TIIE ~OUBNAL OF BIOLOGKZ~L CHEMISTRY Vol.246, No. 9, Issue of May 10,~~. 2780-2795, 1971

Printed in U.S.A.

The Structure of Bovine Proinsulin* (Received for publication, December 17, 1970)

CHRISNOLAN, E. J%ARGOLIASH,JAMES D. PETERSON,AND DONALD F. STEINER*

From the Department of Molecular Biology, Abbott Laboratories, North Chicago, Illinois 60064 and the Department of Biochemistry, University of Chicago, Chicago, Illinois 60037

SUMMARY

Several insulin-related substances, comprising 1 to 2% of the total protein, were isolated from commercial crystalline bovine insulin. The main components of this mixture were: the 81-residue, single chain proinsulin, two degradation products of proinsulin, termed Intermediate Forms I and II, and an insulin dimer, possibly linked by covalent bonds and apparently an artifact of the preparation. Treatment of the biologically inactive proinsulin or intermediate forms with trypsin yielded dealanylinsulin, which is fully biologically active. The amino acid sequence of the intact proinsulin was shown to be bovine insulin B chain-Arg-Arg-Glu-Val- Glu-Gly-Pro-Gln-Val-Gly-Ala-Leu-Glu-Leu-Ala-Gly-Gly- Pro-Gly-Ala-Gly-Gly-Leu-Glu-Gly-Pro-Pro-Gln-Lys-Arg- bovine insulin A chain. The only disulfide bonds in this protein are those in the insulin moiety. Intermediate Form I is a two-chain protein consisting of the insulin B chain ex- tended to the glutaminyl residue preceding the Lys-Arg terminal sequence of the connecting peptide segment, and the A chain, these two chains being held together by the disulfide bonds found in insulin. Intermediate Form II is a similar protein, consisting of the insulin B chain and an A chain ex- tended to the glutamyl residue following the Arg-Arg se- quence at the amino terminus of the connecting segment.

Proinsulin is the biosynthetic precursor of insulin and functions to facilitate the folding of the molecule to yield the correct pairing of cysteinyl residues required to form the disulfide bonds of insulin. The transformation of proinsulin to insulin occurs intracellularly in the p cells of the islets of Langerhans and insulin is the major storage form. The structures of the intermediate forms, the recovery from pancreas of connecting peptide lacking the amino- and car- boxyl-terminal basic dipeptide sequences in molar quantities equal to those of insulin, and the variability of the residue at the carboxyl terminus of the B chain of the insulins of various species, all indicate that the proteolytic enzyme or enzymes responsible for the proinsulin to insulin transformation proba- bly act by virtue of trypsin-like and carboxypeptidase B-like specificities.

Studies of the biosynthesis of insulin by human islet cell adeno- mata (1, 2) and normal islet tissue from rats (2-4) have provided

* A part of this work was supported by Grants AM04931 and AM13914 and a Career Development Award from the National Institutes of Health.

strong evidence that the A and B chains of insulin are assembled as parts of a larger continuous polypeptide, proinsulin. Human proinsulin was found to have amino-terminal phenylalanine, as is the case for the B chain of insulin, indicating that the polypep- tide chain might consist of, from amino- to carboxyl-terminus: B chain-connecting peptide segment-A chain (I). Similar pre- cursor forms have been detected in biosynthetic studies with codfish islets (5), fetal calf pancreas (6), and fetal rat pancreas (7). The insulin precursor protein was very sensitive to trypsin which transformed it to a product having the same electrophoretic mobility as insulin, and showing immunological properties similar to those of the hormone (l-3). Moreover, when tryptic diges- tion was followed by treatment with performic acid, the behavior of the resultant two peptides was indistinguishable from that of the oxidized insulin A and B chains with respect to their gel ex- clusion characteristics, electrophoretic mobilities, and the pro- portion of leucy1 and phenylalanyl residues in them (l-3, 8).

Materials having properties similar to the human and rat pro- insulins were detected as minor contaminants of commercial crystalline insulin preparations (1, 3, 9). Gel filtration of first

crystals of bovine insulin was employed to separate a fraction containing several minor components including intact proinsulin, proteolytic cleavage products of proinsulin, and an aggregated form of insulin (3, lC--12). These components, and related forms which occur in even much smaller quantities in this crude proinsulin-containing fraction, have been resolved essentially to homogeneity by chromatography in urea-containing buffers on columns of carboxymethyl cellulose and DEAE-cellulose. This paper describes the purification procedure and the detailed de- termination of the amino acid sequence of bovine proinsulin and of two of its proteolytic degradation products. Part of this work was previously reported (8, 13).

While these studies were in progress, the amino acid sequence of porcine proinsulin was reported by Chance, Ellis, and Bromer (14). This confirmed the proposed arrangement of the insulin chains within the molecule, showed that the transformation of proinsulin by trypsin yielded the fully biologically active dealanyl insulin, that is, insulin lacking the carboxyl-terminal alanine of the B chain, and made it possible to compare the structures of proinsulin from two relatively closely related species.

EXPERIMENTAL PROCEDURE

Materials

Bovine insulin, crystallized once, and the crude proinsulin frac- tion prepared from such crystalline preparations of insulin were kindly supplied by the Novo Company (Copenhagen). L-l-

tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin

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and preparations of chymotrypsin, pepsin, papain, and diiso- propylphosphofluoridate-treated carboxypeptidases A and B were obtained from Worthington. Commercial DEAE-cellulose (Whatman, DE-23) and Cm-cellulose’ (Whatman, CM-23) were cycled prior to use according to the directions of the manufac- turer. Urea free of cyanate was prepared by passage of freshly dissolved reagent quality urea through a column of mixed bed ion exchanger (Dowex AG-501, 50 mesh; Bio-Rad, Richmond, California) immediately before use (15). Other chemicals were reagent grade.

Isolation of Proinsulin and Intermediate Fractions

One-gram quantities of once crystallized bovine insulin were dissolved in 75 to 100 ml of 1 N acetic acid, applied to a column (8 x 100 cm) of Sephadex G-50, fine bead form (Pharmacia), and eluted with 1 N acetic acid (see Fig. 1). The fractions containing the crude proinsulin (tubes 98 to 114, Fig. 1) were combined and concentrated under reduced pressure at 28” in a rotary evaporator to remove most of the acetic acid, diluted with water, and then lyophilized. This crude fraction was chromatographed in 500 mg portions on a column of Cm-cellulose, as indicated in Fig. 2. The two fractions obtained (I and II, Fig. 2) were each chromato- graphed on a column of DEAE-cellulose, as shown in Figs. 3 and 4. Chromatographic fractions were pooled as indicated by the letters in the figures, acidified with glacial acetic acid to a concen- tration of 1 K, passed through a column (2.5 X 35 cm) of Sepha- dex G-25 in 1 N acetic acid to remove urea and salt, and then concentrated under reduced pressure, diluted with water, and lyophilized, as described above. The dry protein fractions were stored at -18”.

Analytical Procedures

Analytical disc gel electrophoresis was performed on 7.5% poly- acrylamide gels (5 cm in length) at pH 8.9 as described by Orn- stein and Davies (16) or in 7 M urea at pH 4.4 as described by Reisfeld, Lewis, and Williams (17). Gels were removed from the tubes, stained with Amido schwarz in 7.5% (v/v) acetic acid, destained electrophoretically and stored in 7.5% acetic acid.

Amino acid analyses were performed on a Beckman/Spinco model 120B analyzer run in 4-hour cycles or on a Technicon analyzer by the method of Piez and Morris (18). Samples were hydrolyzed in three times distilled constant boiling HCl (5.7 N)

in evacuated Pyrex glass tubes at 105-110”. Peptides were hy- drolyzed for 23 to 25 hours unless otherwise noted. In some in- stances, phenol (0.1%) was added before hydrolysis to prevent destruction of tyrosine. Proinsulin and the intermediate and nonconvertible fractions were hydrolyzed for the times indicated in Table II. For hydrolysis of samples containing S-carboxy- methylcysteine, the procedure of Crestfield, Moore, and Stein (19) was employed. Reduction of disulfide bonds and carboxy- methylation were performed in 8 M urea as described by Crest- field, Moore, and Stein (19). Performic acid oxidation was car- ried out at 2” essentially as described by Hirs (20). The phenol- sulfuric acid procedure (21) was used for carbohydrate analysis with thyroglobulin and glucose as reference materials.

Amino-terminal residues were identified by the dansyl pro- cedure of Gray and Hartley (22) as given by Woods and Wang (23) or by the Edman degradation procedure previously described

1 The abbreviations used are: Cm-cellulose, carboxymethyl- cellulose; dansyl, I-dimethyl aminonaphthalene-5-sulfonyl; PTH, phenylthiohydantoin.

(24). PTH-amino acids were identified by chromatography on thin layers of silica gel containing a fluorescent indicator (East- man Chromagram sheets, type 6060). The chromatograms were developed first with Solvent V and then with Solvent IV, as described by Jeppsson and Sjijquist (25), and the PTH deriva- tives were detected under ultraviolet light. For Peptides T-5-Cl and T-5-C7, in addition to direct identification of the PTH- amino acids, the composition of the residual peptide was deter- mined either by amino acid analysis following acid hydrolysis, or the amino terminus of the residual peptide at each step was identified by the dansyl procedure, or both methods were used together. Digestions with carboxypeptidase A (0.10 mg per ml) or carboxypeptidase B (0.05 mg per ml) were performed at 37” in 0.05 M Tris-HCI buffer at pH 8.0 at a substrate concentration of 10 mg per ml in the case of protein substrates and 1.0 pmole per ml for peptide substrates. Free amino acids in the digestion mixtures were identified by electrophoresis-chromatography, as given below for peptide mapping. The relative amounts of amino acids released by the carboxypeptidases, as well as the PTH and dansyl derivatives, were estimated visually on peptide maps or chromatograms on a scale of +l to f5. Some carboxy- peptidase and trypsin digests were analyzed quantitatively for free amino acids by applying an aliquot diluted in 0.2 M sodium citrate buffer, pH 2.2, to the amino acid analyzer.

Peptide mapping was performed on paper (Whatman No. 3MM, 46 x 57 cm) as previously described (26), or, in most instances, on thin layer cellulose sheets (20 x 20 cm). Electro- phoresis was in pyridine-acetate buffers at pH 6.5 (pyridine, 25; acetic acid, 1; water, 225; v/v) or pH 3.6 (pyridine, 1; acetic acid, 10; water, 89; v/v), at 45 volts per cm for paper and 20 volts per cm for the thin layer sheets. Chromatography was in 1-butanol-pyridine-acetic acid-water (600 :400 : 120 :480, v/v). The maps were developed with ninhydrin reagent and, in some cases, with the Pauly, Sakaguchi, or Ehrlich reagents for the de- tection of tyrosine and histidine, arginine, and tryptophan, respectively, as previously described (26).

