The Amino Acid Sequence of the Tryptic Peptides from Cytochrome b,*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 243, No. 12, Issue of June 25, pp , 1988 Pentea in U.S.A. The Amino Acid Sequence of the Tryptic Peptides from Cytochrome b,* JURIS OZOLS AND PHILIPP STRITTMATTER (Received for publication, January 10, 1968) From the Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri SUMMARY Apocytochrome bs was hydrolyzed with trypsin and the resulting peptides were resolved on Dowex 1 and Dowex 50 columns. The expected 11 peptides were isolated and fragmented further with papain and pepsin. The complete amino acid sequences of these peptides were determined by carboxypeptidase A and B and procedures. Previous studies on calf liver microsomal cytochrome bs have shown that trypsin cleaves a COOH-terminal heptapeptide and an NH2-terminal dipeptide from the heme protein (2). The resulting heme peptide is unaltered in its spectral properties and the remaining lysyl and arginyl residues are resistant to further tryptic hydrolysis (2, 3). In the present study apocytochrome b5 was subjected to tryptic hydrolysis and the resulting peptides were isolated by ion exchange chromatography. The expected tryptic peptides were isolated and their complete amino acid sequences were determined. A preliminary report on the linear arrangement of partially sequenced tryptic peptides has been given (1). The isolation of the chymotryptic peptides and the complete amino acid sequence of the calf liver microsomal cytochrome b5 are presented in the succeeding paper (4). EXPERIMENTAL PROCEDURE &fateriuzs-calf liver cytochrome bs was prepared as previously described (2). Preparation I, the predominant heme protein species in crude cytochrome bb preparations, was used (2). L-(l-Tosylamido-2-phenyl)ethyl chloromethyl ketone-trypsin, chymotrypsin, diisopropyl fluorophosphate-treated carboxypeptidase A and B, papain, pepsin, and diisopropyl fluorophosphate-treated leucine a.minopeptidase (Batch LAP-DFP-GIC) containing 12.6 mg of protein per ml were obtained from Worthington. Dowex 50-X2 and Dowex l-x2 were products of Dow * A preliminary report of this work has appeared (1). This investigation was supported by Research Grant HE from the United States Public Health Service. Chemical Company. Sephadex G-25 was obtained from Pharmacia. Dithioerythritol was obtained from Calbiochem. Phenylisothiocyanate and trifluoroacetic acid were products of Eastman Chemicals. The pyridine used was spectroquality and a product of Matheson Coleman Bell. N-Ethylmorpholine and oc-picoline were redistilled and stored at -20. All other solvents used were reagent grade. Trypsin Hydrolysis of Apocytochrome bs-heme-free protein was prepared by treatment of cytochrome bg with acid-acetone at 0. To 0.5 pmole of cytochrome bs in 0.1 to 0.5 ml of 0.02 M sodium phosphate, ph 7.5, 10 ml of acetone containing 0.2% HCl (v/v) were added. After 10 min, the white precipitate was collected by centrifugation, rapidly dried by a stream of nitrogen, and dissolved in 1.0 ml of 0.1 M (NH&CO+ ph 8.6. A fresh 0.1 To solution of L-(l-tosylamido-2-phenyl)ethyl chloromethyl ketone-trypsin in 0.02 M sodium phosphate, ph 7.5, containing lop3 M calcium chloride, was prepared and 0.05 ml was added to 1.0 ml of the substrate solution. After 3 hours at 25 the hydrolysate was lyophilized. Chymotrypsin HydrolysisOne-tenth milliliter of 0.1 y0 solution of chymotrypsin in N HCl was added to 1.0 ml of peptide (0.2 to 0.5 pmole) in 0.1 M of ammonium bicarbonate, ph 8.5. Incubation was carred out for 2 hours at 25, and was terminated by lyophilization. Pepsin Hydrolysis-The peptide was dissolved in 0.01 N HCl (0.5 pmole per ml) and 0.1% pepsin solution in 0.01 N HCl was added to give a final enzyme concentration of 0.01%. After 15 hours at 25 the digest was lyophilized. Papain Hydrolysis-An aliquot (40 ~1) of the enzyme suspension was added to 1 ml of 0.2 M pyridine-acetate buffer, ph 5.5 (5), containing 0.5 M dithioerythritol. The peptide (0.5 pmole) was dissolved in 1.5 ml of 0.2 M pyridine-acetate buffer, ph 5.5, and 0.10 ml of the diluted enzyme solution was added. Digestion was carried out at 35 for 20 hours. Leucine Aminopeptidase Hydrolysis-The following mixture was incubat,ed at 38 : 0.02 to 0.05 pmole of peptide in 0.1 ml of 0.1 M Tris-HCl buffer, ph 8.5, containing 0.2 M MgCl,, and 5 ~1 of the enzyme suspension. After the indicated time, the contents of the tubes were lyophilized and suspended in cold ph 2.2 citrate buffer, and an aliquot was analyzed immediately on the amino acid analyzer. Since the presence of Tris interferes with accurate arginine determinations, chromatography of aminopep-

