THE JOURNAL cm Bm~oorca~. CHEMISTRY Vol. 242, No. 7, Issue of April 10, pp. 1566-1573, 1967

Printed in U.S.A.

Isolation of Two Large Peptide Fragments from the Amino- and Carboxyl-terminal Positions of Bovine Serum Albumin*

(Received for publication, October 17, 1966)

THEODORE PETERS, JR., AND CYNTHIA HAWN$

From the Mary Imogene Bassett Hospital,$ Cooperstown, New York 133.26

SUMMARY

Bovine serum albumin was treated briefly with pepsin at pH 3 and the digest was fractionated by precipitation with trichloracetic acid, by gel filtration at pH 3, and by zone electrophoresis at pH 5. Two fractions were obtained which appeared pure upon electrophoresis on cellulose polyacetate at pH 5 or pH 8.6, or in polyacrylamide gel at pH 9.3. Evi- dence is presented that these preparations, termed the “Asp” and “Phe” fragments, include the two terminal sites of the albumin molecule.

The amino-terminal or Asp fragment, mol wt 2808, con- tains 24 residues and has the same amino-terminal sequence, Asp-Thr-, as has the parent albumin. Its amino acid composition is: alanine, 1; glycine, 2; valine, 1; leucine, 3; isoleucine, 1; phenylalanine, 2; serine, 1; threonine, 1; arginine, 1; histidine, 3 ; lysine, 3; aspartic acid, 2; and glutamic acid, 3. There is no tyrosine or tryptophan, and no cystine or proline is present to restrict helical folding. The presence of 3 histidine residues and of the free terminal amino group suggests a role in binding small compounds.

The Phe fragment, mol wt 8553, is believed to be the carboxyl-terminal fragment. It contains 77 residues, and has the albumin carboxyl-terminal sequence, -(Ala, Thr)- Leu-Ala. Its composition is: alanine, 9; glycine, 1; valine, 7; leucine, 7; isoleucine, 2; proline, 3; phenylalanine, 5; serine, 1; threonine, 8; half-cystine, 4; methionine, 1; histidine, 2; lysine, 10; aspartic acid, 6; glutamic acid, 11; and amide, 5.

The serum albumin molecule is apparently a long single chain of about 565 amino acid residues stabilized by some 17 disulfide bonds (2). Attempts to isolate large fragments of bovine albu- min after chemical cleavage of peptide bonds at the 2 tryptophyl (3) or 4 methionyl (4) residues have not been productive, even when the cleaved albumin was oxidized or reduced to cleave the disulfide bonds.

* This study was supported by Grants HE-02751 and FR-05498 from the United States Public Health Service. A preliminary report of this work has appeared (1).

$ Present address, Department of Biochemistry, Cornell University, Ithaca, New York 14850.

$ Affiliated with Columbia University.

On the basis of physicochemical changes accompanying the ex- pansion of the albumin molecule at pH 3, Foster (5) has proposed that albumin consists of several globular parts held together by single peptide strands. Weber and Young (6) tested this hypoth- esis by the use of mild peptic digestion at pH 3 in an effort to break the connecting strands. They were able to obtain two fractions differing in amino acid composition and in average molecular weight by chromatography on diethylaminoethyl cellulose.

We have used mild peptic digestion under conditions similar to those used by Weber and Young, in the hope of isolating distinct globular fragments of bovine albumin. Information on the ex- istence of such fragments is of importance to the study of steps in the biosynthesis of the albumin molecule. We have obtained two peptide fragments of molecular weight 2808 and 8553, ac- counting for about 20% of the whole. These peptides appear to represent the amino and carboxyl termini of the intact molecule.

EXPERIMENTAL PROCEDURE

Materials

BSA,’ crystallized, was the generous gift of Armour Pharma- ceutical Company, Kankakee, Illinois. Swine pepsin, twice crystallized, and carboxypeptidase A, crystallized after treat- ment with diisopropyl fluorophosphate, were purchased from Worthington. 1-Anilmonaphthalene-8-sulfonic acid, Eastman, was recrystallized according to the method of Weber and Young (6). Hydrazine, Eastman, was purified by azeotropic distilla- tion with toluene (7) to greater than 98% purity. Phenyl iso- thiocyanate (Eastman), pyridine (Allied Chemical Corporation, General Chemical Division, New York, Code 2165), dioxane, acetone, n-heptane, acetic acid, and triethylamine were redis- tilled. Other solvents were analytical grade. Benzaldehyde and DFB were Eastman.

Methods

Peptic Digestion and Titration-A solution of 5% BSA was adjusted to pH 3.00 with 88% formic acid and allowed to stand for 30 min at 25”. The preparation became cloudy, owing to release of fatty acids. Pepsin in 0.01 N HCl was added in a ratio of 1:3000, pepsin to albumin (w/w), and digestion was carried out for 33 min at 25”. The pH did not increase more than 0.1 unit. Concentrated ammonium hydroxide was added

1 The abbreviations used are: BSA, bovine serum albumin; DFB, 2,4-dinitrofluorobenzene; DNP-, 2,4-dinitrophenvl-: TCA, trichloracetic acid.

