Vol. 241, No. 19, Issue of October 10, PP. 4366-4385, 1966 F'rinted in U.S.A. The Amino Acid Sequence of an Extracellular Nuclease of Staphylococcus aureus I. LINEAR ORDER OF THE FRAGMENTS PRODUCED BY CLEAVAGE WITH CYANOGEN BROMIDE* (Received for publication, March 14, 1966) HIROSHI TANIUCHII AND CHRISTIAN B. ANFINSEN From the Laboratory of Chemical Biology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014 SUMMARY An extracellular nuclease of Staphylococcus aureus, strain V8, was purified to a state of sufficient homogeneity for the study of its covalent structure. The purified enzyme ex- hibits both ribonuclease and deoxyribonuclease activities. The protein contains alanine and glutamine as the ammo- and carboxyl-terminal residues, respectively. Neither cysteine nor cystine is present. Cyanogen bromide digestion of the nuclease yielded five fragments, designated A, B, C, D, and E. These fragments have been purified and analyzed for amino acid composition. Fragments A, B, and C contain amino-terminal alanine, tyrosine, and threonine, respectively. Both D and E have valme as the amino-terminal residue. Homoserine was found in A, B, C, and D, whereas the carboxyl-terminal residue of E was glutamine. Tryptic pep- tides of each of fragments A, C, D, and E have been sep- arated and analyzed for amino acid composition and amino- terminal residues. The tryptic peptides of the nuclease containing methionine have also been isolated and similarly examined. By a consideration of these results and the total amino acid composition of the nuclease, together with exam- ination of tryptic peptide maps, the minimum molecular weight of the nuclease has been calculated to be approxi- mately 17,000, and the cyanogen bromide fragments have been assigned the order A-B-C-D-E. An extracellular nuclease produced during the growth of Staphylococcus aureu.s has been shown to catalyze the hydrolysis of phosphodiester bonds in both ribonucleic and deoxyri- bonucleic acids (3, 4). A constant ratio of specific activities has been observed during the purification procedure, and prepara- tions which appear to be homogeneous on the basis of both chemical and physical tests retain this dual specificity. Chem- ical studies on the amino acid sequence were undertaken because of interest in the structural basis of thii two-fold function and * Preliminary reports of this work have appeared (1, 2). 3 Visiting scientist, on leave of absence from Kyoto University, Faculty of Medicine, Kyoto, Japan because, as is discussed in this communication, the protein has chemical features which may make it of special value in the study of side chain interactions that determine its tertiary structure. The polypeptide is devoid of sulfhydryl groups and disulfide bonds, undergoes a sharp reversible "melting" of native con- formation over a narrow range of temperature, and contains a considerable amount of helical structure on the basis of its optical rotat.ory properties. The polypeptide chain of the highly purified nuclease was subjected t,o cleavage at its four methionyl bonds with cyanogen bromide. The resulting five fragments were separated and, on the basis of amino acid analysis, digestion with trypsin, and end group analyses, were arranged in their proper order along the chain. EXPERIMENTAL PROCEDURE Materials-The filter cake used as the starting material for purification was prepared as described previously (4). Trypsin (Worthington, twice crystallized) was treated with diisopropyl fluorophosphate as described by Potts et. al. (5). Diisopropyl fluorophosphate-t,reated carboxypeptidase A was donated by Dr. J. Potts (5). Samples of calf thymus DNA were gifts of Dr. D. MacD. Green and Dr. M. Nirenberg. Thymine-methyl-3H DNA from Escherichia co& Klz X, was given by Dr. A. Weissbach (6). High molecular weight yeast RNA was obtained from Sigma. Protein crystals made from the purified nuclease de- scribed below were provided by Drs. T. Hazen and A. Cotton. Carboxymethyl cellulose (medium) was obtained from Sigma. Phosphocellulose (Whatman, column chromedia, pll fibrous powder) was obtained from Angel (7.4 meq per g). Sephadex G-50 (medium) was obtained from Phsrmacia (water regain = 5.1 f 0.3 g per g; particle size, 100 to 270 mesh). Dowex AG 5OW-X2 (200 to 400 mesh) was obtained from Calbiochem. All of these ion exchange materials were washed with distilled water, 1 N NaOH, distilled water, 3 N HCl, and distilled water. Other chemical reagents were analytical grade. Cellophane dialysis tubing (I&king) was heated at 80" for 72 hours or boiled in 1 y0 NaHCOt for 10 min' to decrease pore size. Assay of Enzymic Activity-The methods for the determination of ribonuclease and deoxyribonuclease activities, and the def- 1 R. Suriano, personal communication. 4366 Reprinted with permission by the U. S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service Issue of October 10, 1966 H. Taniuchi and C. B. Anfi,nsen 4367 i&ion of units, were described pre\-iously (4). The concentra- wtivities. DNase activity with DNAVH as sub&ate was deter- tion of protein was determined by measuring a.bsorba,nrr nt 280 mined erccrding to the methcd cf Weissbach and Korn (6). nip. The activit,y of the nuclease is espresred in RNase units Yeast RXA was used as inhibitor of DNase activity, and calf unless otherwise specified. One absorbance unit at, 280 rnp was thymus 13N.1 was added as unlabeled carrier DXA after the assumed to be 1 mg of prot,ein per ml for the ce,lculation of specific incubaticu. 6.t 8.0 7.0 6.0 25.0 z g 4.0 z 5 g 3.0 m : 2.0 1.0 0 3 I I I I I I I I I 600 1200 1800 8000 6000 z- =i z 4000 $ 2 5 P 2000 3 EFFLUENT VOLUME, ML I I I I I I I I I 3000 E -r zoo0 -rJ m I) z r z h 1000 2 0 EFFLUENT VOLUME, ML FIG. 1. Chromatographic purification of the nuclease on carboxymethyl cellulose in the first. fractionation (upper) and the second fractionation (lower). The crude extract obtained from 100 g of filter cake (see Table I) was applied to the column in the first frac- tionation. In the second fractionation the fraction containing the nuclease (410,000 units) wzs applied. O--O, absorbance at 280 ma; o---O, RNase activity; A---& pH. 4368 l'artial Sequence oj a Staphylococcal ;Vwlease. I Vol "11 To 19 .