2 3368 Sequence of Tryptic Peptides from Cytochrome bg Vol. 243, No. 12 tidase digests on the short column of the amino acid analyzer was performed only through the position of histidine. Carboxypeptidase A and B Hydrolysis-A stock solution of carboxypeptidase A was prepared by adding 0.1 ml of a 5y0 suspension of the enzyme to 1.0 ml of buffer containing 0.8 ml of 2 M NaCl and 0.2 ml of 1 M sodium phosphate, ph 8.0 (5). A 20-~1 aliquot of this solution was added to 0.1 pmole of peptide in 0.4 ml of 0.2 M sodium bicarbonate, ph 8.3. Digestion was carried out at 38 for the indicated time. Hydrolysis was terminated by adding 0.1 ml of 6 N acetic acid followed by lyophilization. After adding ph 2.2 citrate buffer, an aliquot was analyzed on the amino acid analyzer. Digestions with carboxypeptidase B were performed similarly, except that the enzyme was not diluted but was used directly at the same enzyme to substrate ratio. Dilute Acid Hydrolysis of Peptides-Peptides containing aspartic acid or asparagine were desalted by filtration through a column of Sephadex G-25, 20 X 1 cm, equilibrated with 30% acetic acid. A 0.02~pmole aliquot of the peptide was dissolved in 0.30 ml of 0.03 N HCl and hydrolyzed in an evacuated sealed tube at 107 for 12 to 14 hours (6). The hydrolysate was evaporated to dryness, citrate buffer (ph 2.2) was added, and free amino acids were determined on the amino acid analyzer. Gel Filtration of Peptides-Sephadex G-25, fine grade, was equilibrated with either 0.02 M sodium phosphate, ph 7, or 3Oy, acetic acid. A column (0.9 x 60 cm) was packed from a stirred reservoir containing the gel slurry, and equilibrated overnight with the eluant from a constant pressure flask. A 0.5-ml sample was applied on top of the bed and eluted at a flow rate of 10 ml per hour at 25. Fractions of 1 ml were collected. Chromatographic Separatbns on Dowex 50-Dowex 50-X2, 200 to 400 mesh, was suspended in water and washed with N NaOH, HCl, and pyridine as described by Moore and Stein (7) and Schroeder et al. (8). The resin was stored in pyridine-acetate buffer, ph 3.1, in the resin buffer volume ratio of 2: 1. The column (60 x 0.9 cm) was packed at room temperature under a pressure of 6 p.s.i. of nitrogen and equilibrated overnight with the starting buffer. The flow rate was maintained throughout with an ACCU-Flo Pump (Beckman) at 25 ml per hour, and the column temperature was kept at 38 with a constant temperature circulating bath. A sample of 2.0 ml at ph 2.5, containing 0.3 to 0.6 pmole of peptides, was placed on the column and eluted with a linear gradient of increasing ph and pyridine concentration (see Margoliash and Smith (9)) with the use of the buffer system detailed in the legend to Fig. 1. The mixing chamber and the reservoir were equal diameter flasks connected with a three-way stopcock. The pyridine-acetate buffers used (5) had the following composition per liter of solution: ph 3.1,0.2 M pyridineacetate (16 ml of pyridine and ml of acetic acid); ph 5.7, 8.5 M pyridine-acetate (684 ml of pyridine and 180 ml of acetic acid); ph 4.8, 0.2 M pyridine-acetate (14 ml of pyridine and 15 of acetic acid). Chromatographic Separations on Dowex I-Dowex l-x2, 200 to 400 mesh, was recycled through the chloride form and then converted to the acetate form as described by Schroeder and Robberson (10). The volatile buffers developed by Rudloff and Braunitzer (11) and Schroeder and Robberson (lo), were used. Buffers of ph 9.4, 8.4, and 6.4 contained 15 ml of N-ethylmorpholene, 20 ml of oc-picoline, 10 ml of pyridine per liter, and 0.13, 0.75, and 0.93 ml of acetic acid to give the desired ph. Prior to use, the resin slurry and the buffers were thoroughly degassed under vacuum. A column of resin, 0.9 x 60 cm, was poured at 38 and equilibrated with 400 ml of the ph 9.4 buffer and a 2-ml sample at ph 9.8 containing 0.3 to 0.6 Mumole of peptides was introduced. The column was connected through a pump to a magnetically stirred constant volume mixer of 50-ml volume. The mixing chamber was in turn connected to a reservoir of 300-ml capacity by Teflon tubing (k-inch inside diameter, Beckman). The whole system was filled with the equilibrating buffer and the column was developed at 38 with a decreasing ph gradient, obtained by replacing the solution in the reservoir with corresponding buffers at the fraction numbers indicated in the figure legends. A constant flow rate was maintained at 30 ml per hour. After the chromatogram had been completed, the column was flushed with 30% acetic acid and reused. Examination of Column Fractions-Fractions containing peptides were identified by the ninhydrin reaction after base