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to bring the pH to 7, and the solution was cooled to 0”. In earlier preparations BSA was desalted first with ion retardation resin AGllA8 (Bio-Rad Laboratories, Los Angeles), but omis- sion of this step did not influence the results.

When proton uptake was followed during digestion, the pH was initially adjusted with concentrated HCl, and the pH during digestion was maintained at 2.95 to 3.05 by the addition of 0.1 N

HCl in a pH-stat constructed from a Radiometer pH Meter 22 and a Coleman Autotrator, with a reaction vessel maintained at 25” by a thermostat.

Determination of Fluorescence Eficiency-Aliquots of the di- gestion mixture containing 2.5 mg of albumin were added to 10 ml of 0.1 M sodium phosphate buffer, pH 7.4, containing 0.032 mM l-anilinonaphthalene-8-sulfonic acid. After 60 min the fluorescence efficiency was determined as the transmittance read- ing registered in a Coleman model 9 Nephocolorimeter when the specimen was excited by a long wave ultraviolet lamp (Burton model 1910) directly over the cuvette. The standard curve was linear in the range, 0 to 3 mg of BSA.

Determination of Total Amino GroupsAliquots of the digest were diluted 30-fold with 0.01 M NaH2POh and assayed by reac- tion with ninhydrin with the use of the analytical components of a Technicon automatic amino acid analyzer. The diluted speci- mens were sampled for a 2-min period at 0.1 ml per min and were mixed with 1.06 ml per min of a solution containing 0.4% ninhydrin, 0.03% hydrindantin, and 0.28 N sodium acetate buffer, pH 5.5, in 53% methyl Cellosolve. The mixtures were heated at 95” for 18 min and then cooled, and absorbances at 570 rnp were measured. The standard was 0.1 mM norleucine.

Assays of Amino-terminal Residues and Sequences-Free (Y- amino groups were determined with DFB (8). Reaction was carried out in 1% trimethylamine and the mixture was then lyophilized. Hydrolysis was for 11 hours at 110” in 6 N HCl. A separate hydrolysis for 3 hours in 12 N HCl was used to detect DNP-glycine when pure fractions were assayed. Ether-soluble fractions were chromatographed on the toluene and 2 M phos- phate systems, and aqueous fractions were examined on the tert-amyl alcohol-phthallate system for DNP-arginine (8). Recovery factors, determined by adding the DNP-amino acid to BSA before hydrolysis, were 0.50 for DNP-aspartic acid and 0.60 for DNP-phenylalanine.

The Edman degradation according to the procedure of Schroeder et al. (9) was used for sequence studies. The first and second amino-terminal residues were identified by chromatog- raphy of the phenylthiohydantoins, by use of DFB after cleaving away the phenylthiohydantoins, and, in the case of Asp fragment (see below), by complete amino acid analysis after removal of each of the first 2 residues.

Assays of Carboxyl-terminal Residues and Sequences-Hy- drazinolysis was performed according to the method of Niu and Fraenkel-Conrat (10). The dried specimens were then brought to pH 3 with dilute HCl and extracted twice with 0.4 volume of benzaldehyde and twice with ether, and the free amino acids were determined quantitatively on the Technicon automatic amino acid analyzer. Norleucine was added before hydrazinolysis as an internal standard.

Carboxypeptidase A was used in sequence studies as described previously (II), except that the residual peptide or protein was precipitated with addition of TCA to 10% rather than with acetone at pH 5. Digestions were conducted for various times at 38”, and the amino acids released were identified by assaying

aliquots of the TCA supernatant on the amino acid analyzer, with the use of norleucine as internal standard. (TCA was 6rst removed by extraction with ether.)

EZectrophoresisElectrophoresis, as a criterion of purity, was performed with the use of polyacrylamide gel at pH 9.3 (12) or cellulose polyacetate (13) (Sepraphore III, Gelman Instrument Company) in 0.05 M barbital sodium, pH 8.6, or in 0.05 M sodium acetate, pH 5.0, at 3”. TCA concentration in the ponceau red stain for cellulose polyacetate electrophoresis was increased from 3% to 10% to prevent elution of peptides.

Preparative electrophoresis was performed at 3” on a bed of Sephadex G-25 (fine) (14), 1 x 30 cm, of width about 2.5 cm per ml of sample. The bed was connected to a-liter buffer tanks by eight thicknesses of Whatman No. 3MM filter paper. In order to avoid subsequent dialysis, a volatile buffer, 0.05 N acetic acid-O.048 N pyridine, pH 5.0, was used. The sample, containing about 5% protein, was mixed with dry Sephadex G-25 and ap- plied in a l-cm trough near the anode. Preparations rich in Asp fragment (see below) dissolve poorly at pH 5, and are best taken into solution with NH40H, the pH then being adjusted with dilute acetic acid. The applied potential was 200 volts between the buffer tanks, which resulted in a voltage drop across the bed of about 4 volts per cm. Peptide zones were located by staining prints taken on filter paper strips (14) with ponceau red in 10% TCA. After satisfactory separation was achieved (24 to 70 hours), zones were cut out, the peptide material was eluted with water, and water and buffer were removed by rotary evaporation followed by lyophilization.

