were eluted at


sufficiently low pH values to prevent preci-pitation of the protein.

Insulin samples with larger amounts ofadded alanine {i.e., PAI 5-8) were elutedfrom phosphocellulose at an earlier time thanthe control insulin samples. Amino acid anal-yses of material from the mid-portion of theascending edge of the insulin peak of thesePAI derivatives contained approximately30% more added alanine than material fromthe mid-portion of the descending edge ofthe same peak. The content of alanine in thefraction with the highest insulin concentra-tion, as well as that from the pooled material,was approximately the average of theseother two values. These findings reflect amoderate degree of heterogeneity among thesubstituted insulin molecules.

The number of added alanines, calculatedfrom amino acid analyses of each PAI sam-

TABLE 2. Substitution of alpha and epsilon-amino groups measured in isolated

A and B chains of polyalanyl-insulin derivatives (PAI)

Insulinderiva-

tive

PAI123456789

(control)

Percent of NH2-terminalamino acid substituted

with alanine*

A-chain

72644477469

100930

B-chain

8103625556576970

Percentof lysine

substitutedwith

alanine**

7101316223451520

* In the control sample (PAI 9) 68% of the avail-able glycine and 9 1 % of the available phenylalaninewere removed by a single Edman degradation on theisolated A and B chains. The percentage of NH2-terminal amino acids substituted with alanine inderivatives PAI 1-8 was calculated from the ob-served reduction in the release of glycine and phenyl-alanine following subtractive Edman degradationperformed under identical conditions on the isolatedA and B chains of the derivatives (10).

**The percent of epsilon amino groups substi-tuted with alanine was determined by amino acidanalysis of isolated B chains of the PAI derivativesfollowing deamination with nitrous acid and acidhydrolysis (4).

pie, is listed in the second column of Table 1.The third and fourth columns of the Tableshow the number of added alanines found byanalysis of the isolated A and B chains. Theresults suggest that the alpha amino group ofthe A chain is slightly more reactive with theN-carboxyanhydride than that of the Bchain, which had a portion of its added ala-nines attached to the lysine residue at posi-tion 29. Those lysine residues that hadalanines linked to their epsilon amino groupswere protected from deamination. Their es-timated values are given in Table 2.

The results of the Edman degradation per-formed on A and B chains isolated from thepolyalanyl-insulin derivatives are also sum-marized in Table 2. Glycine and phenylala-nine are the NHb-terminal residues of theA and B chains respectively. In the controlsample 0.68 moles/mole of the glycine and0.91 moles/mole of the phenylalanine wereremoved despite the insolubility of the oxi-dized peptide chains. The percent of normalNBb-terminal groups accessible for removalby the Edman degradation decreased pro-gressively with increasing alanylation. It isapparent that virtually all of the alpha aminogroups were substituted in PAI 7 and 8, yetone-half of the lysines remained unreactive.The lesser reaction of the epsilon aminogroups may be attributed to their loweredreactivity at pH 7.0.

The results of the epididymal fat pad bio-assays (Table 1) indicate that through PAI5 the insulin derivatives were as active asthe standard Boots insulin. The details ofdata for a representative epididymal fat padbioassay of a PAI sample in comparison withstandard Boots insulin are presented inTable 3. Selected polyalanyl insulin sampleswere also assayed for their glucose loweringability in alloxan-treated rats. These sampleswere found to be biologically active in thisexperimental system as well as in the fat padassay. Since the epididymal fat pad assayprovided more quantitative data, this methodwas used for the systematic comparison ofthe biological activity of the insulin deriva-tives. The assay system was designed to as-

January 1972 SYNTHESIS OF 3H-rOLYALANYL INSULIN 117

certain which of the PAI samples, if any,were fully active. Therefore, the data are ex-pressed as the percent of the 14COa releasedby an equal amount of standard insulin.When this ratio is less than one (PAI 6-8),it only indicates that the insulin derivativewas less active than the control. Precise esti-mates of the specific biological activity ofthese less active PAI derivatives would re-quire more extensive testing in view of thelogarithmic dose-response relationship ofthis assay (11,12).

The conditions for the synthesis of triti-ated polyalanyl insulin were chosen to result

in the addition of two or less moles of alanineper mole of insulin in order to preserve fullbiological activity. The high specific activityof the tritiated polyalanyl insulin productmade it impossible to perform a standardamino acid analysis, but estimates of thespecific radioactivity of the tritiated poly-alanyl insulin indicated that it was approxi-mately 20 Ci/mM, or the equivalent of oneadded alanine per mole. Thus the tritiatedsample was approximately the equivalent ofPAI 2, which would have been expected tobe biologically active according to the datashown in Table 1.