hydrolysis. Aliquots of 20 to 150 ~1 were first hydrolyzed with 1.0 ml of 2.5 N sodium hydroxide in 15-ml polypropylene centrifuge tubes (Iva,n Sorvall, Inc., Norwalk, Connecticut) for 2 hours at 100 and then 1.0 ml of 30y0 acetic acid was added to each tube. The ninhydrin reaction was carried out by ad ling 0.5 ml of freshly prepared acetate-cyanide buffer (0.1 ml of 0.01 M sodium cyanide to 10 ml bf 4 N sodium acetate, ph 5.5) followed by 0.5 ml of a solution containing 3% ninhydrin in methyl Cellosolve. The tubes were heated in a boiling water bath for 15 min and the color was determined at 570 rnp in a Bausch and Lomb Spectronic 20 calorimeter. Fractions containing peptides were characterized by amino acid a.nalysis and stored at -20. All glassware was rinsed with deionized water, dried, and stored in dust-free surroundings, and every effort was made to prevent any contamination of the effluent fractions or the buffers with ninhydrin-positive material. Paper electrophoresis of peptides was performed at ph 7.5 (2). Peptides were identified after spraying the paper with 0.5% ninhydrin in acetone, and their mobilities were designated as basic, acid, or neutral. Peptides obtained from tryptic digest are denoted by T and an arbitrary number. Letters Pa and Pe denote peptides obtained by papain and pepsin digestion. Amino Acid Analysis-All a.nalyses were performed by the method of Spackman, Stein, and Moore (12) on a Spinco model 120 automatic amino acid analyzer, equipped with a 6.6-mm flow cuvette and a 4- to 5-mv range recorder, permitting the determination of amino acids in the range of to 0.03 pmole. When it was necessary to use quantities of less than pmole, the amounts of all other trace amino acids present were also included in the calculations. Samples were hydrolyzed routinely in 6 N HCl at 110 in evacuated tubes for 22 hours. A duplicate sample was hydrolyzed for 72 hours whenever valine, isoleucine, or leucine was detected. Amino acids present in yields below 15% of a residue are not repcrted. Tryptophan was identified with the use of the Ehrlich reaction (13) and after base hydrolysis (5 N NaOH, 18 hours, 107 ) or in enzymic hydrolysates with the amino acid analyzer. Glutamine and aspara,gine in enzymic hydrolysates were determined quantitatively in the position of serine on the analyzer. The identity of each was checked by acid hydrolysis of an aliquot of protein-free enzymic hydrolysate to yield the expected free glutamate or aspartate. Determination of Amino Acid Sequences-The sequential degradations of Edman (14) as modified by Konigsberg and Hill (15) were performed. The coupling with phenylisothiocyanate and its cyclization in anhydrous trifluoroacetic acid were carried

3 Issue of June 25, 1968 J. 0~01s and P. Xtrittmatter 3369 A 0 c T-3 T- 5.d, OO II I I I I I I I I FRACTION FIG. 1. A, chromatography of tryptic hydrolysate of 0.5 wmole of apocytochromebb ona Dowex50 column, 60 X 0.90 cm. Gradient elution was begun after 25 ml by allowing 500 ml of 8.5 M pyridineacetate, ph 5.6, to flow into the mixing chamber containing 475 ml of starting buffer, 0.2 M pyridine-acetate, ph 4.8, at a flow rate of 25 ml per hour. Fractions of 3.ml were collected and 100~~1 aliquots were analyzed after alkaline hydrolysis by the ninhydrin method. B, rechromatography of Fractions 60 to 68 (A) on a out under nitrogen. The identity of the NH*-terminal residue was determined by amino acid analysis on a fraction of the residual peptide. The number of micromoles of amino acid found was converted to residues per molecule by assuming that the concentration of one of the acid-stable amino acids represents a single residue in the peptide. The yields of the degradation steps varied from 75 to 97%, and were not reported unless they were outside this range. The residue marked in boldface type corresponds to the residue removed at each step. I I I I I I 1 I I ) NUMBER Dowex 1 column, 60 X 0.9 cm. Elution wasbegunwith theconstant volume mixing chamber and a reservoir containing ph 9.4 buffer. The eluent in the reservoir was then replaced with ph 8.4 and ph 6.3 buffers at Fractions 25 and 55, respectively, followed by 0.5 N acetic acid at Fraction 80 and 2.0 N acetic acid at Fraction 120. Fractions of 1. 