THE Jourttur, or Bto~oatca~ Ctimttsmr Vol. 242, No. 20, Issue of October 25, PP. 4733-4751,1367 Printed in U.S.A. The Amino Acid Sequence of an Extracellular Nuclease of Staphyl'ococcus aureus II. THE AMINO ACID SEQUENCES OF TRYPTIC AND CHYMOTRYPTIC PEPTIDES (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 Instktes of Health, Bethesda, Maryland SO014 SUMMARY Treatment of the extracellular nuclease of Staphylococcus aureus, strain V8, with cyanogen bromide was previously shown to produce five polypeptides which could be arranged in a linear order. Trypsin digestion of these yielded sets of peptides, in each of which the carboxyl-terminal fragment could be identified by the absence of lysine or arginine, or by the presence of homoserine. The amino acid sequence of each tryptic peptide has been determined. These results, together with the isolation and characterization of peptides produced by chymotrypsin digestion of the intact nuclease, account closely for the amino acid composition of the cyano- gen bromide fragments. The linear arrangement of the five peptide fragments pro- duced by cyanogen bromide treatment of the extracellular nuclease of Staphylococcus aureus, strain V8, has been described (1,2). Peptides produced by tryptic digestion of each cyanogen bromide fragment were also separated and subjected to amino acid analyses (2). The present paper describes the isolation and sequence determination of trypsin fragments produced by digestion of the intact nuclease and of the cyanogen bromide fragments. These peptides account for the total amino acid content of the protein. Peptides produced by chymotryptic digest.ion have also been separated and analyzed. The com- bined results of the previous and present studies permit the alignment of all of the trypsin fragments within each cyanogen bromide fragment. The reconstruction of the entire sequence of the nuclease is presented in the succeeding paper in this volume. EXPERIMENTAL PROCEDURE Unless otherwise specified, the materials and procedures used were those presented in a previous report (2). * Visiting Scientist. Chromatography of Typtic Fragments of N&e-Tryptic digestion of the nuclease was performed with approximately 2 pmoles of the protein, and the fragments obtained were frac- tionated on a Dowex 50 column (1 x 94 cm) as previously described (2). The elution pattern is shown in Fig. 1. Frac- tions comprising a peak were pooled and examined for purity by two-dimensional peptide mapping (2). Further purification, when necessary, was performed on Whatman No. 3MM paper as reported previously (2). Chroma&graphy of Chymotryptic Fragments of Nucleate- Chymotryptic digestion of 6 pmoles of nuclease in 5 ml of HzO was carried out with 2 mg of a-chymotrypsin (Worthington, three times crystallized). The pH of the mixture was main- tained at 8 with 1 M NH,OH during the large scale incubation, which was carried out at 37" for 1 hour. The extent of chymo- tryptic cleavage was checked, in preliminary experiments, by two-dimensional peptide mapping of aliquots withdrawn after 0, 20, 60, and 240 min. After 60 min, no residual undigested "core" remained. The digestion mix%ure was lyophiliid, and the dried sample was subjected to chromatographic fractionation (Fig. 2) on a Dowex 50 column (1 x 94 cm) as described pre- viously (2). When necessary, further purification w&s per- formed by paper electrophoresis, paper chromatography, or two-dimensional peptide mapping as described in the previous report (2). Dip.6m.s with Pepttiases-Five milligrams of leucine amino- peptidase (Worthington) were dissolved in 2 ml of 0.05 M NH, HCO, containing 0.01 M MgCb and dialyzed against 3 liters of the same buffer at 4" for 48 hours with three changes. The dialyzed protein solution was incubated at 37-40' for 2 hours with magnesium ions to activate the enzyme and was stored at -2OO.l The leucine aminopeptidase digestion mixture con- sisted of 0.05 to 0.2 pmole of peptide and the peptidase, at a level of 2 to 10% by weight of the substrate, in 0.2 to 0.5 ml of 0.05 M NHJICOaO.O1 Y MgC& buffer, pH 8. Incubations were performed at 37" for 5 to 6 hours, unless otherwise specified. 1 A similar preparation made from diisopropyl fluorophosphate- treated leucine aminopeptidase (Worthington) was provided by Dr. Francesco DeLorenzo and used for some of the digestions. 4736 Issue of October 25, 1967 H. Taniuchi, C. B. Anjzksen, and A. Sodja Purified carboxypeptidase B was donated by Dr. S. Korenman (3). Incubations with carboxypeptidase B were performed as with carboxypeptidase A (2). l'ronase, obtained from Calbiochem (Grade B), was prepared as a 0.3% solution in 0.05 M NHJICOa containing 0.001 M CaC& (4). The incubation mixtures contained 0.1 ml of 0.05 M NHJICOs (pH 8), 0.05 to 0.1 Mmole of peptide, and 5 ~1 of Pronase solution. Incubations were performed at 37" for 16 hours. The amino acid analyses of the peptidase digests were carried out as described previously (2). Blanks, containing approxi- mately 50 fig of each peptidase, were incubated without sub- strate at 37" for 20 hours. The amounts of free amino acids (mainly serine, glutamic acid, and glycine) in these blank in- cubations were less than 0.003 pmole. Dilute dcid Hydrolysis of Peptides-Partial acid hydrolysis of peptides containing aspartic acid or asparagine was performed by the method of Tsung and Fraenkel-Conrat (5). Peptides (0.05 to 0.2 pmole) were incubated in 0.2 to 0.5 ml of 0.03 N HCl in sealed, evacuated tubes at 110' for 15 hours and taken to dryness under reduced pressure over NaOH. The dried material was dissolved in 0.1 to 0.2 ml of H&. Aliquots (containing 0.02 to 0.04 pmole of the original peptide) were removed for determination of free amino acids on the amino acid analyzer, and the production of peptide fragments was monitored by two- dimensional peptide mapping. Free aspartic acid could also be estimated qualitatively on the peptide maps. Peptide fragments were purified by paper electrophoresis, paper chroma- tography, or two-dimensional peptide mapping as described previously (2). HydruzinoZy&--The hydrazinolysis of peptides was performed by the method of Winstead and Wold (6) as reported by Koren- man, Craven, and Anfinsen (3). The dried peptide (0.02 to 0.04 pmole) was incubated with 0.5 ml of anhydrous hydrazine in an evacuated, sealed tube at 110" for 10 hours. After re- moval of excess hydrazine under reduced pressure over concen- trated H$O, , the sample was applied on the amino acid analyzer without previous