Amino-Acid Sequence of Feather Keratin from Fowl

Eur. J Biochem. 132, 501 -507 (1983) ( FEBS 1983

Amino-Acid Sequence of Feather Keratin from Fowl Kunio Murayama ARAI, Rieko TAKAHASHI, Yoshiko YOKOTE, and Kiso AKAHANE Department of Chemistry, Faculty of Science, Josai University (Received August 24/December 6, 1982)

-

EJB 5931

The amino acid sequence of a single polypeptide chain, B-4, from fowl feather barbs has been determined. The B-4 chain was found to consist of 96 amino acid residues and to have a molecular weight of 10206 in the S-carboxymethylated form. The N terminus of this protein was an N-acetylserine residue. The B-4 protein contained seven S-carboxymethylcysteine residues, six of which are located in the N-terminal region (residues 1 - 26), and other one in C terminus. The central region of the peptide chain was rich in hydrophobic residues. There were homologous amino acids at 66 positions in the sequences of the feather keratins of fowl, emu and silver gull. The variation (substitution, deletion and insertion) in sequence was found to be localized in both terminal sections of the polypeptide chain. The B-4 protein structure was predicted to contain fi-sheet (about 30 %), turn and random-coil-like structure, and no a-helix. P-Sheet structure is mostly located in the central region (residues 22-70). On the other hand, both terminal regions are almost devoid of secondary structure. Feather proteins are solubilized in the presence of denaturant by cleavage of disulfide cross-linkages of cystine residues with oxidant or reductant [l - 31. Although soluble feather keratins from various birds have been reported to be relatively homogeneous with respect to molecular weight (about 10000) [4,S], they are at the same time heterogeneous by electrophoresis and chromatography and contain slightly different components [6,7]. It was found that an extract of emu feather gave the simplest electrophoretic pattern and that from silver gull was the next simplest. Thus single polypeptide chains were isolated from feather of these two avian species [8]. The present authors have reported on isolation and characteristics of soluble proteins from fowl feather barbs and calamus [9-111. From these studies it has been found that the components of each feather keratin are almost identical in amino acid composition except for cystine and/or amide content. O’Donnell [12] and O’Donnell and Inglis [13] determined amino acid sequences of the proteins from emu and silver gull feather calami, respectively. It was found that a central section, about 60-residues long, of the polypeptide chain contained predominantly hydrophobic residues and was free of S-carboxymethylcysteine, while both terminal sections were rich in S-carboxymethylcysteine. Fraser et al. estimated from infrared spectra data that about 30 % of the polypeptide chain in seagull feather rachis had an antiparallel pleated sheet conformation [14]. The ultimate purposes of the present study were to elucidate (a) the whole structure of feather keratin and (b) the relation between the feather keratin sequence and the evolutionary developments in Aves. In this paper, the amino acid sequence of the main component of fowl feather barbs is reported. The sequence is compared with those of emu and silver gull feather calami.

MATERIALS A N D METHODS Preparation o j a Single Polypeptide Chain from Fowl Feather Barbs

A single polypetide chain, peak 4 protein, was isolated from S-carboxymethylated proteins of fowl feather barbs, as described in the previous paper [9]. In this paper, peak 4 is newly termed B-4, of which the letter B represents barbs. Materials

Trypsin (treated with diphenylcarbamyl chloride), achymotrypsin, pepstatin A and dansyl chloride were all obtained from Sigma Chemical Co. (St Louis, MO, USA); carboxypeptidase Y from Oriental Yeast Co. (Tokyo, Japan). Acid carboxypeptidase of Penicillium janthinellum was a gift from Prof. Ichishima (Tokyo Noko University, Japan). An acylamino-acid-releasing enzyme was purified from pig liver as described by Tsunasawa et al. [15]. Sephadex G-50 fine was purchased from Pharmacia (Uppsala, Sweden); DEAEcellulose (DE 52) and high-performance liquid chromatography (HPLC) column Partisil 10 ODs-3, from Whatman (Springfield, GB); HPLC packing media Nucleosil 5C, from Macherey-Nagel (Diiren, FRG) ; anion-exhange resin AG 1 x 2 minus 400 mesh from Bio-Rad (Richmond, CA, USA). All reagents employed for the Edman degradation were purchased from Wako Pure Chemicals (Tokyo, Japan). Polyamide layer sheets were obtained from Cheng Chin Trading (Han Kow, Taiwan). Tryptic and Chymotryptic Digestions of Peptide Chains

