Tux JouRNaL or Brotoeica, CHEeMisTRY Vol. 242, No. 20, Issue of October 25, pp. 4752-4758, 1967 Printed in U.S.A. The Amino Acid Sequence of an Extracellular Nuclease of S taphylococcus aureus III. COMPLETE AMINO ACID SEQUENCE (Received for publication, May 3, 1967) Hrrosut Tanrucui,* Curistian B. ANFINSEN, AND ANN SopJa From the Laboratory of Chemical Biology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014 SUMMARY The amino acid sequence of an extracellular nuclease of Staphylococcus aureus, strain V8, is presented. The calcu- lated molecular weight of the nuclease is 16,807. In a previous paper data were presented that permitted the linear arrangement of five fragments produced by cleavage of an extracellular nuclease of Staphylococcus aureus with cyanogen bromide (1). The amino acid sequences of the tryptic peptides prepared from these fragments, together with the partial se- quences of chymotryptic peptides isolated from digests of the intact nuclease, have also been presented (2). In the present communication, the tryptic peptides derived from each cyanogen bromide fragment are arranged in linear order on the basis of the amino acid sequences of the tryptic and chymotryptic cleavage products. In order to obtain supple- mental information necessary for final alignment, certain tryptic fragments of trifluoroacetylated nuclease were separated and examined. The molecular weight calculated from the deduced sequence is consistent with estimates obtained from physico- chemical examination (3) and from x-ray diffraction studies (4). EXPERIMENTAL PROCEDURE The procedures employed have been described in the pre- ceding reports (1, 2), unless otherwise specified. Tryptic Digestion of Trifluoroacetylated Nuclease—The tri- fluoroacetylation of the nuclease was carried out by the method of Goldberger and Anfinsen (5). Approximately 1 umole of the protein was used for each preparation. After trifluoroacetyla- tion, the reaction mixture (5 ml) was dialyzed against 3 liters of 0.1 M acetic acid for 48 hours with five changes, and lyophilized. The trifluoroacetylated nuclease was digested with trypsin at 37° for 3 hours, and the resulting material was treated with * Visiting Scientist. piperidine as described elsewhere (5). The mixture, containing large tryptic fragments, was lyophilized. Approximately 92% of the lysine residues of the nuclease had been trifluoroacetylated as judged by resistance to deamination with NaNO, (6) (Table I) Separation of the fragments was performed by column chromatography either on carboxymethyl Sephadex C-50 (Pharmacia), 1 x 43 cm, or on phosphocellulose P 11 (Whatman Chromedia), 7.4 meq per g, 1 x 14cm (7). Separation by two- dimensional peptide mapping on Whatman No. 3 MM paper was utilized for some of the peptides as described previously (1). The amino acid compositions and NH,-terminal groups of trifluoroacetylnuclease fragments obtained in pure form are presented in Table II. Thermolysin Dige.tion—Purified thermolysin was kindly do- nated by Dr. T. H. Jukes (8, 9). The reaction mixture, con- taining 6 mg of a given peptide, 0.1 mg of thermolysin, and 1 mg of soybean trypsin inhibitor (Worthington) in 1 ml of 2.5 x 10~? m NHiHCO;-2 x 10-4 m CaCh, pH 8, was incubated for 2 hours at, 37°. RESULTS Linear Arrangement of Tryptic Fragments—Designations of peptides and their amino acid sequences, and assignments of tryptic peptides to particular cyanogen bromide fragments, are described in preceding reports (1, 2) unless otherwise specified. , Cyanogen Bromide Fragment A—The relation of peptides as- signed to Fragment A and the deduced amino acid sequence of Fragment A are illustrated in Fig. 1. Peptide T-V-1, containing homoserine, is placed at the COOH terminus. Since the NH:- terminal residue of Fragment A is alanine (1), either Peptide T-V-8a or T-V-2, both of which contain NH,-terminal alanine, should constitute the NH.