extraction of hydraaides. :lmino Acid /lnalyses-Values for amino acids are expressed in micromoles. Analyses were performed by the method of Spackmau, Moore, and Stein (7) as reported previously (2). Values for amino acids present in amounts less than 0.003 I.tmole are not included unless otherwise specified. When it is necessary to present values of less than 0.003 pmole, the observed values of all other amino acids in the peptide are also included. As- paragine and glutamine released by the peptidase digestions were determined quantitatively on the amino acid analyzer, and qual- itatively by paper chromatography as described previously (2). Electropkurelic Jlobility-Paper electrophoresis on Whatman So. 3?tI?tI paper was performed at pH 6.5 (8), at 46 volts per cm for 60 min, to examine the electrophoretic mobilities of peptides and amino acids (releaqd from peptides with peptidase digestion). Known neutral, basic, and acidic amino acids served as standards. The mobilities of the samples are presented, qualitatively, as "basic," "acidic," or "neutral." RESULTS Tryptic and Chymokyptic Peptides of Suclease Tryptic Pep&&+-The amino acid compositions of tryptic fragments purified from the chromatographic fractions (Fig. 1) are presented in Table I, together with their electrophoretic mobilities at pH 6.5. Phenol Red I 50 100 I50 200 FRACTION NUMBER FIG. 1. Chromatography of tryptic peptides of the nuclease. Experimental details are given in "Experimental Procedure." Phenol red was added to the sample as an indicator of the elution front. The pooled fractions comprising the peaks and the valleys are indicated by the arrows. These fractions are designated T-l, T-2, etc., in the text. Another tryptic digest of the nuclease was separated by two- dimensional peptide mapping on four or more sheets of Whatman No. 3MM paper, and the amino acid compositions of the eluted peptides were determined as described previously (2). No peptides were detected in the eluates from the preparative maps that were not also found in the fractions prepared by ion exchange chromatography (Table II). Furthermore, peptides isolated from the tryptic digests of the intact nucleate were completely consistent with those derived from the cyanogen bromide fragments, except of course for those peptides in the latter digests that were derived from portions of the sequence containing methionine. Thus, Peptides T-7b, T-9d, T-15b, and T-17a (or F15, F17, F18, and F19; see Reference 2) were not found after cyanogen bromide treatment. Peptide T-lla could not be clearly located on the peptide maps, presumably because this peptide did not form a discrete, uncontaminated spot. The counterpart of Peptide T-18a (or F9) was not found in the set derived from the cyanogen bromide fragments. How- ever, Peptide T-V-7b was shown to be a part of Peptide T-18a and Peptide T-llb was the sum of Peptide T-V-6, threonine, and lysine, as described below. Minor Components of Typtic Peptide Mtiture-Further purification and amino acid analyses were performed on minor components of the tryptic digests of the cyanogen bromide fragments (2). Cyanogen bromide Fragments A and C were separated in fairly pure form on Sephadex G-50, but Fragments D and E were contaminated with small amounts of Fragments A and C, respectively (2). A few tryptic peptides, derived from the contaminated fragments, were separated as minor components (2). Peptides T-V-6 and T-V-13, were previously assigned to CNBr Fragment C (2). Peptide T-V-6 was found, in the present studies, to be identical with a product of cleavage of the minor component, T-III-8a,* by the intrinsic chymotrypsin- ZLys, 0.016(l); Thr, 0.018(l); Ser, 0.018(l); Glu, 0.021(l); Pro, 0.019(l); Gly, 0.022(l); Ala, 0.041(2); Tyr, 0.017 (1); Phe, 0.033(l); yield, 4%. RPC, 0.40; RpS, 0.42 (see Peptide Fzl in Table II). -I738 Partial Sequence of Staphylococcal Nuclease. II Vol. 242, Xo. 20 TABLE I Amino acid composition of tryptic fragments of nuclease V8 No half-cystine was found in any of the peptides analyzed. indicated. The electrophoretic mobilities at pH 6.5 were ex- When peptides from the chromatographic fractions (see Fig. 1) amined with aliquots of the chromatographic fractions unless were purified further, the purification methods (paper electro- otherwise specified. Tryptophan was not determined, except in phoresis, paper chromatography, and two-dimensional peptide Peptide T-2. With this peptide leucine aminopeptidase digestion mapping) are indicated as E, C, and M, respectively. The yields released free tryptophan in a 1: 1 molar ratio to Ieucine. Fraction were calculated from the amino acid analyses of aliquots of the 22 accounted for several spots on the peptide map. The amino chromatographic fractions. When more than one peptide was acid analyses of the eluted map components indicated that these contained in the same fraction, the yield of each peptide was cal- were large fragments, present in small amounts, that overlapped culated on the basis of amino acid residues specific to each peptide. other peptide fragments Special colors produced by cadmium ninhydrin staining- (i) are Amino acid T-2 T-3 T-4a T-4b Lysine Hiatidinc. Argiaine Aspartie acid Threooine Serine Glutamic acid. Proline. G Iycine Alanine. Vslioe. Methionine, Isoleuoine. Leucine.. Tyrosine. Pheoylalanine. Electrophoretic mo- bility Niohydrm color. Purification method. Yield 0.045(l) 0. M(4) 0.063(2) 0.068(2) 0.042(l) 0.042(l) 0.067(2) 0.040(l) 0.044(l) 0.040(l) 0.041(l) 0.041(l) 0.007(l) 0.007(l) 0.009(l) 0.036(l) 0.010(l) 0.005(l) 0.007(2) O.OCa(l) 0.695(l) 0.oD3(0) 0.008(l) 0.032(l) 0.035(l) O.Oll(3) 0.602(1)~ 0.0030) Acidic Acidic Acidic 82% 1% E 32% Neutral' E 92% Amino acid T-lla T-llb T-llc T-lid - Lysim?. Hiatidiie. Arginine . Aepartic acid. Threonine Se&e. Glutamic acid. Proline. Glycine. Alanine. Vane. Methionine . Isoleucine. Leucine. . Tyrosine.. Phenylalanine. Electrophoretic mo- bility . Ninhydria color. . Purification method. Yield 0.021(2) 0.046(l) 0.118(2) 0.021(l) 0.014(l) 0.005(0) 0.005(O) 0.630(2) 0.042(l) 0.032(l) 0.060(l) 0.060(l) 0.047(l) 0.092(2) 0.os0(1) 0.145(2) 0.010(O) 0.008(0) 0.014(l) 0.034(l) 0.022(l) 0.152(2) 0.012(l) 0.004 (0) 0.014(l) Neutral O.Guo(l) 0.051(l) Neutral Basic C 3% C 73% C 4% Basic YdOW C 92% c , z: -- - T-k T-5b T-6b 0.007(l) 0.007(l) 0.005(l) O.OM(l) 0.007(l) O.ca(l) 0.007(O) 0.013(l) 0.012(l) 0.007(l) 0.006(l) 0.009(l)' Neutral Neutral M M 37% 44% Neutral Basic E 63% T-13a T-13~ T-14~ 0.016(2) 0.015(l) 0.012 (1) 0.011(l) BWiC E 50% 0.031(2) 0.024(l) 0.056(l) 0.065(l) 0.123 (2) 0.127(2) 0.0580) 0.062(l) 0.017(2)' Bfb_iC E 55% Neutral Yellow E 55% T-6c -. 