- )A . Puri$cation of B&ease-All procedures were performed at 4'. One hundred grams of filter cake were routinely used for each preparation. The cake was dissolved in 200 ml of 0.05 M sodium phosphate buffer, pH 6.1, and the solution was dialyzed against 6 liters of the same buffer for 36 hours with four changes. The activity lost through the cellophane tubing during dialysis was about 1.5% of the total activity. The dialysis solution was centrifuged in a Servall Superspeed centrifuge at 4000 rpm for 10 min. The supernatant solution (crude est,ract ) was applied to a carboxymethyl cellulose column. The unadsorbed activit) was 77, of the total activity applied. The procedure for the first fractionation on the carboxymethyl cellulose column (4 x 30 cm) was essentially as described previously (4) except that a two- chamber Varigrad was used to produce a linear gradient, with 1 liter of 0.05 M sodium phosphate, pH 6.1, and 1 liter of 0.15 M potassium phosphate, pH 7.5, as the first and second buffers, respectively. The elution was carried out at a flow rate of 45 to TABLE I Pwi$cation of n&ease from S. aureus Vga Enzyme fraction Crude extracts First carboxymethyl cellulose fraction. Second carboxymethyl cellulose fraction. 1 ~~ __~ ml F70 1,160 39.7 2.9 776,OCW 1.04 530 1,650 1.82 920 877,OOQ 1.65 103 4,750 2.372,OOO 489,000 1.70 a One hundred grams of the filter cake were used as starting material. b The total activity in the crude extracts was variable, pre- sumably due to the extent of removal of inhibitors by dialysis. The total number of units obtained from 100 g of the filter cake was occasionally as high as 2 million. 60 ml per hour. Fraction? were collected every 10 to 15 min. L% typical elution diagram is .shown in Fig. 1.1. The fractions giving specific activities of more than 900 units per mg Tvc're pooled. After adjustment to pH 6 by the addition of phosphoric arid, the pooled fraction was diluted with distilled water to 2.5 times its volume (approximat,r final concentration, 0.05 M phosphate). The acidified solution was applied t,o the ;-ccond carboxymethgl cellulose column (2.2 x 7,5 cm) which was equilibrated with 0.05 M sodium phosphate buffer, t)II 6.1, and gradient elution was carried out as with the first column. Fractions were coll&ed every 30 min at a flow rate of 16 ml per hour. A typical diagram of the second fractionation is shown in Fig. IB. The fractions with constant, specific activity were combined. The combined solution (approsimatley 100 ml) was dialyzed against 6 liters of distilled water with two changes for a total of 20 hours. After lyophilization, the dried material was dissolved in 20 ml of dis- tilled water. The concentrated solution was again dialyzed against 6 liters of distilled water with four changes for 36 hours to remove the last traces of buffer salt. The dried material obtained by 1yophilizat)ion was used in the studies described below. Table I summarizes the results of this purification procedure. Fig. 2 shows the ultraviolet absorption spectrum of the purified nu- clease. Determination of Ultraviolet d bsorbance-A sample of the purified nuclease was dried over P205 under reduced pressure at 25" for 3 days to constant weight. An aqueous solution of the `dried protein, containing 1 .O mg per ml, showed an absorbance at 280 rnp of 1.16. Electrophoresis and Ultracentrifugal zlnalysis-The procedures employed were essent,ially the same as those described by Craven, Steers, and Anfinsen (7). Glycine-HCl buffer of 0.1 ionic strength, for electrophoresis, was made according to the method of Miller and Golder (8). Cyanogen Bromide Digestion-The following modification by Steers et al. (9) of t,he Gross and Wit,kop procedure (10) was utilized. Protein preparat,ions were dissolved in 70yG formic acid 260 270 280 290 n-9 FIG. 2. The ultraviolet absorption spectrum of the nuclease in 0.15 M sodium phosphate buffer, pH 7.2 (see the text). Issue of October 10, 1966 H. Taniuchi ank C. B. Anjinsen 4369 to make a 1% solution. A 30.fold molar excess of CNB? per methionine residue.was added. The reaction mixture was kept at 25" for 20 hours. Distilled water was then added (2 volumes), and the diluted solution was lyophilized. The reaction pro- ceeded to the extent of more than 90yc as judged by amino acid analysis. Digestion with Trypsin--Nuclease and separated cyanogen bromide peptides were incubated with trypsin (1 y0 by weight of substrate) at 37" for 3 hours. The pH during digest'ion was ad- justed to 8.0 with IKH*OH. For amounts of substrate less than 1 mg, 0.05 M ammonium bicarbonate, pH 8.0, was used as buffer. Subtilisin Digestion-One hundred micrograms of peptide were dissolved in 15 /*l of 0.05 M ammonium bicarbonate, and the solution was adjusted to pH 8 (using phenol red) by the addition of 15 pl of 0.01 ?VI NHJOH if necessary. Subtilisin (Nagarse) (1 mg in 5 ,ul of 0.05 M ammonium bicarbonat,e) was added. The reaction mixture was incubated at 37" for 3 hours and lyophilized. Peptide Mapping-Peptide maps were performed as described by Katz, Dreyer, and hnfinsen (11). A small drop of phenol red was added at the origin as a reference standard in the chromato- gral'hic dimension as used by Canfield (12). Free lysine which was released from the nuclease or cyanogen bromide peptides served as a reference standard for the electrophoretic dimension. The peptides were located by spraying with 0.25% ninhydrin in ethyl alcohol. Tryptophan-, arginine-, histidine- and tyrosine-, and methioninc-containing peptides were located on the map with Ehrlich, Sakaguchi, l'auli, and platinum iodide reagents, respec- tively (13). For the preparation of certain purified peptides, aqueous solutions of the trgpsin digest, containing 0.1 to 0.2 pmole of each peptide, were applied on What,man 3MM filter paper. After location by light staining with 0.025%;; ninhydrin solution at 25", spots were cut out and peptides were eluted with 50% aqueous pyridine at 25". The eluates (5 to 10 ml for each spot) were ly- ophilized. The dried materials obtained were dissolved or sus- pended in 0.6 ml of distilled water and frozen. Polyacrylanlide Gel Electrophoresis-Polyacrylamide gel electro- phoresis was performed at 4-6" with 7.5yc "standard gel" and 15~~ "4.3 gel" as described in t'he manual supplied by the Canal Indust,rial Corporation. The direction of the electrical current was reversed when necessary. Samples of 0.1 to 0.3 mg of pro- tein (or peptides) were applied and run at a const'ant current of 3 to 4 ma per tube for 3 to 5 hours. Paper ElectrophoresisPyridinium acetate buffers, pH 3.6 and 6.5 (ll), were used on Whatman No. 3MM paper. Applied voltage was 2500 volts for 80 