TABLE 3. Epididymal fat pad bioassay data for PAI 4

Assay

1

2

3

4

5

6

7

8

9

10

Preparation*

StandardPAI 4BlankPAI 4BlankStandardBlankStandardPAI 4StandardPAI 4BlankPAI 4BlankStandardBlankStandardPAI 4StandardPAI 4BlankPAI 4BlankStandardBlankStandardPAI 4StandardPAI 4Blank

Fat padsection(mg)

42.982.360.151.167.291.951.045.747.445.642.456.149.865.181.562.791.4

113.581.664.887.474.097.393.053.845.146.648.159.660.7

CPM(mg)

28626635

25638

20755

28924431330543

23835

19942

21319314115122

19222

12334

25832234231732

PAI 4**STD

0.92

1.29

0.81

0.97

1.24

0.88

1.08

1.68

1.29

0.92

Assay

11

n

13

14

15

16

17

18

19

20

Preparation*

StandardPAI 4BlankPAI 4BlankStandardBlankStandardPAI 4StandardPAI 4BlankPAI 4BlankStandardBlankStandardPAI 4StandardPAI 4BlankPAI 4BlankStandardBlankStandardPAI 4StandardPAI 4Blank

Fat padsection(mg)

74.166.292.662.165.986.392.777.365.8

100.796.9

103.939.945.569.855.764.663.040.651.934.354.462.168.969.667.359.572.067.898.3

CPM(mg)

24823224

25140

19816

11210411711020

19724

22022

19616613316620

16718

11016

165131146173

14

PAI 4**STD

0.93

1.33

0.92

0.93

0.88

0.83

1.29

1.62

0.77

1.20

* The sequence of preparations was varied in the assays so that anatomical differences in the epididymalfat pads would be averaged (see Methods).

** The biological activity ratio of the PAI derivative is expressed as the ratio of counts liberated fromD-glucose-l-14C by the polyalanyl insulin derivative to the counts liberated from this substrate by an equalweight of the standard insulin after subtraction from each measurement of the counts liberated in a blankcontaining no insulin.


A&B

C&D


The epididymal fat pad assay data con-firmed that the tritiated polyalanyi insulinderivative was biologically active. The valuefor the ratio of 14CO2 released by the 3H-PAIwhen compared to an equal quantity of stan-dard insulin was 1.04 with a standard errorof 0.05. Ten sets of three flasks were used inthis assay. The entire experiment was con-ducted within 24 hours to reduce the possi-bility that the high degree of specific radio-activity would destroy the biological activityof the insulin.

The autoradiographic preparations wereintended to determine the feasibility of usingpreparations of tritiated polyalanyi insulinwith this high level of specific activity tolocalize the hormone in various tissues. Theresults indicate that the method is a usefulone.

Fig. 2 shows the extent and localization oflabeling in four tissues, kidney, liver, cardiacmuscle and skeletal muscle, fixed seven min-utes after intravenous injection of the triti-ated insulin.

In the renal cortex (Fig. 2A) the majorportion of the labeled material is found tohave cleared the glomeruli and localized inthe first portions of the proximal convolutedtubules. The loop of Henle and the distalconvoluted tubules are unlabeled. The extentof clearance of the 3H-insulin by the kidneyin the seven minute period is quite evident.

The liver, on the other hand (Fig. 2B),

shows only slight labeling. This labeling isrestricted to the region immediately sur-rounding the portal vein and to a fewsinusoids radiating from it. The hepatocytes,hepatic venules and canalicular regions areunlabeled.

Both heart muscle (Fig. 2C) and skeletalmuscle (Fig. 2D) show extremely clear theapparently specific localization of the labelat the level of the sarcolemma. In bothcross-sections and longitudinal-sections theindividual muscle fibers are outlined by thesilver grains of the autoradiograph. The ab-sence of silver grains over the blood vesselsin the muscle sections emphasizes the effi-ciency of clearance by the glomeruli (Fig.2A). In addition, despite the high specificactivity of the 8H-PAI, there is very lowbackground in the autoradiographs.

Discussion

In order for a polypeptide hormone tobegin its action, the hormone's three-dimen-sional structure must be recognized by aspecific receptor site in the target cell. Ifthe hormone is bound at cellular sites ofaction for a period of several minutes (incontrast to the more rapid turnover in en-zyme-substrate interactions), the location ofthese receptor sites may be determined byuse of radioactive polypeptide hormones.Such attempts at localization would requirethat the labeled hormone have full biological

FIGS. 2A-D. Autoradiographs prepared from representative sections of kidney, liver, cardiac muscle andskeletal muscle of a rat sacrificed seven minutes after intravenous administration of 100 ng of 3H-polyalanyl insulin.