Trypsin and chymotrypsin were employed for the degradation of the single polypeptide chain, B-4, and the latter Abbreviations. Dansyl, 1-dimethylaminonaphthalene-5-sulphonyl; enzyme was also employed for the subsequent degradation of HPLC, high-performance liquid chromatography. Enzymes. Trypsin (EC 3.4.21.4); a-chymotrypsin (EC 3.4.21 . l ) ; car- the larger fragments from the tryptic hydrolysis. The substrate boxypeptidase Y (EC 3.4.16.1); Penicillium junthinellum acid carboxypep- (B-4, SO mg) was dissolved in 5 ml of 0.1 M (NH,)HCO, (pH 8.0). Digestion by trypsin was carried out at 30 ” C for 3 h tidase (EC 3.4.16.1); acylamino-acid-releasing enzyme (EC 3.4.19.1).

502

and by chymotrypsin at 37°C for 6 h with an enzyme/substrate ratio of 1 : 100 (w/w). Peptide Separation Gel Filtration. Tryptic peptides from B-4 were applied to a column of Sephadex G-50 fine equilibrated with 0.25 M (NH,)HCO, (see Supplement Fig. 1). Anion-Exchange Chromatography. The fraction (TI and T3) corresponding to pool A in Supplement Fig. 1 was purified on a column of DEAE-cellulose equilibrated with 0.05 M (NH,)HCO, (see Supplement Fig.2). On the other hand, chymotryptic peptides derived from B-4 were loaded on an anion-exchange resin AG 1 x 2 column equilibrated with 1 pyridine/l % collidine/acetic acid buffer at pH 8.3 (see Supplement Fig. 7). High-Performance Liquid Chromatography. The fractions (T2, T4 and T5) corresponding to pool B in Supplement Fig. 1 were adjusted to p H 3.0 with phosphoric acid and loaded on an HPLC column of Partisil I0 ODs-3 (see Supplement Fig. 3). Two peptides, TI and T3, indicated by bars in Supplement Fig.2 were subdigested by chromotrypsin and then each of them was applied to HPLC (see Supplement Fig.4 and 5 , respectively).

Sequence Analysis Edman Degradation. Sequence analyses were carried out according to the dansyl-Edman method [16]. The 3-phenyl-2thiohydantoin derivatives of the amino acid residues, including those of amides, were identified by HPLC employing a Shimadzu LC-3A pump and Altex 153 ultraviolet detector as described by Zimmerman et al. [17], except that the elution was stepwise. The reverse-phase packing material used was Nucleosil 5C,, and the column was operated in a stepwise mode with two eluants, the first of which contained 0.01 M sodium acetate/phosphoric acid buffer (pH 4.5) containing 23 % acetonitrile and the second the same buffer containing 47% acetonitrile. The change of eluant was made at the step between the phenylthiohydantoins of glycine and alanine. The dansyl derivatives of the amino acids were identified by thin-layer chromatography on a polyamide sheet ( 5 x 5 cm) [18]. Determination of C-Terminal Sequence with Carboxypeptidase. Carboxypeptidase Y was used to determine the C-terminal sequence for the intact protein B-4 and fragment TICI, and acid carboxypeptidase from P. janthinellum for B-4 and fragment T2. Appropriate quantity of sample (B-4, 7 mg, 0.7 pmol) was dissolved in 7.0 ml of 0.05 M phosphate buffer at pH 5.5, and 0.7 nmol of carboxypeptidase Y and 20 nmol of pepstatin A were added at zero time. The digestion was carried out at 30°C. Aliquots of 1.0 ml were removed at intervals and the reaction was stopped by heat treatment in boiling water. The digests were centrifuged to remove insoluble material. The supernatants were evaporated to dryness in vacuo, dissolved in 0.2 M citrate buffer (pH 2.2) and applied to a Jeol JLC-5AH amino acid analyzer. In the case of acid carboxypeptidase, 0.1 M acetate buffer (pH 3.7) was used [19]. Identification of N-Terminal Blocking Group

The method of Schmer and Kreil [20] was applied to B-4 and to the N-terminal peptide TIC1 with blocked cc-amino group.