5 ml were collected and 100-~1 aliquots analyzed by the ninhydrin method I 10 RESULTS Separation and Composition of Tryptic Peptides The elution pattern of the tryptic peptides from a Dowex 50 column is shown in Fig. IA. This procedure provided Peptides T-l, T-4: T-11, and T-5 in yields above 60%. The yield of T-2 was 30%. Rechromatography of Fractions 60 to 68 on Dowex 1 gave an additional yield of Peptide T-2, and Peptides T-3, T-6, and T-7 (Fig. 1B). Peptides T-8, T-9, and T-10 were not completely resolved. Since 2 eq of Peptide Ser-Lys were found in the tryptic digest of apocytochrome bs, this peptide is designated T-9 and T-10. Fig. 2 shows the elution pattern of the tryptic digest from Sephadex G-25 with 0.02 M phosphate buffer, ph 7. Fractions 5 and 6 contained the 21-amino acid peptide T-l (yield 90%) in sufficiently pure form to be used for the sequence determination. The amino acid composition and electrophoretic mobility of Fractions 8, 9, and 10 indicated a peptide mixture which, upon resolution on Dowex 50, gave equal amounts of Peptides T-2 and T-3. The remaining ninhydrin-positive fractions indicated by FRACTION NUMBER FIG. 2. Gel filtration of a tryptic hydrolysate of 0.5 pmole of apocytochrome on a Sephadex G-25 column, 60 X 0.9 cm, equilibrated with 0.02 M phosphate, ph 7. After 10 ml, l-ml fractions were collected at a flow rate of 10 ml per hour. the solid bar were resolved on a Dowex 1 column and gave an elution pattern identical with Fig. 123, with Peptides T-l, T-2, and T-3 absent as expected. As shown in Fig. 3, Dowex 1 chromatography of the tryptic digest gave essentially a complete resolution of all of the expected tryptic peptides, with the exception of Peptide T-5. The amino acid composition of the peak fraction and the approximate

4 3370 Sequence of Tryptic Peptides from Cytochrome bs Vol. 243, No. 12 T-i & 4.20 NH3 R 6 i 2- i$ 0.80 z : N 2.0 N F ~H8.3 ph6.3 Acetic Acid Acetic Acid T-10 T-9 T-4 T-11 T-8 T-6 T-3 T-2 T-7 h FIG. 3. Chromatography of a tryptic digest of 0.5 rmole of apocytochrome on a Dowex 1 column, 60 X 0.9 cm. Elution was begun with a mixing chamber and reservoir containing ph 9.4 buffer. At the fractions indicated by the arrows on the elution diagram the buffer in the reservoir was replaced by the buffer indicated. Fractions of 1.5 ml were collected and 100-J aliquots were analyzed by the ninhydrin method., OO!,wL, JTbu FRACTION NUMBER TABLE 1 Amino acid composition of tryptic peptides of apocy6ochrome ba isolated from Dowex 1 The number above each peptide represents the peak fraction number in Fig. 3, with the exception of T-5*, which is from Dowex 50 (Fig. IA). The yield of peptide in the indicated fraction was calculated from the micromoles of apoprotein digested. - Amino acid Lysine Histidine Arginine. Aspartic acid. Threonine Serine. Glutamic acid.. Proline. Glycine Alanine. Valine. Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Yield in the above fraction (%) No. of residues T-l T-2 T _ ;y;* h!l 108 T T-l T-i: Total Protein (2) yield of the peptide in this fraction are given in Table I. Two equivalents of Ser-Lys again were found in this tryptic digest (T-9 and T-10). The yields of peptides in Table I are based on the number of micromoles of peptides found in the indicated fraction compared to the number of micromoles of apocytochrome digested. Analysis of the ascending and descending fractions showed no significant differences in the amino acid composition, except when the elution pattern indicated an overlap of peptides. Amino Acid Sequences of Peptides Peptide T-i: Glu-Gln-Ala-Gly-Gly-Asp-Ala-Thr-Glu-Asp- Phe-Glu-Asp-Val-Gly-His-Ser-Thr-Asp-Ala-Arg (Table II)- This peptide was isolated from tryptic hydrolysates by three different chromatography procedures: gel filtration with Sephadex G-25 (Fig. 2), Dowex 50 (Fig. IA), and Dowex 1 (Fig. 3). In addition to an identical amino acid composition, removed only a single glutamate residue from the peptides obtained by the three procedures. on an aliquot (0.1 pmole) established the NH&erminal sequence of 6 residues, and leucine aminopeptidase hydrolysis indicated the presence of glutamine adjacent to the NHz-terminal glutamate. Pepsin digestion of 0.4 pmole of peptide for 15 hours, followed by Dowex 50 chromatography (Fig. 4), gave three peptides with the total amino acid composition equal to Peptide T-l. Peptide Pe-1 had a composition identical with the NH&erminal segment