-terminal sequence of Fragment A. Chymotryptic Peptide C-9c includes Peptide T-V-2, as judged by its partial sequence. It contains the NH:-terminal sequence Ile-Lys, and leucine as the COOH-terminal residue. Upon dilute acid hydrolysis of Peptide C-9c, aspartic acid and glycine 1 Preliminary results of the amino acid analysis of the tryptic fragments of trifluoroacetylated nuclease were consistent with trifluoroacetylation of almost all lysine residues of the nuclease. 4752 Issue of October 25, 1967 were liberated in a molar ratio of 2:1, indicating a sequence (Asp)—Gly-(Asp),? a sequence found in Peptide T-V-2 (2). Thus Peptide T-V-8a must form the NH, terminus of Fragment A. Peptide T-18a, which includes Peptide T-V-7b and contains the 2The abbreviations used are: (Glu) and (Asp), Glu or Gln and Asp or Asn, respectively; TFA-nuclease, trifluoroacetylated nuclease. TaBLe | Deamination of «-amino groups of lysine residues in nuclease before and after trifluoroacetylation The amounts of protein used for deamination (see ‘““Experimen- tal Procedure’’) were 0.5 to lmg. After incubation, the mixtures (0.5 ml) were dialyzed against 2 liters of 0.1 m acetic acid at 4° for 24 hours. The dialysates were lyophilized. The dried samples were subjected to acid hydrolysis and amino acid analysis. Rep- resentative residue numbers, calculated on the basis of 3 phenyl- alanine residues per molecule, are presented. Other amino acids were found in proportions essentially the same as in hydrolysates of native nuclease (1). As a control, trifluoroacetylated nuclease was subjected to hydrolysis without treatment with NaNO:. Samples not Samples treated with NaNOz treated with NaNO2 Ami id . : mmo ach Native Triduoroacetyt: Trifuoroacetyl- wmole lresidues| pmole | residues | pmole residues Lysine.............- 0.012 | 0.48 | 0.199 | 18.3 | 0.400 | 19.6 Histidine........... 0.080 | 3.2 | 0.086 | 2.9 | 0.059 | 2.9 Arginine*........... 0.066 | 2.6 | 0.028! 2.4 | 0.112| 5.5 Methionine*........ 0.068 | 2.7 | 0.085 | 3.2 | 0.083) 4.1 Tyrosine*........... 0.006 | 0.25 | 0.007 | 0.56 | 0.147 | 7.2 Phenylalanine...... 0.076 | @) | 0.036 (3) | 0.061 | (3) H. Taniuchi, C. B. Anfinsen, and A. Sodja 4 Arginine, methionine, and tyrosine are partially destroyed by NaNO. 4753 Taste II Amino acid composition of tryptic peptides obtained from trifluoroacetylated nuclease Peptides obtained by chromatography on either carboxymethyl Sephadex or phosphocellulose are designated CM and P, respec- tively. The peptides obtained by the two-dimensional peptide mapping (see ‘Experimental Procedure’’) are indicated as M. Other details are given in the text. Amino-terminal groups were determined by the dinitrophenylation procedure (1). When identical fragments were obtained by more than one separation method, only one analysis is presented. Amino acid TFA-M7 TFA-P2 TFA-M1 TFA-CM11 Lysine.............- 0.015(1) | 0.039(1) | 0.005(1) | 0.052(4) Histidine........... 0.005(1) Arginine............ 0.016(1) 0.005(1) Aspartic acid....... 0.020(1) | 0.015(0) | 0.014(2) | 0.040(4) Threonine.......... 0.012(1) 0.008 (1) Serine.............. 