0.064(l) ( E -. - T-15b -. ( 0.016(l) ( ( 0.016(l) ( 0.021(l) ( 0.017(l) 0.013(l) I 0.008(1)" ( 0.016(l) Basic E 3% T-7b T-IC T-7d T-9a T-9b 3.025(1)~ ).042(l) ).042(l) l.008(0) ).047(l) M56(1) ).OlO(l)~. Neutral M 37% Neutral YdlOW M 65% Basic M as% Neutral' M 83% T-M T-l'ia T-i7d T-Ma ).028(l)" ).046(l) ).035(l) ,.ola(l)o 1.010(0) ).033(l) l.OlQ(l)d Basic OmWX M 76% 0.022(l) O.MO(l) 0.026(l) 0.011(l) 0.021U) 0.012(l) 0.015(l) 0.609(l) 0.016(l) 0.013 (0) 0.014(I) 0.016(l) 0.015(l) 0.015(1)1 0.0040) 0.006(l) 0.010(l) 0.013(l)' 0.022(2)' Basic Basic Basic E 46% - 0.012(l) 0.011(l) 0.007(l) 0.006(l) NeUtd Neutral 5% 90% T-13b T-20 0.026(l) 0.026(l) 0.020(l) 0.029(l) 0.641(2) 0.022(I) 0.055(2) 0.02611) 0.016(l) 0.008(l) Basic Basic D The compositions of certain peptides eluted from peptide maps after light staining with ninhydrin indicate partial destruction of NH*-terminal and lysine residues (e.g. T-16d, which contains NH*-terminal threonine). b The parent fraction contained methionioe in an amount comparable to other residuea. `See the text and Table II. d The number of residues of tyrosine was estimated from the amino acid analyses of the parent cbromatagrapbio fractions (2). *The mobility WBL) only tentatively judged 88 neutral because of the tailing of the spot. f Subsequent degradations with leucine aminopeptidase and the Edman method indite the sequence of this peptide to be Gin-Gly-Leu-Ala-Lye. The electrophoretic neutrality of Peptide T-Qa may be due to formation of a" NH&erminal pyrrolidonecarboxylio acid residue. 0.4lanine. ieoleucine, and leucine were not determined in this analyak The values obtained with another analysis are presented. The values for threonine and glutamic acid were used for standardization. h The sum of methiooine (0.W) and methionine sulfooae (0.003) is presented. Issue of October 25, 1967 H. Tuna&hi, C. B. Anjinsen, and A. Sodja TABLE II 4739 Comparison of tryptic peptides from native nuclease with those prepared from cyanogen bromide fragments of nuclease The peptides were compared and identified on the basis of amino acid composition, positions on two-dimensional peptide maps, color by niuhydrin staining, and patterns of elution from columns of Dowex 50. The primary data are presented in Table I and Fig. 1 and in the previous report (2). The chromatographic yields from Dowex 50 columns of the tryptic peptides prepared from cyanogen bromide fragments are indicated in parentheses and were calculated as for the tryptic peptides of the native nuclease summarized in Table I. Tryptic peptides from native nuclease Isolated by chro- matogra- Dpohwye% T-2 T-3 T-4a T4b T-5s T-5b T-6b T-6c T-7b T-7c T-7d T-9s. T-9b T-9d T-lla - I _- s&ted by peptide mapping Composition Aspd, Serz, Glu?, Gly, Ala, Ile, Leu, Trp LYS, ASPZ, Thr, Gly, Ala, Val, Ile Ser, Glu, Pro, Gly, Ala, Tyr, Phe Lys, Asp, Thrt, Glu, Pro, Val, LeQ Lys, Thr, Glu, Pro, Ala, Ile, Leu Ala, Val, Tyr Lys, Glu LYS Lys, Asp, Glu, Sla, Val, Met Lys, Glu, Gly, Val Lgs, Thr*, Ser, Ala Lys, Glu, Gly, Ala, Leu Lys, Asp, Gly, Ala, Ile, Tyr Arg, Asp, Glu, Ala, Vab, Met, Leu Lysr, Asp, Glu?, Val, Ile, Phe - Tryptic peptides from cyanogen bromide fragments (2) - II Peptide T-III-2 W%) T-V-2 (50%) T-V-6 (37%) T-V-Sb (25%) T-V-7b G%) T-III-4C W%) T-III-5b (39%) T-V-8b (15%) T-V-8a (37%) T-III-7b (46%) T-VII-5a (42%) T-VII-8a (21%) - C-D C A E D D-E D 0 These components, occasionally observed on peptide maps, were presumably due to instrinsic chymotryptic-like activity in the trypsin preparation (see the text). * Three residues of leucine were assigned on the basis of the amino acid analysis of T-VSb (2). c Both peptides occupied essentially the same position on the like activity of the trypsin preparation (9, 10). Peptide T-V-13 was identical with the minor component, T-III-Qb.3 Peptide T-V-7b (2) was previously found to contain a fraction of 1 eq of histidine. However, Peptide T-5a (Table I), which had the same amino acid composition and essentially the same position on the peptide map as Peptide T-V-7b, contained no histidine and gave a negative Pauly reaction. It was concluded, there- fore, that Peptide T-V-7b does not contain histidine. Peptide T-5a, obtained as a very minor component, was found to be part of Peptide T-18a, as described below. `Lys, 0.018(2); Glu, 0.013(l); Gly, 0.013(l); Val, 0.015(l); yield, 6%. RPC, 0.13; RFB, O.&i. Tryptic peptides fro] native nuclease Isolated by chro- matogra- phy 00 Dowex S T-llb T-llc T-lld T-13a T-13~ T-14s T-15b T-16d T-17a T-17d T-18a T-18b T-20 I I _- - s&ted b! peptide mapping F21 F8 FlO F6 Fb F24 Fl8 F 15 Ff FB FlC Fll - ml Composition Lys, Thr, Ser, Glu, Pro, Gly, Ala,, Tyr, Phe Lysr, Ser, Glun, Ala2 Arg, Gin, Gly Lyst, Glu, Gly, Val Lysr, Glu Lys, Asp, Glyt, Ala2 Ile, Lea, Tyrz Arg, Thr, Glu, Pro, Gly, Met, Leu, Phe Lys, Arg, Asp, Thr, GUY, Tv Lys, Met, Leu, Tyr His, Pro, Lys LY% His, Thr, Glu, Pro, Ala, Ile, Leu, Tyr, GUY, Aw Lys, His, Arg, Asp,, Thr, Gluz, Val, Leu?, Tyr Tryptic peptides from cyanogeo bromide fragments (2) -- - Peptide T-III-8b (59%) T-VII-8b (34% ) T-V-13 6%) T-III-SC (41%) T-VII-9 (7%) T-VII- load T-V-15b' (30%) T-VII-lla (26%) T-III-12 (68%) Parent cyanogen bromide tragment C E D C E D B-C D A-B C A D E peptide map (see Reference 2). The analysis of the sample ob- tained from Component Fir (see Reference 2) gave the sum of their amino acid contents. d See the text. 8 Identified on the basis of the peptide map and amino acid composition of the parent chromatographic fract,ion. This consideration of the minor component is consistent with the assignments of the tryptic peptides to cyanogen bromide fragments listed previously (Table II) (2). Avignment of Tryptic Peptides to Cyancgen Bromide Fragments -The peptides produced by trypsin digestion of the nuclease may, on the basis of the above observations, be assigned to cyanogen bromide Fragments A, B, C, D, and E (2) as summa- rized in Table II. Certain peptides in this table do not contain lysine or arginine and are the result of chymotrypsin-like activity in the trypsin preparation. Four of the peptides, containing the 4 residues of methionine in the nuclease, have been assigned as overlapping peptides that bridge cyanogen 4740 Partial Sequence of Staphylococcal Nuclease. II Vol. 242, No. 20 z z 1.5 2 8 3 I.0 0.5 0 0 50 100 I 50 200 FRACTION NUMBER FIG. 2. Chromatography of chymotrypt.ic peptides