Preparation and Isolation of Peptides

General Fractionation Procedures-Gel filtration of peptide mixtures was performed on columns (1.9 X 155 cm) of Sephadex (G-15, G-25, or G-50, fine bead forms) at room temperature. Columns were eluted with 0.1 M NH~HCOI or 2 M pyridine at about 15 ml per hour and the effluent collected in 2.5-ml frac- tions. With the former eluent, peptides were detected by their absorbance at 220 rnp and with ninhydrin reagent following thin layer chromatography of 50- to loo-p1 aliquots of every other fraction. Only the latter procedure was used when 2 M pyridine was the eluent.

Free flow electrophoresis was performed on a Brinkman model FF-1 apparatus, essentially as described by Hannig (27). The electrode buffer was pyridine-acetic acid-water (5:0.2:95, v/v), pH 6.5; the plate buffer was the same buffer diluted with an equal volume of water. Electrophoresis was at 1500 to 2300 volts and 5-6” with a sample retention time of 60 to 90 min. Fractions of 6 to 7 ml were obtained; 0.2-ml aliquots were used for the detec- tion of peptides with ninhydrin reagent after thin layer electro- phoresis or chromatography as described above.

Cation exchange chromatography was performed on columns (0.9 x 25, 60 or 150 cm) of Dowex AG-50W-X2 (Bio-Rad) or Beckman type AA-15 spherical resin at 40” and a flow rate of 30 ml per hour. Columns were eluted with 0.2 N pyridine-acetic

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2782 Structure sf Bovine Proinsulk Vol. 246, No. 9

acid buffer, pH 3.1, with a linear gradient to 0.5 N, 1.0 N, or 2.0 N

pyridine-acetic acid at pH 5.0. Fractions of 2.5 ml were col- lected, and detection of peptides was by a calorimetric ninhydrin procedure (28) or by thin layer chromatography of aliquots, as described above.

Peptide fmctions collected in 0.1 M NHJHCOJ were concen- trated on a rotary evaporator at 30” and lyophilized; those col- lected in other solvents were concentrated to dryness on the rotary evaporator at 40”.

Tryptic Peptides of Intermediate Fraction-The protein (30 to 50 mg) at a concentration of 20 mg per ml in 0.11\1 NH,HC03 was incubated with trypsin (2 mg per ml) at 38” with frequent stirring until a heavy precipitate which formed at the onset of proteolysis had largely disappeared (1 to 2 hours). After removal of a small amount of insoluble material by centrifugation, the digest was subjected to gel filtration on a Sephadex G-50 column in 0.1 II~ NH4HC03 (see Fig. 8) or 2 M pyridine. Peptides in Fractions I and II (Fig. 8) were resolved by free flow electrophoresis at pH 6.5.

Papain Fragments of Peptides T-4 and T-5-The peptides, at approximately 1 pmole per ml, were incubated with papain (75 pg per ml in 0.01 M NaCN) at pH 7.0 for 25 to 60 min. The reaction was stopped by addition of iodoacetamide to a concen- tration of lop3 RX and lyophilization. The digest was then frac- tionated by free flow electrophoresis at pH 6.5. When proteoly- sis was extensive, it was necessary to further purify some of the electrophoretic fractions by gel filtration or cation exchange chromatography, as described above.

Peptic Peptides of Intermediate Fraction-The protein (60 mg) at a concentration of 10 mg per ml and pepsin (0.20 mg per ml) in 50/, formic acid were incubated at 38” for 5 hours. The digest was lyophilized, redissolved in 0.1 M NH,HCOz with a few drops of 25% trimethylamine, and fractionated on a column (1.9 X 155 cm) of Sephades G-50 in 0.1 M NHdHC03. The first of two arginine-containing fractions was chromatographed on a column (0.9 x 60 cm) of Spinco AA-15 spherical cation exchange resin at 50”, first with 45 ml of 0.2 M pyridine-acetate buffer, pH 3.1, and then with a linear gradient between the starting buffer (500 ml) and 1.0 M pyridine-acetate buffer at pH 5.1 (500 ml). Pep- tide Pe-1 eluted in approximately 0.9 M buffer. Fractionation of the second Sakaguchi-positive peak was unsuccessful.

Chymotryptic Peptides from Oxidized Proinsulin and Tryptic Peptide T-5-Performic acid-oxidized proinsulin (48 mg, 10 mg per ml) and chymotrypsin (0.20 mg per ml) in 0.1 M NH4HC03 were incubated at 25”. After 2.5 hours the digest was lyophi- lized twice and chromatographed on a column (0.9 X 150 cm) of Dowex AG-50W-X2 in 0.2 N pyridine-acetate buffer, pH 3.1, with a linear gradient to 2.0 N pyridine-acetate, pH 5.0, over a total volume of 2 liters. Certain Sakaguchi-positive fractions were further purified on Sephadex G-25 columns (1.9 X 155 cm) in 0.1 M NHhHCOz.

Tryptic peptide T-5 (0.5 pmole) was digested with chymotryp- sin (27, w/w) in 0.2 ml of 0.04 M Tris-WC1 buffer, pH 8.2. Of the four fragments produced, Peptides T-5-Cl and T-5-C2 were isolated by electrophoresis on Whatman No. 3MM paper (5 volts per cm, 10 hours, the pyridine-acetic acid-water buffer, pH 6.5, given above). Peptide T-5-C2 (0.5 pmoles) was further digested with chymotrypsin (5% w/w) in 0.2 ml of 0.04 M Tris-HCl buffer, pH 9.0, containing 3 mM calcium chloride to yield three fragments which were isolated by thin-layer chromatography on cellulose sheets with l-butanol-pyridine-acetic acid-water (60 :40 : 12 :48

v/v) as the solvent. Peptides T-5-C6 and T-5-C7 were utilized for these studies. These and other chymotryptic fragments of the connecting segment of bovine proinsulin have been described by Steiner et al. (29).

RESULTS

Isolation of Bovine Proinsulin and Related Proteins-Gel filtra- tion of first crystals of bovine insulin on a column of Sephadex G-50 in 1 N acetic acid (Fig. 1) showed the presence of a minor fraction, referred to as ‘(crude proinsulin” (V,/V, = 1.39), which eluted ahead of insulin (V,/V, = 1.72) and accounted for 2 to 30/, of the total protein. Disc gel electrophoresis of this crude proinsulin gave three major bands, each of which was accom- panied by minor, satellite bands (Fig. 1).

Chromatography of the crude proinsulin preparation on Cm- cellulose in a buffer containing 7 M urea resolved the mixture into two fractions (Fig. 2). Peak I (Fraction BCM-I) corresponded to the middle major band of the disc gel electrophoretogram while the components corresponding to the slower and faster migrating bands eluted together in the second peak (Fraction BCAI-II). Further fractionation of each of these mixtures by DEA4E-cellu- lose chromatography, again in buffers containing 7 M urea, gave the results shown in Figs. 3 and 4. The major component of Fraction BCM-I (Fraction BCM-ID) was obtained essentially free of other components as evidenced by disc gel electrophoresis at pH 8.9 (Fig. 3) and at pH 4.4. The two major components of Fraction BCM-IL emerged in Peaks C (Fraction BCM-IIC) and H (Fraction BCM-HH), which correspond, respectively, to the slowest and fastest migrating major bands in the crude pro- insulin disc gel electrophoretogram (Fig. 4). The electrophoreto- grams showed that each of these two fractions still contained a minor protein contaminant amounting to not more than 2 to 3% of the total, as estimated by visual comparison with electro- phoretograms of mixtures of measured amounts of purified com- ponents. Recoveries of Fractions BCM-ID, BCMIIC, and BCM-IIH from the crystalline insulin preparations were approxi- mately 0.6 to 0.87(,, 0.3 to 0.4cj,, and 0.4 to O.S%, respectively. These preparations were used without further purification.

Characterization of Chromatographic Fractions-Previous stud- ies have shown that Fractions BCXIIC and BCh/l-ID, which have only low insulin activity, can be converted to fully active dealanylinsulin by limited hydrolysis with trypsin (8, 13, 14). The third major fraction, BCM-III-I, was similarly less active than insulin, but could not be converted by tryptic digestion to a fully active form behaving like insulin on gel filtration. It is designated the “nonconvertible” fraction. In studies described below, the trypsin-convertible fractions, BCM-IIC and BCMID, were identified by amino acid sequence analyses, respectively, as proinsulin and a mixture of two partially degraded forms of proinsulin, referred to as the “intermediate” forms. Amino acid and end group analyses of the nonconveritble fraction, together with its gel filtration, chromatographic, and electrophoretic prop- erties have identified it as an insulin aggregate, presumably a dimer. Preliminary studies indicate that the minor fraction BCM-IIB (Peak B, Fig. 4) and the minor component of the in- tact proinsulin-containing fraction (Peak 3, Fig. 4) are single chain structures that are indistinguishable from proinsulin as regards amino acid composition and amino-terminal residue.2

2 J. L. Clark and D. F. Steiner, unpublished data.

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The other minor fractions (Figs. 3 and 4) have not been charac- phenylalanine from the B chain and glycine from the A chain, terized. while carboxypeptidase B released asparagine from the A chain

End Group Annlyses-These arc summarized in Table I. In- and alanine and a smaller amount of lysine from the carboxyl- sulin was used as a control. It gave the expected amino-terminal terminal and penultimate positions of the B chain, respectively.

12.0

11.0

10.0

2 9.0

s 8.0

2 7.0 8 z 4: 6.0 9 $ 5.0 -2

4.0

3.0

2.0

1.0

PROINSULIN PROINSULIN PROINSULIN

FRACTION NUMBER

. I u

0.2M NaCl

10 20 30 40 50 60 7 FRACTION NUMBER

J

6 10 20 30 40 -50 60 70 80 ‘ii FRACTION NUMBER

FIG. 1. (top left). Gel filtration of first crystals of bovine in- sulin on a column (8 X 100 cm) of Sephadex G-50. The column was eluted with 1 N acetic acid at 25” at a flow rate of 150 ml per hour. Fractions of 24 ml were collected. A disc gel electropho- resis pattern of the crude proinsulin fraction is shoxn. Electro- phoresis was at pH 8.9 with migration toward the anode at the bottom.

FIG. 2. (bottom left). Cm-cellulose chromatography of crude bovine proinsulin. A 500-mg sample was chromatographed on a column (1.8 X 40 cm) at 4” with a flow rate of 40 ml per hour. The column was eluted first with 0.01 M sodium citrate buffer at, pH 5.50, containing 7 M urea, and then with the same buffer con- taining 0.2 M NaCI, as indicated. The effluent was collected in 6-ml fractions. The disc gel electrophoresis patterns are, from left IO right those of the crude proinsulin and Peaks I (Fraction BCM-I) and II (Fraction BCM-II). Electrophoresis was as described in the legend to Fig. 1.