0.010(0) 0.024(2) Glutamic acid...... 0.043(1) | 0.015(2) | 0.071(5-6) Proline............. (1)*} 0.004(0) Glycine............. 0.024(1) | 0.029(1) 0.006 (1) Alanine............. 0.057 (2) 0.032(3) Valine.............. 0.081(1) | 0.005(1) Igsoleucine.......... 0.010(1) Leucine............ 0.037(1) | 0.015(2) | 0.013(1) Tyrosine. .......... 0.014(1) | 0.025(1) | 0.006(1) NH.-terminal resi- due............5.- Thr Glu Val Lys Assigned to cyano- gen bromide frag- ment............. D E E E « Partial destruction of NH.-terminal residue by ninhydrin staining. » Qualitative determination. LEU-HIS-LYS-((GLU), PRO, ALA)- THR -(LEU, ILE)-LYS T-184 ALA- THR- SER-THR -LYS T-V-8a T-V-7b 1 10 GLU -PRO-ALA - THR-(LEU, ILE)-LY5 ALA-ILE - ASP -GLY- ASP - THR -VAL-LYS{LEU-HOMOSER T-V-2 T-V-1 20 26 [aLa- THR - SER-THR -LYS -LYS-LEU-HIS-LYS- GLU -PRO-ALA -THR- LEU-ILE -LYS- ALA-ILE - ASP -GLY- ASP -THR -VAL-LYS- LEU -MET| ALA- THR-(SER, THR)-LYS, C-6b C-15a ALA-(THR, SER, THR -LYS)-LYS-LEU C-15¢ HIS -LYS-((GLU). PRO, ALA)- THR- LEU ILE -LYS- (ALA, ILE) - ((ASP)- GLY-- (ASP), THR)- VAL -LYS-LEU ja Cem C-9¢ Fra. 1. Diagram of the sequence of cyanogen bromide Fragment A. The deduced sequence is shown in the enclosed middle line. Tryp- tic and chymotryptic fragments are shown above and below the deduced sequence, respectively. The vertical arrows above and below the sequence indicate the bonds cleaved by trypsin and chymotrypsin, respectively. 4754 NH,-terminal sequence Leu-His-Lys, may be arranged in rela- tion to Peptide T-V-8a by consideration of the structure of chymotryptic Peptide C-15c. Chymotryptic Peptide C-6b was identical with tryptic Peptide T-V-8a as judged by the amino acid sequences (2). The reason for the unusual cleavage pro- duced during chymotryptic digestion is unknown. Chymotryptic Peptide C-15a lacks the COOH terminus of tryptic Peptide T-18a, —Ile-Lys, which is the NH_-terminal part of Peptide C-9c as described above. Thus Peptide T-V-7b is connected to Peptide T-V-2, and the COOH-terminal sequence of Peptide T-V-7b is deduced to be -Leu-Ile~Lys. The COOH- terminal leucine residue of Peptide C-9c is assigned as the NH, terminus of Peptide T-V-1. In Fig. 1, the tryptic fragment (Lys, Leu, His, Lys) is indi- cated as missing. Although adding only tentative evidence, the rather small chromatographic fraction, T-V-18, of the tryptic digest of cyanogen bromide Fragment A (see Reference 1) had the qualitative amino acid composition (Lys: His, Leu), and contained NH.-terminal lysine upon dinitrophenylation. we A B c- THR-PHE - ARG T-V-15a LEU -HOMOSER T-V-1 LEU-(MET, TYR)-LYS|GLY-(PRO,GLN, MET, THR, PHE)- ARG FS Fg 27 32 TYR -LYS-GLY- PRO-GLN-MET MET-TYR |(LYS, GLY, PRO, GLN)-MET C-7e C-9d Fia. 2. Diagram of the sequence of cyanogen bromide Frag- ment B (see legend to Fig. 1). A, B, and C represent cyanogen bromide Fragments A, B, and C. Sequence of Staphylococcal Nuclease. lit Vol. 242, No. 20 Cyanogen Bromide Fragment B—The partial sequence, Tyr- Lys-Gly ((Glu), Pro) homoserine, of Fragment B was deduced by the arrangement of tryptic Peptides F15 and F18, both of which contain a methionine residue (1) (Fig. 2). The NH,-terminal residue was shown to be tyrosine by dinitrophenylation (1). Edman degradation indicated the NH,-terminal sequence to be Tyr-Lys-Gly. Carboxypeptidase A digestion (24 hours, 18% yield) released only homoserine. The electrophoretic mobility of Fragment B at pH 6.5 was consistent with amidation of the glutamic acid residue. The liberation of homoserine