Amino Acid Analysis

Amino acid analyses were carried out with a Jeol JLC-5AH amino acid analyzer. Peptide samples were hydrolyzed with 6 M HCI containing 3 ”/, thioglycolic acid in vucuo at 110‘ C for 24 h. Peptide Nomenclature

Fragments obtained from tryptic and chymotryptic digests are designated by letters T and C, respectively, and numbered according to the position in the complete sequence, counting from the N terminus. In the case of peptides derived from chymotryptic subdigestion of tryptic fragments, a system of nomenclature as seen in the following example was used. T3C2 represents the second chymotryptic peptide in the tryptic fragment number 3. A symbol of plus for numbers indicates an overlapping peptide in which one or more cleavage sites remained intact, e.g. T3C(3 +4).

RESULTS Fig. 1 shows the amino acid sequence of fowl feather protein B-4. Tryptic digestion gave five fragments, TI, T2, T3, T4 and T5, two of which, T I and T3, were sequenced after subdigestion with chymotrypsin. Overlapping peptides were obtained from the chymotryptic digest of intact B-4. Characterization of’Intact Protein B-4 Amino Acid Composition. Table 1 summarizes the amino acid composition of B-4. It is noteworthy that protein B-4 is rich in serine and proline residues, and lacks tryptophan, lysine, histidine and methionine residues. B-4 has five arginine residues susceptible to trypsin digestion. Molecular Weight. The molecular weight of fowl feather keratin had been estimated to be in the range of 10000- 11000 [4]. In this work, the B-4 chain was found to contains 96 amino acid residues and to have a molecular weight of 10206 in the S-carboxymethylated form. N-Terminal Blocking Group. Component B-4 from fowl feather barbs resisted dansylation and Edman degradation. This suggests that the protein has a blocked N terminus. When B-4 was treated with hydrazine and dansyl chloride, the dansyl derivative of acetylhydrazine was identified by thin-layer chromatography. From these results, it was concluded that the N terminus of B-4 was acetylated. C-Terminal Residue. The data from carboxypeptidase Y (Supplement Fig. 8) indicated that the C-terminal sequence of B-4 was -(Arg,Tyr)-(Pro,Pro,Cys). The exact sequence was obtained from Edman degradation of fragment T5 (see later). B-4 had the C-terminal sequence of -Tyr-Pro-Pro-Cys. Tryptic Fragmen fs

Tryptic digestion of B-4 gave five fragments. Fragments T1 and T3 (24 and 58 residues, respectively) were isolated by gel filtration of Sephadex G-50 (pool A in Supplement Fig. 1) and DEAE-cellulose chromatography (Supplement Fig. 2). Fragments T2, T4 and T5 (6, 4 and 4 residues, respectively) were isolated by gel filtration (pool B in Supplement Fig. 1) and HPLC on a reverse-phase column (Supplement Fig. 3). The amino acid compositions of these five fragments are given in Supplement Table 1.

503 c2

r I

rT l C l I

c3

1 1

TlC(2+3) T1C2

t

I - - I T 1C3

T1

va 1 Leu Phe

va 1

-T2

T3

-I

Pro V a l V a l V a l T h r Leu P r o Gly P r o I l e L e u P r o V a l V a l V a l T h r L e u P r o Gly P r o I l e L e u T h r V a l V a l V a l T h r L e u P r o Gly Pro I l e L e

-,

Sex S e r Phe S e r Ser P h e

ser

u i S e r Phe

C(8+9)

I c7 I I C8

T3C(3+4)

~

T3 C 3

T3C(5+6)

- 11

T3C4

-1 I

I

' -I

'

I

-

T3C5 1

T3

T3

1

7

T

4

7

I T5 I

P - _ -----A Fig. I . Amino acid sequence ofprotein B-4 from,fowl feather barbs arid comparison with those of emu and silver gullfeather calami [12,13] andprediction of secorzdarystructure offowlandenmfeatherproteins. Peptides obtained after enzymatic digestion are indicated by lines and the appropriate peptide symbols. The numbering is that of the fowl feather sequence and gaps have been left where necessary to give maximum homology. The homologous amino acids in the three sequences are enclosed with frames and homologous S-carboxymethylcysteine with double frames. S-Carboxymethylcysteine residues are represented as Cys. On prediction of secondary structure, residues are represented in their respective conformational state: P-sheet (zig-zag line), turn (stepped line) and coil (straight line)

504 Table 1. Amino acid composition of protein B-4from fowl feather hurhs The values from amino acid analysis (average of duplicate 24-h hydrolyses) are based on 1.0 for tyrosine Amount by