FIG. 3 (lop right). Gradient elution chromatography of 250 mg of Fraction BCM-I (Peak I in Fig. 2) on a column (1.8 X 40 cm)

10 20 30 40 50 60 70 80 90 100 110 120 FRACTION NUMBER

T &

2, r

z 0

If.

of DEAE-cellulose. The column was eluted at, 4” at 40 ml per hour, first with 0.04 M Tris-HCl, pH 7.6,’ containing 7 M urea, and then with a linear gradient bet’ween that buffer (200 ml) and the same buffer containing 0.2 M NaCl (200 ml), as shown. Fractions of 5 ml were collect.ed. Djsc gel electrophoretograms are inserted over t’he corresponding regions of the chromatograph. The electrophoretogram on the left is t,hat of the crude proinsulin prepa- ration. Electrophoresis was as described in the legend to Fig. 1.

FIG. 4. (bottom right). Gradient elution chromatography of Fraction BCM-II (Peak II in Fig. 2) on a DEAE-cellulose column, 1.8 X 40 cm. Elution was at 4’ and 40 ml per hour, first with 0.02 M Tris-HCI, pH 7.4, containing 7 M urea, and t,hen with a linear gradient between the starting buffer (200 ml) and the same buffer containing 0.2 M NaCl (200 ml). Fractions of 6 ml were collected. Disc gel electrophoretograms are shown above the corresponding regions of the chromatogram; that of Peak C (Fraction BCM-II-C) is immediately to the left of the peak. The electrophoretogram of the crude proinsulin is shown at the extreme left. Electrophoresis was as described in the legend to Fig. 1.

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2754 Xtructure of Bovine Proinsulin Vol. 246, No. 9

The nonconvertible fraction showed the same amino- and car- boxyl-terminal residues as insulin, and in approximately the same

molar proportions. The end groups of the intermediate fraction differed qualitatively from these only in that this fraction con-

tained a.n additional amino-terminal residue, glutamic acid; an

additional carboxyl-terminal residue was not observed. How- ever, the amounts of amino-terminal glycine and glutamic acid

TABLE I

End group analyses of chromatographic fractions

Amino-terminal analyses were performed by the dansyl method. The carboxyl-terminal residues listed are those released by di- gestion with carboxypeptidase B for 2 hours. The relative

amounts of the end groups (given in parentheses) were estimated visually after separation on thin layer polyamide (dansyl deriva- tives) or thin layer cellulose (amino acids). All carboxypeptidase B digests were performed under the same conditions on equimolar quantities of protein

Insulin Phe (+5) Ala (+5), LYS (+3) W (f5) Asn (f3)

Proinsulin (BCM-IIC) Phe Asn

Nonconvertible fraction Phe (+5) Ala (+5), LYS (+2) (BCM-IIH) GUY (f5) Asn (f3)

Intermediate fraction (BCM-ID)

Phe (f5) Gls (+3‘,

Glu (f3)

Ala (+3), LYS (+2)

Asn ($3)

.-

-

.lOO

.025

E' 0 R PI s .04

i3

2 .02

t5

s 0

.l

.05

0

Amino-terminal residues

-

-

Carboxyl-terminal residues (released by

carboxypeptidase B)

12 16 20 24 28 32 FRACTION NUMBER

FIG. 5. Gel filtration of the performic acid oxidation products of bovine proinsulin (Fraction BCM-IIC, Fig. 4)) the intermediate fraction (Fraction BCM-ID, Fig. 3), and insulin on a Sephadex G-75 column (1 X 50 cm) in 50% acetic acid. Fraction volumes were 7 ml. The column void volume, vo, and the elution posi- tions of insulin and the oxidized A and B (A and B) chains are indicated.

were smaller than the amount of amino-terminal phenylalanine; the amount of alanine relative to the amount of asparagine re- leased by carboxypeptidase B was also smaller. By contrast, proinsulin showed only one amino-terminal residue, phenylala- nine, and a single carboxyl-terminal residue, asparagine.

Performic Acid Oxidation Products of Proinsulin and Interme- diate Fraction-Gel filtration of the performic acid oxidation product of proinsulin (Fig. 5) gave only one peak, which eluted ahead of oxidized A and B chains of insulin in essentially the same position as the untreated protein. The elution diagram of the oxidation products of the intermediate fraction on the other hand, showed two major peaks (Fig. 5), one of which eluted in the same position as oxidized A chain; the other eluted ahead of oxidized B chain. A shoulder on the descending limb of the lat- ter peak indicated that there were more than two components present. The oxidized intermediate fraction was subsequently resolved electrophoretically into four components, as described below.

TABLE 11

Amino acid compositions of bovine proinsulin and intermediate and

convertible fractions

The compositions were calculated from the average residue

values determined by the analyses and the assumed amino acid compositions. The values given for proinsulin and the inter- mediate fraction represent average or extrapolated values ob-

tained from duplicate analyses of 20-, 40-, and loo-hour acid hydrolysates. The composition of the nonconvertible fraction is based on a single determination on a 30-hour hydrolysate. The values in parentheses are the nearest whole number of residues, except in the case of half-cystine in the intermediate and non- convertible fractions which were assumed to contain the same

number of half-cystines as proinsulin. Tryptophan was absent from all three protein preparations (see the text), as was methio- nine. The amino acid composition of bovine insulin is taken

from Rvle et al. (30).

Residue

Lysine. 1 Histidine 2

Arginine . 1 Aspartic acid. 3 Threonine . 1

Serine 3 Glutamic acid.. . 7 Proline. 1

Glycine. 4 Alanine.......... 3 Half-cystine. 6

Valine 5 Isoleucine. 1 Leucine. 6

Tyrosine. 4

Phenylalanine.. . 3

Totals. 51

E i

1

I

-

2.3 (2)

2.0 (2) 4.0 (4)

3.2 (3) l.Oa (1) 2.90 (3) 3.0 (13)

4.7 (5) 2.2 (12)

6.1 (6) 5.9 (6)b 6.7c (7) l.OC (1) 9.2 (9)

3.9 (4) 3.0 (3)

81 30 7X-80

residues/molecule

-

1 1.4 (l-2) 0 2.1 (2) 3 2.6 (2-3) 0 3.0 (3) 0 1.05 (1)

0 2.6a (3) 6 1 2.8 (13) 4 4.8 (5) 8 1 2.0 (12) 3 6.2 (6) 0 4.6 (6) 2 6.6” (7) 0 l.OC (1) 3 9.2 (9)

0 3.9 (4) 0 3.0 (3)

c

( _-

NIXI- vnvertible

fraction BCM-IIH)

RtXidUtX/ insulin

fmmomer

1.1 (1) 2.1 (2)

1.4 (1) 3.0 (3) 1.0 (1)

2.5 (3) 6.6 (7) 1.1 (1)

4.0 (4) 2.9 (3) 4.5 (6)

4.8 (5) 1.0 (1) 5.6 (6) 3.8 (4)

3.0 (3)

a Extrapolated values.

b Determined as S-carboxymethylcysteine. c The valine and isoleucine values are those obtained from the

loo-hour hydrolysates.

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Issue of May lo,1971 C. Nolan, E. Margoliash, J. D. Peterson, and D. F. Xteiner 2785

FIG. 6. The amino acid sequence of bovine proinsulin. Proinsulin Intermediate Form I lacks the Lys-Arg sequence (Residues 59 through BO), shown in bold&e circles at the carboxyl terminus of the connect.ing segment. Intermediate Form II lacks the 2 arginyl residues (nositions 31 and 32) at the amino terminus of the connecting segment. The structures of the A and B chains are according t,o Ryle et al. (30).

Amino Acid Competitions-The amino acid compositions of proinsulin and the intermediate and nonconvertible fractions are given in Table II. The analyses indicated that bovine proin- sulin contains 30 residues in excess of those present in bovine insulin, giving a total of 81. The intermediate fraction differs from proinsulin only in that it contains less than 3, rather than 4, residues of arginine and approximately 1.4, rather than 2, lysyl residues. Studies described below confirmed the presence of nonstoichiometric amounts of lysine and arginine. As seen in Table II, the nonconvertible fraction contains the same amino acids as bovine insulin in essentially the same molar ratios. Tryptophan could not be detected with the Ehrlich reagent on tryptic peptide maps of proinsulin or the intermediate fraction. Moreover, the molar extinction coefficient of proinsulin at 275 rnp, based on a molecular weight of 8684 calculated from the composition given in Table II, is the same as that of bovine in- sulin (5.7 x 103), thus confirming the absence in proinsulin of either tryptophan or additional residues of tyrosine and phen- ylalanine in excess of those present in insulin.

Analyses by the phenol-sulfuric acid method indicated the ab- sence of carbohydrate moieties in these three protein preparations since only traces of carbohydrate equivalent to <O.l mole per mole of protein were detected.

Primary Structures of Proinsulin and Intermediate Forms-The amino acid sequence of bovine proinsulin is given in Fig. 6. It consists of a single chain, &residue structure, the amino-termi- nal segment of which is the insulin B chain linked by a 30-residue segment, the “connecting segment,” to the A chain that forms the carboxyl-terminal portion of the molecule. The intermediate fraction was found to be composed of two partially degraded forms of proinsulin, the structures of which are also indicated in Fig. 6. Intermediate Form I differs from proinsulin in that it lacks the dipeptide sequence, Lys-Arg (Residues 59 through 60)) at the carboxyl terminus of the connecting segment, leaving a two-chain structure consisting of a normal A chain (Residues 61 through 81) linked to an elongated B chain (Residues 1 through 58) by t,he disulfide bonds of insulin. Intermediate Form II

lacks the Arg-Arg sequence (Residues 31 and 32) at the amino terminus of the connecting segment, leaving a normal B chain in disulfide linkage with an elongated A chain.

The amino acid sequence of the connecting segment was de- termined by studies of tryptic peptides from proinsulin and the intermediate fraction, chymotryptic peptides from performic acid-oxidized proinsulin, a peptic peptide from the intermediate fraction, and by characterization of the performic acid oxidation products of the intermediate fraction. The sequence, B chain- connecting segment-A chain, indicated by the end group analyses reported above, was confirmed by the isolation of peptides which overlap the connecting segment and the A or B chains.

Tryptic Peptides-Peptide maps of tryptic hydrolysates of the intermediate fraction (Fig. 7) and of proinsulin appeared qualita- tively identical except for the presence of two components, T-2 and T-5, in the hydrolysate of the intermediate fraction which were not observed in the proinsulin hydrolysate. Gel filtration of a tryptic digest of the intermediate fraction in 0.1 M NHJHCOI (Fig. 8) yielded two fractions. The peptides of each were sepa- rated by free flow electrophoresis in pyridine-acetic acid buffer at pH 6.5. When the gel filtration was performed in 2 M pyridine rather than 0.1 M NH4HC03, Peptide T-2 emerged later and was separated from Peptides T-l, T-3, and T-4. In addition to the soluble peptides, T-5 and T-6, the first peak also contained a sub- stance which was poorly soluble in the electrophoresis buffer and was partially removed from the mixture by centrifugation prior to electrophoresis. This material migrated on a peptide map to a position corresponding to that of Peptide T-7 in Fig. 7 and, similarly, stained with both the Pauly and Sakaguchi reagents. It was assumed to be insulin from which the terminal portion of the B chain following arginyl Residue 22 had been cleaved, and was not characterized further. The failure of this fraction to migrate during electrophoresis (Fig. 7) is presumably due to its poor solubility in the pH 6.5 buffer.