min. Preparative Electrophoresis-A Brinkman continuous flow electrophoretic separator was employed for preparative electro- phoresis. The samples to be applied were dissolved in 0.01 M ammonium bicarbonat,e, pH 8.7, to make 1 to 2% solutions. The same buffer was utilized for making the buffer curtain. Temper- ature was maintained at 6". The applied voltage was 2000 volts, and the current was 120 ma. The minimum rate of dosage of the sample was used. Phenol red was added to the sample solution as an internal electrophoretic reference material. Gel Filtration of Cyanogen Bromide PeptidesAll procedures were carried out at 25". A column of Sephadex G-50 (3 X 260 cm) was poured from a thin slurry, which was allowed to settle 2 The abbreviat,ions used are: CNBr, cyanogen bromide; UNP-, 2,4-dinitrophenyl-; IIFB, 1-fluoro-2,4-dlnltrobenzene. FIG. 3. Schlieren pattern of the purified nuclease obtained in the Spinco model E ultracentrifuge. The photograph was taken 200 min after reaching a speed of 59,780. The enzyme had been previously dialyzed against 0.05 M Tris-HCl buffer, pH 6.9, con- taining 0.1 M NaCl and 0.001 M ethylenediaminetetraacetic acid. Protein concentration was 6.1 mg per ml. without extra packing pressure. The column was equilibrated with 0.01 N acetic acid containing 0.1 7, ammonium acetate.3 The sample, an 87, solution of the peptide mixture in the same buffer, was applied to the column and washed in with three l-ml applications of the buffer. Elution was carried out with the same buffer at a flow rate of 9.6 ml per hour. Fractions were collected every 30 min and assayed by measuring absorbance at 280 rnp. Suitable aliquots (15 to 100 ~1) were subjected to alkaline hydrolysis, and hydrolysates were examined by the ninhydrin color reaction according to the method of Hirs, Moore, and Stein (14, 15). Peptide Separation on Dowex 5&The procedures described by Canfield and Anfinsen (12, 16) have been employed. A Dowex AG 5OW-X2 column, 0.9 x 93 cm, equilibrated at pH 3.8, was used. Gradient elution was performed at a flow rate of 23 ml per hour with a Sigma motor pump, model T8. The buffer volume in each chamber of the Varigrad was 150 ml. Fractions were collected every 10 min. Fractions comprising each peak were pooled and lyophilized. The dried materials obtained were dissolved in 0.6 ml of distilled water and frozen. 3 L. C. Craig, Rockefeller University, personal communication. Partial Sequence of a Staphylococcal N&ease. I Vol. 241, h'o. 19 Further Puri$cation oj Peptides--All of the tryptic peptides that separated and the subtilisin peptides derived from one of them were examined for purity by t'wo-dimensional mapping as described above. Larger aliquot,s of the fractions that contained more than one t,ryptic peptide were separated by paper electro- phoresis at pH 3.6 or chromatography of wide bands on Whatman 3MM. The purified peptide components were eluted with 50% aqueous pyridine and lyophilized. Aliquots of the pooled fractions cf the c?-enogen bromide pep- tide mixture were examined by paper electrophoresis (2500 volts) at pH 6.5 for 90 min. Large1 aliquots of each cyanogen bromide peptide fyaction w-ere applied as wide bands. After location by staining cf a side strip with ninhydrin the components were eluted with 50y0 aqueous pyridine and lyophilized. dmino ilcid dnalysis-Samples were hydrolyzed in 0.5 ml of con&ant boiling HCl in evacuated, sealed tubes at 110" for 20 hours. After removal of HCl by rapid evacuation over NaOH pellets, amino acid analyses were performed by the method of Spackman, Moore, and Stein (17) on a Spinro model 120 amino acid analyzer equipped with an Infctronics model CRA-1OA integrator. The recorder of the analyzer was equipped with a 4- to 5-mv resistor card (Minneapolis-Honeywell, kit part No. FII x 4. Moving boundary electrophoresis of the purified nuclease, obtained in the Perkin-Elmer model 238 electrophoresis appara Prott $1 concentrat,ion was 6.1 mg per ml, in glycine-HCl buffer (0.1 ionic strength), pH 3.51 (see "Experimental Procedure"); ( ducti Ivity, 6.2 mmhos. The photograph was taken after 115 min at a constant voltage of 100 volts. A similar symmetrical schlic pattc !rn was obtained with free electrophoresis of the purified nuclease in 0.05 M citrate, pH 7.5, which was kindly carried out by Willi am R. Carroll. :on- :ren Dr. Issue of October 10, 1966 H. Taniuchi and C. B. iZn,finsen 437 1 in FIG. 5. Comparison of purity of the crude extracts (Crude), the purified nuclease (%nd CLW), and a mixture of these. The total num- ber of nuclease units applied to gels was much higher with the purified nuclease than with the crude extracts so that separable im- purities, if any, could be readily seen. The gels used here were standard gels (see the text) with both normal and reversed polarities. 365928-999). A series of analyses on the acid hydrolysates of the nuclease containing between 0.01 and 0.1 Mumole of each amino acid showed that analyses at these low levels were reproducible to within 10%. The analytical values shown (see Table V and Reference 7) have, therefore, been reported to three significant figures in many cases. The procedure of Dreyer and Bynum (18) was also employed for amino acid analysis in some cases as reported by Canfield (19). A voltage gradient of 25 volts per cm was applied for 2 hours at 44-48". Determination of Cysteine and Cysline-The nuclease, oxidized with HCOOOH according to the method of Hirs (20), was ana- lyzed for cysteic acid. Reduction and subsequent alkylaticn with iodoacetate-W (21) was also applied to the nuclease prepa- ration. Radioactive iodoacetate (specific activity 16,500 cpm per pmole; New England Nuclear) was employed. The reaction mixture contained, in a total volume of 10 ml, 4.8 g of urea, ap- proximately 17 mg cf nuclease, 50 ~1 of 2-mercaptoethanol, and 0.1 M Tris buffer, pH 8.0. One drop of phenol red was added to furnish internal monitoring of pH. Tryptophan-The spectrophotometric methods of Goodwin and Morton (22) and Bencze and Schmid (23) were used to determine the ccntent of tryptophan, with the use of 0.1 N NaOH solutions of the nuclease. Alkaline hydrolysis of the nuclease was performed in evacuat,ed, sealed tubes at 110" for various lengths of time by a modificat,ion of the method of Lugg (24). The hydrolysate was analyzed with the amino acid analyzer. The tryptic peptide containing trypt,ophan was detect,ed on two- dimensional pel)tide maps with