THE JOURNAL orB~o~.oamx~C~~xma~ Vo1.242. No. 20,Issue of Oct.&m 26,~~. 4762-4758.1967 Printed in U.S.A. The Amino Acid Sequence of an Extracellular Nuclease of Staphylococcus aureus III. COMPLETE AMINO ACID SEQUENCE (Received for publication, May 3, 1967) HIROSHI TANIUCHI,* CHRISTIAN B. ANFINSEN, AND ANN SODJA From the Laboratory of Chemical Biology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland i?OOlJ SUMMARY The amino acid sequence of an extracellular nuclease of Staphylococcus aureus, strain V8, is presented. The caku- 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 s-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 Trifbroacetylated NucEeaseThe tri- fluoroacetylation of the nuclease was carried out by the method of Goldberger and Anfinsen (5). Approsimately 1 pmole of the protein was used for each preparation. After trtiuoroacetyla- 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 wa.s 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 nucleate had been trifluoroacetylated as judged by resistrtnce to deamination with NaNO2 (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 14 cm (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 trifluoroacetylnucleae fragments obtained in pure form are presented in Table II. Thermolysin Diga thin-Purified thennolysin 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, tend 1 mg of soybean trypsin inhibitor (Worthington) in 1 ml of 2.5 X 10-* M NH.&EOa-2 x 10-' M CaCl*, pH 8, was incubated for 2 hours at 37". RESULTS Linear Arrangement of Typtic 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 Fragmeti 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-l, containing homoserine, is placed at the COOH terminus. Since the NH*- terminal residue of Fragment ,4 is alanine (l), either Peptide T-V-8a or T-V-2, both of which contain NHz-terminal alanine, should constitute the NHz-terminal sequence of Fragment 8. Chymotryptic Peptide C-9c includes Peptide T-V-2, as judged by its partial sequence. It contains the NHz-terminal sequence Ile-Lys, and leucine ss the COOH-terminal residue. Upon dilute acid hydrolysis of Peptide C-SC, aspartic acid and glycine 1 Preliminary results of the amino acid analysis of the tryptic fragments of trifluoroacetylated nucleate were consistent with trifluoroacetylation of almost all lysine residues of the nuclease. 4752 Issue of October 25, 1967 H. Taniuchi, C. B. An$nsen, and A. Sodja 4753 were liberated in a molar ratio of 2: 1, indicating a sequence TABLE II (Asp)-Gly-(Asp),2 a sequence found in PepGde T-V-2 (2). Thus Amino acid composition of tryplic peptides obtained from Peptide T-V-8a must form the NH* terminus of Fragment A. trifluoroacetylated nuclease Peptide T-lSa, which includes Peptide T-V-7b and contains the Peptides obtained by chromatography on either carboxymethyl * The abbreviations used are: (Glu) and (Asp), Glu or Gln Sephadex or phosphocellulose are designated CM and P, respec- and Asp or Asn, respectively; TFA-nuclease, trifluoroacetylated tively. The peptides obtained by the two-dimensional peptide nucleaee. mapping (see "Experimental Procedure") are indicated as M. Other details are given in the text. Amino-terminal groups were TABLE I det,ermined by the dinitrophenylation procedure (1). When Deamination of c-amino groups of lysine residues in m&ease before identical fragments were obtained by more than one separation method. onlv one analvsis is nresented. and ajfer trijluoroacetylation The amounts of protein used for deamination (see "Experimen- tal Procedure") were 0.5 to 1 mg. 