The compositions of the purified tryptic peptides are given in Table III. The heptapeptide, T-l, corresponds to Residues 23 through 29 obtained from tryptic hydrolyses at arginine Residue

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2756 Structure of Bovine Proinsulin Vol. 246, No. 9

3 e ELECTROPHORESIS - 0

ORIGIN

T-4

UIVD 0 T-3

T-l e

FIG. 7. Peptide map on paper of a a-hour tryptic digest of the intermediate fraction. Electrophoresis (at pH 6.5) and chroma- tography were performed as described in the text. Components which gave a positive Pauly test are marked with horizontal lines; those which were Sakaguchi-positive have vertical lines.

I 1 0.0

T-l T-2 T-3 T-4

80 100 120 140 160 180 200

FRACTION NUMBER

FIG. 8. Gel filtration of a 2-hour tryptic digest of the inter- mediate fraction (40 mg) on a column (1.9 X 155 cm) of Sephadex G-50. The column was eluted with 0.1 M NH~HCOZ at 15 ml per hour and the eifluent collected in 2.5 ml fractions.

22 and lysine Residue 29. T-l stained yellow with ninhydrin as expected with amino-terminal glycine, and was not characterized further. Peptides T-2 and T-4 were identified as free alanine and arginine, respectively, by peptide mapping with the authentic amino acids and by amino acid analysis before and after acid hy- drolysis. A 3-hour tryptic digest of proinsulin (performed as described for the intermediate fraction under “Experimental Procedure”), analyzed directly, gave 2.2 moles of aginine per mole of proinsulin. No free alanine was detected. The acidic peptides, T-5 and T-6, had compositions which differed only in that the more acidic peptide, T-5, lacked the single lysyl residue of T-6, and, in fact, lacked any residue at which trypsin normally splits.

Considering that Peptide T-4 arises from two different residues of arginine (Residues 32 and 60), this peptide and Peptides T-3 (Residues 30 and 31) and T-6 (Residues 33 through 59) together contain 31 amino acid residues and account for all of the 30 resi-

TABLE III

Compcsitions and properties of “soluble” tryptic peptides from intermediate fraction and/or proinsulin

The values in parentheses are the nearest whole number of

residues. The residue positions each peptide occupies in the proinsulin molecule is given in parentheses at the top of each column.

Peptide

Residue T-l T-3 T-4 T-5 T-6 (Residues T-2 (Residues (Residues (Residues (Residues

23 (Residue 30 32 33 33 I through 30) through and

29) through through

31) 60) 58) 59) I

Lysine. . 1.0 (1 Arginine 0.1

Aspartic acid. Threonine.. 1.0 (1 Serine............

Glutamic acid. Proline.. 0.8 (1 Glycine. . 1.0 (1

Alanine Valine.. 0.1 Leucine

Tyrosine. 0.7 (1 Phenylalanine 2.0 (2 Yield (%) 44

Electrophoretic mobility6 (cm/ volt/min X 105). +7.1

Ninhydrin stain.. Yel- 10s

Other staining re- actions,. Pauly

I.3b (1:

b

to.5

Blue

residues/molecule

I.9 (l)~2.2.(2)~o,2 1;;; (l)

I IO.2 IO.1

6.2 (6)6.2 (6) 3.7 (4)3.9 (4) 8.1 (8)g.l (8)

1.1 (1) 2.9 (3) 2.8 (3) 1.9 (2)2.1 (2) 3.3 (3)3.1 (3)

+14.3 +21.0 -11.4 -14.6

Blue Blue Blue Blue

Saks- Saka-

guchi guchi

a The value for arginine (2.2) is the number of moles of free arginine per mole of protein released by digestion of proinsulin with trypsin for 3 hours, as given under “Experimental Proced-

ure.” b The alanine value (0.3) is the number of moles of free alanine

per mole of protein released from the intermediate fraction by

extensive tryptic hydrolysis (5% trypsin, w/w, 0.05 M Tris-HCI buffer, pH 9.0, containing 0.3 mM calcium chloride, 5 hours, 37’).

c The electrophoret,ic mobilities are the average mobilities in

the free flow electrophoresis apparatus at pH 6.5. The plus (+)

and minus (-) signs indicate migration toward the cathode and anode, respectively.

dues found in proinsulin in excess of those present in insulin. The additional residue is the alanyl residue of Peptide T-3, which was identified, as described below, as the same residue as alanine T-l and arises from the carboxyl terminus of the B chain (Resi- due 30) by tryptic cleavage at lysine Residue 29.

The amino acid sequence of Peptide T-3 was established by one step of Edman degradation as Ala-Arg, as predicted from tryptic specificity. Amino acid sequence studies on Peptides T-5 and T-6 are summarized in Table IV and Fig. 9. Digestion of Pep- tide T-6 with carboxypeptidase B for 2 hours released only lysine, thus placing the single lysyl residue at the carboxyl terminus. No detectable amino acids were released from Peptide T-5 after incubation with carboxypeptidase A for 2 hours. This, together with the compositions, indicated that Peptides T-5 and T-6 were identical except for the carboxyl-terminal lysine of T-6. To

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TABLE IV

Summary of amino acid sequence studies on tryptic Peptides T-5 and T-6 T, P, and C refer to peptides isolated from trypsin digests, and from papain and chymotrypsin digests of tryptic peptides, respec-

tively. The numbers given in parentheses with the analytical compositions of the peptides are the nearest integral values. In

cases in which more than one PTH or dansyl derivative was observed, the relative amounts are indicated in parentheses on a scale of +l to +5. The method of isolation, approximate electrophoretic mobility (el) in centimeters per volt per min X 106 at pH 6.5, and percentage of yield of each papain peptide are indicated in that order in brackets following the amino acid composition. F, G-25, G-50, and AG-50 designate isolation by free flow electrophoresis, gel filtration on Sephadex G-25 or G-50, and ion exchange chromatography on AG-50 resin, respectively. A plus sign indicates electrophoretic migration toward the cathode at pH 6.5 and a

minus sign migration toward the anode. N denotes an electrophoretically neutral peptide. For the three chymotryptic fragments of T-5, the methods of purification are given under "Experimental Procedure." Paper electrophoresis at pH 6.5 was employed to classifythesepeptides as acidic,basic, or neutral, and the yield is indicated as for the papain peptides listed. ,

Peptide T-5 (Residues 33-58): (Glu6, Pro4, Gly8, Ala3, Val2, Leu3) Carboxypeptidase A (2 hr): no amino

acids released.

Peptide T-6 (Residues 33-59): (Glu6, Pro4, Glyg, Ala3,‘Va12, Leu3, ~ysl) Carbozycypeptidase B (2 hr): Lysine only released; Edman degradation of T-5 + T-6 (9 steps): Step 7,: PTH-Glu; Step 2: PTH-Val; Step 3: PTH-Glu; Step 4: PTH-Gly; Step 5: PTH-Pro; Step 6: PTH-Gln (+5), PTH-Glu (+l); Step 7: PTH-Val; Step 8: PTH-Gly; Step 9: PTH-Ala (+2), PTH-Gly (+l).

Peptide T-5-Pl (Residues 33-40): Glu, 3.2 (3); Pro, 1.0 (1); Gly, 2.1 (2); Val, 1.8 (2); Ala, 0.2; Leu, 0.1 [F, G-50; el, -17.0; 43%].

Peptide T-5-Pla (Residues 36-40): Glu, 0.9 (1); Pro, 1.1 (1); Gly, 2.2 (2); Val, 0.9 (1); Ala, 0.1; Leu, 0.1 [F, AG-50; el, N; 12%] Edman degradation (4 steps): Step 1: PTH-Gly; Step 2: PTH-Pro; Step 3: PTH-Gln; Step 4: PTH-Val.

Peptide T-5-P2 (Residues 41-58): Glu, 3.1 (3); Pro, 2.5 (3); Gly, 6.0 (6); Ala, 2.9 (3); Leu, 3.1 (3) tF; el, -7.9; 31%] E&an degradation (13 steps): Step Z: PTH-Ala; Step 2: PTH-Leu; Step 3: PTH-Glu;

Step 4: PTH-Leu; Step 5: PTH-Ala; Step 6: PTH-Gly; Step 7: PTH-Gly; Step 8: PTH-Pro (+4), PTH-Gly (+l); Step 9: PTH-Gly (+3), PTH-Pro (+l); Step 20: PTH-Ala (+l), PTH-Gly (+3); Step 22: PTH-Gly (+3), PTH-Ala (+l); Step 2.2: PTH-Gly (+3), PTH-Ala (+l); Step 23: PTH-Gly (+3), PTH-Leu (+l).

Peptide T-5-P2a (Residues 41-43): Gh, 1.2 (1); Ala, 0.9 (1); Leu, 0.9 (1) [F; el, -14.3; 28%1 E&an degradation (3 steps): Step Z: PTH-Ala; Step 2: PTH-Leu; Step 3: PTH-Glu.

Peptide T-5-P2b (Residues 44-58): Gh, 2.1 (2); Pro, 3.1 (3); Gly, 6.4 (6); Ala, 2.0 (2); Leu, 1.8 (2) [F, G-25; el, -7.7; 6%] Edman degradation (2 steps): Step 1: PTH-Leu; Step 2: PTH-Ala.

Peptide T-5-P2c (Residues 44-52): Pro, 1.1 (1); Gly, 5.3 (5); Ala, 2.0 (2); Leu, 0.8 (1) [F, AG-50; el, N; 5%].

Peptide T-5-P2d (Residues 44-53): Pro, 1.1 (1); Gly, 5.4 (5); Ala, 2.1 (2); Leu, 1.6 (2); Glu, 0.1 [F, AG-50; el, N; 3%] Ednan degradation (2 steps): Step 1: PTH-Leu; Step 2: PTH-Ala.

Peptide T-5-P2e (Residues 55-58): Glu, 1.0 (1); Pro, 2.0 (2); Gly, 1.5 (1); Ala, 0.2; Leu, 0.1 [F, AG-50; eZ, N; 12%; yellow ninhydrin color] Edman degradation (4 steps): Step l: PTH-Gly; Step 2: PTH- Pro; Step 3: PTH-Pro; Step 4: PTH-Pro (+l), PTH-Gln (<+l).