Amine Acid Analysis-Free amino acids and hydrolysates of proteins and peptides were assayed on a Technicon automatic amino acid analyzer (Technicon Chromatography Corporation, Chauncey, New York), with the use of a single column of 17-p beads of sulfonated polystyrene resin, 8% cross-linked. Nor- leucine was added as an internal standard. Specimens from carboxypeptidase or hydrazinolysis procedures were assayed with a gradient which permitted detection of asparagine and gluta- mine.

RESULTS

Digestion of BSA with Pepsin-Proton uptake, increase in color yield with ninhydrin, and decrease in fluorescence with 1-anilinonaphthalene-8-sulfonic acid of BSA after peptic diges- tion at 25” are shown in Fig. 1. I f intended for fractionation, the digestion was terminated at 33 mm. Fluorescence efficiency had then fallen to 26% of its initial value, which was the criterion established by Weber and Young (6). At this time approxi- mately 5 protons had been taken up per molecule of BSA. If the pK of the new cy-carboxyl groups is taken as 3.1 to 3.3, this would indicate cleavage of an average of 6 to 9 peptide bonds per molecule of BSA.

The initial color with ninhydrin was 34 norleucine units per mole of albumin, increasing to 37 at the termination of digestion. Amino groups of albumin apparently are not fully reactive with ninhydrin, since albumin contains 1 a-amino group and 56 lysine e-amino groups. Hence the increase of 3 norleucine units may represent more than 3 peptide bonds cleaved per molecule of BSA.

New a-amino groups, shown in Fig. 2, were found to be valine, phenylalanine, and isoleucine. Approximately 1 mole of each of these three residues appeared per mole of albumin, plus a small amount of serine.

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1568 Peptide Fragments of Bovine Albumin Vol. 242, No. 7

The major new a-carboxyl group which appeared with diges- tion was that of leucine (Fig. 3), about 3 moles per mole of albu- min. Small amounts of terminal threonine and glutamic acid were also detected.

Electrophoresis on cellulose acetate showed the gradual ap- pearance of several zones moving more slowly than albumin at pH 8.6 (Fig. 4), plus unresolved components causing smearing of the leading portion of the pattern. The electrophoretic pattern, change in fluorescence efficiency, and new terminal amino acid residues were the same when digestion was performed for 133 min at 38” instead of for 33 min at 25’, or when the pepsin to albumin ratio was varied from 1: 1,000 to 1:30,000 with digestion time adjusted accordingly. Similarly, substitution for formic acid of citrate-phosphate buffer, used by Weber and Young (6), or control of pH with HCl in a pH-stat did not affect the results.

Fractiona;Cion of Pepsin-B&~ Digest-The crude digest was first fractionated with a phosphate gradient at pH 6 on diethylamino- ethyl cellulose, exactly as described by Weber and Young (6).

EO 0 IO 20 30 40

DIGESTION TIME AT 25’, mtnutes

FIG. 1. Proton consumption, increase in color yield with ninhydrin, and decrease in fluorescence efficiency with l-anilino- naphthalene-S-sulfonic acid (AN& during digestion of BSA with pepsin at pH 3. BSA concentration was 5%, and the pepsin to BSA ratio was 1:3OUO by weight. Fluorescence efficiency is expressed on a linear scale relative to a value of 5 for undigested BSA. The arrow indicates the termination of digestion prior to fractionation.

0 to 20 30

DIGESTION TIME AT 25: mtnutrr

FIG. 2. Appearance of new amino-terminal residues upon digestion of BSA by pepsin at pH 3. Amino-terminal residues were determined by the DFB procedure (8). Isoleucine was differentiated from leucine by automatic amino acid analysis after hydrolysis of the DNP- derivative in 15 N NHdOH. Yield of DNP-aspartic acid relative to BSA did not increase by more than lo’%, so that figures represent approximate yield per mole of BSA.

0 z Thr Y

$0 e

GIU I 4 0 IO 20 30

DIGESTION TIME AT 25; mtnutsr

FIG. 3. Appearance of new carboxyl-terminal residues upon digestion of BSA by pepsin at pH 3. Carboxyl-terminal residues were determined by hydrazinolysis followed by automatic amino acid analysis. Yield of alanine relative to BSA did not increase by more than lo’%, so that figures represent approximate yields per mole of BSA.

i --_ PEPSM-BSA DFAE ,,, FRACTtONS - 1.i’5~;y&

CONTROL DIGESY f Ilo ffb FRACTlOkI

\

FIG. 4. Electrophoresis on cellulose acetate at pH 8.6 of the pepsin-BSA digest and various fractions thereof. The Control was an unincubated BSA-pepsin mixture. Digest was 5% BSA treated with pepsin for 33 min at 25” (ratio of pepsin to BSA, 1:3000, w/w). DEAE Fractions I, IIa, and IIb were obtained by the use of diethylaminoethyl cellulose according to the method of Weber and Young (6). TCA Fraction is the portion of the whole digest which was soluble at 1.75% TCA and insoluble at 10% TCA, when the protein concentration was 1%.