by carboxypeptidase A indicated the presence of penultimate glu- tamic acid or glutamine on the basis of the specificity of car- boxypeptidase A, which would not hydrolyze a COOH-terminal bend involving proline (10). Chymotryptic Peptide C-9d was assigned to Fragment B on the basis of its amino acid composition, and this supported (2) the amidation of glutamic acid and the location of the proline residue (Fig. 2). Another chymotryptic peptide, C-7e, is com- posed of the carboxyl- and amino-terminal residues of Fragments A and B, respectively (Fig. 2). Cyanogen Bromide Fragment C—Tryptic peptides assigned to this fragment (1, 2) were arranged in the linear order shown in Fig. 3, as follows. Since the NH,-terminal residue of this frag- ment was threonine (1), Peptide T-V-15a occupies the NHb- terminal position (1). Peptide T-V-10, containing homoserine, forms the COOH terminus of Fragment C. Tryptic fragments of chymotryptic Peptides C-18a and C-20a had amino acid com- positions identical with those of most of the tryptic peptides as- signed to Fragment C (1, 2). These fragments could be ar- ranged in order as follows. Peptide C-20a had the same amino acid composition as Peptide C-18a, except that glycine, proline, glutamic acid, 2 alanine residues, serine, and phenylalanine were absent from Peptide C-20a. Since a tryptic fragment (C-18a- TIV) of Peptide C-18a, containing these amino acids in addition to tyrosine, lacked lysine, Fragment C-18a-TIV may be placed at the COOH terminus of Peptide C-18a. Fragment C-18a-TI (C-20a-TI), which lacked the NH,-terminal leucine residues of Peptide T-V-5b, is the NH, terminus of Peptide C-18a (C-20a). Fragment C-18a-TII (C-20a-TII) was connected to Fragment {TYR ,GLY,PRO, GLU ,ALA)-(SER, ALA}-PHE - THR-LYS F21 THR- PHE-ARG|LEU-LEU-LEU-VAL- ASP -THR-PRO- GLN - THR -LYS| HIS-PRO-LYS | LYS-GLY-VAL- GLU -LYS| TYR -GLY-PRO- GLU -ALA - SER-ALA -PHE [THR-LYS-LYS-HOMOSER T-¥-150 T-V-5b F2 T-¥-13 TV-6 T-¥-10 33 40 50 60 65 [THR-PHE- ARG-LEU-LEU-LEU-VAL- ASP -THR-PRO- GLN -THR -LYS- HIS-PRO-LYS - LYS-GLY-VAL- GLU -LYS- TYR -GLY-PRO- GLU -ALA - SER-ALA -PHE THR- PHEJARG-LEU-LEUKLEU, VAL, (ASP), THR, PRO, (GLU), THR)-LYS, (HIS, PRO, LYS), (LYS,GLY,VAL,(GLU) ,LYS, TYR)|GLY-PRO- GLU -ALA - SER-ALA C-4 C-19¢ C-200 LEU -VAL-((ASP), THR, PRO, (GLU), THR)-LYS, (HIS, PRO,LYS),(LYS,GLY, VAL ,(GLU)}-LYS,(TYR , GLY, PRO. (GLU), ALA , SER, ALA , PHE) C-18e C-18a-TI C.184-TH - THR -LYS-LYS- MET | ~PHE b-——-C.22 C-2 C-180-T I! C-180-TIV (€-200-Tl) (C-20a-T Il} C-180-TV (C-200-T IH) Fig. 3. Diagram of the sequence of cyanogen bromide Fragment C (see legend to Fig. 1) ccoly VAL-ASN-GLU- ALA-LEU- VAL-ARG THR -(ASP)-(LYS - TYR, GLY)- ARG|(GLY ,LEU,ALA)-TYR-(ILE, TYR, ALA, ASP, GLY)-LYS T-ViN-100 F 24 VAL- GLU - ASN -ALA-LYS|LYS- ILE- GLU -VAL-(GLU, PHE)}- ASN -LYS] GLY. GLN - ARG] THR- ASP - LYS |] TYR-GLY - ARG] GLY -LEU-ALA -TYR]| ILE-TYR- ALA- ASP-GLY -LYS JHOMOSER T-Vi-3b T-Vil-Sa T-VIN-3c 66 70 T-VIL-8e T-Vi-8b 80 T-VIL-2¢ T-Vil-lla 90 98 [vat - GLU - ASN -ALA-LYS-LYS- ILE- GLU -VAL- GLU -PHE - ASN -LYS- GLY- GLN “ARG. THR- ASP - LYS -TYR-GLY - ARG. GLY -LEU-ALA -TYR- ILE-TYR- ALA -ASP-GLY -LYS -MET| (ASP)-LYS- (GLY , (GLU))- ARG -(THR, (ASP)- LYS)-TYRIGLY -(ARG, GLY)-LEUIALA - TYR C-170 C-15e (ALA, ASP,GLY ,LYS)-MET C59 C-Sd (THR, LYS)-LYS- (MET , VAL , (GLU))- (ASP)- ALA-LYS-LYS-(ILE, (GLU), VAL, (GLU) , PHE)- (ASP) - LYS-(GLY , (GLU)) -ARG- THR-(ASP)- LYS -TYRIGLY -(ARG, GLY ,LEU,ALA)-TYR C-10b ~—C-22-TI 100 C-22 C-22-TIV C-22-TIb C-22-Titl C-19e