Ehrlirh's rea.gent. The Ehrlich- positive tryptic peptide was eluted as described above and di- gested further by subtilisin. The resulting fragments were sep- arated on two-dimensional peptide maps and located by staining with Ehrlich's reagent. Tctal Amide Nitrogen Determination-The amide ammonia of the nucleate was determined a.ccording to the method of Dr. Robert Hill." Dried nuclease, 6.6 mg, was dissolved in 0.60 ml of constant boiling HCl. Aliquot's of 0.10 ml each were placed in hydrolysis tubes, and 0.20 ml of distilled water was added to each tube. Acid hydrolysis was performed in evacuat,ed, sealed tubes at 110" fcr various lengths of time. Hydrolysis was stopped by immersing tubes in powdered Dry Ice. As a refer- ence, a 0.10.ml aliquot was subjected to ccmplete hydrolysis by the method described above and analyzed on the amino acid analyzer. NH&rminal Residues by Dinitrophenylation-The NHz-termi- nal residues of the nuclease and of the cyanogen bromide peptides were quantitatively determined by the modified Levy procedure (25). An unpublished modification, kindly made available by E. Bynum, W. Dreyer, and C. Bennett, was applied with tryptic pep- tides as follows: samples of 0.05 to 0.1 pmole, dissolved in 20 ~1 of water, were placed in test tubes (1.5 x 15 cm), and 200 ~1 of 1 M t,rimethylamine acetate, pH 9.7, were added. One volume of l-fluoro-2,4-dinitrobenzene was dissolved in 20 volumes of anhydrous ethyl alcohol. Aliquots of 5 ~1 of the fluorodinitro- benzene solution were mixed with the sample solutions. The 4 R. L. Hill, Duke University, personal communication. 4372 Partial Sequence 0s a Staphylococcal Nuc2ease. I Vol. 241, No. 19 reaction mixtures were incubated for 3 hours at 37" and lyophil- chIoroform-formic acid (20: 1). The chromatographic plates ized. The dried mixtures were put in a vacuum oven over NaOH were made with MN-cellulose powder 300 (Machery, Nagel and pellet, and sublimation was performed at 60" for 4 hours. The Company). The spots were located with iodine azide spray residues were estracted with et,her and again placed in a vacuum (27). The NHz-terminal residues of tryptic peptides and of a oven at 60" for 3 hours. The dried materials were dissolved in small cyanogen bromide fragment of the nuclease were also de- 0.5 ml of con&ant boiling HCl and hydrolyzed in evacuated t,ermined by the following modified Edman degradation (28-30). sealed tubes at 110" for 16 hours. The hydrolysates were sub- The reaction mixtures consisted of 0.2 t,o 0.d pmole of peptide in jetted to two-dimensional chromatography by the modified Levy 50 ~1 of distilled water, 50 ~1 of 27, phenylisothiocyanate in py- procedure. All manilmlations were performed in a darkened ridine, and 5 ~1 of 25yG aqueous trimethylaminr. The mixture laboratory. The two-dimensional chromatographic separation was placed in a conical 8-ml glass tube fitted with a standard of ether-soluble 11X1'-amino acids was achieved with the tert-amyl taper glass plug which had two openings, one stoppered with a alcohol-l M NH&H (4 : I) system in the first direction and 1.5 M rubber vaccine port and one with a ground glass stopper. After potassium phosphate, pH 6.0, in the second (19). The water- t,he tubes were flushed with Orfree nitrogen through a hypoder- soluble DNP-amino acids were extracted with n-butyl alcohol mic needle for 2 min, the vessel was sealed and incubated at 40" and examined by paper rlcct,rophoresis at pH 6.5. for 4 hours. The mixture was extracted five times with 0.5 ml Edman Degradation-The Edman procedure modified by of thiophene-free benzene under nitrogen. The upper layers Margoliash (26) was employed for the determination of the NH2- were discarded following each extraction after centrifugation in terminal residue cf the nuclease. Phenylthiohydantoins of an International clinical cent,rifuge. The residual solutions were amino acids were identified by thin layer chrqmatograms with rapidly taken to dryness under reduced pressure. Cyclization two different solvent systems; chloroform-methanol (9:l) and of the phenylthiocarbamyl.derivativei was performed by adding Origin 1, Skmdar gel FIG. (i. Electrophoretic pattern of crystalline nuclease on acrylamide gel elect,rophoresis (standard gel). A, needles; B, hexagonal crystals; &4i~, a mixture of both samples. I'lectrophoretic behavior of the purified nuclease at pH 4.3 (see the text) is labeled "4.3 gel." Issue of October 10, 1966 H. !&miuchi and C. B. Anjinsen 4373 0.3 ml of trifluoracetic acid an&incubating the resulting solution at 40" for 15 min under nitrogen. Aft,er having been dried under reduced pressure, the residue was extracted four times with 0.5 ml of et,hylcne chloride. The solvent was removed by Pasteur pipette. The extracts and the residual material were taken to dryness under reduced pressure over NaOH pellets at 25". The dried extracts were dissolved in 0.5 ml of 307, aqueous alcohol, adjusted to pH 1 by the addition of concentrated HCl, and in- cubated at 80" for 1 hour for the cyclization of the thiazolinone derivative, and the solutions were then lyophilized. The dried materials were dissolved in 0.3 ml of 0.1 N NaOH and hydrolyzed in the sealed, evacuated tubes at 110" for 16 hours to form the free amino acids from phenylthiohydantoin derivatives. The dry residual material, containing the protein or tryptides in which the original NH, terminals had been removed, was dissolved in 200 ~1 of 5070 aqueous pyridine. Two aliquots of 10 ~1 were removed and placed in a hydrolysis tube. The aliquots and the residual solutions were lyophilized. The aliquots were then hy 4 rolyzed in evacuated, sealed tubes with 0.5 ml of constant bo' ing HCl at 110' for 16 hours. Amino acid analyses of the h$drolysates were performed with the automatic amino acid analyzer, and by paper electrophoresis by the method of Dreyer ' and Bynum w described above. Carboxyl-terminal Residue Determination-A solution of car- boxypeptidase A, prepared as described by Potts et al. (5), was employed. When serial carboxypeptidase digestion was per- formed to obtain information of the carboxyl-terminal sequence, 0.1 to 0.5 pmole of the nuclease or peptide was dissolved in dis- tilled water to make a 2% solution (w/v). A one-tenth volume of carboxypeptidase A solution was added. The incubations were performed at 25" and 37". Aliquots of 0.1 volume of the reaction mixture were removed at timed intervals. These were immediately acidified