of the nuclease. Experimental details are given in "Experimental Procedure." The designations of the fractions are the same as described in Fig. 1, except that C-l, C-2, etc., are used in the text. bromide fragments (2). Peptide T-18a (F9), obtained in good yield, contains Peptide T-5a (F20) and the fragment (Leu, His) Lys, which was not recovered from the chromatographic columns but has been prepared from T-18a by digestion with trypsin. Chynlotryptic Peptides-The amino acid compositions, electro- phoretic mobilities at pH 6.5, and colors produced by cadmium ninhydrin of purified chymotryptic peptides obtained from the chromatographic fractions (Fig. 2) are presented in Table III. -% reconstructed peptide map is shown in Fig. 3. Presentation of Sequence Analyses The amino acid composition of each peptide is presented in Tables II and III, unless specified otherwise. In reporting the results of Edman degradations, the recovery at each stage was calculated from the analysis of the previous stage, not on the basis of the starting material. The amino acid residue used as a basis for calculation of the recovery of residual peptide at each stage of degradation is indicated in parentheses. The elii- inated residues are indicated in boldface. The results of peptidase digestions are presented in a similar manner. Amino acid analyses of the hydrolysates of phenylthiohydantoins are reported only qualitatively. The specificities of proteolytic enzymes used appeared to agree throughout with general experience (11). Peptide bonds involving proline were not susceptible to any of the enzymes used except Pronase (12), which was able occasionally to cleave the peptide bond involving the imino group of proliie, as has been observed with pepsin (13). When peptides were fragmented by proteolytic or acid hydrolysis, the resulting fragments were purified on Whatman No. 3pIIM paper by two-dimensional mapping, chromatography, or electrophoresis as described previously (2). The positions on two-dimensional peptide maps of such fragments are indicated in parentheses by RF values in the chromatographic direction (Rw) and the electrophoretic direction (RpE), with phenol red and lysine as standards, respectively (2). Free lysine moved 33 cm, at-pH 3.6 and 46 volts per cm, in 70 min (2). No cor- rection was made for movement due to electroosmosis. The RF values were reproducible to approximately 10%. When the presence or absence of an amide group w&s uncertain, the available evidence is presented in the text, and the questionable (amide) residue is enclosed within parentheses in the sequence (see Footnote 5). Dinitrophenyl end groups, which were reported previously (2), are listed with each peptide unless otherwise specified. Amino Acid Sequence of Tryptic Peptides The analyses of sequences were performed either on the tryptic peptides obtained from the cyanogen bromide fragments described in a previous report (2) or on those derived from the intact nuclease. Consideration of the peptides is in accordance with the linear order of the peptides in the nuclease chain, the complete structure of which is presented in the following paper. T-V-8a: Ala-Thr-Ser-Thr-Lys (Residues 1 to 6)- Dinitrophenyl end group: Ala. Edman degradation: Stage 1 (91%): Ala, 0.001; Thr, 0.014(2); (Ser), 0.007(l); Lys, 0.007(l). Stage 2 (85%): Ala, 0.001; Thr, 0.010(l); (Ser), 0.006(l); Lys, 0.012(l). Carboxypeptidase B (2 hours): Lys, 0.017; Ser, 0.003. Carboxypeptidase A (1 hour) : Lys, 0.016; Thr, 0.009; Ser, 0.002. Carboxypeptidase A (2 hours): Lys, 0.022; Thr, 0.012; Ser, 0.003. (Carboxypeptidase A was added after carboxypepti- dase B.) Carboxypeptidase B released only free lysine. A broad peak at the serine position was observed in this analysis. The amount of material in the serine position remained essentially constant after the addition of carboxypeptidase A, as did the quantity of lysine, while threonine appeared at more than half the level of lysine. These observations suggest that the peak at the serine position was not due to serine itself but probably to a peptide fragment. Neither the undigested peptide nor a blank of carboxypeptidase B showed any significant amounts of free amino acids. T-V-%: Glu-Pro-Ala-Thr (Leu, Ile)-Lys (Residues 10 to f6)- Dinitrophenyl end group : Glu. This peptide, because of its availability in only small amounts, was not used for subtractive Edman degradation. However, the phenylthiohydantoins of glutamic acid, proline, and alanine were shown to be released after the first, second, and third stages of the degradation, respectively. Edman degradation (phenylthiohydantoin hydrolysis), three cycles: St,age 1; Glu; Stage 2, Pro; Stage 3, Ala. Carboxypeptidase B: Lys, 0.019; Ala, Ile, Leu < 0.002. Carboxypeptidase B and -2 (5 min) : Lys, O.OOi; Ile, 0.007; Leu, 0.007; Thr, 0.002; Ala,4 0.004. Carboxypeptidase B and A (30 min): Lys, 0.020; Ile, 0.014; IAX, 0.018; Thr, 0.016; hla,4 0.010. This portion of the nuclease sequence was further established by other studies given below. The presence of a free carboxyl group on the glutamic acid residue was indicated by t,he electro- phoretic neutrality of the peptide. 4 This digestion was performed with chromatographic Fraction T-5, which contained Peptides T-5a (T-V-7b) and T-5b (T'al- Ala-Tyr) (see Table II). Released alanine was derived from Peptide T5b together with small amounts of valine and tyrosine. Issue of October 25, 1967 H. Taniuchi, C. B. Anfinsen, and A. Sodja 4741 TABLE III Amino acid composition of chymotryptic peptides The ahromatographic yield of each peptide was calculated from Partial destruction of NH?-terminal residues, lysine, and tyrosine the amino acid analysis of an aliquot of the fraction obtained by occurred with some peptides as the result of ninhydrin staining, chromatography on Dowex 50 (see Fig. 2) as described in Table I. elution, and acid hydrolysis. This is indicated by asterisks. As- The purification method, the electrophoretic mobility, and the signment of residue numbers wss qualitative with some peptides. color produced by cadmium ninhydrin staining are indicated as in The number listed are based on subsequently confirmed sequences Table- I. No half-cystine was found in any of the components. (see the text). - --7 C-Sg C-6a C-6b C-7e C-9C C-9d C-lob -- -- _- -_ 0.151(2) 0.016(1: 0.003(O) 0.003 (0) 0.141(2) 0.664(l) O.oD6(1) D.OWl) D.O14(2) 0.030(2) 0.016(l) 0.010(0) 0.042(l) 0.005(O) O.O29(1j 0.031(1; 0.028(l) 0.016(l) ( ).0.39(l) 0.0730) O.D90(1) O.@?