Peptide T-5-Cl (Residues 45-58): Glu, 2.1 (2); Pro, 3.1 (3); Gly, 6.0 (6); Ala, 1.8 (2); Leu, 1.0 (1) [acidic; 45%] Edman degradation (9 steps): Step 2: PTH-Ala, dansyl-Ala (+3), dansyl-Gly (+l);

Step 2: PTH-Gly, dansyl-Gly (+4), dansyl-Ala (+l); Step 3: PTH-Gly, dansyl-Gly (+4), dansyl-Ala (trace): Step 4: PTH-Pro (+5), PTH-Gly (+l), dansyl-Pro (+4), dansyl-Gly (+2); Step 5: PTH-Gly (+5), PTH-Pro (trace), dansyl-Gly; Residuea: Glu, 2.0; Pro, 2.5; Gly, 4.0; Ala, 1.1; Leu, 1.0; Step 6: PTH-Ala (+4), PTH-Gly (+2), dansyl-Ala (+4), dansyl-Gly (+l); Residue: Glu, 2.0; Pro, 2.2; Gly, 3.6; Ala, 0.6; Leu, 1.0; Step 7: PTH-Gly (+4), PTH-Ala (trace), dansyl-Gly (+4), dansyl-Ala (+l); Residue: Glu, 2.0; Pro, 2.3; Gly, 3.4; Ala, 0.6; Leu, 1.0; Step 8: PTH-Gly, dansyl-Gly; Residue: Glu, 2.0; Pro, 2.3; Gly, 3.1; Ala, 0.6; Leu, 1.0; Step 9: PTH-Leu, dansyl-Leu (+4), dansyl-Gly (+l).

Peptide T-5-C6 (Residues 45-53): Glu, 0.2 (0); Pro, 1.0 (1); Gly, 4.9 (5); Ala, 2.0 (2); Val, 0.1 (0) [acidic; 30%] Amino terminus: dansyl-Ala; Carboxypeptidase A (0.8 mg/ml), 20 min: Leu, 0.84; 3 hr: Leu, 1.0; Gly, 0.13; Ala, 0.07; 8 hr: Leu, 1.0; Gly, 0.17; Ala, 0.08.

Peptide T-5-C7 (Residues 54-58): Glu, 2.0 (2); Pro, 1.9 (2); Gly, 1.0 (1) [acidic; 40%] Edman de- gradation (5 steps): Step l: PTH-Glu, dansyl-Glu; Step 2: PTH-Gly, dansyl-Gly; Step 3: PTH-Pro, dan- syl-Pro (+4), dansyl-Gly (+l); Step 4: PTH-Pro (+4), dansyl-Pro (+4), dansyl-Gly (+l); Step 5: PTH-Gin (+3), PTH-Glu (+l), PTH-Pro (+l), dansyl-Glu (+4), dansyl-Pro (+4), dansyl-Gly (+l).

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2788 Structure of Bovine Proinsulin Vol. 246, No. 9

TABLE IV-continued

Peptide T-6-P1 (Residues 33-40): Glu, 3.0 (3); Pro, 1.2 (1); Gly, 1.9 (2); Val, 1.7 (2) [p; el, -15.7; 55%1.

Peptide T-6-P2 (Residues 41-59): Glu, 2.8 (3); Pro, 3.0 (3); Gly, 6.9 (7); Ala, 3.0 (3); Leu, 2.8 (3); LYS, 0.8 (1); Ser, 0.3 [F; el, -4.9; 16%1.

Peptide T-6-P2a (Residues 41-43): Glu, 1.1 (1); Ala, 1.0 (1); Leu, 0.9 (1) [F; el, -13.0; 25%1.

Peptide T-6-P2e (Residues 55-59): Glu, 1.0 (1); Pro, 1.8 (2); Gly, 1.4 (1); Lys, 1.1 (1); Ser, 0.2; Ala, 0.1; Leu, 0.1 [F; el, +10.8; 4%].

(1 These values were calculated assuming the presence of 1 leucyl and 2 glutamyl residues in the residual peptides, inasmuch as no glutamic acid or leucine was removed until position 9.

I -

p T-S-Cl --e--e--- T-S-C6 T-S-C7

cc-c-----

oc-2

IT-34 OC-2-T4

Arg-61y-lla-Val-6lu-6-~~SO,H-~~SO,H-AI~-Ser-~~-CySO,H-Ser-leu~-~- Asn-COOH

tT-4jjA CHAIN I

FIG. 9. Summary of amino acid sequence studies on the con- left. OC refers to chymotryptic peptides from oxidized proinsulin, netting segment of proinsulin. Peptides are represented by T to tryptic peptides, P and C to peptides from papain and lines above and below the amino acid sequence. Residues placed by chymotryptic digests, respectively, of tryptic peptides, and Edman degradation are indicated by arrows to the right and those Pe-1 to a peptide from a pepsin digest of the intermediate frac- positioned by digestion with carboxypeptidase by arrows to the tion.

conserve material, Edman degradation was performed on a mix- ture (3:2 molar ratio) of T-5 and T-6. This established the sequences of the first 9 residues and showed them to be identical in the two peptides.

A limited papain hydrolysate (25 min) of Peptide T-5 pro- duced four fragments, T-5-PI (Residues 33 through 40), T-5-P2 (Residues 41 through 58), T-5-P2a (Residues 41 through 43), and T-5-P2b (Residues 44 through 58), as determined by peptide mappings. The peptide mixture was fractionated initially by free flow electrophoresis at pH 6.5. Further purification, where required, was achieved as indicated in Table IV. The composi-

tions of Peptides T-5-PI (8 residues) and T-5-P2 (18 residues) account for all the residues in the parent peptide, T-5 (26 resi- dues). The residues of Peptides T-5-P2a (3 residues) and T-5- P2b (15 residues) indicate that they arose from a secondary hy- drolysis of Peptide T-5-P2. Additional peptides, listed in Table IV, were isolated from more extensive papain hydrolysates (50 to 60 min) of Peptide T-5.

Similarly, papain hydrolysis of Peptide T-6 produced Peptides T-6-PI (Residues 33 through 40) and T-6-P2 (Residues 41 through 59), which account for the total composition of the 27- residue parent peptide. Peptides T-6-P2a (Residues 41 through

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Issue of May lo,1971 C. Nolan, E. Margoliash, J. D. Peterson, and D. F. Steiner 2759

43) and T-6-P2e (Residues 55 through 59) were also obtained. Peptides T-6-Pl and T-6-P2a have compositions identical with those of Peptides T-5-PI and T-5-P2a, respectively. However, the compositions of T-6-P2 and T-6-P2e differ from those of the corresponding peptides from T-5 (T-5-P2 and T-5-P2e) in con- taining the additional carboxyl-terminal lysine present in T-6. The analytical data (Table IV) suggest that Peptide T-6-P2 con- tains 7 residues of glycine rather than the 6 found in the corre- sponding peptide, T-5-P2. However, the difference in glycine content between the parent peptide, T-6 (8 residues), and T-6-PI (2 residues) indicates the presence of 6 rather than 7 glycines. This composition was confirmed by subsequent sequence studies.

The compositions of the heptapeptides, T-5-PI and T-6-Pl, identified them as the amino-terminal portion of their respective parent peptides. Edman degradation of the parent peptides (9 steps) provided a l-residue overlap between Peptides T-5-PI and T-5-P2 as well as between the corresponding peptides from T-6. Edman degradation of Peptide T-5-Pla (Residues 36 through 40) (Table IV) and its identification as an electrophoretically neutral peptide at pH 6.5 confirmed the sequence assigned to positions 36 through 40, including the glutamine residue at position 38.

Edman degradation of Peptide T-5-P2a (3 steps) (Fig. 9 and Table IV) confirmed its identification as the amino-terminal tri- peptide sequence of T-5-P2. The sequence of the first 9 residues of T-5-P2 was unequivocally established by Edman degradation. However, due to the large amount of residual PTH-glycine, it was difficult to interpret the results from Steps 10 through 13 (Resi- dues 50 through 54, Fig. 9). Because of this difficulty, the alanyl and glycyl residues at positions 50 and 52 were interchanged in the preliminary reports of the amino acid sequence of bovine pro- insulin (8, 13). However, a peptide containing this segment, T-5-Cl, was isolated from a chymotryptic digest of Peptide T-5 (Fig. 9 and Table IV). Edman degradation of this peptide through nine steps yielded the required sequence (Fig. 9). Fur- ther, carbosypeptidase -4 digestion of Peptide T-5-C6, derived from T-5-C2 by vigorous chymotryptic digestion, confirmed the order of the residues in this region (Table IV and Fig. 9). Ed- man degradation (two steps), carboxypeptidase digestion, and its unique composition identified Peptide T-5-P2d as the amino- terminal portion of T-5-P2b. The composition of a similar peptide, T-5-P2c, confirmed the presence of 5 glycyl residues in the segment. It was isolated in very small amounts and was not characterized further. In Fig. 9 it is assigned to posi- tions 44 through 52 on the basis of its composition, but could represent Residues 45 through 53 equally as well since the single leucyl residue could be at either end.

The presence of a lysyl residue in the basic Peptide T-6-P2e (Residues 55 through 59) identified it as the carboxyl-terminal pentapeptide segment of Peptide T-6-P2, and thus, of T-6. Peptide T-5-P2e, a neutral peptide, was assigned to the carboxyl terminus of T-5 on the basis of its unique composition, which is identical with that of T-6-P2e except that it lacks the terminal lysine. Edman degradation established the sequence of T-5- P2e as Gly-Pro-Pro-Gin. The identification of the carboxyl- terminal glutamine is in accord with the electrophoretic neu- trality of the peptide at pH 6.5 and the net basic charge of the lysine-containing peptide, T-6-P2e.

The above information places all the residues in Peptides T-5 and T-6, except for a single glutamic acid residue. This was placed by difference in position 54 on the basis of the glutamic acid contents of Peptides T-5-P2, T-5-P2b, T-6-P2, and a chymo-

tryptic peptide, OC-2, isolated from oxidized proinsulin (Table V), described below. Since Peptide T-5-PPb was an acidic peptide and contained only two potential glutamic acid residues, one of which was found to be the carboxyl-terminal glutamine, glutamic acid rather than glutamine was assigned to position 54. This assignment was confirmed by Edman degradation of Pep- tide T&C7 (Table IV and Fig. 9).

Chymotryptic Peptides OC-i (Residues 27 through 44) and OC-2 (Residues 45 through 73) from Perjormic Acid-oxidized Proin- s&&-Overlaps between the connecting segment and the A and B chains were sought from a chymotryptic digest of performic acid-oxidized proinsulin. Arginine-containing peptides were selected, since the experiments described above showed that arginyl residues occurred at the amino- and carboxyl-terminal ends of the connecting segment. Two such fragments were obtained by column chromatography, as described under “Ex- perimental Procedure”: Peptide OC-2, which eluted in the starting buffer (0.2 M pyridine acetate, pH 3.1), and Peptide OC-1, which eluted in approximately 1.1 M pyridine-acetate, pH 5.1. Peptide OC-2 was further purified by gel filtration. The compositions of these peptides are given in Table V. The unique composition of OC-1 (Residues 27 through 44), an electrophoretically neutral peptide at pH 6.5, readily identified it as the carboxyl-terminal portion of the B chain (Residues 27 through 30) and the amino-terminal portion of the connecting segment (Residues 31 through 44) (Fig. 9), since it contained the single threonyl residue and 1 of the 2 lysyl residues of proinsulin as well as 2 arginyl residues. Digestion of this peptide with carboxypeptidase A for 30 min released leucine and a trace of glutamic acid.