This procedure yields three fractions, termed I, IIa, and IIb, eluting at 0, 0.08, and 0.28 M phosphate, respectively. These fractions all had multiple amino-terminal residues by the DFB method and were heterogeneous on electrophoresis (Fig. 4). Fraction I contained the slower zones seen in the pattern of the whole digest.

Fractionation of the digest with TCA instead of diethylamino- ethyl cellulose was then tested in an effort to simplify the initial steps. At 1% protein concentration and 0”, about 30% of the material absorbing at 230 rnp was soluble at 1.75% TCA, and about 6% remained in solution at 10% TCA. The fraction precipitating between 1.75 and 10% TCA appeared on electro- phoresis to be very similar to Fraction I (Fig. 4). Therefore, TCA fractionation was chosen as a first step in preference to the use of diethylaminoethyl cellulose because the former is less cumbersome when scaled up to large (20-g) quantities of protein. Digested albumin was diluted to 1% protein, and 40% TCA solution was added at 0” to a final TCA concentration of 1.75%.

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The precipitate was removed by centrifugation, and solid TCA was added to bring the supernatant to 10% TCA. This second precipitate was washed and was extracted several times with ether to remove most of the TCA.

Gel filtration of this 1.75 to 10% TCA fraction on Sephadex G-75 at pH 3 is shown in Fig. 5. The liquid medium was 0.6 N

acetic acid-O.01 N pyridine, pH 3.0, chosen as a volatile buffer containing some salt to decrease adsorption to the gel and as maintaining a pH at which aggregation of albumin fragments would be minimized (6). Six peaks resulted, of which two, peaks 4 and 5, exhibited no appreciable absorption at 280 rnp and were detectable only by staining filter papers spotted with the fractions with a reagent such as light green or ninhydrin. Peaks 1, 2, 3, and 6 have not as yet yielded any fractions ho- mogeneous to amino end group determination and electrophoretic examination. Peaks 4 and 5, eluting at 0.6 and 0.7 column volumes, respectively, were purified by one or two preparative electrophoretic procedures on a bed of Sephadex G-25 at pH 5. Electrophoresis for 30 hours at 4 volts per cm sufficed to purify the Peak 5 material, but with Peak 4 material electrophoresis for 60 to 70 hours was required to separate the main component from a zone which moved more rapidly toward the cathode at pH 5 (see Fig. 11 and “Component Closely Associated with Phe Fragment,” below). The result was two fractions pure by elec- trophoretic criteria and exhibiting single amino-terminal residues of phenylalanine and aspartic acid, obtained from Peaks 4 and 5, respectively. These were termed the Phe and Asp fragments, and were further analyzed as described below. Table I shows a flow diagram of the isolation procedure. In the earlier prepara- tions a final precipitation at 10% TCA was used to remove im- purities retained from the buffers. However, passage through Sephadex G-10 in distilled water was later substituted, as the last traces of TCA were difficult to remove.

Purity and Molecular Weight of Isolated Asp and Phe Frq- me&-Both the Asp and Phe fragments were essentially ho-

100 200 300

Elution volume, ml. 400

FIG. 5. Gel filtration of 1.75 to 10% TCA fraction of a pepsin- BSA digest. Approximately 1 g of the fraction was applied to a column (2.5 X 84 cm) of Sephadex G-75,60 to 400 p, at 24”. The column had been equilibrated with 0.6 N acetic acid-O.01 N pyri- dine, pH 3.0, and was eluted with the same buffer at 20 ml per hour. Output was monitored at 286 rnrc, and 2O-min fractions were collected in a Gilson Medical Electronics fraction collector. Fractions were assayed for peptides by spotting on filter paper and staining the paper with 1% ethanolic ninhydrin or 1% light green SF yellowish (Allied Chemical Corporation) in 1% acetic acid, after heating at 105” for 30 min. Molecular weight was estimated by comparing relative column volume at which the fraction eluted with that of proteins of known molecular weight, as described by Whitaker (15).

TABLE I

Outline of procedure for isolation of fragments

1. 5% BSA, pH 3.00, 25”; add pepsin (pepsin to BSA, 1:3090,

w/w). 2. After 33 min add concentrated NHIOH to pH 7; dilute to 1%

protein; chill to 0”. 3. Add 40% TCA to give 1.75’$$ TCA; centrifuge; wash precipitate

with 1.75’% TCA.

4. Add solid TCA to bring supernatant to 10% TCA; centrifuge; wash precipitate with 10% TCA.

5. Suspend precipitate in water and extract TCA with ether.

6. Dissolve precipitate in 0.6 N acetic acid-O.01 N pyridine, pH 3; pass through Sephadex G-75 column, 24”; collect Peak 4

(Phe fragment) at 0.6 column volume and Peak 5 (Asp frag-

ment) at 0.7 column volume; lyophiliae. 7. Purify separately by zone electrophoresis on Sephadex G-25,

3”, in 0.05 N acetic acid-O.648 N pyridine, pH 5; elute main zone (slower of two zones seen with Peak 4 material) and lyophilize.