5 Issue of June 25, 1968 J. 0x01s and P. Xtrittnzatter 3371 TABLE II Amino acid composition and sequence studies of Peptide T-l Glu~G1~~-Ala-Gly-Gly-Asp~Ala-Thr-G1u-Asp-Phe-G1~~~Asp-Val-Gly-His-Ser-Thr-Asp-Ala-Arg I( Pe-l-pJ-Pe-2- < Pe-3 > Peptide -I- Peptide T-l His, 1.09; Arg, 1.06; Asp,4.05; Thr, 1.85; Ser, ; Glu,4.03; Gly,3.03; Ala, 2.94; Val, 1.09; Phe, 1.03 His, 0.95; Arg, 1.10; Asp, 3.94; Thr, 1.90; Ser, 0.98; Glu, 3.10; Gly, 3.20; Ala, 3.20; Val, ; Phe, 0.94 His, 1.20; Arg, 1.10; Asp, 3.80; Thr, 1.86; Ser, 0.96; GILI, 2.20; Gly, 3.00; Ala, 2.88; Val, ; Phe, 0.86 His, 1.03; Arg, 0.90; Asp, 3.70; Thr, 1.70; Ser, 0.83; Glu, 2.20; Gly, 2.80; Ala, 1.98; Val, ; Phe, 1.03 Step 5 His, 0.83; Arg, ; Asp, 3.90; Thr, 1.63; Ser, 0.94; Glu, 1.96; Gly, 2.20; Ala, 1.80; Val, 1.03; Phe, 0.98 His, l.c3; Arg, ; Asp, 3.90; Thr, 1.84; Ser, 0.92; Glu, 2.10; Gly, 1.38; Ala, 2.16; Val, 0.94; Phe, 1.02 Step 6 His, 0.94; Arg, ; Asp, 3.21; Thr, 1.85; Ser, 0.95; Glu, 2.14; Gly, 1.30; Ala, 1.81; Val, 0.83; Phe, 1.08 Leucine aminopeptidase, 1 hr Glu, 1.0; amides, 0.9; Ala, 0.7; Gly, 0.3 Carboxypeptidase B, 30 min Arg, 1.0 Pepsin peptides Pe-1 Lencine aminopeptidase, Asp, 1.04; Glu, 2.03; Gly, 1.97; Ala, Glu, 1.0; amides, 0.7; Ala, min Carboxypeptidase A, 1.5 hre Asp, 0.75 Pe-2 Asp, 1.06; Thr, 0.98; Glu, ; Ala, ; Phe, Asp, ; Thr, 0.92; Glu, ; Ala, 0.05; Phe, 0.99 Asp, ; Thr, 0.08; Glu, 1.01; Phe, 0.90 Asp, ; Glu, 0.20; Phe, Asp, 0.09; Phe, Pe-3 His, 1.06; Arg, ; Asp, 2.01; Thr,0.99; Ser, 1.02; Glu, 1.07; Gly, 1.01; Ala, ; Val, 0.89 His, ; Arg, ; Asp, 1.94; Thr, 1.02; Ser, 1.02; Gl U, 0.20; Gly, ; Ala, ; Val, 0.80 His, ; Arg, ; Asp, 1.14; Thr, 0.99; Ser, 1.01; Gly, 1.07; Ala, 0.92; Val, 1.01 His, ; Arg, 0.88; Asp, 1.10; Thr, 0.93; Ser, 1.04; Gly, 1.04; Ala, 0.93; Val, 0.05 His, 0.85; Arg, 1.10; Asp, 1.08; Thr, ; Ser, ; Gly, 0.30; Ala, 0.96 Step 5 His, 0.30; Arg, l.co; Asp, 1.20; Thr, ; Ser, 0.93; Ala, 1.10 Step 6 Asp, 1.06; Thr, 0.80; Ser, 0.30; Ala, ; Arg, N.D.* Step 7 Asp, 1.10; Thr, 0.38; Ser, 0.33; Ala, ; Arg, N.D. Step 8 His, 0.15; Arg, 0.82; Asp, 0.60; Ser, 0.25; Thr, 0.30; Ala, Leucine aminopeptidase 10 min 2 hrs Asp, 0.4: Glu, 0.6 Asp, 1.5; Thr, 0.8; Ser, 0.8; Glu, ; Gly, 0.9; Ala, 0.5; Val, 1.0; His, Arg, N.D. Carboxypeptidases 13 and A Arg, 1.0; Ala, min 0.03 N HCl, 14 hrs, 107 Asp, 1.8; Glu, 0.7 Composition Q N.D., not determined. of the parent peptide. Aminopeptidase digestion of Peptide Pe-1 confirmed the NH&erminal Glu-Gln sequence and digestion with carboxypeptidase A released aspartate, clearly indicating this hexapeptide to be the NHz-terminal segment of Peptide T-l. The structure of Peptide Pe-2 was established by Edman degradation as Ala-Thr-Glu-Asp-Phe. After the third Edman degradation step the acidic electrophoretic mobility of the residual peptide indicated the presence of aspartic acid. Since repeated amino acid analysis of Peptide Pe-2 showed less than 1 eq of ammonia, the absence of amide nitrogen on glutamate was indicated. Peptide Pe-3 had arginine at the COOH-terminal position and therefore represents the COOH terminus of T-l. This decapeptide was taken through eight cycles of Edman degradation as shown in Table II. While the eighth step of the degradation indicated the disappearance of aspartic acid, the decrease was only 0.4 to 0.5 of a residue. Carboxypeptidases B and A and the dilute acid hydrolysis experiments, however, clearly indicated an Asp-Ala-z4rg sequence and aminopeptidase hydrolysis experiments showed the absence of amide groups in this peptide. Peptide T-2: Phe-Leu-Glu-Glu-His-Pro-Gly-Gly-Glu-Glu- Val-Leu-Arg (Table III)-This peptide was isolated by Dowex 50 or Dowex 1 chromatography of the tryptic digest (Fig. 1). The peptide was also found in a fraction of the Sephadex column eluate and was quantitatively recovered by chromatography of Fractions 8 and 9 (Fig. 2) on Dowex 50. of 0.2 pmole of T-2 indicated a Phe-Leu-Glu-Glu-His sequence. Papain digestion of 0.4 pmole of Peptide T-2 yielded arginine and three peptides which were separated by Dowex 50 chromatography (Fig. 5). The sum of amino acids of the isolated peptides and the free arginine accounted for the entire composition of T-2. Peptide Pa-2 is a tripeptide with phenylalanine as the NH, terminus and thus represents the 3 NHe-terminal residues of T-2. Carboxypeptidase A hydrolysis of Pa-2 con-

6 3372 Sequence of Tryptic Peptides from Cytochrome bg Vol. 243, No. 12 firmed the presence of a COOH-terminal glutamate. Three steps of of Peptide Pa-l gave a Glu-His-Pro- Gly sequence, and a l-hour aminopeptidase digestion released glutamic acid and 1 eq of proline, histidine, and glycine. The release of proline by leucine aminopeptidase preparations has also been observed by Groskopf et al. (16). Peptide Pa-3 is a pentapeptide and four steps formulated the & 8 Os60- T-l,Pe-1 lo I 8 p : cr, Q T T-l W- I T-l,Pe-2 T-l, Pe-3 OO FRACTION NUMBER FIG. 4. Chromatography of a peptic hydrolysate of Peptide T-l on a Dowex 50 column, 60 X 0.9 cm. Gradient elution was begun after 10 ml by allowing 400 ml of 8.5 M pyridine-acetate to flow into the mixing chamber containing 390 ml of starting buffer (0.2 M pyridine-acetate, ph 3.1). Fractions of 3 ml were collected at a flow rate of 25 ml per hour. Peptide Peptide T-2 Step 5 Papain peptides Pa-2 Carboxypeptidase A, 1.5 hrs Pa-l Leucine aminopeptidase, 1 