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, calculat,ed on the baais of 3 phenyl- alanine residues per molecule, are presented. Other amino acids were found in proportions essentially the same as in hydrolysates of native nucleate (1). As a control, trifluoroacetylated nuclease was subjected to hydrolysis without treatment with NaN02. 1 Amino acid I -_ Lysine . Histidine Arginine. Aspartic acid. Threonine Serine............. Glutamic acid. Proline. Glvcine Samples treated with NaNOl Samples not treated with Alanine. Nan`02 Valine. Amino acid Isoleucine . Native Trikmcetyl- Trif?uoroacetyl- ated Leucine . Tyrosine . pmole residncs @de rcsiducs ~molc residues NH*-terminal resi- ------ due. . Lysine.............. 0.012 0.48 0.199 18.3 0.406 19.6 Assigned to cyano- Hi&dine. 0.080 3.2 0.036 2.9 0.059 2.9 gen bromide frag ArginineO. 0.066 2.6 0.023 2.4 0.112 5.5 merit............. :- TFA-M7 0.015(l) 0.016(l) 0.020(l) 0.012(l) 0.024(l) 0.014(l) Thr D - -- a - TFA-PZ 0.039(l) 0.015(O) 0.010(0) 0.043(l) 0.029(l) 0.057(2) 0.031(l) 0.037(l) 0.025(l) Glu E T _- TFA-Ml TFA-CM11 0.005(l) 0.005(l) 0.005(l) 0.014(2) 0.008(l) 0.052(4) 0.040(4) 0.015(2) (1) 0.024(2) 0.071(56) 0.004(0) 0.006(l) 0.032(3) 0.010(l) 0.013(1) LYS E 0 Partial destruction of NH*-terminal residue by ninhydrin b Qualitative determination. a Arginine. methionine, and tyrosine are partially destroyed byNaN02. ALA. THR. SER.THR -LYS T-V-8a - 10 20 + 26 I IALA- 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\ EU-HIS-LYS-((GLU), PRO,ALA)-THR-(LEU, ILE)-LYS T-180 GLU -PRO-ALA -THR-(LEU,ILE)-LYS T-V-7 b ALA-ILE - ASP -GLY- ASP -THR -VAL-LYSILEU-HOMOSER T-V-2 I ALA. THR - (SER , THR) - LY S , C-b b ALA-(THR, SER,THR ,LYS)-LYS-LEL c-15c HIS-LYS-((GLU),PRO ,ALA)-THR- LEI J ILE . LYS. (ALA, ILE) -((ASP). GLY.- (ASP), THR) -VAL - LYS- LEU c-150 c-9c 4 A c C-7e- Fro. 1. Diagram of t.he sequence of cyanogen bromide Fragment A. The deducedsequence 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 Sequence of Staphylococcal Nuclease. III Vol. 242, No. 20 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-Xc. Chymotryptic Peptide C-6b was identical with tryptic Peptide T-V-8a as judged by the ammo 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, -IleLys, which is the NH2-terminal part of Peptide C-SC as described above. Thus Peptide T-V-7b is connected to Peptide T-V-2, and the COOH-terminal sequence of Peptide T-V-Sb is deduced to be -Leu-Ile-Lys. The COOH- terminal leucine residue of Peptide C-9c is assigned as the NH2 terminus of Peptide T-V-l. 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 ammo acid composition (Lysz, His, Leu), and contained NH*-terminal lysine upon dinitrophenylation. -A 8 C- LEU - HOMOSER THR-PHE -ARG T-V-l T-V-15a LEU-(MET,TYR)-LYS GLY-(PRO,GLN,hiET,THR,PHE)-ARG F15 , I F18 27 32 TYR -LYS-GLY- PRO-GLN-MET MET-TYR I (LYS,GLY, PRO.GLN)-MET I C-7e C-9d FIG. 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. Qanogen 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 bond 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&erminal residue of this frag- ment was threonine (l), Peptide T-V-15a occupies the NH,- 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 ammo acid composition as Peptide C-18a, except that glycine, proline, glutamic acid, 2 alanine residues, se&e, and phenylalanine were absent from Peptide C-20a. Since a tryptic fragment (C-18a- TIV) of Peptide C-18a, containing these ammo 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-26a-TI), which lacked the NH&erminal 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-ARC; 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-V-15~ T.V.5 b F2 T.V.13 T.V.6 T.V.10 33 I 40 v 50 t 60 I 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.LYS.LYS.MET I , A t THR.PHE ARG-LEU-LEU~LEU,VAL.