Fic. 4. Diagram of the sequence of cyanogen bromide fragment D (see legend to Fig. 1). Subfragments of chymotryptic peptide C-22 are indicated by horizontal arrows. T-ll-6 TFA-CMI1 TFA-P2 T-IU-7b 110 GLN-GLY -LEU-ALA-LYS] VAL-ALA - TYR T-llt-4e TFA-M1 VAL- TYR-LYS-PRO-ASN- ASN- THR - HIS-GLU-GLN-LEU-LEU- ARG T-H-2 120 T-IN-Ge T-U-8 130 LYS -GLU-LYS| LYS-SER-GLU-ALA-GLN-ALA-LYS|LEU-ASN-ILE-TRP-SER-GLU-{ASP)- ASP-(ALA ,(ASP))-SER- GLY -GLN T-U-2 140 149 [VAL -ASN-GLU-ALA-LEU- VAL-ARG - GLN-GLY -LEU-ALA-LYS- VAL-ALA -TYR- VAL-TYR-LYS-PRO-ASN- ASN- THR - HIS-GLU-GLN-LEU-LEU-ARG-LY$-GLU-LYS-LYS-SER-GLU-ALA-GLN-ALA-LYS-LEU-ASN-ILE-TRP-SER-GLU- ASN - ASP- ALA - ASP -SER- GLY -GLN —C-10 (VAL, ARG)-(GLN, GLY)-LEU| ALA-LYS-(VAL, ALA), TYR C-15b Cl4c C-6a VAL- TYRILYS-PRO-ASN- (ASN, THR)- (HIS, GLU, GLN)- LEU C140 SER-GLU- ASN -(ASP, ALA)- ASP -SER - (GLY ,GLN) C1 C-20b Fic. 5. Diagram of the sequence of cyanogen bromide Fragment E (see legend to Fig. 1). Peptide T-111-12 is described as T-I[I-12a in the text. 4756 ee Sequence of Staphylococcal Nuclease. TFA-CM11 If Vol. 242, No. 20 ———— TFA -I1- Thi A LYS-GLU-LYS-LYS-SER “GLU-ALA-GLN-ALA-LYS) aT - Il -9e T-UN-8 137 TFA-H- Th! 25-——_—_——___—> 140 EU-ASN-ILE-TRP-SER-GLU-ASN-ASP-ALA-ASP- SER -GLY -GLN B LYS-SER -GLU-ALA-GLN-ALA-LYS -LYS -GLU-LYS T-1-8 T-11I-9e T-U-2 Fig. 6. Diagram of the linear order of Peptides T-III-9e and T-III-8. The bonds cleaved by thermolysin are indicated by the vertica arrows. Other details are given in the text. Fragment TFA-II-Thl-26, which was obtained by the same procedure as that used to prepare Fragments TFA-II-Thl-5,6 and 25, appeared to be identical with Peptide T-III-2 on the basis of qualitative amino acid com- position and position on two-dimensional peptide maps. Tasue IIT Thermolysin fragments of Peptide TFA-CM11 The mixture of tryptic fragments obtained from TFA-nuclease was fractionated on Sephadex G-25. The fraction containing Peptide TFA-11 was digested with thermolysin for 2 hours. The fragments thus formed were separated by two-dimensional pep- tide mapping. An aliquot of the eluate obtained from each nin- hydrin-positive spot was analyzed for amino acid composition, and from these results Fragments TFA-JI-Thl5, Thl6, and Thl25 were shown to be derived from TFA-11. Amino acid TFA-II-Thl-5 TFA-II-Thl-6 TFA-II-ThI-25 Lysine................ 0.016 (4) 0.024(4) Aspartic acid......... 0.006(1) 0.068 (3) Serine................ 0.005(1) 0.005(1) 0.085(2) Glutamie acid.........| 0.014(3) 0.022(3) 0.050(2) Glyecine............... 0.005(0)° 0.004(0)® 0.029(1) Alanine............... 0.011(2) 0.013 (2) 0.026(1) Isoleucine............. 0.015(1)2 Leucine............... 0.006 (1) Tryptophan........... (ye * Partial destruction by ninhydrin staining. > This amount of glycine was often found as a contaminant in samples eluted from paper. ¢ Determined by the amino acid analysis of a digest by leucine aminopeptidase. C-18a-TI (C-20a-T]) by Fragment C-18a-TV (C-20a-TIII). Thus the corresponding Peptides T-V-5b, F2, T-V-13, and T- V-6 were placed in order and tyrosine was deduced to be the COOH terminus of Peptide C-20a, consistent with the specificity of chymotrypsin. Chymotryptic Peptide C-19f connected Peptides T-V-15a and T-V-5b. Tryptic Peptide F21 gave the connection between Peptides T-V-6 and T-V-10. Cyanogen Bromide Fragment D—Fig. 4 shows the relations of tryptic and chymotryptic peptides assigned to Fragment D. Digestion of chymotryptie Peptide C-22 with trypsin produced many of the originally