hr Pa-3 Leucine aminopeptidase, 1 hr I I TABLE III Amino acid composition and sequence studies of Peptide T-2 Phe-Leu-Glu-Glu-His-Pro-Gly-G1y-Glu-Glu-Val-Leu-Arg I+---- Pa-2+1 -Pa-l Pa-3F 1 sequence as Gly-Glu-Glu-Val-Leu. Amino acid analysis of the leucine aminopeptidase digest showed that both glutamyl residues are not amidated. Since steps of Peptide T-2 overlapped Pa-2 and included the 2 NHz-terminal residues of Pa-l, the order of papain peptides is known and the sequence of Peptide T-2 is established. Peptide T-S: Thr-Phe-Ile-Ile4ly-Glu-Leu-H&Pro-Asp- Asp-Arg (Table IV)-This peptide was obtained by Dowex 1 fractionation of the tryptic digest (Fig. 3). Chromatography of the unresolved Dowex 50 fractions (Fig. 1) or Sephadex G-25 fractions (Fig. 2) on Dowex 1 each gave the expected yield of Peptide T-3. studies established the sequence of the terminal eight amino acids and a prolonged carboxypeptidase B and A digestion released only arginine. Partial acid hydrolysis of Peptide T-3 released 1.98 residues of aspartic acid and 0.99 residue of arginine, thus establishing the COOH-terminal as Asp-Asp-Arg. A complete aminopeptidase digest of Peptide T-3 yielded 2 aspartyl and 1 glutamyl residues, thus excluding the presence of amide groups. The single proline residue of this peptide was again quantitatively released by the amidopeptidase preparation. Peptide T-4: Glu-Leu-Ser-Lys-This neutral peptide (Glu, 1.0; Leu, 0.95; Ser, ; Lys, 0.97) was eluted from Dowex 1 or Dowex 50 columns. Three steps of the established the sequence Glu-Leu-Ser-Lys: : Lys, 0.93; Ser, ; Glu, 0.0; Leu, 0.99 : Lys, 0.74; Ser, ; Leu, 0.0 : Lys, ; Ser, 0.1 Composition His, ; Arg, ; Glu, 3.90; Pro, 0.90; Gly, 2.03; Val, ; Leu, 1.91; Phe, His, 1.03; Arg, 0.85; Glu, 4.20; Pro, 1.0; Gly, 2.00; Val, 1.01; Leu, 2.00; Phe, 0.1 His, 0.87; Arg, 0.87; Glu, 4.05; Pro, 1.0; Gly, 1.97; Val, 1.01; Leu, His, 0.92; Arg, 0.93; Glu, 3.20; Pro, 1.0; Gly, 2.00; Val, 1.15; Leu, 0.90 His, 0.82; Arg, 0.89; Glu, 2.28; Pro, 1.0; Gly, 2.00; Val, 0.89 His, 0.20; Arg, 0.90; Glu, 2.30; Pro, 1.0; Gly, 2.20; Val, ; Leu, Glu, ; Leu, ; Phe, Glu, 0.98; Leu, ; Phe, 0.0 Glu, 0.50 His, ; Glu, 0.95; Pro, 1.03; Gly, His, 0.90; Glu, 0.0; Pro, ; Gly, 0.98 His, 0.0; Pro, 1.03; Gly, Pro, 0.1; Gly, 1.0 Glu, 1.0; Pro, 1.0; Gly, 1.0; His, 1.2 Glu, 1.84; Gly, 1.01; Val, 1.02; Leu, 0.98 Glu, 2.00; Gly, 0.20; Val, ; Leu, Glu, 1.17; Gly, 0.2; Val, 0.94; Leu, Glu, 0.3; Val, ; Leu, Val, 0.2; Leu, Glu, 2.0; Gly, 1.0; Val, 1.0; Leu, 1.0

7 Issue of June 25, 1968 J. 0x01s and P. Xtrittmatter 3373 FIG. 5. Chromatography of a papain hy drolysate of T-2 on a Dowex 1 column, 60 X 0.9 cm. Elution was begun with a mixing chamber containing ph 9.4 buffer and a reservoir containing ph 6.3 buffer. At the fractions indicated by arrows on the elution diagram, the buffer in the reservoir was replaced by the buffer indicated. The flow rate was 30 ml per hour and 1.5-ml fractions were collected. r-- Ars JoO FRACTION NUMBER TABLE IV Amino acid composition and sequence studies of Peptide T-5 Thr-Phe-Ile-Ile-Gly-Glu-Leu-His-Pro-Asp-Asp-Arg Peptide Peptide T-3 His, 0.97; Arg, ; Asp, 2.00; Thr, 0.91; Glu, 1.01; Pro, 1.0; Gly, 0.94;Ile, 1.90; Leu, 0.99; Phe, His, 0.89; Arg, 0.99; Asp, 1.98; Thr, 0.0; Glu, 1.03; Pro, 1.0; Gly, 1.01; Ile, 1.90; Leu, ; Phe, His, 0.93; Arg, ; Asp, 2.06; Glu, ; Pro, 1.0; Gly, 1.05; Ile, 1.90; Leu, 1.04; Phe, 0.0 His, 0.95; Arg, 1.05; Asp, 2.04; Glu, 1.09; Pro, 1.0; Gly, 1.13; Ile, 1.09; Leu, His, 0.75; Arg, 0.95; Asp, 2.01; Glu, 1.08; Pro, 1.0; Gly, 1.07; He, 0.20; Leu, Step 5 His, 0.90; Arg, ; Asp, 1.98; Glu, 1.03; Pro, 1.0; Gly, 0.30; Leu, 1.06 Step 6 His, ; Arg, ; Asp, 1.94; Glu, 0.31; Pro, 1.0; Gly, 0.15; Leu, Step 7 His, 0.76; Arg, 0.86; Asp, 2.00; Glu, 0.30; Pro, 1.0; Leu, 0.40; Step 8 (yield, 67y0) His, 0.40; Arg, 0.92; Asp, 2.13: Glu, 0.30; Pro, 1.0; Leu, 0.40 Carboxypeptidases I3 and A 30 min Arg, hrs Arg, N HCl, 14 hrs, 107 Arg, 0.99; Asp, 1.98 Leucine aminopeptidase, 2 hrs His, 0.78; Arg, 1.0; Asp, 1.8; Thr, 0.95; Pro, 1.0; Gly, 1.0; Ile, 1.73; Leu, 1.1; Phe, 0.92 Leucine aminopeptidase hydrolysis gave glutamic acid as expected from the electrophoretic mobility of the peptide. Peplide T-5: His-Asn-Asn-Ser-Trp-Lys-This basic peptide was obtained by Dowex 50 column chromatography (Fig. 1A) and is the only peptide from the tryptic digest of apocytochrome that could not be found in a Dowex 1 column eluate. The composition of T-5 is as follows: Lys, 0.99; His, 1.03; Asp, 1.99; Ser, 1.01; Trp, 1 gave the following results (N.D., not determined) : : Lys, 0.87; His, 0.0; Asp, 2.13; Ser, ; Trp, N.D. : Lys, 0.70; Asp, 1.30; Ser, 0.98 : Lys, 0.65; Asp, 0.35; Ser, Although the yields