8. Repeat Step 7. 9. Dissolve in water at 0”; pass through Sephadex G-10 column

with distilled water; collect fraction eluting at 0.3 to 0.5

column volume; lyophilize.

mogeneous upon electrophoresis on cellulose acetate at pH 8.6 or 5.0, or in polyacrylamide gel at pH 9.3 (Fig. 6). Two-dimen- sional paper chromatographs of the ether-soluble DNP-deriva- tives of the amino-terminal residues are shown in Fig. 7. These also attest to the purity of the preparations.

Molecular weights of the Asp and Phe fragments were esti- mated as 2300 and 7600, respectively, by their relative elution volumes from Sephadex G-75 at pH 3 (Fig. 8). With increasing pH, aggregation apparently occurs, as estimated molecular weights were higher at pH 7. The molecular weights at pH 3 agreed reasonably well with minimum molecular weights of 2808 and 8553, respectively, obtained from ammo acid analysis by finding the value for total residues which gave the closest ap- proach to integral values for all the amino acids.

These molecular weights, 2808 and 8553, were assumed in calculating yields of terminal amino acids (Table II). Support- ing these choices are the near integer values obtained for DNP- aspartic acid and DNP-phenylalanine with the DFB procedure; for leucine and valine from the Asp fragment and for the penulti- mate leucine from the Phe fragment with carboxypeptidase (see below); and for alanine from the Phe fragment upon hydrazinoly- Sk

Average amino acid composition of five preparations of each of the two fragments is presented in Table III, together with standard deviations and nearest integer values. Each of the in- dividual preparations yielded the same integer values as the av- erage of the five, excepting one preparation of the Phe fragment, which had 6.13 leucine residues instead of 7. The preliminary report (1) of the composition of the Phe fragment represented that preparation, so that the present figures, which are more ac- curate, differ from the earlier ones in showing 7 instead of 6 leucine residues, a total of 77 instead of 76 residues, and a molecu- lar weight of 8553 instead of 8440 for the Phe fragment.

Yield of Asp fragment was about 0.50/, of the original albumin, or about 12% on a molar basis. Yield of Phe fragment was about 1.2% of the albumin, or about 9% on a molar basis.

Characteristics of Asp Fragment-The Asp fragment showed

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1570 Peptide Fragments of Bovine Albumin Vol. 242, No. 7

FI& 6. Electrophoresis to test purity of the two isolated acrylamide gel patterns of the Phe and Asp fragments is brom- fractions, designated Phe and Asp fragments. The positive pole phenol blue, which was added to indicate the extent of migration was at the lop in each case. Direction of migration is indicated of anions. This sharp zone does not appear in the BSA pattern, by orro~s. The sharply defined zone at the top of the poly- since intact BSA adsorbs the bromphenol blue.

Fxo. 7. Chromatography of ether-soluble DNP- derivatives of the Asp and Phe fragments of BSA. Samples were applied at the upper left-hand corner of the paper and were developed in the horizontal direction with the toluene system (8) and in the vertical direction in 2.0 M phosphate, pH 6. The single spots seen are in the positions of (a) DNP-aspartic acid and (6) DNP-phenylala- nine.

amino-terminal aspartic acid by both the DFB and Edman pro- cedures, and the latter technique revealed that the penultimate residue is threonine. The amino-terminal sequence, Asp-Thr-, was also found in intact bovine albumin, in agreement with the results of Thompson (17). New aspartic acid amino-terminal residues were not observed to appear during peptic digestion (within an error of about lo%), so it seems reasonable to conclude that the Asp fragment arises from the amino terminus of the albumin molecule. Further evidence for this conclusion is that

5

s

$4

c3 s

3 * 1 I I

15 20 25 30 35

I I I I

. Pepsin

\ &ytochrome C

“Phe +X II\

fragment \+ulin \

\

“&x fragment

Elution volume, ml.

FIG. 8. Estimation of molecular weights of Asp and Phe frag- ments of BSA on Sephadex G-75 at pH 3, by the technique of Whitaker (15). Samples of 1 to 2 mg were applied to a column, 1.2 X 55 cm, of Sephadax G-75 and eluted with 0.6 N acetic acid- 0.01 N pyridine, pH 3. Calibrating proteins were pepsin (mol Wt 33,500), cytochrome c (Nutritional Biochemicals) (mol wt 13,600), and insulin (Eli Lilly and Company, crystallized five times) (mol wt 6,060).

both albumin and Asp fragment bind a single atom of copper(I1) in a unique manner, as reported in the following paper (18).

The carboxyl-terminal sequence determined with carboxypep- tidase is -Leu-Val-Leu (Fig. 9). Leucine was the predominant amino acid found upon hydrazinolysis, although the yield was only 0.2 mole per mole (Table II). The Asp fragment contains 24 amino acid residues, and has a calculated molecular weight of 2808. It contains no proline, tyrosine, tryptophan, cysteine, cystine, methionine, or amide groups. There are 3 histidine residues among its 24 total residues.