isolated tryptic peptides. The amino acid analyses and end groups of these fragments of Peptide C-22 permitted the ordering of the tryptic peptides (2). Thus Pep- tides T-VII-3e, T-VII-8a, T-VII-8b, and T-VIT-2c (see Fig. 4) can be placed in order. The NH,-terminal part of Peptide C-22 (Thr, Lys, Lys, Met) was found in the COOH-terminal sequence of Fragment C (see Fig. 3), further confirming the juxtaposition of cyanogen bromide Fragments C and D. Another chymo- tryptic peptide, C-17a, included Peptides T-VII-8b and T-VII- 2c and had, in addition, the NH,-terminal sequence (Asp)—Lys (2). Since Peptide T-VII-8a is adjacent to Peptide T-VIJ-8b (as discussed above), the sequence (Asp)—Lys is the COOH terminus of Peptide T-VII-8a, consistent with the known se- quence —Asn-Lys (2). The undetermined part of the sequence of Peptide T-VII-8a (Glu, Phe) could be deduced, since the bond involving the amino group of asparagine should be susceptible to chymotrypsin. Thus phenylalanine, rather than glutamic acid, may be placed adjacent to the asparagine residue. Peptide T-VII-lla was connected to Peptide T-VII-2c by consideration of the COOH terminus of Peptide C-22. This arrangement was confirmed by an overlapping tryptic peptide, T-VI-10a. Chymotryptic Peptide C-19e (2) connected Pep- tide T-VII-1la to T-VII-3b, consistent with chymotryptic Pep- tides C-15e and C-5g. A tryptic peptide (TFA-M14),? identical with peptide T-VII-3b, was obtained from tryptic digest of TFA-nuclease, together with TFA-M7 (identical with Peptide T-VII-10a) (Table II). These findings are consistent with the indicated positions of the two arginine residues. Tryptic Peptide Fy, included Peptides T-VII-3b and T-VH-5a. Chy- motryptic Peptide C-5d lacked only isoleucine and tyrosine from the amino acid composition of Peptide T-VII-5a. A chymotryptic peptide corresponding to residues 92 and 93, Tle-Tyr (Fig. 4), has not been found. Cyanogen Bromide Fragment E—Fig. 5 summarizes the relationships of tryptic and chymotryptic peptides assigned to Fragment E. Since the COOH-terminal residue of Fragment E was glutamine, Peptide T-III-2 may be assigned to the COOH terminus (1, 2). Chymotryptic Peptide C-15b connected Peptide T-III-7b to Peptide T-III-6, which is assigned to the NH, terminus of Fragment E (1). Another chymotryptic peptide, C-10b, included the COOH terminus of cyanogen bromide Fragment D, Peptide T-III-6, and the NH, terminus of Peptide T-III-7b, further confirming this arrangement. Chymotryptic Peptide C-5e (2) included residues 94 to 103 (not shown in Fig. 5). Chymotryptie Peptide C-14c connected 3 Gly, 0.011; Ala, 0.018; Leu, 0.011; Tyr, 0.008. Issue of October 25, 1967 H. Taniuchi, C. B. Anfinsen, and A. Sodja 4757 * GOOOOGOOGOOOESEOOGOOS EXAMI OIELHOEMVAMNI MOOI SOSOSIOOSGHOOSOOSOSSON DR POMROQOQIVMM AISI ISICON GOOBOSOSIGEOSOSSOSSSOM HOQOUMOYOVIAIEH INEM IGE II OMITS FQGGI@OSOSSOSEOSSES—« Fig. 7. Proposed amino acid sequence of nuclease V-8 TasLe IV Amino acid composition of cyanogen bromide fragments of nuclease V8 calculated from sequences The amino acid compositions reported previously (1) are shown in parentheses. Amino acid A B c D E Sum Acid hydroly- Lysine... 00.0. 0c0ccecec ees eset ees 5(4-5) 1(1) 6 (5-6) 5(4-5) 6(5) 23 (21.4) Histidine. ...........0... 200-2000 -- 1(1) 0(0) 11) 0(0) 1q) 3 (3.0) Arginine .................0502 2s eee 0(0) 0(0) 1(1) 2(1-2) 2(2) 5 (4.8) Aspartic acid. ............-..5-005- 2 0 1 2 2 ~ 7 Asparagine...............06s00c2 ee i @) o (0) a (1) , G4) (6-7) i (14.6) Threonine... ............. 