with 5 N acetic acid to yield 0.3 N acetic acid and left at 25" for 30 min. Five microliters of the acidified aliquots were removed for assay of enzymic activity, and the residual solutions were lyophilized to be analyzed for released amino acids with the amino acid analyzer. Ascending chromato- grams at 25" on Whatman No. 1, with 80% aqueous pyridine as solvent, were also used for qualitative analysis. Determination of Sugar Content-The phenol-sulfuric acid method of Dubois et al. (31) was used to examine the nucle- ase for carbohydrate, with n-glucose as a standard. Determination of Radioactivity-A Packard Tri-Carb liquid scintillation spectrometer, equipped with an automatic sample changer, was utilized to determine the radioactivity of samples dissolved in Bray's solution (32). The radioactivity of 1 ab- sorbance unit of DNA-3H was found to be 14 x 10' cpm. RESULTS Purity oj Nuclease Preparation-The purified nuclease de- scribed above appeared to be homogeneous on the basis of ultra- centrifugation,5 free boundary electrophoresis,5 and acrylamide gel electrophoresis (Figs. 3, 4, and 5). The two small compo- nents previously observed during free boundary electrophoresis (4) had been removed. When the preparation was examined by electrophoresis on polyacrylamide gel, two bands were occasion- ally observed, even with crystalline preparations, as shown in Fig. 6. The fast running band was dominant on standard gel L These experiments were performed with the cooperation of Dr. R. Suriano. and the slow one on 4.3 gel. To examine the nature of this phenomenon, a nuclease preparation which showed such hetero- geneity was studied in the Brinkmann preparat,ive electrophoresis apparatus (Fig. 7). Aliquots taken across the effluent curtain were subjected to polyacrylamide gel electrophoresis. Partial separation of the two components was achieved. The almost homogeneous materials in tubes 12 and 24 retained their electro- phoretic behavior when rerun on standard gel (Fig. 8), but both yielded a mixture of the fast and slow components when run on the 4.3 gel. Chromatography of material from tube 12 on carboxymethyl cellulose columns (as described under "Experi- mental Procedure") also produced both the fast and slow com- ponents in proportion to that shown by the original nuclease preparation. Thus the two forms of the nuclease appear to be interconvertible. The basis for this interconversion is unknown at present. The two forms were examined by amino acid and carbohydrate analysis (Table II), pept'ide mapping of trypsin digests, poly- acrylamide gel electrophoresis of CNBr digests, and measure- ments of specific activities against RNA and DNA. None of these procedures showed significant diffeiences. Together with the end group analyses, which indicate single NH2- and COOH- terminal residues, and the partial sequence analysis described below, these data indicate that the nuclease preparation is homo- geneous in the covalent sense and adequate for structural studies. RNase and DNase Activities-As illustrated in Fig. 9, the linear ranges of activity with respect to enzyme concentration are limited for both RNase and DNase activity, particularly the latter. Furthermore, the activities are sensitive to low levels of certain anions such as citrate and phosphate (Table III). Com- parisons of the ratios of the two activities during purification therefore required estimations at several levels of enzyme concentration under carefully standardized conditions. The constant ratios of the activities previously found during the purification procedure (2) suggested that both RNA and DNA serve as substrates for the nuclease, and this conclusion is sup- ported by the data shown in Fig. 10 where constant relative activities were observed across the entire effluent peak in a typical `d )JLfL- + 0 IO 20 30 40 50 FRACTION NUMBER FIG. 7. Preparative eleetrophoresis of the purified nuclease. Twenty milliliters of the nuclease solution (460 mg of protein) were applied at the port indicated by the arrow. Other details are given in "Experimental Procedure." 12 14 16 18 20 22 2 Origin Mix. FIG. 8. Upper, polyacrylamide gel electrophoresis pattern (standard gel) of fractions from preparative electrophoresis (Fig. 7). Suit- able aliquots from alternate tubes were lyophilized to be used for the electrophoresis. Lower, electrophoretic behavior on polyacryl- amide gel of two forms of the nuclease separaf,ed by preparative electrophoresis. A and R show the mobilities on standard gel, of samples obtained from tubes 12 and 24, respectively (see Fig. 7); and Miz represent,s the pattern obtained from a mixture of the two. 4374 Issue of October 10, 1966 H. Taniwhi and C. B. Anjinsen 1375 TABLE II TABLE III Comparison of a&no acid compositions and sugar contents of two components obtained etectrophoreticallg front nuclease preparation Efiect of phosphate and citrate ions on RXase and DNase actiuity - Activity Addition DNase RNase % oj conlrol % oj confrol -Norma.......................... 100 100 Phosphate, 5 X 1W M. . 117 105 Phosphate, 5 X lO+ M. . 228 Citrate, 5 X 1W M. 337 239 Citrate, 5 X 10-a M. . . . . 542 184 a The concentrations of contaminating phosphate ions from the original enzyme solution were 1.2 X 1W5 M and 5 X 1W8 M in the reaction mixtures for the DNase and RNase assays, respectively. ___. Lys .............. His .......... : .. Arg ............. ASP ........... Thr 1: ........... Ser ................ Glu ............... Pro ............... Gly ................ Ala ............. cys. ............... I Val .............. Met ................ Ile. ................ Leu ............... Tyr ................ Phe ................ Glucose equivalents' Component I" fimolc 0.270 0.030 0.055 0.166 0.122 0.062 0.197 0.071 0.113 0.173 0.000 rmolc 70 15..7 1.7 3.2 9.7 7.1 3.6 11.5 4.1 6.6 10.1 0.0 pm&? 0.303 0.040 0.065 0.187 0.127 0.062 0.240 0.087 0.138 0.185 0.000 0.116 6.7 0.121 0.026 1.5 0.036 0.070 4.1 0.065 0.132 7.7 0.155 0.076 4.4 0.088 0.040 2.3 0.041 mole/mole profcin 0.23c - Component II" rmle % 15.6 2.0 3.4 9.6 6.5 3.2 12.4 4.5 7.1 9.5 0.0 6.2 1.9 3.4 8.0 4.5 2.1 0 The fast and slow running components on standard acrylamide gel are designated as Components I and II and were obtained from tubes 12 and 24 in Fig. 7, respectively. 