Amino acid

- -- - - -

__

amino acid analysis

0 0 0 4.5 4.8"

TrP LYs His Arg Asp Asn Thr Ser Glu Gln Pro GlY

-

4.7 27.2 7.4b -

11.1 11.3 4.7 6.7 7.4 0 4.6 7.0 1.o 3.6

Ala

Cys' Val Met Ile Leu TYr Phe

-

96.0

Total ~~~~

~~~~~

a

Valuc is for aspartic acid and asparagine. Value is for glutamic acid and glutamine. Determined as S-carboxymethylcysteine.

- _ _ - - -sequence analysis 0

0 0 5 2 3 4 16 2 5 12 11

4 7 9 0 5 6 I 4 ~

96

Pro-Leu. TIC2 and T l C a are clearly homologous and two substitutions by serine have occurred in T I C a at the first and fourth residues from the N terminus. It was also found that Pro-21 in T I C 3 had been replaced by alanine in TICP and by valine in T I Cy, respectively. T 2 (residues 25-30). This fragment was also resistant to the Edman degradation. This suggests that the N terminus of T2 may be a glutamine residue, which is converted into pyrrolidone carboxylic acid during the purification procedures. The partial sequence of four residues -Gln-Asp-Ser-Arg, along the T2 chain from the C terminus was determined by digestion with acid carboxypeptidase (Supplement Fig, 9). The sequence of the two remaining residues were obtained from the data of chymotryptic fragment C3 of B-4 (see later). It was concluded that T2 had a sequence of Gln-Cys-Gln-Asp-Ser-Arg. T3 (residues 31-88). On elution from the DEAEcellulosse column, this fragment appeared near the void volume (Supplement Fig.2). Fragment T3 was free from S-carboxymethylcysteine residues and had a very small number of charged residues, i.e. Val-31 (N terminus), Glu-72 and Arg-88 (C terminus). The first 23 residues in T3 were directly sequenced by the dansyl-Edman method. Further information for the sequence of T3 was obtained from the data of chymotryptic subdigestion of T3. Ten peptides were purified by HPLC from chymotryptic subdigests of T3 (Supplement Fig. 5 and 6 and Supplement Table 3). Fragments T3C1 (residues 31 - 40), T3C3 (48-61) and T3C5 (69-75) were found to have Cterminal residues of Val, Ser and Ile, respectively. These results indicated that chyniotrypsin cleaved not only phenylalanyl and leucyl bonds but VaI4'-Thr4', Ser6'-Ala6' and Ile75-Ser7h bonds. In particular, the cleavage of Val-Thr bond was complete; T3C1 was isolated in good yield, whereas an overlapping peptide between T3C1 and T3C2 was never obtained. The Jle-Ser bond was only cleaved to a small extent. The tripeptide chain (residues 86 - 88), corresponding to T3C8, was not isolated from chymotryptic digest. However, the sequence data of T3C(7+8), which was obtained in small amounts, filled this section. T4 (residues 89-92), This fragment was a tetrapeptide which had phenylalanine as the N-terminal residue and arginine as the C-terminal residue. TS (residues 93- 96). This was a tetrapeptide which had the sequence Tyr-Pro-Pro-Cys. This result was consistent with the data from intact B-4 by carboxypeptidase Y digestion, and elucidated the C-terminal sequence of B-4.

TI (residues I - 24). This fragment was resistant to Edman degradation. Therefore, it was cleaved into smaller fragments, TIC1, TlC2, TIC3 and TlC(2 + 3), by chymotryptic digestion. These enzymatic peptides were purified by HPLC (Supplement Fig. 4 and Supplement Table 2). The N-blocked terminal fragment, T l C l (residues 1 - 3), was a tripeptide consisting of phenylalanine, serine and S-carboxymethylcysteine. Phenylalanine was expected to be the C terminus, from the specificity of the enzyme. Confirmation was obtained by digesting T l C l with carboxypeptidase Y. The penultimate residue, however, was not liberated. T l C l was further digested with acylamino-acid-releasing enzyme from pig liver. The N-terminal residue of the resulting peptide was found to be S-carboxymethylcysteine by the dansyl method. From these Chymotryptic Fragments results it was concluded that TIC1 had an N-terminal sequence C3 (residues