of the stages were above 80% there was a gradual decrease in lysine content. Analysis of a 2-hour carboxypeptidase B and A digest showed 1 eq of lysine and tryptophan. Leucine aminopeptidase hydrolysis for 2 hours liberated His (1.O), Asn (2.0)) and Ser (0.9)) in agreement with the electrophoretic mobility of T-5, but no lysine or tryptophan. Peptide T-6: Val-Tyr-Asp-Leu-Thr-Lys-Three steps of on T-6 (Val, 0.98; Tyr, 1.05; Asp, 1.OO; Leu, Composition 1.02; Thr, 0.91; Lys, ) were as follows. : Val, 0.0; Tyr, ; Asp, 1.02; Leu, ; Thr, ; Lys, : Tyr, 0.0; Asp, ; Leu, 0.95; Thr, 0.91; Lys, : Asp, 0.25; Leu, 0.95; Thr,. Carboxypeptidase B digestion indicated the sequence to be Leu-Thr-Lys (30 min, 40 -Lys, 0.7; Thr, 0.6; Leu, 0.4; 2 hours, 40 -Lys, 1.0; Thr, 0.97; Leu, 0.93; Asp, 0.50). A complete leucine aminopeptidase digest of the peptide showed that it contained aspartic acid and not asparagine (2 hours, 40, moles per mole of peptide-lys, N.D.; Asp, 1.01; Thr, 1.0; Val, 0.9; Leu, 1.0; Tyr, 1.1). Peptide T-? : Tyr-Tyr-Thr-Leu-(Glu,Gln)-Glu-Ile-Lys (Table I/ )-This acidic peptide was isolated from tryptic digests of cytochrome by Dowex 1 chromatography (Figs. 1B and 3). Six steps of established the amino-terminal sequence of this nonapeptide, and carboxypeptidase B and A hydrolysis results positioned the remaining three amino acids in the sequence Glu-Be-Lys. Analysis of a complete leucine aminopeptidase digest showed 1 eq of glutamine. Peptide T-8: Ala-Val-Lys; (T-9, T-10) : Ser-Lys-These were the only peptides from the tryptic digest that Dowex 1 chromatography initially failed to resolve completely. Analysis of the amino acid composition of effluent Fractions 60 to 70 (Fig. 1B) and Fractions 56 to 64 (Fig. 3) revealed a mixture of two

8 3374 Sequence of Tryptic Peptides from Cytochrome bg Vol. 243, No. 12 TABLE Amino acid composition and sequence studies of Peptide T-7 Tyr-Tyr-Thr-Leu-(Glu, Gln-Glu-Ile-Lys V Peptide Composition Peptide T-7 Lys, ; Thr, 0.93; Glu, 3.04; Ile, 0.99; Leu, 1.03; Tyr, 2.00 Lys, 1.10; Thr, 0.96; G111, 2.98; Ile, 0.99; Lell, 0.97; Tyr, 0.95 Lys, N.D.a; Thr, 0.95; Glu, 2.98: Ile, 0.94; Tyr, 0.0 Lys, N.D.; Thr, 0.0; Glu, 3.00; Ile, 0.99; Lerl, 1.02 Lys, N.D.; Glu, 3.06; Ile, 0.95; Leu, 0.0 Step 5 Lys, N.D.; Glu, 2.20; Ile, Step 6 Lys, 0.75; Glu, 1.30; Ile, Carboxypeptidases B and A, Lys, 1.0; Ile, 0.95; Glu, hr Leucine aminopeptidase, 2 hrs Lys, 1.0; Thr, 0.9; Glu, 1.95; Ile, 0.90; Leu, 1.13; Tyr, / 2.00; amides, 0.9 Q N.D., not determined. lysine-containing peptides. Fractions on the ascending edge of this peak contained serine and lysine, and the descending edge contained predominant.ly alanine, valine, and lysine. Examination of the entire peak composition revealed 2 eq of serine per eq of alanine and valine, to give serine-lysine and alanine-valinelysine ratios of 2: 1, or 2 eq of Ser-Lys per mole of apocytochrome. of the mixture clearly indicated serine and alanine as the NH&erminals, as one step of the peptide mixture (Lys, 1.25; Ser, 0.35; Ala, ; Val, ) gave the following results: Lys, 1.20; Ser, 0.0; Ala, 0.11; Val,. Since tryptic digestion specificity requires lysine to be positioned at the COOH-terminal, the Ser-Lys and Ala-Val- Lys sequences are thus established in accord with the previously determined cytochrome b5 NH&erminal Ser-Lys-Ala sequence (3). Peptide T-11: Ile-Thr-Lys-ProSer-Glu-Xer-This pepide was isolated from the tryptic digest by Dowex 1 and Dowex 50 chromatography (Figs. 1 and 3). The composition of T-11 (Lys, 0.96; Thr, ; Ser, 1.84; Glu, 1.02; Pro, 1.0; Ile, ) was identical with the previously reported cytochrome bg COOHterminal heptapeptide. Since its structure has been established, no sequence studies mere performed (2). DISCUSSION The primary objective of this study was to provide an essential part of the information required for an unambiguous determination of the primary structure of cytochrome bg. These data and the additional data on the chymotryptic peptides presented in the following paper (4) are utilized to deduce the primary structure of this heme protein. These experiments also provide the experimental design for recognizing the amino acid sequence variations in the minor heme protein components of cytochrome bs preparations (2) and in the phylogenetic spectrum of this heme protein. Since the quantity of pure heme protein components from calf liver and from various species is limited (2)) the development of micro procedures was essential. Thus, although 2.5 pmoles of cytochrome bb were available only 1.3 pmoles of the protein were sufficient to establish the sequence of the tryptic peptides. As seen in Table I, the sum of the amino acid compositions