(l) 0.021(l) 0.140(2) 0.097(1 j D.O13(2) D.O12(2) D.Oll(2) 0.012(2) D.OOP(l) 0.014(2) ).037(l) 0.037(l) c Neutral ! Yeutral Basic 0.020(l) 0.008(O) 0.031(l) Neutral Slightly basic Basic Basic Basic C E 29% 28% f= C-17b c-a E 2% C-l% 0.013 (1) 0.019(2)' 0.014(l) 0.014u) 0.011u) BE& YdlOW C 73% z= _- , ' I I C 69% C-19f E 63% E 59% -- C ( I.068 1.014 0.086 0.014 0.014 I.025 I.032 I.017 I.055 I.001 I.037 I.039 I. 049 0.006 1.013 I.014 I.020 Basic 0.043 Basic !- C 75% C 66% - 2: .- , / / I , , f / , , I ( ( I - C-208 C-ZOb D.051 0.027 0.036 cl.007 0.048 0.025 D.017 D. 016 D. 038 0.022 D.009 0.020 D.022 D.012 Not de- ter- mined D.OC4 D.051 - I I ( ( I ( ( ( 4 0.019 0.044 0.030 I.033 3.060 0.065 3.017 3.033 3.008 I.011 0.023 0.024 0.036 0.018 0.013 Not de- ter- mined 0.016 0.017 Basic E E E 34% 26% 19% - - -. =: -. ( ( ( . ( - C-2 c-4 c-148 C-Sd c-o C-l O.D38(3) 0.062(3) 0.022(2) 0.037(2) 0.028(2) 0.043(2) 0.016(l) 0.019(l) 0.014(l) 0.023(l) 0.011(l) 0.029(2) 0.048(l) 0.021(l) O.O2l3(1) 0.028(l) 0.0260) 0.028(l) 0.015(l) 0.012(l) 0.007(l) Acidic Acidic OWlge 1% c-14c 23% 0.0350) 0.068(l) Acidic Neutral YE3lOW OWlge 31% @% 81% C-1Sa C-15b c-15c C-l% Amino acid Lysine ............... Histidme ............ A&nine. ............ .4apnrtic acid. ....... Threonine ........... Se&e ............... Glutamic acid. ...... Proline ............... Glycim ............. Alanine. ............ Vsline .............. Methionine .......... I~le"cina. ........... Leu&e .............. Tynuline. ............ Phenylalanine ....... Elmtrophoretic mm bility .............. Ninhydrin color ..... Purification method. Yield ................ Amino acid Lyaine. ........... Hiatidine .......... Arginiie ............. Aspartio acid. ...... Tbreonine. ......... Serine ................ Glutamic acid ....... Proline ............... Glycine. ............ Alanine. ............. Valise ............... Methionine .......... Isoleucine. ........... Leuoine .............. Tyrosine. ............ Phenylalaoine ....... Eleotrophoretic mm bility .............. Ninhydrin color. ... Purification method. Yield ................ 0.026(l) o.oao(l)a 0.043(2) 0.022(l) 0.048(2) 0.047(2) 0.027(l) 0.036(Z) 0.035(l) 0.020(l) 0.041(2) 0.018(l) 0.038(l) 0.031(l) 0.030(1 0.040(2) 0.013(l)' 0.035(1 0.026(l) 0.032(l) Basic Basic B&c M 57% M 21% YdlOW M 15% 0.015(l) 0.018(l) 0.02ac2) 0.016(l) 0.010(l) 0.018(l) Slightly acidic ).021(l) 3.011(l) ).042(2) ).025(l) ).016(l)" Basic C 34% 0.071(2) 0.027(l) O.oaO(2) 0.035U) O.cQa(O) 0.053(l) 0.036(l) 0.011(O) 0.0290) Basic M `38% n These residues were confirmed by staining the peptides with Pauly reagent. b Determined 88 methionine sulfone. No methionine WBB found. * Peptide T-18a: Leu-His-Lys ((G~u),~ Pro, Ala) Thr (Leu, Ile) i,ys (Residues 7 to IS&This peptide was obtained from the tryptic digest of the intact nuclease (see Table II). Dinitrophenyl end group: Leu or Ile. Edman degradation (phenylthiohydantoin hydrolysis) : Leu. The amino acid composition suggested that this peptide included Pept,ide T-V-ib and t.he peptide (Leu, His) Lys. This w&s confirmed by tryptic digestion of Peptide T-18a, which 6 The abbreviations used are: (Glu) and (Asp), Glu or Gin and Asp or Asn, respect.ively, where amide groups were not de- termined; DNP-, dinitrophenyl-. yielded two fragments, T-18a-TI ((Leu, His) Lys) and T-18a- TII. The position of the latter fragment on a peptide map was the same as that of Peptide T-V-7b. Trypsin Fragments: T-Ma-T1 (Pauly-positive): Lys, 0.016; His, 0.017; Leu, 0.014; Ser, Glu, Gly, Ala, < 0.004. RFC, 0.14; RpE, 1.16. T-18a-TII (Pauly-negative) : RFc ,0.70; RFB, 0.55. Carhoxypeptidase B and A (10 min) (34%): (Lys), 0.010; Be, 0.011; Leu, 0.018. Carboxypeptidase B and A (30 min) (96%): Lys), 0.026; Ile, 0.019; Leu, 0.029; Thr, 0.017. 4712 Partial Sequence of Staphylococcal Nuclease. II Vol. 242, No. 20 I r I ' I ' I 1 1'1' 1 I .o - Lysine @ _ 6 15c I - a 191 o ?? ? ? ?? 4 1 , Phenol Red o-a""""Q"" 0.2 0.4 0.6 0.0 I 1.2 1.4 RF (CHROMATOGRAPHY 1 FIG. 3. Reconstructed map of the peptides obtained from the chymotryptic digest of the nuclease. The reference standards are the same as described in the text. Fragment obtained after carbosypeptidase B and A: Lys, 0.01g6; His, 0.026; Glu, 0.031; Pro, 0.037; Ala, 0.032; Leu, 0.014.' RPC, 0.17; Rm, 0.90. T-V-2: Ala-Ile-Asp-Gly-drp-Thr-Val-Lys (Residues 17 to 24)-- Dinitrophenyl end group: Ala. Edman degradation:' Stage 1 (98%: Ala, 0.661; Be, 0.015(l); Asp, 0.028(2); Gly, 0.014(l); (Thr), 0.016(l); Val, 0.016(l); phenylthiohydantoin hydrolysis, Ala. Stage 2 (100%): Ala, 0.002; Ile, 0.002; Asp, 0.027(2); Gly, 0.015(l); (Thr), 0.016(l); Val, 0.018(l); phenylthiohydantoin hydrolysis, Be. Stage 3 (86%): Ala, 0.000; Ile, 0.000; Asp, 0.015(l); Gly, 0.013(l); (Thr), 0.014(l); Val, 0.009(l); phenylthiohydantoin hydrolysis, hsp. Stage 4 (107%): .Ua, 0.601; Ile, 0.000; Asp, 0.017(l); Gly, 0.008; (Thr), 0.015(l); Val, 0.014(l). Stage 5 (89%): Ala, 0.001; Be, 0.001; Asp, 0.010; Gly, 0.006; (Thr), 0.012(l); Val, 0.014(l). Stage 6 (9OyJ: Ala, 0.000; Ile, 0.002; i4sp, 0.008; Gly, 0.005; Thr, 0.007; (Val), 0.012(l). Electrophoretic mobility: .i\cidic (aspartic acid residues not amidated). Leucine aminopeptidwe (19 hours): Ala, 0.043; Ile, 0.040. T-V-1: k-u-1lomoPaine (Residues 25 and 26)-Dinitrophen- ylation gave DNP-leucine (2). T-V-15a: Thr-Plx-;lrg (Residues 33 to 35)8-This peptide 6 Low yield was due to loss from ninhydrin staining of map. r Analysis by paper electrophoresis showed the presence of 1 mole of lysine t,hroughout the degradation. * Residues 27 through 32 correspond to cyanogen bromide Fragment B (2) (see the following paper (14)). was purified by paper electrophoresis. Dinitrophenylation gave DNP-threonine (2). T - V-5b: Leu-Leu-Leu-Val-Asp-Thr-PreGln-Thr-Lys (Rer- idues 36 to 45)- Dinitrophenyl end group: Leu or Ile. Edman degradation (phenylthiohydantoin hydrolysis), five cycles: Stage 1, Leu; Stage 2, Lou; Stage 3, Leu; Stage 4, Val; Stage 5, Asp. Electrophoretic mobility: Neutral (suggesting one amide) (see Table I, Footnote e). Leucine aminopeptidase (11 hours): Leu, 0.073; Val, 0.029 (Lys, Thr, Ser, Ala, Tyr < 0.01). Pronase digestion: Free Asp, Lys, Thr, Val, Leu. Pronase fragment: T-V-W-PI, RPC, 0.45; RPB, 0.03. Thr, 0.004; Glu, 0.005; Pro, 0.006. Pronase digestion produced free aspartic acid. Therefore, glutamic acid appears to be present as glutamine. In addition Pronase released lysine, threonine, valine, and leucine. The amount of threonine determined was approximately equal to lysine