(ASP).THR,PRO,(GLU).THR).LYS.(HlS,PRO.LYS),(LYS,GLY,VAL,(GLU) ,LYS. TYRI GLY-PRO. GLU -ALA - SER.ALA -PHE - c-22 - c-4 c-191 c-20 0 c-2 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-180 C.l8..TI C.lfi..Tll-C-lBn.TIII C-IB.,.TIV K.ZOa.TI) (C.2Oe. T II) - .I_ (C-230.TIII) FIG. 3. Diagram of the sequence of cyanogen bromide Fragment C (see legend to Fig. 1) THR-(ASP).(LYS .TYR,CLY)- ARC (GLY .LEU.ALA)-TYR-(ILE.TYR. ALA,ASP.GLY)-LYS T.VII-100 F 24 VAL- GLU ASN .ALA-LYS LYS. (LE. GLU .VAL.(GLll, PHE). ASN .LY$ GLY.GLN ARC THR- ASP. LYS TYR-GLY .ARG GLY -LEU-ALA .TYR ILE.TYR- ALA. ASP-GLY -LYS HOMOSER T.Vll.3c I T.Vll.8a T-VII.8 b T.VII-2c T-VII.ll o T.VII-3b T-VII-So 66 70 80 VAL- GLU - ASN-ALA.LYS.LYS. ILE. CLU -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). c.,7o (THR, LYS).LYS-(MET, VAL, (GLU)).(ASP)-ALA.LYS-LYS.(lLE, (GLU), VAL, (GLU), PHE). (ASP).LYS-(GLY, (GLU)). ARG- TtlR.(ASP). c.22 c-190 FIG. 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. VAL.ASN-GLU.ALA-LEU. VAL-ARC T-III-6 r GLN-GLY -LEU-ALA-LYS VAL-ALA -TYR "AL.TYR.LYS-PRO-ASN.ASN-THR-HIS-GLU.GLN.LEU-LEU-ARG LYS.GLU.LYS LYS-SER-GLU-ALA-GLN-ALA.LYS LEU.ASN.ILE-TRP-SER-GLU-(ASP)- ASP.(ALA ,(ASP))-SER- GLY.GLN T.lll.rn TFA.pzF:::; TFA-CU'l T-Ill-2 100 110 I 120 I 130 I 160 149 VAL-ASN-GLU.ALA-LEU. VAL-ARG. GLN.GLY -LEU-ALA.LYS- VAL-ALA -TYR-"AL-TYR-LYS-PRO-ASN. ASN.THR- HIS.GLU-GLN LEU-LEU-ARG.LYS-GLU.LYS-LYS-SER-GLU-ALA.GLN.ALA.LYS-LEU~ASN.lLE.TRP~SER~GLU. ASN. ASP- ALA. ASP -SER. CLY.GLN 6 -C.lOb WAL.ARG).(GLN.GLY)-LEU ALA.LYS-(VAL,ALA).TYR VAL-TYR LYS.PRO-ASN.(ASN,THR)-(HIS.GLU,GLN)-LEU SER-GLU- ASN -(ASP, ALA). ASP .SER-(CCY,GLN) C.lSb c-lk C.60 c-140 Cl r C.Mb FIG. 5. Diagram of the sequence of cyanogen bromide Fragment E (see legend to Fig. 1). Peptide T-III-12 is described as T-III-12a in the text. 4756 Sequence of Staphylococcal Nuclease. III Vol. 242, No. 20 TFA-CM11 c 4 t t A LYS-GLU-LYS-LYS-SER-GLU-ALA-GLN-ALA-LYS cT-III-9c--r: T-Ill-8 137 :- J 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-Ill-8 T-111.9~ T-Ill-2 L FIQ. 6. Diagramof thelinear order of Peptides T-III-Se and T-111-8. The bonds cleaved by thermolysin are indicated by the uerlica 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. TABLE III 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-II-Thl5, Th16, and Th125 were shown to be derived from TFA-11. Amino acid TFA-II-TM-5 TFA-II-TM-6 TFA-II-Thl-25 Lysine 0.016(4)" 0.024(4) Aspartic acid. 0.006(l) 0.068(3) Serine 0.005(l) 0.005(l) 0.035(2) Glutamic acid.. 0.014(3) 0.022(3) 0.050(2) Glycine 0.005(0)b O.o04(0)b 0.029(l) Alanine. O.Oll(2) 0.013(2) 0.026(l) Isoleucine. 0.015(l)" Leucine. 0.006(l) Tryptophan. UP (1 Partial destruction by ninhydrin staining. b This amount of glycine was often found as a contaminant in samples eluted from paper. c Determined by the amino acid analysis of a digest by leucine aminopeptidase. C-l&-T1 (C-2Oa-TI) by Fragment C-Ha-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-2Oa, consistent with the specificity of chymotrypsin. Chymotryptic Peptide C19f connected Peptides T-V-15a and T-V-W. Tryptic Peptide F21 gav-e the connection between Peptides T-V-6 and T-V-10. Cyanogen Braide Fragment D-Fig. 4 shows the relations of tryptic and chymotryptic peptides assigned