Churacteristks of Ph.e Fragment-The Phe fragment, so named because of its amino-terminal residue, was calculated to contain

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77 amino acid residues and to have a molecular weight of 8553. It lacks tyrosine and tryptophan, as does the Asp fragment, and arginine is also missing. There are 3 proline residues and 2 cystine bridges.

The carboxyl-terminal residue found by hydrazinolysis (Table II) is alanine, the same as in intact BSA. Treatment with car- boxypeptidase yields a pattern of release of free amino acids (Fig. lob) resembling that obtained with BSA (Fig. lOa). The results with BSA are in good agreement with those of White, Shields, and Robbins (19)) and suggest the sequence, -(Gly , Ser , Val)-(Ala , Thr)-Leu-Ala. Since the carboxyl-terminal se- quences of the peptide and of albumin are similar, and since the increase in carboxyl-terminal alanine with peptic digestion was less than 0.1 mole per mole, it seems probable that the Phe frag- ment represents the carboxyl-terminal segment of albumin.

TABLE III Amino acid composition of isolated fragments

Samples of each of five preparations were hydrolyzed (6 N HCI, sealed tube under reduced pressure, 110”) and taken to dryness. BSA and one preparation of each fragment were hydrolyzed for 24, 48, or 96 hours; values for serine, threonine, and amide NH3 were obtained by extrapolation to zero hydrolysis time; values for valine and isoleucine are the average of the two highest figures. Other preparations were hydrolyzed for 24 hours, and values for serine, threonine, and valine were multiplied by the observed factors of 1.13, 1.05, and 1.10, respectively. Tryptophan and cysteine were determined on intact protein or peptide (16) on one preparation of each fragment.

-7 -

-

1

Asp fragment* Phe fragmentC

Component Closely Associated with Phe Fragment-Material migrating just ahead of the Phe fragment upon electrophoresis at pH 5 (Fig. 11) has been isolated in apparently pure form in one instance. It is remarkably similar to the Phe fragment in the following respects. (a) It is eluted from Sephadex G-75 at the same volume, so its size is probably about the same. (5) It shows, as amino-terminal residue, only phenylalanine. (c) Its amino acid composition is almost identical with that of the Phe fragment. Hydrazinolysis, however, shows as carboxyl-terminal residues 0.67 leucine and 0.22 alanine per 8500 molecular weight, compared to 0.15 leucine and 0.77 ahaine for the Phe fragment (Table II). Carboxypeptidase treatment indicates the sequence, -(Thr,Ala)-Leu. This suggests the possibility that this faster component is the Phe fragment with its carboxyl-terminal alanine residue removed. Indeed, treatment of a Phe fragment prepara- tion with pepsin could be shown to cause a decrease of carboxyl- terminal alanine and an increase of carboxyl-terminal leucine, as determined by hydrazinolysis. Amino acid analysis of the faster migrating component confirmed the expected absence of 1

1

t -_

Residue (in-

eger) Aver- I ! S.D.

In- we teger

res- idues

46 16 35 61 13 28 26 19 2

26 32

1 34 4

22 17 56 53

(ii;

566

9 idues

1.02 0.11 1 1.87 0.12 2 1.11 0.10 1 3.00 0.25 3 1.02 0.03 1 0.05 0.05 0 2.02 0.12 2 0.05 0.02 0 0.01 0.01 0 1.18 0.08 1 1.03 0.11 1 0.00 0.00 0 0.02 0.02 0 0.01 0.01 0 1.05 0.08 1 2.78 0.11 3 2.81 0.13 3 1.92 0.09 2 2.99 0.14 3

(0.21 (0)

24

residues

9.32 0.18 9 1.15 0.07 1 6.79 0.13 7 6.80 0.39 7 1.89 0.13 2 3.17 0.19 3 5.02 0.08 5 0.05 0.03 0 0.01 0.01 0 1.04 0.16 1 8.04 0.26 8 0.01 0.01 0 3.69 0.26 4 0.91 0.09 1 0.04 0.02 0 2.11 0.05 2

10.06 0.18 10 5.97 0.24 6

10.96 0.15 11 (5.2) (5)

77 77

Alanine. Glycine Valine Leucine Isoleucine Proline Phenylalanine. Tyrosine. . . Tryptophan. Serine . Threonine Cysteine............ Half-cystine Methionine Arginine . . Histidine Lysine Aspartic acid. Glutamic acid.. Amide NHad TABLE II

Molecular weights and terminal seqz Lences of isolated fragments

I - -

Total............... Phe fragment

-

.- Asp fragment -

7600

8553

a One preparation, six analyses. * Five preparations, 15 analyses. c Five preparations, 10 analyses. d Values in parentheses were not included in the totals