6 See "Experimental Procedure." c The amounts of protein used for analysis were 0.060 and 0.12 pmole for Components I and II, respectively. ENZYME CONCENTRATION FIG. 9. RNase and DNase activities of the nuclease as a func- tion of enzyme concentration. The methods of assay are described "Experimental Procedure." O--O, RNase; O---O, iiNat3e. =.O~ E" 8 8 N 2.0- jq % 5 5 E? s: s I.O- O+-- I boo 1200 1500 I800 3000 2000 5 7 3 I 1000 I- O EFFLUENT VOLUME, ML FIG. 10. RNase and DNase activities of chromatographic frac- tions of the nuclease from ,carboxymethyI cellulose. O---O, absorbance at 280 w; 0- - -0, RNase; A-.-.& DNase. The procedure for chromatography was the same as that described for the second fractionation on carboxymethyl cellulose in "Experi- mental Procedure." Approximately 250 mg of the protein were applied. Aliquots of the fractions comprising the peak in ab- sorbance at 280 mr~ were diluted lOO-fold with 0.05 M sodium phos- phate buffer, pH 6.1. From each diluted solution, 5- and 10.~1 aliquots were incubated for assay of DNase and RNase activi- ties, respectively. The values obtained were plotted without correction for the effect of enzyme concentration (see Fig. 9). The low specific RNase activity is probably due to the nature of the RNA substrate used in this experiment. chromatographic experiment. These observations, together with the evidence for the purity of the nuclease discussed above, strongly suggest that a single enzyme is responsible for both en- zymic activities. Competitive inhibition tests of DNase activity in the presence of added RNA gave result's consistent with this conclusion (Table IV) and confirm the results found with another staphylococcal nuclease preparation by Alexander, Heppel, and Hurwitz (3). Amino Acid Composition-The results of analyses of timed hydrolysates are summarized in Table V. The half-cystine content, previously reported to be zero, was checked by anal- -137(i I'nrlicll Sequelzce f!/' a Ai'taphylococcal ~l~uclease. I \'()I. 211 ) so. 19 TABLE I\ value of 7 rwiduc+ of tyrosinc Iwr molwulc (Tahl~ VII). This l?flect of It.v.4 on D2Yccrr nctitit:c/ was checked further by staining pcf~title maf)s of tr!-pain and cyanogm bromide dig&s ivith Ehrlich'n reagent,. Both of tht Concentration of RNA I Concentration of DNA-JH DNase activity digests shoned only a single Ehrlirh-fmsitive component. The absorbance rrnifslml cPm Ehrlich-positive tryptic peptide was isolated and subjected t.o 0 0.25 3535 further digestion with subtilisin as described above, giving t,wo 0 0.75 0700 fragments. Only one fragment, with a high RF value on the pa- 90 0.25 1387 per chromatogram, showed a positive reaction with Ehrlich rea- 90 0.75 4222 gent. These results indicat,e that 1 residue of tryptophan is present per molecule of the nuclease. TABLE V Residue I- Amino acid composition of nuclease Amino acid content after acid hvdrolvsis foP I I AVerage 17 hrs 34 hrs 57 hrs Calculated moles per 3 moles of phenylalanine6 jmuh Lys. ............. His. ............. Arg ............ Asp ............ Thr ........... Ser. ............. Glu ............. Pro. ............. Gly ............. Ala. ............ cys I Val Met ...... ...... lle .............. Leu ............. Tyr ............. Phe ............. Trp ............ 0.148 0.144 0.148 0.143 0.024 0.022 0.024 0.022 0.039 0.038 0.039 0.036 0.113 0.113 0.112 0.112 0.077 0.075 0.078 0.076 0.040 0.037 0.038 0.039 0.145 0.148 0.154 0.144 0.041 0.041 0.040 0.036 0.064 0.064 0.064 0.062 0.113 0.116 0.112 0.112 0.000 0.004 0.000 0.000 0.069 0.068 0.076 0.071 0.071 0.077 0.074 9.2 0.02B 0.028 0.029 0.027 0.026 0.028 0.028 3.5 0.038 0.038 0.042 0.038 0.040 0.040 0.040 5.0 0.091 0.091 0.098 0.088 0.090 0.094 0.093 11.6 0.049 0.049 0.054 0.049 0.052 0.054 0.053 6.6 0.023 0.018 0.025 0.021 0.025 0.023 0.024 (3.0) 0.000 0.000 0.000 0.000 0.000 0.000 0.000 16 pwlc 0.138 0.150 0.023 0.025 0.038 0.039 0.105 0.108 0.073 0.075 0.032 0.033 0.144 0.148 0.039 0.043 0.063 0.063 0.110 0.114 0.000 0.000 pmole 0.147 0.024 0.038 0.117 0.077* 0.041* 0.146 0.041 0.063 0.115 0.000 18.4 (21.4c) 3.0 4.8 14.6 9.G 5.1 18.3 ,5.1 7.9 14.4 0.0 0 The results of duplicate analyses are given (see the text). b Three tryptic peptides containing phenylalanine were obtained from tryptic digests of cyanogen bromide fragments by separation on Dowex 50 columns (see the text). c The lysine content calculated in this series of analyses was lower than the usual observed value, since the standard lysine gave an unusually high constant. The value shown in parentheses, which was obtained from analyses of 20-hour hydrolysates of several different samples of the nuclease, is considered a better value. * Corrected for slight destruction during hydrolysis. 8 See the text. ysis for cysteic acid after performic acid oxidation and by The amide-NH3 content, determined as described above, was radioactive measurements after alkylation with W-iodoacetic shown to be 11 moles per mole of nuclease, based on an assumed acid. Cysteic acid levels were less than 0.08 mole per mole of value of 3 residues of phenylalanine per mole (Table V). nuclease (Table VI). Although approximately 1 mole of iodo- acetic acid-l-W was bound per mole of nuclease, subsequent experiments demonstrating slight alkylation of e-amino groups of lysine and imidazole nitrogens of histidine residues appear to account for this radioactivity. Furthermore, the direct analysis and sequence determination of tryptic fragment9 of the nuclease, accounting for the total amino acid content of the protein, indi- cate the complete absence of half-cystine residues in the poly- peptide chain. The amino acid analyses permit the calculation of a minimum molecular weight for the protein of approximately 17,000.7 Preliminary studies on the amino acid content of the separated tryptic peptides are in good agreement with this figure, as de- scribed below. Both spectrophotometric methods described under "Experi- mental Procedure" indicated the presence of approximately 1 mole of tryptophan per mole (Table VII), based on an assumed 6 To be published NHt-terminal Residue-The DFB method yielded alanine ex- clusively as the NH*-terminal residue in a yield of 80 to 90%. The. phenylthiohydantoin derivative of alanine, obtained in a yield of 90% by the Edman procedure, was identified in two dif- 7 The results of high speed equilibrium studies in t,he ultracen- trifuge, together with determinations of partial specific volume in density gradiems, yield a molecular weight. value in agreement, with this number within experimental error; J. Heins, R. Suriano, H. Taniuchi, and C. Aufinsen, unpublished results. frrcrlt, 5r,lvc>Ilt, \!.