and aspartic acid, suggesting that 1 of the 2 threonine residues was not released. Accordingly, it is likely that 1 threonine residue is adjacent to the prolime residue, which would be cleaved very slowly or not at all by Pronase. The Pronase digest was examined for peptide fragments by mapping, and ammo acid analysis was carried out on the eluted peptides. One of these components (T-V-5b-PI) gave, qualita- tively, the composition (Thr, Glu, Pro). Acid hydrolysis of T-V-5b for 7 hours with 0.03 N HCl pro- duced two fragments, T-V-5b--41 and T-V-Sb-AD, as well as free aspartic acid. These fragments were purified by paper electrophoresis, and amino acid analyses were carried out on the eluted samples. Fragment T-V-5b-AI (RFB, 0.39) appeared to give the composition (Leur, Val), and Fragment T-V-W-AI1 (RF&, 0.54) gave (Thr, Thr, Glu, Pro, Lys). Therefore it was evident that Fragments T-V-Sb-AI and AI.1 were NHz-terminal and COOH-terminal, respectively, a conclusion consistent with the position of the aspartic acid residue determined by Edman degradation. The orange color developed by cadmium ninhy- drin staining of Fragment T-V-W-AH suggested an NHz-termi- nal threonine residue. Another fragment (T-V-5b-AIII) (RFC , 0.98; RFB, 0.20) was obtained by longer digestion (20 hours) with 0.03 N HCl. Its ammo acid composition was shown to be (Thr, Pro). Accordingly, the partial sequence .4sp (Thr, Pro) Glu-Thr-Lys may be deduced, based on the significant suscepti- bility of the glutamic acid residue to the dilute acid hydrolysis (15). Together with the results of Pronase digestion of Peptide T-Vdb and the color produced by cadmium ninhydrin staining, the sequence of Fragment T-V-Sb-AI1 may be deduced to be Thr-Pro-Gln-Thr-Lys. Support for the presence of threonine in the penultimate position of Peptide T-Vdb was obtained by the following experiment. Peptide T-V-5b was digested wit,h carboxypeptidase B for 6 hours. Free lysine w-as released in a yield of 23% as judged by ammo acid analysis of an aliquot. Hydrazinolysis was performed with the rest of the digest mixture. Free threonine was found as well as free lysine, the former in a yield of 36% based on the recovery of lysine after carboxypep- tidase B. Further information on this portion of the sequence is given below (chymotryptic fragments). FP: His-Pro-Lys (Redues 46 to 48)-The COOH-terminal ly- sine residue was assigned on the basis of the specificity of trypsin. Staining of this peptide with cadmium ninhydrin did not give the yellow color characteristic of prolme. Issue of October 25, 1967 H. Tankhi, C. B. Anjinsen, and A. Sodja 4743 T-V-13: Lys-Gly-Val-Glu-Lys (Residues @ to 53) Dinitrophenyl end group: Lys. Edman degradation: Stage 1 (67%): Lys, 0.008(l); Gly, 0.010(l); (Val), 0.009(l); Glu, 0.010(l). Stage 2 (104%): Lys, 0.011(l); Gly, 0.004; (Val), 0.009(l); Glu, 0.009(l). Stage 3 (86%): (Lys), 0.011(l); Gly, 0.004; Val, 0.006; Glu, 0.012(l). Stage 4 (82%): (Lys), 0.011(l); Gly, 0.004; Val, 0.004; Glu, 0.008. Leucine aminopeptidase (68%): Lys, 0.035(2); (Glu), 0.017(l); Gly, 0.016(l); Val, 0.018(l). T-V-6: Tyr-Gly-Pro-Glu-Ala-Ser-Ala-Phe (Residues 54 to 61)- Dinitrophenyl end group: Tyr. Edman degradation : Stage 1 (80%): Tyr, 0.001; Gly, 0.008(l); Pro, 0.010(l); Glu, 0.009(l); (Ala), 0.017(2); Ser, 0.007(l); Phe, 0.008(l); phenyl- thiohydantoin hydrolysis, Tyr. Stage 2 (88%): Tyr, 0.000; Gly, 0.002; Pro, 0.008(l); Glu, 0.008(l); (Ala), 0.015(2); Ser, 0.006(l); Phe, 0.008(l); phenylthiohydantoin hydrolysis, Gly. Stage 3 (102 %) : Tyr, 0.000; Gly, 0.002; Pro, 0.004; Glu, 0.009(l); (-4la), 0.015(2); Ser, 0.008(l); Phe, 0.010(l); phenylthiohy- dantoin hydrolysis, Pro. Stage 4 (96%): Tyr, 0.000; Gly, 0.003; Pro, 0.003; Glu, 0.002; (Ala), 0.015(2); Ser, 0.009(l); Phe, 0.006(l). Stage 5 (118%): Tyr, O.ooO; Gly, 0.002; Pro, 0.000; Glu, 0.003; Ala, 0.009(l); Ser, 0.006(l); (Phe), 0.007(l). Stage 6 (78%): Tyr, 0.000; Gly, 0.003; Pro, 0.003; Glu, 0.005; Ala, 0.019(l); Ser, 0.005; (Phe), 0.011(l). Phenylalanine was assigned to the COOH-terminal position on the basis of the known contamination of the trypsin prepara- tion with chymotryptic-like activity. The electrophoretic mobility indicated the presence of nonamidated glutamic acid. T-V-10: Thr-Lys-Lys-Homoserine (Residues 6.9 to 66)-The amino acid composition of this peptide was previously reported (2) as (Lysz, Thr, Glu). However, further examination, in- cluding leucine aminopeptidase digestion and Edman degrada- tion, revealed that the residue originally identified as glutamic acid was homoserine. Dinitrophenyl end group: Thr. Amino acid composition: Lys, 0.028; Thr, 0.014; homoserine, 0.015; (Gly, Ser), ~0.004. Leucine aminopeptidase (69 %) : Lys, 0.046; Thr, 0.026; (homo- serine), 0.024. Edman degradation: Stage 1: (Lys), 0.009(Z) ; Thr, 0.000; homoserine, 0.0040). T-llb: (Tyr, Gly, Pro, Glu, Ala) (Ser, Ala) Phe-Thr-Lys (Residues 54 to 63)-The amino acid composition of this peptide was the sum of those of Peptide T-V-6 and Thr-Lys, which ap- pear to be fragments produced by the chymotryptic-lie action of the trypsin preparation on Peptide T-llb. This assumption was supported by carboxypeptidase B and A digestions of Pep- tide T-llb. Free lysine, threonine, serine, alanine, and phenyl- alanine were released as follows. Carboxypeptidase B (3 hours) (57%): (Lys), 0.013; Thr, 0.009; Phe, 0.011. Carboxypeptidase B and A (16 hours) (100%): (Lys), 0.023; Thr, 0.018; Ser, 0.010; Ah, 0.022; Phe, 0.018. Electrophoretic mobility: Neutral (glutamic acid). T-VII-SC: Val-Glu-Asn-Ala-Lys (Residues 66 to 70)- Dinitrophenyl end group: Val. Electrophoretic mobility: Neutral (one free carboxyl group). Edman degradation: The degradations of the above peptide and of T-VII-3b (Gly-Leu-Ala-Tyr; residues 88 to 91) could be carried out simultaneously on an unresolved mixture (2) of the two. These results, and analyses of a leucine aminopepti- dase digest, are summarized in Table IV. The latter data show that the glutamic and aspartic acid residues are present as glu- tamic acid and asparagine, respectively. TABLE IV Sequence studies on mixture of Peptides TVII-3b and TVII-k (see Reference .??) Amino acid Valine.. Glutamic acid..... Aspartic acid..... Alanine. . Lysine . Glycine.. Leucine Tyrosine Phenylthi- ohydan- toin hy- drolysis . Yield.. -7- -- - Total Edman Edman Edman composition stage 1 stage 2 stage 3 3c 3b 3c - .- 04 3b 3c 0.000 3b 3c 3b ___~ 0.089 0.068 (0.105) 0.079 0.036 0.048 0.048 0.604 0.031(l) 0.020(l) (1) (0. 