00000020 4 (3-4) 0(0) 4(3) 11) 1(1) 10 (9.6) Serine...0.. 0... eee 1(1) 0(0) 1(1) 0(0) 3 (2-3) 5 (5.1) Glutamic acid... 00.2.2... eee eee 1 0 2 3 5 ll Glutamine. ..........0...0.... 2000+ 0 (1) i (1) 4] 3) 1 4) ; ) ; (18.3) Proline. ........s0eeeceeveeeveeee- 101) 1(1) 3(3) 0(0) 1(1) 6 (5.1) Glyeine....0.00.0.00.0 0c cece eee 1(1) 1(1) 2(2) 4(4) 2(2) 10 (7.9) Alanine. ............ 0.002000. e ee eee 3 (2-3) 0(0) 2(2) 3(3) 6(6) 14 (14.4) Half-cystine............0...--2 025 0(0) 0(0) 0(0) 0(0) 0(0) 0 (0.0) Valine... 0... eee ees 1(1) 0(0) 2(2) 2(2) 4(3) 9 (9.2) Methionine.................2.205-- 1(1)« 1(1)¢ 1(1)4 1(1) 0(0) 4 (3.5) Isoleucine... 0... 0000.0 0. eee eee 2(2) 0(0) O(1) 2(1-2) 1(1) 5 (5.0) Leucine. ...... 0... eee 3(3) 0(0) 3 (2-3) 1(1) 5(5) 12 (11.6) Tyrosine... 00.02.06. e eee 0(0) 1(1) 1(1) 3(3) 2(2) 7 (6.6) Phenylalanine. ................-+-- 0(0) 0(0) 2(1-2) 1(1) 0(0) 3 (3.0) Tryptophan...................-0-5. 0(0) 0(0) 0(0) 0(0) 1(i) 1 (ye Total. oo. ee eee 26 6 33 33 51 149 Molecular weight................-- 16,807¢ * Determined as homoserine (1). > See Reference 1. ¢ On the basis of this molecular weight, the absorbance at 280 my of an aqueous solution containing 1.0 mg of nuclease V8 in 1.0 ml was 0.92 (see References 1 and 11). The concentration of protein was determined by amino acid analysis. Peptides T-III-7b and T-III-4c. The juxtaposition of Pep- tides T-IlJ-4c and T-III-12a was discussed in the preceding report (2). Chymotryptic Peptide C-6a should be the NEH.- terminal part of Peptide T-III-12a, compatible with the above arrangement from the standpoint of the specificity of chyvmo- trypsin. A tryptic fragment (TFA-P2) obtained from TFA- nuclease overlapped Peptides T-IJI-7b and T-IJI-4c. Another peptide, TFA-M1, was identical with Peptide T-III-12a. The latter peptide furnishes a second example of chymotryptic cleavage between Peptides C-14e and C-6a and further indicates 4758 the intrinsic chymotryptic activity of the trypsin preparation (2). Chymotryptic Peptide C-14a provided confirmation for the distribution of amide groups in Peptide T-IJ]-12a. A large chymotryptic peptide, C-20b, forms the COOH-terminal part of Fragment E, including the COOH terminus of Peptide T-IJT-12a, together with peptides T-ITI-9c, T-I11-8, and T-III-2. The composition of a tryptic fragment of Peptide C-20b was identical with that of Peptide T-III-2. Peptide TFA-CM11, obtained from TFA-nuclease, appears to overlap Peptides T-Il]-9c, T-III-8, and 'T-III-2 (Table II). Since Peptide T-ITI-2 should be the COOH terminus of Fragment E, only the order of Peptides T-ITI-9c and T-III-8 remained to be deter- mined. When Peptide TFA-CM11 was digested with thermo- lysin, which preferentially cleaves bonds involving the NH; groups of leucine and isoleucine (9), fragments that overlapped Peptides T-IT]-9c and T-III-8 were obtained (see Fig. 6 and Table III; Peptides TFA-II-Thl-5 and TFA-II-Thl-6). These fragments were subjected to combined digestion with carboxy- peptidases A and B. Lysine, alanine, and glutamine were released with the former peptide, and leucine and asparagine were liberated, in addition to the above amino acids, with the latter peptide. No glutamic acid was released in either incuba- tion. Peptide TFA-II-Thl-5 (5 hours): Lys, 0.008; Ala, 0.004. Peptide TFA-II-Thl-6 (8.5 hours): Lys, 0.011; Ser, Gln, and Asn (as serine), 0.010; Ala, 0.006; Leu, 0.008 (Thr, Glu, Gly < 0.003). These observations suggested the order of