of the isolated tryptic peptides is in good agreement with the previously reported (2) a.mino acid composition of the predominant cytochrome bg species. The uncorrected minimal yields of the peak fraction given in Table I indicate a good peptide recovery. As expected from peptide elution patterns of Dowex 1 columns (8), the basic peptides emerge first followed by the neutral and finally by the acidic peptides (Figs. 3 and 5). An exception was the basic, tryptophan-containing Peptide T-5, which was irreversibly adsorbed by the resin. Considerable retention of Peptide T-5 was also observed on the Dowex 50 column (Fig. 1A). The proposed amino acid sequence of the tryptic peptides is primarily based on the data of substractive. The commonly observed cyclization to pyrrolidine-carboxylic acid of the amino-terminal asparagine and glutamine residues (5, 16, 17) was not encountered in this sequence study. Edman degradation continued past the 2 asparagine residues in Peptide T-5; similarly, in Peptides T-l and T-7, containing glutamylglutaminyl sequences, a termination of was not observed. It is noteworthy that the procedure served to detect an error in the amino acid composition of a peptide. Analysis of the residual T-3 peptide, after the third degradation stage, indicated 95y0 recovery but a loss of only 0.1 residue of isoleucine. Further s, however, proceeded normally. This observation led to analysis of samples hydrolyzed for 12- to 72-hour periods. The result was an increase in isoleucine from 1.1 residues in the 22-hour hydrolysate to 1.95 residues in the 72-hour hydrolysate. A repeated of Peptide T-3, with residual peptides hydrolyzed for 72 hours, confirmed the presence of an Ile-Ile sequence. The unexpectedly broad specificity of the leucine aminopeptidase preparation provided a simple procedure for the assignment of amide groups. As shown in Table III and in the sequence data of Peptide T-3, aminopeptidase quantitatively hydrolyzed Pro-Gly- and Pro-Asp bonds. A similar observation has also been made by Groskopf et al. (16). This enzyme, however, failed to hydrolyze the Trp-Lys bond in Peptide T-5. Failure of prolonged carboxypeptidase B and A digestion to release the aspartic residue from Peptide T-3 resulted in an initial erroneous placement of this amino acid (1). A check of the Peptide T-3 sequence by the dilute acid hydrolysis method of Tsung and Frankel-Conrat (6) indicated the COOHterminal sequence of T-3 to be Pro-Asp-Asp-Arg instead of the previousiy reported Asp-Asp-Pro-Arg (1). The ease of quantitative isolation of all of the tryptic peptides from cytochrome b5 now permits a more detailed correlation of

9 Issue of June 25, 1968 J. 0x01s and P. Xtrittmatte~ 3375 the effects of modified amino acid residues on the protein struc- 6. TSUNG, C. M., AND FRAENKEL-CONRAT, H., Biochemistry, 4, ture and function. Thus it will be possible, for example, to 793 (1965). 7. MOORE, S., AND STEIN, W. H., J. Biol. Chem., 192, 663 (1951). identify the imidazole residue which is most reactive with 8. SCHROEDER, W. A., JONES, R. T., CORMICK, J., AND MCCALLA, diazotized sulfonic acid and essential for heme binding (18, 19) K., Anal. Chem., 34, 1570 (1962). and to detect the lysyl residues in the heme peptide which are 9. MARGOLIASH, E., AND SMITH, E. L., Nature, 192, 1121 (1961). trinitrophenylated most readily (3). 10. SCHROEDER, W. A., AND ROBBERSON, B., Anal. Chem., 37, 1583 (1965). Acknowledgment-The expert technical assistance of Mrs. 11. RUDLOFF, V., AND BRAUNITZER, G.; Z. Physiol. Chem., 323, Janice Van Buren is gratefully acknowledged. 129 (1961). 12. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem., REFERENCES 30, 1190 (1958). 1. OZOLS. J., AND STRITTMATTER, P., Proc. Nat. Acad. Sci. U. S. A., 13. SMITH, I., ivatu$e, 171, 43 (1953). 68, 264 (1967). 14. EDMAN, P., Acta Chem. Scud, 7, 700 (1953). 2. STRITTMATTER, P., AND OZOLS, J., J. Biol. Chem., 241, KONIGSBERG, W., AND HILL, R. J., J. BioZ. Chem., 237, 2547 (1966). (1962). 3. OZOLS, J., AND STRITTMATTER, P., J. Biol. Chem., 241, GROSKOPF, W. R., HOLLEMAN, J. W., MARGOLIASH, E., AND (1966). KLOTZ, I., Biochemistry, 6, 3783 (1966). 4. OZOLS, J., AND STRITTMATTER, P., J. Biol. Chem., 243, HILL, R. L., Advance. Protein Chem., 20, 37 (1965). (1968). 18. STRITTMATTER, P., J. BioZ. Chem., 235, 2492 (1960). 5. LIU, T. Y., STEIN, W. H., MOORE, S., AND ELLIOTT, S. D., J. 19. OZOLS, J., AND STRITTMATTER, P., J. BioZ. Chem., 239, 1018 Biol. Chem., 240, 1143 (1965). (1964).

10 The Amino Acid Sequence of the Tryptic Peptides from Cytochrome b 5 Juris Ozols and Philipp Strittmatter J. Biol. Chem. 1968, 243: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at