The composition of OC-2 (Residues 45 through 73), which includes the single isoleucine of proinsulin located at position 3 of the A chain (Residue 63, Fig. 9), clearly identified it as a peptide which overlaps the connecting segment and the A chain (Fig. 9). Amino-terminal analysis by the Edman procedure gave PTH-alanine (+5) and a trace of PTH-glutamic acid (+I). The latter was attributed to a minor, more acidic contaminant of the Peptide OC-2 preparation observed on a peptide map. Without further purification, Peptide OC-2 was digested with trypsin at 39” for 3 hours and the digest was fractionated on a column (0.9 x 19 cm) of Dowex AG-50W-X2. Elution was with 0.2 M pyridine-acetate, pH 3.1 (125 ml), with a linear gradient to 0.5 M pyridine-acetate, pH 5.1 (125 ml). Four peptides, with the compositions given in Table V, were eluted in the order OC-2-T4 (Residues 61 through 73), OC-2-Tl (Residues 43 through 59), OC-2-T2 (Residues 45 through 59), and OC-2-T3 (Residue 60) in approximately 0.20, 0.21, 0.21, and 0.40 M buffer, respectively. Peptide OC-2-Tl apparently arose from the minor contaminant of OC-2 which had yielded the PTH-glutamic acid mentioned above, as it differed from OC-2-T2 in containing 1 extra residue each of glutamic acid and leucine (Residues 43 and 44, Fig. 9).

Peptic Peptide Pe-1 (Residues 26 through 40) from Intermediate Fraction-Peptide Pe-1 (Table V) was isolated from a peptic digest of the intermediate fraction as described under “Experi- mental Procedure.” It gave a positive Sakaguchi test for arginine and was positively charged at pH 6.5. Bmino-terminal analysis by the Edman method gave PTH-tyrosine. Pro- longed digestion (16 hours) with carboxypeptidase A released approximately equimolar quantities of glycine and valine, as estimated from a peptide map of the digest. From its com-

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2790 Vol. 246, No. 9 Structure of Bovine Proinsulin

TABLE V

Peptic and chymotryptic peptide overlaps between connecting segment

and A and B chains and derived peptides

Pe-1 is a peptic peptide from the intermediate fraction, OC-1 and OC-2 are chymotryptic peptides from performic acid-oxidized

proinsulin, and the remaining peptides were derived from OC-2 by tryptic hydrolysis. The values in parentheses are the assumed integral numbers of residues

Residue

Lysine.............................

Arginine . Aspartic acid. . Threonine..........................

Serine Glutamic acid...................... Proline.

Glycine............................ Alanine............... _.___..._... Half-cystine”.

Valine Isoleucine.. Leucine............................

Tyrosine...........................

Yield (%). Electrophoretic migration (cm)@.

Ninhydrin stain.. Other staining reactions..

-

Pe-1 oc-1 (Residues 26 (Residues through 40) 27 through 44)

1.1 (1)

1.8 (2)

1.0 (1)

3.2 (3) 1.7 (2)

2.2 (2) 1.0 (I)

2.1 (2)

0.8 (1)

10

+2.0 Purple Sakaguchi,

Pauly -

0.9 (1) 2.2 (2)

0.2 0.8 (1) 0.2 4.3 (4)

1.8 (2) 2.4 (2) 2.0 (2)

1.0 (1) 1.0 (1)

1.9 (2)

2.1 (2)

1.8 (2) 4.2 (4)

2.8 (3) 7.1 (7) 2.7 (3) 2.7 (3)

1.2 (2)d 0.3 (l)d 2.2 (2)

11 19

N -5.5 Purple Purple Sakaguchi Sakaguchi

a The lysine value was not determined but is assumed to be 1

based on the compositions of the parent peptide, OC-2, and of Pep- tide OC-2-Tl.

b OC-2-T3 is free arginine and is assigned a value of 1 residue

based on the composition of the parent peptide, OC-2. c Determined as cysteic acid. d The low analytical values for isoleucine and valine in Peptides

OC-2 and OC-2-T4 a.re attributed to incomplete hydrolysis of the

ORIGIN I

FIG. 10. Paper electrophoretogram of the performic acid oxidat,ion products of the intermediate fraction and of insulin. Electrophoresis was for 5 hours at 5 volts per cm in pyridine-acetic acid buffer, pH G.5, containing 7 M urea. The urea was removed from the dried paper by repeated washings with 95yo ethanol. The spots were detected with ninhydrin and Pauly reagents. The darker spots (oxidized B chain and components B-l and B-2) stained orange with Pauly reagent. The lightly stippled spots (oxidized A chain and components A-l and A-2) gave a purple Pauly stain.

position (Table V), this peptide represents an overlap between the B chain and the connecting segment. A peptide overlap of the connecting segment and the A chain was not isolated from the peptic digest. Such a peptide may have been present in a mixture of two arginine-containing peptides obtained from the cation exchange chromatography step which were not adequately resolved.

- I oc-2 OC-Z-T1

(Residues (Residues 43 1.5 through 73) through 59)

-7

1.3 (1) VP

0.2 0.1 0.2

3.2 (3) 2.8 (3) 6.1 (6) 2.0 (2)

0.2 2.0 (2)

3.3 (3) 5.6 (6) 1.8 (2)

2.0 (2) 1.0 (1)

6 36 -0.7 N

Purple Purple

OC-2.T2 (Residues 45 through 59)

OC-2-T3 (Residue 60)

(l)b

45

$5.5 Purple Sakaguchi

OC-2-T4 (Residues 61 through 73)

1.9 (2) 2.1 (2)

1.2 (1) 1.0 (1)

2.7 (3) 1.2 (2)d 0.3 (1)d

0.9 (1)

63

Yellow

Ile-Val bond at positions 62 and 63 during the 24-hour hydrolysis period.

e Electrophoretic migration is the distance migrated in centi- meters relative to alanine on thin layer cellulose sheets (20 X 20

cm) in pH 6.5 pyridine-acetic acid buffer at 400 volts for 1 hour. The plus and minus signs indicate migration toward the cathode and anode, respectively. N denotes electrophoretic neutrality.

Isolation of Performic Acid Oxidation Products of Intermediate Fraction-Attempts to resolve the unmodified intermediate fraction were unsuccessful. It behaved as a homogeneous substance by chromatography on columns of Cm- and DEAE- cellulose (Figs. 2 and 3), by disc gel electrophoresis at pH 8.9 (Fig. 3), or in 7 M urea at pH 4.4. Initial attempts to separate the expected four performic acid oxidation products of the fraction were also unsuccessful. Only partial separation was obtained by gel filtration (Fig. 5) and by paper electrophoresis in 30% formic acid at pH 0.9. Subsequently, however, the performic acid-oxidized intermediate fraction was resolved into four components by paper electrophoresis in pyridine-acetate buffer, pH 6.5, containing 7 M urea (Fig. 10). One of these, B-l, migrated with oxidized B chain and another, A-2, with oxidized A chain. Components B-l and B-2 gave an orange Pauly stain characteristic of the B chain, which contains both types of Pauly-positive residues, tyrosine and histidine, whereas A-l and A-2 gave a purple characteristic of the A chain, which contains tyrosine but no histidine.

The components were eluted from the paper with 50% acetic acid and analyzed. Amino-terminal analyses by the dansyl method showed phenylalanine in B-l and B-2, glutamic acid and a trace of glycine in A-l and glycine in A-2. This accounts for the three types of amino-terminal residues detected in the inter-

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mediate fraction (Table I). The trace of amino-terminal glycine observed in A-l was attributed to minor contamination with the A-2 fraction. Together with the compositions of these components (Table VI), this information identifies B-l as the oxidized B chain of insulin (Residues 1 through 30, Fig. 6), B-2 as the oxidized extended B chain (Residues 1 through 58), A-l as the oxidized extended A chain (Residues 33 through 81), and A-2 as the oxidized A chain of insulin (Residues 61 through 81).

Summary of Amino Acid Sequence Studies and Conclusions- The over-all structures of proinsulin and Intermediate Forms I and II, illustrated schematically in Fig. 11, were deduced as follows. End group analyses (Table I), amino acid analyses (Table II), and performic acid oxidation experiments (Fig. 5) demonstrated that bovine proinsulin is a single chain structure containing 30 amino acid residues in addition to those present in bovine insulin. Since the B chain of insulin has amino-ter- minal phenylalanine and the A chain has carboxyl-terminal asparagine, the identification of phenylalanine at the amino terminus of proinsulin and asparagine at the carboxyl terminus strongly suggested that the B chain forms the amino-terminal segment of the molecule and the A chain the carboxyl-terminal segment, the two being linked by a segment consisting of the 30 additional residues in proinsulin. These tentative conclusions were confirmed by the amino acid sequence studies.

As shown above, tryptic peptides T-3, T-4, and T-6 from the intermediate fraction together contain 31 amino acid residues which account for all the 30 residues found in proinsulin in excess of those present in insulin. The additional residue is alanine. Since from the Lys-Ala sequence at the carboxyl terminus of the B chain (Residues 29 through 30), one would expect a tryptic peptide containing amino-terminal alanine and a portion of the connecting segment, the dipeptide Ala-Arg (T-3) was placed at positions 30 and 31 (Fig. 9). The order of the re- maining tryptic peptides, T-4 and T-6, could not be assigned on the basis of the tryptic peptide data. The three possible orders were T3-Arg-Arg-T6, T3-T6-Arg-Arg, and TY-Arg-TG-Arg, the 2 arginyl residues represented by T-4 being either contiguous or separated by T-6. Thus, the possible structures of proinsulin were B chain-Arg-Arg-Arg-T6-A chain, B chain-Arg-TG-Arg- Arg-A chain, and B chain-Arg-Arg-T6-Arg-A chain. This last was shown to be the correct structure. Indeed, the amino acid compositions and end groups (Table V) clearly identified Pep- tides OC-1 (Residues 27 through 44) and Pe-1 (Residues 26 through 40) as overlaps of the connecting segment and the B chain, and OC-2 (Residues 45 through 73) as an overlap of the connecting segment and the A chain, and are in agreement with the amino acid sequences assigned to the corresponding seg- ments of the protein (Fig. 9). Peptide OC-2 contains only 1 arginyl residue (Residue 60), whereas the other two each con- tain 2 (Residues 31 and 32).