Peptides T-II]-9c and T-III-8 by the following reason. If Peptide T-III-9c is the NH, terminus of Peptide TFA-11, as illustrated in Sequence -4 in Fig. 6, the COOH termini of Fragments TFA-IT-Thl-5 and TFA-II-Thl-6 should be -Ala-Gln-Ala-Lys and —Ala-Gin- Ala-Lys-Leu-Asn, respectively. Carboxypeptidases A and B should release these amino acid residues, but not glutamic acid, at least at early stages of digestion. If Sequence B in Fig. 6 were the case, carboxypeptidase A and B digestion should release glutamic acid from either Fragment TFA-II-Thl-5 or TFA-II-Thl-6 before the liberation of alanine. Chymotryptic Peptide C-1 provided information on the distribution of amide groups, not available from studies on Peptide T-III-2. The comparison of the partial sequences of these two peptides permitted construction of the complete se- quence shown in Fig. 5. imino Acid Sequence of Nuclease—The deduction of the amino acid sequence of each cyanogen bromide fragment made it possible to place 149 amino acid residues in order. The Sequence of Staphylococcal Nuclease. II Vol. 242, No. 20 resulting sequence furnishes a working hypothesis for the covalent structure of the nuclease illustrated in Fig. 7. All major frag- ments obtained from tryptic and chymotryptic digests of the nuclease were found in this structure, as discussed above. The amino acid compositions of the nuclease and of the cyanogen bromide fragments that were reported previously (1) were found to be closely compatible with those calculated from the sequence (Table IV). The difference between the numbers of glycine residues calculated from the reconstructed sequence and from direct amino acid analysis of the nuclease (1) was somewhat large (Table IV). However, the glycine content was well defined in the tryptic peptides and the consistency between the tryptic and chymotryptic peptides that cover the entire sequence was satisfactory.4 Acknowledgments—We would like to acknowledge the ex- cellent assistance of Mr. Clifford Lee in the amino acid analyses. One of the authors (H. Taniuchi) wishes to express his appreci- ation to the China Medical Board, New York, Inc., for pro- viding a fellowship to begin this series of studies in 1963. His appreciation also goes to Kyoto University, Faculty of Medi- cine, for granting a leave of absence, and to Dr. Masana Ogata for his continuing encouragement. REFERENCES 1. Tanrucni, H., anp ANnFINsEN, C. B., J. Biol. Chem., 241, 4366 (1966). 2. Tantucnt, H., ANFINsEN, C. B., anp Sonya, A., J. Biol. Chem., 242, 4736 (1967). 3. Heins, J. N., Surtano, J. R., Tantucut, H., anp ANFINSEN, C. B., J. Biol. Chem., 242, 1016 (1967). 4. Cotron, F. A., Hazen, E. E., Jz., anp Ricuarpson, D. C., J. Biol. Chem., 241, 4389 (1966). 5. GOLDBERGER, R. F., anp ANFINSEN, C. B., Biochemisiry, 1, 401 (1962). 6. ANFINSEN, C. B., Seva, M., anp Cooxg, J. P., J. Biol. Chen., 237, 1825 (1962). 7. CanFIELD, R. E., AnD ANFINSEN, C. B., J. Biol. Chem., 288, 268 (1963). 8. Envo, 8., Hakko Kogaku Zasshi, 40, 346 (1962); Chem. Absir., 62, 5504 (1965). 9. Matsuspara, H., Stncer, A., Sasaki, R., anp Jukes, T. H., Biochem. Biophys. Res. Commun., 24, 242 (1965). 10. Neuratu, H., in P. D. Borer, H. Larpy, anp K. Myrpicx (Editors), The enzymes, Vol. 4, Academic Press, New York, 1960, p. 11. 11. Fucus, 8., Cuarrecasas, P., anp ANFINSEN, C. B., J. Biol. Chem., 242, 4768 (1967). * Amino acid analyses of 20-hour hydrolysates of several dif- ferent samples of the nuclease showed 10 moles of glycine per 3 moles of phenylalanine.