>l t'lllc: (ill lllill layer ~hl,onlrttclg~aIn~. Alkaline h>-drol>-sib of the intclrmcdiatc thiazolinone, formrtl by cyclization of t.he original l)hcr1~lthiocsrt~amyi nuclease, yielded alaninr, ~~itli a trace amount of glycine upon amino acid analyhii. COOKterminal Residlte-Treatment with carboxypeptidase A liberated glutamine in a yiclld of approximately 80y0 after incuba- tion at both 25" and 37" for 24 hours. Glutamine was identified qualitatively on arcending chromatograms with SOFO pyridine as solvent (RF, 0.21) (asparginr, R F, 0.14; serine, 0.35). No other amino acids were detected either on t)he automatic amino acid analyzer or on ~)aper chromatograms. After 24 hours of incuba- tion with the enzyme at 25" and 37", respectively, 85 and 7Syc of the specific enzymic activity of the nucleasc remained. Peptide Maps-Fig. 11 .shows a peptide map obtained wit.h a mist.urr of peptides produced by trypsin digestion of the nuclease at 37" for 3 hours. Major spots, numbered F2 to F25, were highly reproducible from preparation to preparat.ion. Only F25 gave a positi\,e Ehrlich reaction for tryptophan. Components FITi, Fli, FM, and F19 were clearly bleached by the platinum iodide test for methionine. F17 and F19 also gave a posit,ive Sakaguchi test for argmme. F2 produced the pink color char- acteristic of histidine residues when treated with the Pauli re- agent. F4 occupied the same position as free lysine as deter- mined by a mixed pept,ide map of an eluted sample of the peptide with this amino acid. The components numbered F15, F17, F18, and F19 were eluted from eight peptide maps after location by light staining wit,h ninhydrin, the individual eluates were pooled, and samples were hydrolyzed for automatic quantitative amino acid analysis. Aliquots were also submitted to end group analysis by the DFB technique as described under "Experimental Procedure." These results are shown in Table VIII. The four peptides that gave positive reactions with the platinum iodide met,hod con- tained methionine as expect~ed and account for the 4 residues of this amino acid indicat'ed by the amino acid analyses shown in Table V. When, as described below, peptide maps were prepared with trypsin digests of CNBr-treated nuclease, the four methio- nine peptides had disappeared and several new peptides were found as expected. Amino acid analysis of acid hydrolysates of samples F2 and F4 eluted from the paper by t,he same method as above indicated the compositions (His, Pro, Lys) and (Lys), respectively. Cyanogen Bromide Fragment-If the nuclease contains 4 residues of methionine, five fragments should be formed after cleavage with cyanogen bromide. End group analysis by t,he DFB method of CNBr digests showed t,he presence of 2 moles of NH&erminal valine and 1 each of alanine, threonine, and tyro- sine per mole of nuclease (Table IX). The yields of the latter two DNP-amino acids were unaccountably low in this experiment but have approached 1 residue per molecule in others. Among a variety of methods tried, the most successful for the separation of the cyanogen bromide cleavage products was gel filtration on Sephadex G-50, the results of which are summarized in Fig. 12. The over-all recovery of material applied to the column (based on absorbance at 280 mN) was 65%. An aiiquot of every sixth effluent sample was examined by paper electrophoresis at pH 6.5. On the basis of these qualitative tests, fractions were pooled as shown in Table X and lyophilized. These pooled fractions were again examined by paper and polyacrylamide gel electrophoreses and by end group analysis (Table X). Fractions III, VII, and VIII, which were relat,ively homogeneous on t'he basis of these Lys fiis Arg Cysteic acid.. Methionine sulfone Asp...... Thr.. ..,... Per .._... ml Pro.. cay Ala. cys.. I-al Met. Ile. Leu Tyr Phe. ........ ........ ........ ........ ........ ........ Amino acid content @mole 0.825 0.100 0.175 0.003 0.135 0.502 0.341 0.166 0.664 0.217 0.360 0.511 0.000 22.2 2.7 4.i 0.1 3.G 13.F 9.2 4.5 17.9 5.9 9.7 13.8 0.0 0.325 8.8 0.000 0.0 0.175 4.7 0.4X 11.5 0.196 5.3 0.111 (3) TABLE l-11 Spectrophotometric analysis of mole ratio of tyrosine to tryptophan Method of analysis Ratio of tyrosine to tryptophan Method of Bencze and Schmid (23). 5 Method of Goodwin and Morton (22). 6.6 Amino acid analysis after alkaline hydrolysis. 8.7" a This value was obtained from a sample subjected to 44 hours of hydrolysis, which gave the maximum yield of tryptophan in the series of 20-, 44., and G-hour hydrolysates. tests, were further purified by preparative paper electrophoresis at pH 6.5. The purified components after elution were hydro- lyzed and subjected to amino acid analysis. Fraction V, which contained both KHz-terminal threonine and alanine, was subjected to preparative electrophoresis in t,he Brinkmann apparatus (Fig. 13) as described in "Experimental Procedure." Aliquots of the effluent fractions were examined by gel electrophoresis, with standard gel. On the basis of these tests, Fractions 27 to 31 (Fraction Va) and 38 to 40 (Fraction Vb) were pooled and examined for NHn-terminal end groups and amino acid composition. Fraction I, as indicated in Table X, contained all of the amino- terminal residues of the entire CNBr digest and produced a peptide map after further digestion with trypsin which indicated the presence of all the components of the CNBr digest. This fract,ion was not examined further since it appeared to consist of an aggregated mixture of all the peptide components. A summary of the properties of the five large fragments pro- duced by CNBr and their designations is given in Table XI. As shown, homoserine is present in Fractions Va and Vb, VII, and VIII (Peptides A, C, D, and B, respectively). Fract'ion III (Peptide E), lacking this residue, must be COOH-terminal in the nuclease; and Fraction Va (Peptide A), which contains an h-Hz- 4378 Partial Sequence of a Staphylococcal Nuclease. I Vol. 241, No. 19 FIG. 11. Peptide map obtained from a trypsin digest of the nuclease. The digest, containing 1 mg of protein, was applied to What- man No. 3MM paper for two-dimensional separation. Electrophoresis was carried out at 2500 volts for 80 min. The peptides giving a positive platinum iodide reaction are indicated as MET. The spots indicated by Y gave yellow color upon staining with ninhydrin- alcohol solution. Ehrlich indicates the spot with