(A top, left) The heaviest labeling is found in the kidney. In the renal cortex the major portion of thelabeled material is shown to have cleared the glomeruli and to be localized in the proximal tubules. Thedistal tubules and the ascending limb of the loop of Henle are unlabeled. X100.

(B top, right) In the liver there is very little labeling. This labeling is restricted to the region immediatelysurrounding the portal vein and to a few sinusoids radiating from it. Hepatocytes, hepatic venules andcanaliculi are unlabeled. X25O.

(C bottom, left) Cardiac mustle and (D bottom, right) skeletal muscle show extremely clear and ap-parently specific localization of the 8H-PAI at the sarcolemma. The individual muscle fibers are outlined bythe silver grains. The absence of silver grains over the blood vessels in these muscle sections emphasizes theefficiency of renal clearance indicated by the absence of label over the glomeruli in Fig 2A. There is alsoextremely low background and no diffuse labeling despite the very high specific activity of the BH-PAI.X2S0.


activity as well as sufficient specific radio-activity to permit experiments with reason-able pharmacologic doses.

Because there is considerable knowledgeconcerning the chemistry and mode of actionof insulin (15), many studies with radioac-tive insulin have been attempted (16-21).Most of these studies, however, have usediodinated insulin. There is doubt whethersuch labeled molecules retain full biologicalactivity, so there is reason to evaluate othermethods of preparing labeled protein hor-mones. Arquilla et al. have reviewed the evi-dence concerning the effect of iodination andreport that purified iodoinsulin derivativeshave a markedly attenuated biological ac-tivity (22). A recent report suggests thatthe technique of preparing iodinated insulinmay account for the loss of biological ac-tivity (23), and iodinated insulin prepara-tions have been successfully employed instudies of membrane binding in fat cellsand liver (23-25).

As noted earlier, the addition of poly-alanyl side chains to the amino groups ofantibodies and enzymes frequently produceslittle, if any, alteration in the biological ac-tion of these molecules. Presumably, the hy-drophilic polyalanyl side chains which areadded to amino groups at the surface of theprotein serve to complement the existing hy-drophilic exterior shell of the molecule (26).In contrast, the tyrosine residues are fre-quently a part of the hydrophobic interior ofthe protein where there is no free space, sothat the addition of a bulky iodine atommight be disruptive to the architecture of theprotein. Thus, the addition of radioactiveatoms by reaction with the N-carboxy-D,L-alanine anhydride offers some theoretical ap-peal (7). In addition to the insulin studiesreported here, biologically active prepara-tions of polyalanyl human growth hormone(7) and of polyalanyl human chorionic go-nadotropin (27) have been prepared. How-ever, the studies with polyalanyl enzymes andantibodies involved molecules in which thebiological activity was measured against sol-uble substrates or antigens. It is possible

that the polyalanyl side chains on hormonescould inhibit their "fit" into a receptor siteat the surface of the cell, leaving open thepossibility that there may be a limit tothe total number of alanine residues thatcan be added.

In 1953 Fraenkel-Conrat showed thatinsulin would react with the N-carboxyan-hydride of leucine and, while no specificbioassay was reported, he noted that theinsulin derivative possessed some activity(28). The development of techniques toprepare tritiated alanine with a high specificradioactivity by reduction of N-acetyldehy-droalanine made it possible to design experi-ments using this amino acid. The D,L alaninemixture is customarily employed to preventthe formation of side chain alpha heliceswhich might reduce the solubility of themodified protein. Another advantage of theD,L polyalanyl side chain is that circulatingexopeptidases are not likely to release theadded alanines (29).

The results of the bioassay of unlabeledpolyalanyl insulin derivatives indicated thatup to 3.75 moles of alanine could be addedper mole of insulin (PAI-5) without reduc-tion of the biological activity of the hormonewhen it was compared to the starting ma-terial. The A chains consistently receivedmore alanine than the B chains, despite thefact that the latter possessed two types ofreactive sites. The control insulin samplethat was subjected to all the proceduresincluding the addition of dioxane did notlose biological activity when compared withthe starting material.

The Edman degradation data for the iso-lated A and B chains indicated that themajority of the NHo-terminal glycine andphenylalanine residues in PAI-5 were pro-tected from removal by the addition of ala-nines. It is concluded that at least 74% ofthe A chains of insulin molecules in PAI-5possessed at least one added alanine residueindicating that insulin molecules with min-imal alanine addition are biologically active.Furthermore, since over half of the B chainsin PAI-5 were also shown to be substituted


with alanine, it is likely that over one half ofthose insulin molecules that did not havealanine added to the A chain {i.e., 26%) hadat least one added alanine on their B chains.Therefore, the percentage of substituted in-sulin molecules in PAI-5 is probably close to87%, and the remaining 13% of unsubsti-tuted molecules are insufficient to account forthe finding of full biological activity in thesample (11,12).