IS-30). This fragment provided the conof AcSer-Cys-Phe. TIC2 (residues 4- 14) contained a partial sequence of -Arg7-Pro8-, which was insusceptible to tryp- nection between fragments T1C3 and T2. In addition, this sin. Fragment T1C3 (residues 15-24) was a decapeptide with sequence proved that the N-terminal residue of T2 was arginine as the C terminus. Fragment T1 C(2 + 3 ) was a larger glutamine. It was unexpected that this peptide had an arginine peptide which overlapped fragments T1 C2 and T1 C3. The sum residue as the C terminus. C I I (residues 86-89) and C12 (residues 90-96). The of the number of amino acid residues for the three chymotryptic fragments (TlC1, T1C2 and TlC3) accounted for the amino sequences of fragments C11 and C12 provided overlaps acid composition of the parent fragment TI. These data between T3C(7+8) and T 4 and between T4 and T5, allowed the arrangement of parent fragment TI to be assigned. respectively. The striking characteristic of the TI fragment is the high content of S-carboxymethylcysteme residues, that is, 5/24 Alignment of Fragments of total number of residues in TI and 5/7 of total number of The five tryptic fragments, T1 -T5, covered the entire S-carboxymethylcysteine in B-4. Another remarkable feature of the TI fragment is the variable locus. As shown in sequence of B-4 from fowl feather barbs. The sum of the Supplement Fig.4, elution from a Partisil 10 ODS-3 column number of amino acid residues of the five tryptic fragments gave three unexpected peaks, TICa, T l C j and TICy. T l C a accounts for the amino acid composition of B-4, as shown in Table 1. The chymotryptic fragments C3, C11 and C12 were has the sequence of Ser-Leu-Cys-Ser-Pro-Cys-Gly-Pro-Thr-

505

available for arrangement of the tryptic fragments. Although overlapping peptides in four positions, Phe3-Asp4, Arg”Leu6’ -Ser69 and Pheso-Glysl, have not been obtained, the peptides were placed in order by comparison with the emu [I21 and silver gull [13] feather keratins.

Table 2. Hydrophobicity q f vurious secrions of B-4from fowl feather burbs The relative hydrophobicity of uncharged amino acids as defined by Segrest and Feldman [21] is used. Values for aspartic acid and glutamic acid were assumed to be equal to those for their amides, -1.5 and - 1.0, respectively. The value for arginine was assumed to be 1.5, based on a relative value to alanine compiled by Bigelow and Channon [22]

DISCUSSION

Section (position in sequence)

Hydrophobicity index

1-21 1-30 9-22 22 - 70 31 88 38-52 71-96 89 - 96

0.69 0.55 0.36 1.16 1.32 1.93 1.35 1.69

Peptide fragments from the B-4 molecule were separated by a combination of gel filtration, ion-exchange chromatography and HPLC. Because the central portion of B-4 was rich in hydrophobic residues, a reverse-phase column, separating peptide fragments according to hydrophobicity, was found to be more effective than high-voltage paper electrophoresis based on differences in charge. The sequences from three avian species showed that there were homologous amino acids at 66 posittions (homology of 69%). Comparison in dual sets are as follows: fowl/emu, 79 positions (82 %); emu/silver gull, 73 positions (74 %); silver gull/fowl, 74 positions (78 %). It is noteworthy that homology (82 %) between fowl barbs and emu calamus is higher than that (74%) between emu calamus and silver gull calamus. This suggests that the difference between tissues in feather is smaller than that between species in Aves. Fowl feather keratin, B-4, contains seven S-carboxymethylcysteine residues. Six of those are included in the N-terminal region and are conserved in all the three avian species. This constancy suggests that the position of cysteine in the N-terminal region is important for formation of structure of feather. On the other hand, cysteine residue in the C-terminal region are variable as shown in Fig. 1. In addition, it has been