to Fragment D. Digestion of chymotryptic 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-3c, T-VII-8a, T-VII-8b, and T-VII-2c (see Fig. 4) can be placed in order. The NH&erminal 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-VII-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 phenykdanine, 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-VII-1Oa. Chymotryptic Peptide C-19e (2) connected Pep- tide T-VII-lla to T-VII-3b, consistent with chymotryptic Pep- tides C-15e and C-5g. -4 tryptic peptide (TFA-M14),3 identical with peptide T-VII-3b, was obtained from tryptic digest of TFA-nuclease, together with TFA-M7 (identical with Peptide T-VII-1Oa) (Table II). These findings are consistent with the indicated positions of the two arginine residues. Tryptic Peptide Fzr included Peptides T-VII-3b and T-VII-5a. Chy- motryptic Peptide C-5d lacked only isoleucine and tyrosine from the ammo acid composition of Peptide T-VII-5a. A chymotryptic peptide corresponding to residues 92 and 93, He-Tyr (Fig. 4), has not been found. Cyarwgen 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-111-6, which is assigned to the NH2 terminus of Fragment E (1). Another chymotryptic peptide, C-lob, included the COOH terminus of cyanogen bromide Fragment D, Peptide T-111-6, and the NH2 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). Chymotryptic Peptide C-14~ connected 3 Gly, 0.011; Ala, 0.018; Leu, 0.011; Tyr, 0.008. Issue of October 25, 1967 H. Tan&hi, C. B. Anfinsen, and il. Sodja 4757 FIG. 7. Proposed amino acid sequence of n&ease V-8 TABLE 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 Lysine ........................... Hi&dine. ...................... Arginine .......................... Aspartic acid. .................... Asparagine, ....................... Threonine ........................ Serine. ............................ Glutamic acid. ................... Glutamine. ....................... Proline. .......................... Glycine ........................... Alanine ........................... Half-cystine ...................... Vslirie ............................ Met hionine ....................... Isolencine ........................ Leucine. ....... .................. Tyrosine. ......................... Phenylalanine .................... Tryptophan ....................... Total ............................ Molecular weight. ................ * Determined as homoserine (1). * See Reference 1. - - A 5 (4-5 j l(l) O(O) ;} (2) 4 (3-4) 10) ;} (1) 10) 10) 3 (2.-3) O(O) l(1) l(l)" 2 67 3(3) NO) O(O) O(O) 20 B 10) l(1) O(O) O(O) O(O) l(lP O(O) O(O) l(l) O(O) O(O! 6 C 6645) l(1) 10) 1 o (1) I 4(3) 10) 4 (3) I 3(3) 2(2) 2(2) O(O) 2Gv l(1)" 00) 3 (2-3) 10) 2 (l-2) O(O) 33 - D 5(4-5) O(O) 2(1-2) ; (3-4) i 1'u 1 O(O) ; (4) I 0) 4(4) 3(3) O(O) 2@) l(l)@ 2(1-2) l(l) 3(3) 1(l) O(O) 33 - E 6(5) l(1) 2(2) "5 (G-7) ) 10) 3 (2-3) ; (9) I 10) 2(Q) 603) O(O) 4(3) O(O) 10) 5(5) 2@) O(O) 10) 51 - Sum 23 3 5 7 I 7 10 5 11 I 7 6 10 14 0 9 4 5 12 7 3 1 149 16,807c Acid hydroly- de of nuclease (21.4) (3.0) (4.8) (14.6) (9.6) (5.1) (18.3) (5.1) (7.9) (14.4) (0.0) (9.2) (3.5) (5.0) (11.6) (6.6) (3.0) cl)* c On the basis of this molecular weight, the absorbance at 280 rnp of an aqueous solution containing 1.0 mg of nuclease VS 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-111-6. The juxtaposition of Pep- trypsin. A tryptic fragment (TFA-PP) obtained from TFA- tides T-113& and T-III-129 was discussed in the preceding nucleate overlapped Peptides T-III-7b and T-III-4-c. Another report (2). ChJmotryptic Peptide C-6a should be the NH,- peptide, TFA-Ml, was identical with Peptide T-111-12~~ The terminal part of Peptide T-III-12a, compatible with the above latter peptide furnishes a second example of chymotryptic arrangement from the standpoint of the specificity of chpmo- cleavage between Peptides C-lb and C-6a and further indicates 4758 Sequence of Staphylococcal Nudease. III Vol. 242, No. 20 the intrinsic chymotryptic activity of the trypsin preparation (2). Chymotryptic Peptide C-14a provided confirmation for the distribution of amide groups in Peptide T-III-12a. A large chymotryptic peptide, C-20b, forms the COOH-terminal part of Fragment E, including the COOH terminus of Peptide T-III-12a, together with peptides T-III-SC, T-111-8, and T-III-P. The composition of a tryptic fragment of Peptide C-20b was identical aith that of Peptide T-111-2. Peptide TFA-C&Ill, obtained from TF.4-nuclease, appears to overlap Peptides T-III-SC, T-111-8, and T-III-2 (Table II). Since Peptide T-III-2 should be the COOH terminus of Fragment E, only the order of Peptides T-III-SC 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 leucme and isoleucine (9), fragments that overlapped Peptides T-III-SC 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 TF.4-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-111-9~ and T-III-8 by the following reason. If Peptide T-III-SC is the NH, terminus of Peptide TFA-11, as illustrated in Sequence -1 in Fig. 6, the COOH termini of Fragments TFA-II-Thld and TF.4-II-Thld should be -.4la-Gln-Ala-Lys and -Ala-Gln- Ala-Lys-Leu-;lsn, respectively. Carboxypeptidases A and B should release these ammo 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 TF~4-II-Thl6 before the liberation of alanine. Chymotryptic Peptide C-l provided information on the distribution of amide groups, not available from studies on Peptide T-111-2. The comparison of the partial sequences of these two peptides permitted construction of the complete se- quence shown in Fig. 5. Amino dcid Sequence of Nucleuse-The deduction of the amino acid sequence of each cyanogen bromide fragment made ,t possible to place 149 amino acid residues in order. The 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 nucleate were found in this structure, as discussed above. The ammo 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 es- 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. 2. 3. 4. 5. 6. 7. 8. 9. 10. TANIUCFII, H., AND ANFINSEN, C. B., J. Biol. Chem., 241, 4366 (1966). TANIUCHI, H., ANFINSEN, C. B., AND SODJA, A., J. Biol. Chem., 242, 4736 (1967). HEINS, J. N., SURIANO, J. R., TANIUCHI, H., AND ANFINSEX, C. B., J. Biol. Chem., 242, 1016 (1967). COTTON, F. A., HAZEN, E. E., JR., AND RICHARDSON, D. C., J. Biol. Chem., 241. 4389 (1966). GOLDBERGER, R. F., AND ANFINSEN, C. B., Biochemistry, 1. 401 (1962). ANFINSEN, C. B., SELA, M., AND COOKE, J. P., J. Biol. Chem., 237, 18% (1962). CANFIELD, R. E., AND ANFINSEN, C. B., J. Biol. Chem., 238, 268 (1963). 11. ENDO, S., Hakko Kogaku Zasshi, 40, 346 (1962); Chem. Abstr., 62. 5504 (1965). MATSUBAK~, H., SINGER, A., SASAKI, R., AND JUKES, T. H., Biochem. Biophys. Res. Commun., 24, 242 (1965). NEURATH, H., in P. D. BOYER, H. LARDY, AND K. MYRB.XCK (Editors), The enzymes, VoZ. 4, Academic Press, New York, 1960, p. 11. FUCHS, S., C~ATRECASAS, P., AND ANFINSEN, C. B., J. Biol. Chem., 242, 4768 (1967). "Amino acid analyses of 26hour hydrolysates of several dif- ferent samples of the nuclease showed 10 moles of glycine per 3 moles of phenylalanine.