Once it was established that biologicallyactive polyalanyl insulin derivatives couldbe prepared, the second step was to synthe-size a tritiated polyalanyl insulin and toevaluate such a derivative in autoradio-graphic localization studies.

While the unlabeled N-carboxyanhydrideof D,L alanine is commonly available, thetritiated reagent is not. However, techniquesto prepare tritiated alanine with high spe-cific activity have been developed in studiesof labeled antigens. These techniques wereduplicated on a reduced scale for the insulinstudies described here (8). Several pilot ex-periments were conducted until conditionswere established for the synthesis of a poly-alanyl insulin derivative that possessed be-tween one and two moles of labeled alanineper mole of insulin. These conditions werethen employed using the high specific ac-tivity, tritiated D,L alanine (20 Ci/mM). Adetermination of the specific radioactivity ofthe purified product indicated that it had re-ceived approximately one added mole ofalanine per mole of insulin. This figure mustbe taken as approximate because it is basedon the vendor's estimate of the specific ac-tivity of the tritiated alanine and also be-cause multiple dilutions were necessary toobtain satisfactory concentrations to deter-mine radioactivity. However, the estimatesof concentrations of the tritiated polyalanylinsulin for the bioassay and tail vein injec-tion were accurate because they were basedon the optical density of the 3H-PAI solutionmeasured at 280 nm.

The autoradiographs shown in Fig. 2indicate that tritiated polyalanyl insulin de-rivatives can be used effectively for such

studies. Because of the low energy of itsbeta decay, tritium provides excellent reso-lution for autoradiography (30,31). Equallyclear results probably would have been ob-tained if the animal had received 10 ug of3H-PAI, i.e., an amount closer to a physio-logical level.

While the results illustrated in Fig. 2 areprimarily intended to demonstrate the feasi-bility of this method for preparing labeledinsulin, they also provide an interesting com-parison with earlier studies which employediodinated insulin.

Several investigators have reported thatiodinated insulin molecules are concentratedby kidney proximal tubule cells, and haveconcluded that the labeled insulin is filteredat the glomerulus and reabsorbed in theproximal tubule (16,19,21). Presumably theappearance of the labeled insulin moleculesin the glomerular filtrate is a function ofmolecular size and would occur whether ornot the insulin derivative was biologicallyactive. Thus, proximal tubule localization ofgrains in Fig. 2 A is consistent with theseearlier autoradiographic studies and alsowith other studies concerning the role ofthe kidney in insulin metabolism (32,33).The tritiated insulin, however, gives a muchclearer autoradiograph than iodinated insulineven where the 3H-PAI is present in suchhigh concentrations as shown in Fig. 2A.

The almost uniform distribution of grainsalong the sarcolemmal membranes in thecross-section of the rat's striated muscle andin the section of cardiac muscle providesgraphic confirmation of the cell fractionationdata of Edelman and Schwartz (20). Theseauthors observed that virtually all of the131I-insulin in rat striated muscle could befound attached to sarcolemmal tubules orreleased into a soluble fraction. This pat-tern of the 3H-PAI labeling contrasts withthe diffuse localization obtained by previousworkers with 131I- or 125I-insulin (17-19).This diffuse labeling had been interpretedby some workers as evidence for intracellularpenetration of the iodinated insulin (18).In addition to the autoradiographic and bio-


chemical data which indicate that insulin isbound and acts at membrane in muscle cells,Cuatrecasas has shown that insulin coval-ently bound to Sepharose is capable of inter-acting with superficial membrane structuresof isolated fat cells to initiate transport andother metabolic functions (34).

Since insulin normally reaches the livervia the portal circulation and since the la-beled insulin was administered in this studyby injection into the peripheral circulation,it is difficult to interpret the paucity ofgrains in the liver section. Diminished liveruptake of insulin following peripheral in-jection has been reported (35). It shouldbe noted in this regard, that in the experi-ments reported here all tissues were takenfrom the same animal at a single time pointso that observations of the relative distri-bution of labeling are valid.

The present study demonstrates, therefore,that a biologically active tritiated polyalanylinsulin (3H-PAI) can be synthesized andused effectively in autoradiographic studiesof the cellular localization of this polypep-tide hormone.

Acknowledgments

The authors are indebted to Dr. Michael Sela formany useful discussions and to Dr. Israel Schechterfor the gift of N-acetyldehydroalanine.

References

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the preparation and evaluation of tritiated polyalanyl insulin

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