1) 0) 0.029(l) 0.066 0.018(l) 0.015(l) Sl%(LYS) 0.000 0.008 0.009 0.015(l) 0.011 (1) (0.023) (1) 0.024(l) o.llOo 0.018(l) 0.025(l) 0.000 0.000 0.603 0.006 0.008(l) 0.009(1 Glo Leu Ala ~~%(LYs) 113%(LYf3) - -_ -_ 1 - Edman stage 4 3c 0.000 0.009 0.014 0.024(1 Ala - -_ ) 3b 0.000 0.000 0.000 0.000 W%(LYS) - _- . _ - Leucine aminopeptidase digestion 3c 3b 0.158 0.143 0.087" (0.196) 0.116 0.069 0.081 0.080 86% O-93) 1027, (Tyr) 0 As asparagine, in the position of serine on the analyzer; identity checked by paper chromatography. 4744 Partial Sequence of Staphylococcal N&ease. II TABLE V Sequence studies on mixture of Peptides T-VIZ& and T-VII-86 Vol. 242, No. 20 Amino acid - -- Lysine................... Arginine Aspartic acid. Serine................... Glutamic acid. Glycine.................. Valine Isoleucine. Phenylalanine . Phenylthiohydantoin hy- drolysis. Yield.................... 8a 8b 88 8b aa 8b sa 8b sa sa 8b -- -- 0.020 0.666(l) 0.006(l) 0.005(1 -1 0.035(l) 0.035 0.025 0.017(l) 0.011(l) 0.002 0.132 0.027 0.006(l) 0.006(l) 0.006(1 1 0.004(l) 0.050" 0.071 (2)0.023(l)* 0.013 (2) 0.000 O.Oll(l 1 0.000 0.007(l) 0.060" 0.636 0.092 0.000 0.000 0.665 0.018 0.006(l) 0.006(l) 0.007(1 1 0.006 0.046 0.015 0.005(l) 0.990 0.030 0.017 0.004(l) 0.005(l) O.OlO(l 1 0.005(l) 0.028 LYS GUY Ile Glu C&l Val 6%(AsP)d 11% il Lap)* 89%&p)* 17% (Asp) a As glutamine, in the position of serine on the analyzer; identity checked by paper chromatography and with pure peptide. n Assigned to both peptides. - Edman Stage I Edman Stage 2 Leucine aminopeptidase digestion c The presence of 2 moles of glutamic acid was confirmed with the purified peptide by digestion with leucine aminopeptidase. * The parentheses indicate the residue on the basis of which the yields were calculated (see the text "Presentation of Amino Acid Sequence"). T-VII-8a: Lys-Zle-Glu-Val (Glu, Phe) Am-Lys (Residues 71 Dinitrophenyl end group: Thr (confirmed by Edman degra- to 78)- d&ion and leucine aminopeptidase digestion). Dinitrophenyl end group: Lys. Acid hydrolysis (0.03 N HCl): Three components on the pep- Electrophoretic mobility: Neutral (two nonamidated carboxyl tide map. Elution, hydrolysis, and analysis yielded (a) Thr, groups). 0.018; Asp, Gly < 0.004; (5) a4sp; (c) Lys, 0.019r"; Arg, 0.045; Edman degradation: Carried out on a mixture (2) of T-VII-8a Gly, 0.053; Tyr, 0.0251o (RpC, 0.27; Rra, 0.98). and Peptide T-VII-8b (Gly-Gln-A4rg; residues 79 to 81). T-VII-Sb: Gly-Leu-Ala-Tyr (Residues S8 to 91)- These results are summarized in Table V. Dinitrophenyl end group: Gly; yellow color with cadmium Leucine aminopeptidase: Alsc summarized in Table V. Two ninhydrin. moles of glutamic acid were released together with 1 mole of Edman degradation: See Table IV. isoleucine, valine, and phenylaknine. Only 1 mole of lysine T-VIZ-6a: Ile-Tyr-=ila-iiqp-Gly-Lys (Residues 9% to 97)- appeared to be present in the digest. Neither aspartic acid nor Dinitrophenyl end group: Leu or Ile. asparagine was found. An undigested fragment, Asn-Lys, Edman degradatio+ : presumably remained. Data to be presented below support the presence of a penultimate asparaginyl residue. Stage 1 (69%): De, 0.661; Tyr, 0.021(l); .4la, 0.025(l); (Asp), T-VIZ-8b: Gly-Gln-.-lrg (Residues 79 to Sl>- 0.018(l); Gly, 0.029(l); phenylthiohydantoin hydrolysis, Ile. Dinitrophenyl end group: Gly; cadmium ninhydrin staining Stage 2 (115%): Ile, 0.001; Tyr, 0.992; .4la, 0.018(l); (Asp), yielded a yellow color. 0.019(l); Gly, 0.017(l); phenylthiohydantoin hydrolysis, Tyr. Edman degradation: See Table V. Stage 3 (81%): Ile, 0.001; Tyr, 0.002; Ala, 0.005; (Asp), 0.017; Electrophoretic mobility: Basic. Gly, 0.024(l); phenylthiohydantoin hydrolysis, Ala. T-VIZ-k: Thr-Asp-Lys (Residues 89 to 84)- Stage 4 (83%): Ile, 0.000; Tyr, 0.001; Ala, 0.003; Asp, 0.006; Dinitrophenyl end group: Thr; orange color with cadmium (Gly), 0.020(l). ninhydrin. Leucine aminopeptidase (19 hours) (107%) : (Ala), 0.061; Leucine aminopeptidase : Thr (96 %) . Ile, 0.049; Tyr, 0.089. Electrophoretic mobility: Neutral. Electrophoretic mobility: neutral. T-VII-11~ Tyr-Gly-;lrg (Residues 86 to S7)- Fzr: (Gly, Le-u, dla) Tyr We, Tyr, .ila, Asp, Gly) Lys (Residues Dinitrophenyl end group: Tyr. 88 to 97)-The amino acid composition of this peptide was equal Further information on this portion of the sequence is given to the sum of the compositions of Peptides T-VII-3b and T-VII- below in connection with the chymotryptic fragment, C-15e. 5a. The latter were presumably formed by the chymotryptic- T-VIZ-lOa? Thr-(Asp)-(Lys, Tyr, Gly)-Arg (Residues 82 to 87) like activity in the trypsin preparation. 9 The peptide map of Fraction T-VII-10 (2) showed a single lo Low yield was due to ninhydrin staining and destruction Pauly-positive spot (2). However, the amino acid analysis of during hydrolysis. Peptide T-VII-10s (ion exchange chromatographic yield, 26%) *i The basic amino acids were not determined on the analyzer. was only qualitative, because of contamination of Fraction T- Analyses by paper electrophoresis showed 1 eq of lysine through- VII-10 with minor component,s. out. Issue of October 25, 1967 H. Taniuchi, C. B. Anfinsen, and A. Sodja 4745 T-NI-6: Va~-~~~~lu-.~la-Leu-Val-.-l~g (Residues 99 to 106)`2- Dinitrophenyl end group: Val. Edman degradation13: Stage 1 (104%): Val, 0.036(l); Asp, 0.018(l); Glu, 0.025(l); Ala, 0.029(l); (Leu), 0.025(l); phenylthiohydantoin hydroly- sis, Val. Stage 2 (89%): Val, 0.019(l); Asp, 0.005; Glu, 0.020(l); Ala, 0.020(l); (Leu), 0.021(l); phenylthiohydantoin hydrolysis, Asp. Stage 3 (89%): Val, 0.022(l); Asp, 0.002; Glu, 0.006; (Ala), 0.020(l); Leu, not determined; phenylthiohydantoin hydroly- sis, Glu. Stage 4 (96%): (Val), 0.021(l); Asp, 0.004; Glu, 0.008; Ala, 0.010; Leu, 0.014(l). Leucine aminopeptidase": Asn, 0.054; Glu, 0.065; Ala, 0.066; Val, 0.208; Leu, 0.964. Carboxypeptidase B (30 min): ,4rg, 0.063; Val, 0.013. Carboxypeptidase B (30 min) followed by carboxypeptidase A (20 min): Arg, 0.066; Val, 0.075; hu, 0.082. T-III-?