found in silver gull feather that there is a certain component in which deletion of the C-terminal end group, S-carboxymethylcysteine, occurs [13]. Amino acid compositions of several keratin components have been obtained from three species and those in each species have been found to give very similar compositions, apart from cysteine and/or amide contents [9,12,13]. These data suggest that the amino acid sequences are almost constant for various components from feather keratins, except for the distribution of cysteine residues in the C-terminal region. When a number of feather components other than B-4 are sequenced, further information on the distribution of cysteine residues will be obtained. A native feather keratin which is not modified chemically is poor in charged amino acids. The section comprising residues Va131-Gln70 contains no charged amino acids. The hydrophobicity indices [21] of various sections obtained from B-4 are given in Table 2. It is clear that hydrophobicity in the central region and the C-terminal region of the chain is higher than that in the N-terminal region. There is a marked difference in hydrophobicity between N-terminal and C-terminal regions though both are rich in cysteine and poor in p-structure (see Fig. 1). Prediction of the secondary structure was attempted by methods proposed by Lewis et al. [23] and Chou and Fasman 124, 251 (Fig. 1); the P-sheet contents of fowl and emu feather keratin were estimated to be about 30%. These results agree very well with the value obtained for seagull feather rachis from infrared study by Fraser et al. [14]. In the sequence shown in Fig. 1, the P-structurre-rich region of fowl keratin starts from position 22 and continues up to around position 70. The P-sheet content of this region was about 43 %. On the other hand, both terminal regions (residues 1 - 21 and 71 - 96) are almost devoid of secondary structure (P-sheet content of about 10 %). In the central region, variation (substitution, deletion and insertion)

+

~

(N-terminal region) (TI T2) (common sequence) (central region) (T3) (common sequence) (C-terminal region) (T4 TS)

+

+

of sequence is less (1 1 %), while in both terminal regions more is found (38 %). The characteristic, that a variable region in amino acid sequence is localized in a part of molecule, has been observed in C-peptide of proinsulin [26], fibrinopeptide of fibrinogen [27] and activation peptide of pepsinogen [28]. Since these variable regions are unnecessary after activation, the terminal regions may not be essential for the structure of feather keratin. I t is noteworthy that P-sheet and turn occur alternately in the central region. Units of four amino acid residues of b-sheet and turn repeat regularly three times in the region of Cys2’Pro4’. The section C y . ~ ~ ‘ - - S eisr ~favorable ~ for forming a normal p-turn [29] and it is expected that the sections CysZ2Gln2’ and Arg30-Ile33 constitute an antiparallel pleated sheet. On the other hand, the section of Va13*-Thr41 is unfavorable for constituting a pleated sheet with the above two sections, because the G l n ” - P r ~ ~section, ~ containing a sequence of the Pro-Xaa-Pro type, cannot form normal pturn. The same turns occur in three sections of feather keratin, positions 11 - 13, 35 - 37 and 43 - 45. Since a turn of this type restricts strictly the direction of the peptide backbone, it must play a significant role in the formation of keratin conformation, particularly in the assembly of a pleated sheet structure. It is obvious that situations of pleated sheet structure and cysteine cross-linkage, whether intrachain or interchain, are essential to the whole structure of feather keratin. The existence of homologous peptides in the N-terminal region was indicated in this paper. Since, in the present study, a mixture of feather from several individuals was used, it is ambiguous whether the