Amino acid analyses (Table II) indicated that the composition of the intermediate fraction differed from that of proinsulin only in that it contained approximately 0.6 residues less of lysine and 1.4 residues less of arginine. This, and the detection of three end groups (Table I) and four performic acid oxidation products from the intermediate fraction (Fig. 5), suggested that it contained partially degraded forms of proinsulin resulting from the excision of lysyl and arginyl residues from the polypeptide chain. Since the amino and carboxyl termini of the connecting segment had been shown to be Arg-Arg and Lys-Srg, respec- tively, and since some of the end groups of the intermediate

TABLE VI

Compositions of fragments isolated from performic acid-oxidized bovine proinsulin intermediate fraction

Fragments B-l, B-2, A-l, and A-2 were separated electrophoreti- tally as described in the text (see Fig. 10). The urea was removed from the dried paper electrophoretogram with 957, ethanol, and

the peptides were eluted with 50yo acetic acid. Samples were hydrolyzed for 30 hours at 110” in 5.8 N HCl. Numbers in paren- theses indicate the assumed composition of each peptide.

Residue I

Lysine. . Histidine. Arginine Aspartic acid.

Threonine. . Serine. Glutamic acid

Proline. Glycine. Alanine. . Half-cystine” Valine.. . Isoleucine . Leucine . . Tyrosine

Phenylalanine Amino terminus. Residues’ positions in

B-l B-Z A-l / A-2

residue/moZeczde

0.92 (1) 1.03 (1) 1.00 (1) 1.60 (2) 2.11 (2)

1.04 (1) 3.09 (3) 1.10 (1)

0.92 (1) 1.06 (1) 2.42 (2) 2.07 (2)

0.83 (1) 0.91 (1)

0.70 (1) 0.91 (1) 2.00 (2) 1.68 (2)

3.17 (3) 8.77 (9) 9.53 (10) 3.71 (4)

1.13 (1) 4.94 (5) 3.68 (4)

3.30 (3) 10.25 (11) 8.42 (9) 1.21 (1)

1.96 (2) 4.80 (5) 3.89 (4) 1.10 (1)

1.70 (2) 2.00 (2) 4.26 (4) 4.03 (4)

2.70 (3) 4.70 (5) 3.47 (4) 1.57 (2)

0.84 (1) 0.67 (1)

3.43 (4) 7.00 (7) 4.32 (5) 1.89 (2)

1.78 (2) 1.80 (2) 2.11 (2) 1.79 (2)

2.70 (3) 2.71 (3) Phe Phe Glu GUY

proinsulin. 130 (B chain)

1-58 33-81 61-81 (A chain)

a Determined as cysteic acid.

PROINSULIN

INTERMEDIATE FORM I

NtiI-Phe&-~~~$,-Am-CIJON

+A CHAIN+

INTERMEDIATE FORM II

FIG. 11. Schematic representation of the structures of proin- sulin and Intermediate Forms I and II. The tryptic peptides derived from each of the three proteins are indicated. The two polypeptide chains of Forms I and II are linked by the two disul- fide bonds between the A and B chains.

fraction were those of insulin (Table I), it appeared that this fraction consisted in part of chains from which the Lys-Arg sequence had been removed (Intermediate Form I) and in part of chains which lacked the Arg-Arg sequence (Intermediate Form II) (see Fig. 11). Direct evidence for such structures was provided by the amino acid sequences of Peptides T-2 and T-5

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2792 Structure of Bovine Proinsulin Vol. 246, No. 9

(Table III and Fig. 9) which were isolated from tryptic hydroly- sates of the intermediate fraction but were not observed in tryptic digests of proinsulin. Peptide T-2 was free alanine and could have arisen by a tryptic split at lysyl Residue 29, only if alanyl Residue 30 occupied the carboxyl-terminal position postulated for Intermediate Form II, since the Ala-Arg bond (Residues 30 and 31) would not be susceptible to trypsin. The dipeptide Ala-Arg (T-3) is attributed to Residues 30 and 31 of the alternate intermediate form (Form I), as discussed above for proinsulin. The amino acid sequence of Peptide T-5, which had carboxyl-terminal glutamine, was identical with that of T-6 except that T-5 lacked the carboxyl-terminal lysine. Since trypsin would not split a peptide bond at the carboxyl end of glutamine, the glutamingl residue must have been terminal in the intermediate fraction and is attributed to Form I. The origin of Peptide T-6, which was common to tryptic digests of proinsulin and the intermediate fraction, is attributed to tryptic cleavage of Form II at lysine Residue 59. The absence of this lysyl residue from Form I accounts for the decreased amount of lysine in the intermediate fraction (Table II). Carboxyl-ter- minal glutamine was not detected in carboxypeptidase digests of the intermediate fraction (Table I) since the carboxyl-terminal Pro-Pro-Gln sequence of Peptide T-5 is resistant to the enzyme (Table IV). The amino-terminal glutamic acid in the inter- mediate fraction is ascribed to the amino-terminal glutamic acid residue of Peptide T-6 (Residue 33) in Form II. The yields of Peptides T-5 (51%) and T-6 (24%) from the inter- mediate fraction (Table III) indicate that the molar ratio of Form I to Form II was approximately 2 : 1, This is in good agreement with the yields of peptide fragments isolated from performic acid-oxidized intermediate fraction and of free alanine, T-2 (0.3 mole per mole of protein), which was estimated by analysis of the complete tryptic digest of the intermediate fraction and is a measure of the amount of Form II in the inter- mediate fraction. Based on a molar ratio of 2: 1 of Forms I and II in the intermediate fraction and the assigned amino acid sequences (Fig. 6), the calculated lysine and arginine contents of the intermediate fraction would be lf and 2% residues, re- spectively, which is in substantial agreement with the amino acid analysis of the intermediate fraction (Table II) which gave 1.4 and 2.6 residues, respectively. Thus, the assigned sequences are in quantitative agreement with the end group and amino acid composition data.

Final confirmation of the structures of the intermediate forms came from the isolation of the performic acid oxidation products of the intermediate fraction. Based on the proposed disulfide-bonded, two-chain structures of the intermediate forms (see Fig. II), performic acid oxidation of the intermediate fraction should produce four polypeptide fragments correspond- ing to Residues 1 through 30 (the B chain) and 33 through 81 of Form II and Residues 1 through 58 and 61 through 81 (the A chain) of Form I. Such fragments were, in fact, isolated and identified (Fig. 10 and Table VI). Two of these fragments, B-2 and A-l, together completely overlap the connecting seg- ment and provide additional confirmation of the amino acid sequence assigned to bovine proinsulin in Fig. 6.

The disulfide linkages assigned to proinsulin (Fig. 6), con- necting half-cystine Residues 7 and 67, 19 and 80, and 66 and 71, are assumed from the structure of insulin (30). Preliminary studies of the disulfide bonds3 by the diagonal paper electro-

3 C. Birdwell and D. F. Steiner, unpublished data.

phoresis technique (31), using peptic digests of proinsulin, the intermediate fraction and insulin, provided evidence that the disulfide bonds in all three cases are the same. Indeed, the electrophoretic patterns of the disulfide peptides in the peptic digests and their performic acid oxidation products appeared to be identical. It was not possible, however, to distinguish between the possible isomers involving half-cystine Residues 7, 66, 67, and 71 by this technique since all four of these residues were present in the same peptic peptide.

The properties of the nonconvertible fraction strongly indicate that it is an insulin aggregate, probably a dimer. End group analyses revealed the same end groups as insulin (Table I) and the molar ratios of the component amino acids are in good agree- ment with the composition of bovine insulin (Table II). In the purification procedure, it eluted from a Sephadex G-50 column in 1 N acetic acid ahead of insulin in the crude proinsulin peak (Fig. 1) and eluted from the Cm-cellulose column in 7 M urea later than added authentic insulin. Migration of the fraction on polyacrylamide disc gel electrophoretograms (7.5% cross- linked gel) at pH 8.9 was essentially identical to that of insulin. Moreover, incubation with trypsin did not alter its elution posi- tion in a Sephadex G-50 column, in contrast to the changes ob- served with proinsulin and the intermediate fraction. The fact that insulin and the nonconvertible fraction behaved dif- ferently in 1 IU acetic acid and 7 M urea indicates a highly stable aggregate, possibly involving covalent linkage of unknown nature between the insulin monomeric units. Since the non- convertible fraction was obtained from commercial prepara- tions of insulin, but could not be detected as a biosynthetic product (8), it most probably represents a preparation artifact.

DISCUSSION

It is now well established that proinsulin is the biosynthetic precursor of insulin. Studies in vitro with human islet cell tumors (1, 2), isolated islets of Langerhans from rats and mice2 (2-4), codfish (5), and angler fish (32) islets, and fetal calf pan- creas (6, 33) all indicate that proinsulin is the earliest labeled protein structurally related to insulin that can be detected during incubation with labeled amino acids. We have been unable to obtain any evidence for the biosynthesis in rat islets of peptide material having the exclusion characteristics of insulin A or B chains on Sephadex columns.4 Therefore, the amino acid sequence of proinsulin must be encoded in the DNA of the structural gene or genes for insulin in all the vertebrate species studied thus far.

It is evident that one of the important functions of proin- sulin, if not perhaps its sole function, is to facilitate the correct pairing of cysteinyl residues necessary for the formation of the three characteristic disulfide bonds of insulin. This process has been demonstrated by reoxidation of the fully reduced and un- folded polypeptide chain of proinsulin in vitro. The regenera- tion of the native structure in such experiments proceeds effi- ciently to levels as high as 70 to 80% (34) or even higher (35), under conditions in which the reduced chains of insulin recombine only to an extent of 1 to 2%. Likewise, the intermediate frac- tion of proinsulin, consisting of material cleaved either at the A chain or the B chain junctions with the connecting segment, shows no greater refolding ability than separated insulin chains (34). These results indicate that the integrity of the proinsulin polypeptide chain is a necessary prerequisite for efficient folding.