`b: Gin-Gly-Lm-*Ma-Lys (Rekdues 106 to llO)- Dinitrophenyl end group: Glu. Edman degradation: Stage 1 (86%) Glu, 0.002; Gly, 0.005(l); Leu, 0.007(l); Ala, 0.006(l); (Lys), 0.006(l); phenylthiohydantoin hydrolysis, Glu. Leucine aminopeptidase (10 hours) (60%): Gln, 0.015; Gly, 0.014; (Ala), 0.018; Leu, 0.016. Carboxypeptidase B (3 hours) (667,) : (Lys), 0.018; Ala, 0.005. Carboxypeptidase B and d (5 hours) (1037,) : (Lys), 0.031; Ala, 0.032; Leu, 0.026; Gly, 0.008; Gin, 0.008. Since glutamine was known to be the ?rTHz terminus, carboxy- peptidase digestion provided the above sequence. The basic amino acids in the leucine aminopeptidase hydrolysate were not determined on the analyzer, but lysine was found on paper chromatography. Glutamine, which appeared as serine on the analyzer, was identified by paper chromatography. T-III-,@: Vd--Ala-Tyr (Residues 111 to 1 IS)-This peptide, upon dinitrophenylation, gave DNP-valine. The carboxyl- terminal tyrosine residue is consistent with the chymotryptic- like activity of the trypsin preparation. T - I II - 1%~ Val-Tyr-Lys-Pr~dsn-;l sn-Thr-His-Glu-Gln- Let-Leu-.irg (Residues 114 to I,%)-Fraction T-III-12 gave a single ninhydrin-positive spot on the pcptide map. Dinitro- phenylation of this component gave esclusively valine as the dinitrophenyl end group (2). Leucine aminopeptidase released valine and tyrosine and a smaller, but significant, amount of alanine. (Alanine was found in the hydrolysate of Fraction T-111-12, but was not assigned to Peptide T-III-12 in the pre- vious report (2).) Pronase digestion also yielded alanine in the same relative amount as did leucine aminopeptidase digestion. Peptide T-20 (see Table II) had the same amino acid composition la Residue 98 was deduced to be methionine (14). Free homo- serine was found in the tryptic digest of cyanogen bromide frag- ment D by Peptide mapping and amino acid analysis. 1s The basic amino acids RTere determined by paper electro- phoresis. Approximately 1 eq of arginine was present through all stages. lIThe basic amino acids were not determined. Asparagine, which appeared as serine 011 the amino acid analyzer, was identified by paper chromatography. aa Fraction T-III-12 except that alanine was missing. Free valine and tyrosine were again produced by leucine aminopepti- dase digestion. Peptide T-III&, Val-Ala-Tyr (residues 111 to 113; see above), was found in a relatively low yield (16%), compared with other peptides (30 to 70%) in the tryptic digest of cyanogen bromide Fragment E (see Table II). The same peptide was found (as T-5b and F2,) in the tryptic digest of the nuclease (see Tables 1 and II). The presence of alanine in Fraction T-III-12 u-as therefore interpreted as follows. Fraction T-III-12 contained two peptides, T-III-12a and T-III-12b, which had occupied closely neighboring positions on the peptide map. Peptide T-III-12a had the NH,-terminal sequence Val-Tyr-. Peptide T-III-12b, with the NHz-terminal sequence Val-Ala-Tyr-Val- Tyr-, overlaps Peptides T-111-4~ and T-111-12s. These con- clusions were supported by the following experiment. Fraction T-III-12 was incubated with trypsin for 22 hours and peptide maps were prepared. A new Pauly-positive spot was found at the position on the map corresponding to that of Peptide T-III- 4c!. The further study of the sequence of Peptide T-III-12a was made on both Fraction T-III-12 and Peptide T-20. Carbosy- peptidase B released only arginine. Further incubation nith carboxypeptidase A released 2 moles of leucine. Thus the COOH-terminal sequence was interpreted to be -Leu-Leu-.1rg. Dilute acid hydrolysis with 0.03 N HCl produced free aspartic acid in a yield of 83% and two fragments, T-20-AI and T-20-.111 (see Table VI), which were purified by two-dimensional map- ping. The amino acid compositions of these fragments were (Val, Tyr, Leu, Pro) and (His, Arg, Thr, Glu, Glu, Leu, Leu), respectively. Fragment T-20-AI is evidently the NHs-terminal portion of Peptide T-III-12a. Since leucine aminopeptidase digestion of Peptide T-III-12a released valine and tyrosine reai- dues as described above, the proline residue should not be nest to valine and tyrosine. Thus, the sequence of Fragment T-20-&41 was deduced as Val-Tyr-Lys-Pro. Fragment T-20-AI1 was subjected to timed digestion with leucine aminopeptidase to determine the NHz-terminal sequence. Threonine was released first,, followed by histidine. This is consistent aith the orange color developed by staining Fragment T-20-X11 with cadmium ninhydrin. From the above results, the sequence Val-Tyr-L-s- Pr*(Asp)-(Asp)-Thr-His-(Glu)-(Glu)-Leu-Leu-Arg was de- duced for Peptide T-111-12&. Since the electrophoretic mobility of Peptide T-III-12a iyas basic at pH 6.5, at least two carbosyl groups of four should be amidated. The digestion of Peptide T-III-12a with Prona..e released a very small amount of histidine. Neither glutamic acid nor proline was found. However, valine, tryrosine, lysine, asparagine, threonine, glutamine, and leucine were released. Glutamine and asparagine were id-ntified by ascending paper chromatography M ith 80 y0 aqueous pyridine and subsequent paper electrophoresis at pH 6.5. Since Pronase did not cleave the peptide bond involving the carbosyl group of proline, the released asparagine residue must be adjacent to threonine. The mixed digestion of Peptide T-III-12a with Pronase and leucine aminopeptidase released equimolar amounts of glutamic acid and histidine in addition to all ot.her residues released by Pronase alone. However, proline was not found. These results indicated that the fragment His-Glu remained almost intact during Pronase digestion, as well as Pro-Asp (or Pro-&n). Since no acidic 4746 Partial Sequence of Staphylococcal Nuclease. II Vol. 242, No. 20 TABLE VI Sequence studies on Peptide T-III-lfia 1. Leucine aminopepti- Ala, 0.013;b (Val), 0.093 ; Tyr, 0.051 dase digestion,a 19 hrs; 8641, 2. 1 + carboxypeptidase Ala, 0.009;b (Val), 0.040; Tyr, 0.027; B, 80 min; 130yo Arg, 0.025 3. 2 + carboxypeptidase Ala, 0.007;b (Val), 0.032; Tyr, 0.031; A, B hrs; 110% Arg, 0.019; Leu, 0.055 4. Pronase, 20 hrs; 52% Lys, 0.047; His, 0.005; Arg, 0.092; Thr, 0.@47; Ser,c 0.041; Ala,' 0.019; (Val), 0.097; Met <0.003; Leu, 0.104; Tyr, 0.094 Acid fragmentsd T-20-AI RF~, 0.45; RFB, Lys, 0.031(l); Pro, 0.017(l); Gly, 0.73) 0.005(O); Val, 0.031(l) : Leu, 0.003(O); Tyr, 0.042(l) T-20-AI1 RFC, 0.47; Rpg, Lys, 0.015(O); His, 0.041(l); Arg, O&l) 0.049(l); Asp, 0.005(O); Thr, 0.022(l); Glu, 0.091(2); Gly, 0.005(O); Val, 0.012(O); Leu, O.lOO(2j ; Tyr, 0.018(O) 0 Basic amino acids were not determined. b See the text. c The peak at the serine position was supposedly due to aspara- gine, glutamine, or both (see the text). d Peptide T-20 was used for dilute acid hydrolysis (see Table I). components were found upon electrophoresis of the Pronase digest at pH 6.5, the latter fragment must contain asparagine. Thus the distribution of amide groups in Peptide T-III-12a is as shown above. Pertinent results are summarized in Table VI. T-III-9c: Lys-GEu-Lys (Re