Molecular weight Sephadex G-75,

PH 3 Minimal, amino

acid analysis Amino-terminal

Residue, DFB, mole per mole5

Sequence, Edman degradation

Carboxyl-terminal Residue, hydra-

zinolysis, mole per molea

2300

2808

0.9 Asp 0.92 Asp Asp-Thr- Asp-Thr-

0.83 Ala 0.26 Gly 0.22 Ser 0.13 Phe 0.09 Leu

-(Gly,Ser,- Val)-(Ala, Thr)-Leu- Ala

0.20 Leu

0.10 Gly 0.06 Ser

-Leu-Val- Leu

0.9 Phe

0.77 Ala

0.15 Leu 0.11 Ser 0.10 Gly

Sequence, car- boxypeptidase (Figs. 9 and 10)

-(Ser,Val,- Gly)-(Ala, Thr)-Leu- Ala

0

I NC”BA&I TIME, I

hours

a Based on molecular weights of 65,000, 2,808, and 8,553, re- FIG. 9. Release of free amino acids from Asp fragment with

carboxypeptidase. For details, see “Methods.” Ratio of carbox- spectively. ypeptidase to peptide was 1:23 by weight.

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1572 Peptide Fragments of Bovine Albumin Vol. 242, No. 7

INCUBATION TIME, hours

(a) BSA (b) Phe FRAGMENT

FIG. 10. Release of free amino acids from intact BSA (a) and from Phe fragment (5) with carboxypeptidase. For details, see “Methods.” Ratio of carboxypeptidase to protein was 1:70 for BSA and 1:2O for the Phe fragment.

of the 9 alanine residues, but also indicated that possibly it con- tains 1 less residue, each, of threonine and valine than the Phe fragment. Hence, the question still remains whether this faster migrating component arises from cleavage of the terminal alanine from the Phe fragment or whether it is a very similar segment situated in an internal location of the albumin chain.

DISCUSSION

That cleavage of bovine albumin by pepsin proceeds via frag- ments of intermediate size has been documented previously. Annau (20) found that brief peptic digestion at pH 3 caused the appearance of several peaks on electrophoresis. Smet, Lontie, and Preaux (21) isolated an immunologically active fragment of estimated molecular weight 35,000 after prolonged digestion at pH 4.4. Schlamowitz, Peterson, and Wissler (22) studied the FIG. 11. Electrophoresis on cellulose acetate at pH 5 showing

peptic fragments in the ultracentrifuge and by fractionation with the relationship of purified Phe fragment to the second component

solutions of TCA in urea. They detected a new amino-terminal closely associated with it. The second component is the lower zone in the rig/+Aand figure.

valine or isoleucine bond at an early stage of digestion, which is in accordance with our findings (Fig. 2).

Leu-Val-Leu “Asp” fragment 24 residues

Ile/Val

Known internal sequences:

-Trp-Ser-Val-Ala-(Gly , Ala)-(Ser , Glu/Gln)-

-Trp-Gly-Phe-Leu-

-Leu-Gln-Asp-Glu-Gln-Glu-Cys-Pro-Phe-

m)

(2’3

(27)

. Leu

Remainder of albumin molecule 465 residues ; 15 S-S ; 1 -SH

. I- -s-A

. . . . . . . . . . . . , .,. . Phe S-S

. -(Ser , Val)-(Ala , Thr)-Leu-Ala

“Phe” fragment 77 residues; 2 S-S

FIG. 12. Proposed relationship of the Asp and Phe fragments to the remainder of the BSA molecule. Areas are scaled approximately to molecu lar weights. Known amino acid sequences of BSA are also listed.

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Issue of April 10, 1967 T. Peters, Jr., and C. Hawn 1573

The action of pepsin at pH 3, however, is obviously not simply to cleave BSA into three major globular parts. The Asp and Phe fragments resulted from cleavage of bonds readily accessible to pepsin, and yet they compose together only about one-fifth of the molecule. Furthermore, the remaining Sephadex G-75 and electrophoretic fractions are polydisperse on examination by electrophoresis and end group assay.

Enzymes other than pepsin can also produce fragments of BSA. Adkins and Foster (23) recently obtained two compo- nents of molecular weight 31,000 and 38,000 after treatment with subtilisin. Porter (24), using chymotrypsin, obtained an immunologically active fragment of molecular weight about 12,000.

It is not possible at the present state of knowledge to correlate the Asp and Phe fragments with these various larger components. Fig. 12 shows a simple proposed relationship of the terminal Asp and Phe fragments to the remainder of the molecule. Even the Phe fragment must be judged too small (about one-eighth of the molecule) to represent one of the three major globular components proposed on the basis of physicochemical data (5, 6). Bloomfield (25) has calculated these components to be of the relative sizes t : # : 4.

Fig. 12 also summarizes known data on sequences of BSA. The third residue of the Asp fragment is shown as histidine, evidence for which is presented in the following article (18). Three internal peptide sequences have been identified by their content of tryptophan (26) or cyst&e (27). Overlap of any of these three peptides with either the Asp or Phe fragment is excluded, since neither fragment contains tryptophan or cys- teine, and none of the three peptides contains any of the se- quences (Leu-Ile, Leu-Val, or Leu-Phe) believed to be cleaved by pepsin in the production of the Asp and Phe fragments. The 12,000 molecular weight fragment of Porter (24) must in- clude the g-residue peptide isolated by Witter and Tuppy (27), because both contain the single cysteine residue of BSA. Por- ter’s fragment may overlap with the Asp or Phe fragments, but it cannot contain either of these fragments in its entirety: Por- ter’s fragment has amino-terminal phenylalanine, and the Phe fragment has 2 cystine residues while Porter’s fragment has but 1.