4 E. Schlenck and D. F. Steiner, unpublished data.

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Such observations, however, do not allow a distinction between the possibilities that, on the one hand, the connecting segment may specifically interact with regions in the A and B chain portions of the molecule to align these properly or that, on the other hand, this segment may merely provide a highly flexible connector that converts the process of chain combination from a bimolecular to a more efficient unimolecular reaction. The much larger species variation observed between the connecting segments of porcine, bovine, and human proinsulin (17 of 35 residues) (see Fig. 12) than between the corresponding insulins (3 of 51 residues) supports the latter view. However, from the recent crystallographic determination of the three-dimensional structure of insulin (36), it is clear that the carboxyl-terminal end of the B chain and the amino-terminal end of the A chain are close enough to be spanned by a peptide chain of only 3 residues. The fact that the connecting segments are much longer may indicate the existence of more specific requirements for function. Among the 18 residues that are identical in the three comrecting segments (Fig. 12) are the 4 glutamyl residues and 6 of the 7 or 8 glycines. Similarly, 2 of the 2 to 4 glutamines are invariant, suggesting that several of these residues provide interactions necessary for the three-dimensional structure of proinsulin. Moreover, the remarkable similarity and restriction in composition in human (37), monkey (37), porcine (14), murine (38), codfish (5), and angler fish (32) proinsulin con- necting segments also indicates a requirement for particular amino acid side chains, perhaps, as suggested recently (39), with respect to maintaining a balance between hydrophilicity and hydrophobicity in the molecule as a whole. Indeed, where substitutions do occur there is considerable conservatism with regard to polar and nonpolar side chains. Most of the polar residues are concentrated near the ends of the connecting seg- ment. Such a distribution could influence the folding of the molecule so as to maintain these regions on the surface for ease of proteolysis in the proinsulin to insulin transformation. Further studies of the refolding properties of native as well as altered or synthetic proinsulins should help to determine the mechanisms responsible for this phenomenon.

Despite the numerous differences (Fig. 12), the three se- quences exhibit considerable similarity throughout. The minimal number of nucleotide exchanges needed to interchange the corresponding gene segments is well below the level expected for random peptides of the same composition (40). Moreover, all the residue substitutions except two can be accomplished by single nucleotide changes. The high degree of variability among these peptides is similar to that seen in the fibrinopeptides (41, 42), which also vary in length and sequence but retain the Arg-Gly structure for cleavage by thrombin.

Various biosynthetic studies indicate that proinsulin is trans- formed to insulin within the islet cells and that the major storage form in the pancreas is insulin rather than proinsulin (2-6). Consistent with this is the relatively low content of proinsulin and related substances (1 to 2y0) present in crystalline insulin preparations from rats (38), cattle, and pigs (14) obtained by techniques which do not readily separate insulin from proinsulin. To accomplish the transformation of proinsulin to insulin in the @ cells, the existence of a unique system of intracellular proteo- lytic enzymes must be postulated. If one assumes that the intermediate fractions are in fact biosynthetic intermediates in this transformation, their structures provide clues regarding the nature of this mechanism. The elimination of both basic resi-

31 35 40 45

50 55 60

FIG. 12. Comparison of the amino acid sequences of the con- necting segments of porcine (14) and bovine proinsulins and the C peptide of human proinsulin. Positions with identical residues in all three proteins are enclosed in rectangles. Residue gaps have been arbitrarily placed to maximize similarity. The Arg- Arg and Lys-Arg sequences in the human protein have been placed by analogy to the other proinsulins, since in the human protein only the structure of C peptide, the composition of the proinsulin, and the order of the chain fragments have been deter- mined (37).

dues at the sites of cleavage in Intermediate Forms I and II (Fig. 6) could not be accomplished by any known protease acting alone. Such cleavage could result, however, from the sequential actions of an enzyme or enzymes having trypsin-like and carboxypeptidase B-like activities. Since the Arg-Gly (Residues 60 and 61) and Arg-Glu (Residues 31 and 32) bonds in proinsulin are cleaved much more rapidly by trypsin than any other susceptible bonds (14), these could well be split by the trypsin-like enzyme to yield the free peptide extending from Residue 32 to Residue 60 of bovine proinsulin (Fig. 6), bearing carboxyl-terminal lysyl and arginyl residues, and insulin bearing 2 carboxyl-terminal arginines attached to the B chain. The rapid formation of these intermediates in the tryptic conversion of proinsulin can be readily demonstrated by paper (11) or disc gel electrophoresis (43). The carboxypeptidase B-like enzyme could then remove the basic residues from the extended carboxyl end of the insulin B chain, leaving it to terminate with the alanine, serine, threonine, or aspartic acid characteristic of the particular mammalian species considered. The variability of insulins at this position argues against the existence of a novel protease, such as a dipeptidase which might cleave on either side of the pairs of basic residues at either end of the connecting segment.

Further evidence supporting the action of trypsin-like and carboxypeptidase-B-like activities in the conversion in vivo is the recent isolation from human, monkey, and bovine pancreas of the C peptide, namely, the proinsulin connecting segment from which all four terminal basic residues are absent, in equimolar amounts to the insulin contained in the pancreas (29, 37, 44). The production of equal amounts of this peptide and of native insulin from proinsulin, as demonstrated directly with isolated rat islets (45), would be an expected consequence of the action of the postulated carboxypeptidase B-like and trypsin-like en- zymes. Inasmuch as the islet tissue is closely associated ana- tomically and developmentally with the exocrine pancreatic tis- sue, which produces both trypsin and carboxypeptidase B, it is perhaps not difficult to imagine that similar enzymes could have evolved in islet tissue for the specific purpose of trans- forming proinsulin to insulin (7). It is of interest in this con- nection that most fish insulins have B chains with carboxyl- terminal lysine and are analogous in structure to dealanyl

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2794 Xtructure of Bovine Proinsulin Vol. 246, No. 9

porcine or bovine insulin (46). Thus, although certain species of fish have been reported to have pancreatic carboxypeptidase B (47), it appears likely that a trypsin-like enzyme alone may suffice to accomplish the transformation of fish proinsulin, just as trypsin alone produces biologically active dealanyl insulin from the mammalian proinsulins. It must be emphasized, however, that at this time such proteolytic enzymes have not been isolated from islet tissue.

Intact proinsulin and the intermediate forms, described in detail here, account for most of the proinsulin-related protein separated along with the insulin dimer or “nonconvertible” fraction by gel filtration of crystalline bovine insulin. Several satellite bands migrating either slightly faster or slower than the major bands of proinsulin or the intermediate fraction can be seen on polyacrylamide gel electrophoretograms (Figs. 1 through 4). Although these components have not yet been extensively characterized, their amino acid compositions5 indicate that all are closely related to proinsulin, being either single-chain or two-chain degradation products of proinsulin. The relatively small amounts of these materials suggest that they are either artifacts of acid-ethanol extraction, i.e. deamidated or esterified forms, or secondary products arising in viva or from autolytic cleavages occurring during the purification of the insulin.

hexamers similar to those formed by insulin (50), and the asym- metric unit in proinsulin crystals is a dimer (51). Thus, it is probable that proinsulin can cocrystallize readily with insulin when present in small amounts. Moreover, it has been shown by electron microscopy (52) that the insulin packaged in the granules of p cells is in the same crystalline state as ordinary zinc insulin crystals (36). It is possible that such crystallization prevents the continued action of the proteolytic system respon- sible for the proinsulin to insulin conversion. Such a hypothesis would account for the presence of zinc in /? granules (since the metal is required for the crystallization), for the retention of the small amount of proinsulin and intermediate forms (because they are cocrystallized with the insulin), and possibly for the fact that the insulin itself is not degraded. This hypothesis is consistent with the observation that zinc is not found in guinea pig /3 cells (53) and that guinea pig insulin, unlike other mamma- lian insulins, lacks the histidyl residue at position 10 of the B chain (46) that is important for zinc binding in the crystal (36).

It is remarkable that all these insulin-related components are carried along with the insulin during the usual extraction and crystallization procedures. It has been noted that repeated crgstallizations of insulin, as many as 10 times, lower the content of these minor components by only about half.6 Several factors probably contribute to the similarity in properties of proinsulin and insulin necessary to account for this phenomenon. Most important is the likelihood that the tertiary structure of the insulin moiety in proinsulin is nearly identical with that in insulin, as evidenced by optical (48) and immunological (49) comparisons. Another reason may lie in the fact that the cal- culated isoelectric pH of proinsulin is only 0.16 pH unit higher than that of insulin, pH 5.3.% Proinsulin would then be expected to exhibit solubility properties quite similar to those of insulin, with a minimum of solubility at about pH 5.45. However, the isoelectric pH of both the intermediate forms is considerably lower than that of insulin (0.58 pH unit lower) which explains their failure to bind strongly to the carboxymethylcellulose columns at pH 5.5, while both proinsulin and the insulin-like dimer were retained (Fig. 2).

The biological activity of proinsulin is dependent upon the particular assay method used to measure it and ranges between 2 and 20% of that of crystalline insulin (25 units per mg), while the intermediate fraction (both intermediate forms together) shows an activity of 15 to 50% that of insulin (10, 54, 55). All these substances can be converted by mild trypsin treatment to fully active dealanylinsulin.

Even though most of the proinsulin appears to be converted to insulin in the islet cells and stored as such in the fi granules, some of it appears in the circulation under normal or pathological conditions (56). However, the /3 cell secretion consists mostly of equimolar amounts of insulin and C peptide (56), as well as zinc. This raises the question as to whether the C peptide has any hormonal or metabolic function. The large variability of the C peptide among various species appears to argue against such a possibility. However, that this deduction is not neces- sarily correct is indicated, for example, by the large degree of variability among highly active calcitonins of different species (57). Immunological assays which discriminate either com- pletely or partially between proinsulin and insulin have been developed and used to determine the proinsulin and C peptide contents of biological fluids from human subjects under normal and pathological conditions (55, 56).

The relative mobilities of proinsulin, the intermediate forms and insulin on polyacrylamide disc gel electrophoresis cannot be explained only by differences in charge. This is particularly evident in the case of the intermediate forms which have a considerably larger net negative charge at pH 8.9 than does insulin.2 Correction to charge per unit of mass fails to account for the slower migration of the intermediate forms, and therefore a molecular sieving effect must also be invoked to explain the observed mobilities of both the proinsulin and this fraction. Whether some self-association to dimers of these various pro- teins may occur under the conditions of electrophoresis is not known.

The quantitative refolding of fully reduced proinsulin would appear to provide a suitable means for an efficient chemical synthesis of insulin. The feasibility of this approach is currently under investigation.

The phenomena of self-association and crystallization of insulin and of proinsulin may well be of physiological importance. Indeed, proinsulin forms dimers and, in the presence of zinc,

Acknowledgments-The authors thank Dr. J. Schlichtkrull and Mr. Henrik Ege of the Novo Company, Copenhagen, for generous supplies of bovine insulin and of bovine crude proin- sulin fractions. We thank Dr. A. J. Moody of the Novo Insti- tute, Copenhagen, Dr. J. Gliemann of the University of Copen- hagen, and Drs. H. T. Narahara and L. S. Weis of the Depart- ment of Biological Chemistry, Washington University, St. Louis, for performing bioassays on the various fractions. Mrs. Claudia Svensen, Miss Sooja Cho, and Mr. L. J. Weiss provided expert technical assistance.

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5 S. Cho and D. F. Steiner, unpublished data. 1. STEINER, D. F., AND OYER, P., Proc. Nat. Acad. Sci. c’. S. A., 0 J. Schlichtkrull, personal communication. 62, 473 (1967).

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Chris Nolan, E. Margoliash, James D. Peterson and Donald F. SteinerThe Structure of Bovine Proinsulin

1971, 246:2780-2795.J. Biol. Chem. 

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