existence of homologous peptides is attributable to variation in individuals or to microheterogeneity in various components from an individual. The sequence analysis of feather keratin from one fowl should give the answer to this question and is in progress. We wish to thank Prof. Haruhiko Noda (The Univerersity of ElectroCommunications) and Prof. Sumiko Narise (Josai University) for helpful and critical discussions and for the preparation of the manuscript. We also thank Prof. Eiji Ichichima (Tokyo Noko University) for a generous gift of acid carboxypeptidase from P . junthinellum.

REFERENCES 1. Goddard, D. R. & Michaelis, L. (1934) J. Biol. &hem. 106, 605-614. 2. Jones, C.B. & Mecham, D.K. (1944) Arch. Biochem. Biophys. 3, 193-202. 3. Woodin, A . M . (1954) Biochem. J . 57, 99-109.

506 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18.

Harrap, B.S. &Woods, E . F . (1964) Biochem. J . 92, 19-26. Jeffrey, P. D. (1970) Aust. J . Biol. Sci. 23, 809-819. Harrap, B. S. & Woods, E. F. (1964) Biochem. J . 92, 8- 18. Woods, E.F. (1971) Comp. Biochem. Physiol. 39A, 325-331. O'Donnell, I . J. (1973) Aust. J . B i d . Sci. 26, 401-413. Murayama, K., Akahane, K. & Murozono, S. (1977) J . Biochem. (Tokyo) 81, 19-24. Akahane, K., Murozono, S. & Murayama, K. (1977) J . Biocheni. (Tokyo) 81, 11-18. Muzorono, S . , Murayama, K. & Akahane, K. (1977) J. Biochcm. (Tokyo) 82, 53-58. O'Donnell, I.J. (1973) Aust. 1. Biol. Sci. 26, 415-437. O'Donnell, I. J. & Inglis, A. S. (1974) Aust. J . Biol. Sci. 27, 369-382. Fraser, R. D. B., MacRae, T. P., Parry, D. A. D. & Suzuki, E. (1971) Polymer, 12, 35 - 56. Tsunasawa, S., Narita, K. &Ogata, K. (1975) J . Biochem. ( T o k y o ) 77, 89- 102. Gray, W. R. & Smith, J . F. (1970) Anal. Biochem. 33, 36-42. Zimmerman, C. L., Appella, E. & Pisano, J. J . (1977) Anal. Biochem. 77, 569-573. Woods, K . R. & Wang, K. T. (1967) Biochim. Biophys. Actu, 133, 369- 370.

19. Yokoyama, S., Oobayashi, A,, Tanabe, 0. & Ichishima, E. (1975) Biochim. Biophys. Acta, 397, 443 - 448. 20. Schmer, G. & Kreil, G. (1969) Anal. Biochem. 29, 186- 192. 21. Segrest, J. P. & Feldmann, R. J. (1974) J. M o l . Biol. 87, 853- 858. 22. Bigelow, C. C. & Channon, M. (1976) Handbook of Biochemistry and Moleculur Biology, 3rd edn, vol. 1, pp. 209, CRC Press. Cleveland, OH. 23. Lewis, P. N., Momany, F. A. & Scheraga, H.A. (1971) Proc. Nut1 Acud. Sci. USA, 68, 2293 - 2297. 24. Chou, P. Y. & Fasman, G. D. (1974) Biochemi,rtry, 13, 21 1 - 221. 25. Chou, P . Y . & Fasman, G. D. (1974) Biochemistry, 13, 222-245. 26. Dayhoff, M. 0. (1976) Atlas of Protein Sequence and Structure, vol. 5 , Supplement 2, pp. 127, National Biomedical Research Foundation, Silver Spring, MD. 27. Dayhoff, M. 0. (1972) Atlas of Protein Sequence and Structure, vol. 5, pp. D-92, National Biomedical Research Foundation, Silver Spring, MD. 28. Kageyama, T. & Takahashi, K. (1980) J . Biochem. (Tokyo) 88, 571 -582. 29. Dickerson, R. E., Takano, T., Eisenberg, D., Kalloi, O., Sumson, L., Cooper, A. & Margoliash, E. (1971) J. Biol. Chem. 246,151 1 - 1533.

K. M. Arai, R. Takahashi, Y. Yokote, and K. Akdhdlle, Department of Chemistry, Faculty of Science, Josai University, Sakado, Saitama, Japan 350-02

Supplemental Material to AMINO ACID SEQUENCE OF FEATHER KERATIN FROM FOWL K. M. Arai, R. Takahashi, Y. Yokote and K. Akahane

RESULTS

T1.3

Tlme (min)

..

.

of acet&itrile at a flow rate Of O.Sml/min. was monitored at 230nm.

Elution of pepcides

Froction Nmber Supplement Fig. 1. Gel filtration of tryptic diaest Of f o w l feather keratin 8 - 4 on Sephader G-50 fine. The columnl2.5r85cm) w a s eluted with 0.25M (NHd)HCO, (oH 8.0) at flow rate of 35ml/h. Fractions of 3ml were collected and measurement was made at 230nm. The bars indicate pooled fractions.

>

Supplement Fig. 4 . HPLC of ch otr tic di est of fra ment TI on Partieil 10 005-3. legend to Supplement Fig. 3 except for a linear gradient with 1-propanol. I