The Phe fragment, mol wt 8553, is probably the carboxyl terminus of BSA. It is unusual only in lacking tyrosine, trypto- phan, and arginine. It would therefore not be detected by the usual ultraviolet absorption techniques. Its cystine and proline contents, 2 and 3 residues, respectively, are in proportion to those of albumin. Although easily large enough to have anti- genie properties, the Phe fragment caused no blocking of the reaction between BSA and its rabbit antibody in preliminary tests with agar diffusion.

The amino-terminal or Asp fragment, mol wt 2808, has several interesting properties which make it worthy of further study.

Besides lacking tyrosine, tryptophan, methionine, cysteine, asparagine, and glutamine, it has no proline or cystine residues to restrict coiling into helical form. Its histidine residues (3 out of a total of 24 residues) and its free aspartyl a-amino group are likely binding sites for smaller molecules. Chelation of copper at the terminal site does occur; further data on this phenomenon are reported separately (18).

Acknowledgments-Assistance by Mrs. Margaret Elkan and Mrs. Dorothy Moakler in the laboratory and by Mrs. Elizabeth Felts in a secretarial capacity is greatly appreciated.

1. 2.

PETERS, T., JR., J. Biol. Chem., 240, PC1866 (1965). HUNTER, M. J., AND MCDUFFIE, F. C., J. Amer. Chem. Sot.,

81, 1400 (1959). 3. PETERS, T. JR., Compt. Rend. Trav. Lab. Carlsberg, 31, 227

(1959). 4.

5.

PETERS, T., JR., Abstracts Sixth International Congress of Biochemisiry, 1964, IUB Vol. S??, p. 174, 11-151. - -

FOSTER. J. F.. in F. W. PUTNAM (Editor). The vlasma vroteins.

6. Vol. i, Academic Press, New ?ork, l&O, p.*179. A ’

WEBER, G., AND YOUNG, L. B., J. Biol. Chem., 239, 1415, 1424 (1964).

7. 8.

9.

10.

KUSAMA, K., J. Biochem. (Tokyo), 44, 375 (1957). FRAENE~L-CONRAT, H., I%ARE&,. J. I., AND LEVY, A. L.,

Method. Biochem. Anal., 2, 359 (1955). SCHROEDER, W. A., SHELTON, J. R., SHELTON, J. B., CORMICK,

J., AND JONES, R. T., Biochemistry, 2, 992 (1963). NIU. C.. AND FFUENKEL-CONRAT. H.. J. Amer. Chem. Sot..

11.

12. 13.

77; 5882 (1955). I I

PETERS, T., LOGAN, A. C., AND SANFORD, C. A., Biochim. Biophys. Acta, 30, 88 (1958).

DAVIS, B. J., Ann. N. Y. Acad. Sci., 121, 401 (1964). RITTS, R. E., JR., AND ONDRICK, F. W., Amer. J. Clin. Pathol.,

41, 321 (1964). 14. BROWN, J. R., GREENSHIELDS, R. N., YAMASAKI, M., AND

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NE&ATH, H., Biochemistry, a, 867 (1963). ’ . WHITAKER, J. R., Anal. Chem., 36, 1950 (1963). PETERS, T., JR., J. Biol. Chem., 237, 2182 (1962). THOMPSON, E. 0. P., J. Biol. Chem., 208,566 (1954). PETERS. T.. JR.. AND BLUMENSTOCK. F. A.. J. Biol. Chem..

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WHITE, W. F., SHIELDS, J., AND ROBBINS, K. C., J. Amer. Chem. sot., 77, 1267 (1955).

ANNAU, E., Nature (London), 183. 190 (1959). SMET, l?., ~ONTIE, R:, AND P&AUX, G., ih H.@EETERS (Editor),

Protides of the biolooical fluids. Vol. II. American Elsevier Publishing Company Inc.“, New York, l&3, p. 119.

SCHLAMO~ITZ, M., PETERSON, L. U., AND WISSLER, F. C., Arch. Biochem. Biophys., 92, 58 (1961).

ADKINS, B. J., AND FOSTER, J. F., Biochemistry, 6,2579 (1966). PORTER, R. R., Biochem. J., 66, 677 (1957). BLOOMFIELD, V., Biochemistry, 6, 684 (1966). SUQAE, K., AND JIRQENSONS, B., Tokyo, J. Biochem., 66, 457

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REFERENCES

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Theodore Peters, Jr. and Cynthia HawnCarboxyl-terminal Positions of Bovine Serum Albumin

Isolation of Two Large Peptide Fragments from the Amino- and

1967, 242:1566-1573.J. Biol. Chem. 

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