I

F m c t l o n Number

Supplement Fig. 2. Chromatoqraphy of fraqments Tl and T3(pool A in S~pplementFiq. 1) on D E A E - c ~ ~ ~ u ~ oThe s ~ column(DE . 52, l.5x20cm)

was equilibrated with 0.05M lNHn)HC07 (oH 8 . 2 ) and eluted with a linea; Salt gradient of 0 - 0.4; KCldin-the same solution at a flow rate of 20ml/h.

Fractions Of 4ml were collected and measurement The arrow indicates the Start Of the l i n e a r gradent and the bars indicate pooled fractions.

w a s made at 230nm.

Time (mi111 Supplement fig. 5. HPLC of chymotryptic diqest of fraqment T3 on Partisil 1 0 ODs-3. Conditions were the same a s described in the legend to Supplement Fig. 3 . The fractions A and B indicated by bars were rechromatographed in Supplement Fig. 6.

Supplement Table 1. Amino acid compositions of tryptic peptides of feather barbs B-4. Relative molar quantities are given f o r each peptide and the integral values in parentheses. are based on the sequence.

Gln Pro Gly Ala

3.914) 1.211) 1.311)

- 12)

- 13) 5.916) 9.4i9) 3.1131

2.2(2)

4.3(51 3.914)

0.9111

1.512)

0.9(1)

58

4

1le

Leu TYI Phe

1.8i2) 1.0i1)

0.911) ~~

Number of residues

Time (rninl

24

6

4

a) Values are for aspartic acid and asparagine.

bl Values are for glutamic acid and glutamine. c ) Determined as S-carboxymethylcysteine.

(b): fraction B.

e

Supplement Table 2 . Amino acid com~ositionsof chymotryptic subfraqments of T1. Relative molar quantitles are given for each peptide and the integral values in parentheses are based on the sequence.

h

:0,8 u

Amino acid

0

$0.4

TlCl

TlCZ

TIC3

T1Cm

TlC6

TlCI

n

0

300

200

100 FmCtIOn

Number

buffer at pH'8.3 and eluted with'iinear gradient of the same b u f f e r (400ml) and 0.5M acetic acid(400ml). The limiting solution was replaced successively by 2M. 5M and 17Mlglacial) acetic acid at the posotions indicated by arrows. Elution of peptides was monitored by ninhydrin reaction.

Thr Ser Glu PTO GlY Ala Cysb Val Leu P he

0.8(11 1.0i1) 3.3i3) 0.711) 1.8i2)

11

0.9(1) 1.7(2)

-

12)

1.011) 1.211)

12)

1.011) 1.0(1)

3.4i31 0.911)

1.1(1) 2.112) 1.111)

l.9(2)

Number of residues

-

- 12) 1.0i1) 1.111) 1.111)

1.712) 2.1i2) 1.1(1)

1.8(2) 2.0(2)

10

11

10

10

a1 Values are for aspartic acid and asparagine. b) Determined a5 S-carboxymethylcysteine.

Supplement Table 3. Amino acid compositions of chwotryptic subfraqments o f T3. Relative molar quantitles are given for each peptide and the integral values in parentheses are based on the sequence. Amino acld

T3C1

T3CZ

T3C3

T3C4

1.2a

T3C(3+4) 1.2a - I11

- 11) Time l h )

?

Supplement Fig. 8. Rate of release of amino acids from intact B-4 by carboxypeptidase Y. The reaction was carried out in 0.05M phosphate buffellpH 5.5) at 30'C with an enzyme to substrate molar zatio of 1:lOOO~ The reaction mixture contained pepstatin A in excess of 30-fold molar ratio to enzyme. 0 : proline, a: carboxymethy1cysteine.A: tyrosine, A:arginlne.

.

Gln PTO

Glv Ala Val Ile Leu Phe Number of residues

Amino acid

- (1) 2.212)

1.8(2) 1.211)

1.1(1) 3.9(51

- (1) 1.311) 1.8i1) l.Zi1) 1.2i1)

1.011) 1.9121

1.211) 1.712) 1.1(1) 1.0(1) 1.0111

. .

~

1.211) lo

T3C5

7

T3CG

- (1) 1;5(1) 2.4121 23.313) .2(2) 1.2(1) 1.2111 i.oiii

14

7

21

T3C(5t6)

T3C7

T3C(7+8)

Arq

0.8(1)

ASP ASn

Thr

ser

1.2(1)

1.812)

Glu

1.5b(l) - 11) 1.0(1) 1.8(1)

2.112)

Gln Pro

PtY

Supplement Fig. 5. Rate of release of amino acids from fragment TZ by acid carboxypeptidase from Penxillium ianthmellum. The reaction was carried out in 0.1M acetate bufferlDH 3.7) at 30°C with an enzyme to substrate molar ratio o f 1:lOO. 0 : arginine, a: serine, A: aspartic acid. A: glutamine. ~~

~~

Number of reaianps

5

3.213) i.9b(ii - 11) 1.1i1) 3.113)

0.911)

3.012)

2.1i2)

3.113)

12

5

8

a) Values are for aspartic acid and asparagine.

b) Values are for glutamic acid and glutamine.