a positive Ehrlich reaction. Among those spots giving a positive Sakaguchi reaction, only spots clearly identified on the original map are shown (as iirg). terminal alanine residue, must, be the NH%-terminal peptide. Trypsin Digestion of CNBr Fragments-Samples of cyanogen Only Fraction III (Peptide E) gave a positive test for tryptophan bromide peptide Fractions III, V, and VII were digested with and, as discussed below, yielded a fragment, upon trypsin diges- trypsin, and the resulting mixtures of pept,ides were subjected to tion which corresponded to the single Ehrlich-positive compo- separation on Dowex 50-X2 columns & described under "Experi- nents on the peptide map. The sum of the residues determined mental Procedure." The chromatographic patterns are shown for the individual CNBr fragments corresponds well with the in Fig. 14. The purity of the separated peptides was examined total amino acid content presented in Table V above. by two-dimensional peptide mapping, amino acid analysis, and Issue of October 10, 1966 H. Taniuchi an8 C. B. An&men 4379 end group determinations by both the DFB and the Edman procedures. Further purification, when necessary, was performed by either paper tilectrophoresis or paper chromatography as described in "Experimental Procedure." Table XII summarizes TABLE VIII Amino acid compositions and amino-terminal residues of tryptic peptides giving positive reaction for methionine The values reported are in micromoles, and the assumed number of residues is given in parentheses. Where no numbers are given, the values obtained were equal to or less than 0.002 rmole. Lys ............ His. ........... Arg ........... Asp. .......... Thr ............ Ser ............. Glu ......... Pro. .......... Gly ........... Ala .......... cys. ........... I Val ............ Met. ......... Ile ... ......... Leu ......... Tyr ........ Phe 1:. ...... NH*-terminal residuea. -i Designation on peptide map 0.003 (0) 0.004 (0) 0.010 (1) 0.009 (1) 0.007 (1) Leucine 1 F-17 0.025 (1) 0.003 (0) 0.004 (0) 0.023 (1) 0.003 (0) 0.009 (0) 0.028 (1) 0.007 (0) 0.011 (0) 0.023 (1) 0.021 (1) 0.013 (1) 0.004 (0) Methionin F-18 0.003 (0) 0.015 (1) 0.005 (0) 0.016 (1) 0.004 (0) 0.015 (1) 0.015 (1) 0.013 (1) 0.004 (0) 0.012 (1) 0.015 (1) Glycine F-19 0.015 (1) 0.020 (1) 0.007 (0) 0.017 (1) 0.006 (0) 0.018 (1) 0.029 (2) 0.007 (1) 0.017 (1) Methionine a The analysis was qualitative, but only a single ether-soluble DNP-amino acid was found for each peptide. the data obtained on these tryptic fragments. This table con- tains only the data on peptides which were obtained in significant amounts as judged from the amino acid analyses. The cal- culated yields of these peptides after chromatography on Dowex 50 ranged from 30 to 90%, except those peptides derived from the contaminated fragment described below. The trypsin digest of purified Peptide C (obtained by prepara- tive electrophoresis of Fraction V; see Table XI) was subjected to peptide mapping, and the position of each component was used to assign, by difference, the map positions of the tryptic fragments of Peptide A, the other CNBr component in Fraction V. Trypsin digests of Fraction VII contained, as minor com- ponents, tryptic Peptides TVII-1 and VII-4b (in yields of less than 10% of each peptide). These could be assigned to CNBr Peptide A, which contaminates Fraction VII. They did not TABLE IX Amino terminal analysis of cyanogen bromide fragments of n&ease v&h DNP-method Sample 1, intact nuclease; Sample 2, nuclease treated with 70% HCOOH (without CNBr); Sample 3, nuclease treated with CNBr in 70yo HCOOH. Each sample contained 0.12 pmole of the nu- clease. DNP-amino acid founda Sample Alanine Valiine Threonine Tyrosine #mole pwle pmole pm& 1 0.10 2 0.11 3 0.12 0.24 O.Olb 0.026 a No water-soluble DNP-amino acids, other than e-DNP- lysine, could be detected by paper electrophoresis in pyridinium acetate, pH 6.5. b The low recovery of these end groups in the present experiment is discussed in the text. TUBE NUMBER FIG. 12. Gel filtration pattern of cyanogen bromide peptides prepared from the nuclease. The column was loaded with 360 mg of cyanogen bromide digest. Other details are given in "Experimental Procedure." O--O, absorbance at 280 mp; O-- -0, ab- sorbance at 570 mp. 4380 Partial Seqwgux of a Staphylococcal Nuclease. I voi. 241, so. 19 apprar in the peptir!- rnnj)? of Peptide C. Cyanogen bromide Reconstruction of Trgp+ .?epk& Alo>~ The map j)<~sil itil:: of E'rac:lr:en+ R (obtairlc,c! from Fraction VIII of the Se&&x G-50 separated tryptic pept ides of each cyanc,g+aLl bromide pt. ,,: i:l.,: yr-t::`e s;cj~:~".:`i~lrl) did noi l`s nprrwnt further ujron trypsin as tested by determined on t\Vo-dir;;ei`,-i~,llal j)eptick [:~a;)>. A I'CCOII~-*.! l.ii ~,`d jzptiric ,naj~ping. `I`t ie r*omposition of thp CNBr fragmenk is peptide map wad prepared as shown in Kg. 15. The tr>.ptic compnrt,d with tbr sum of the amino acid compositions of the trypsin fragments assignrd to them in Table XIII. The tot,al 0.5r I I I amino acid analysis of the original nuclease is included in this +' table for over-all comparison. *3 *J--7 TABLE X p 0.4 Qualitative summary of seweral sets of quantitative analyses for is amino-terminal residues of cyanogen bromide fragments * 0.3 pTeparkd by Sephadex G-60 gel jiltration t Fraction Pooled tubes" I III IV V VI VII VIII 128-145 151-168 169-173 174-191 192-195 196-213 272-295 I a See Fig. 10. FIG. 13. Preparative electrophoresis of cyanogen bromide frag- b Only ether-soluble DNP-amino acids were analyzed (see Ta- ment Fraction V, obtained by gel filtration. Approximately 10 ble IX). The relative intensities of the DNP-amino acids are pmoles of peptides, dissolved in 0.5 ml of 0.05 M NH,HCOJ, were qualitatively indicated by plus signs. Ether-soluble DNP-amino applied through the entry port above tube 30. Other details of acids, other than those shown in this table, were not found. the procedures are described in "Experimental Procedure." Pooled fractions are indicated by horizontal arrows. DNP-amino acid foundb Valine +:+ ++ Trace ++ +++ - T .- `breonine + - : + +++ + - - $ 0.2 ryrosine P PHENOL RED + E: 4 - 5 0.1 +++ TUBE NUMBER - ' + Trace TABLE XI Approximate amino acid composition of cyanogen bromide fragments Values are reported in micromoles. The values in parentheses reDresent assumed number of residues. Lys His Arg Asp. Thr Ser Glu Pro. Gly Ala cys Val Met. Ile Leu. Tyr Phe Trp Homoserine, NH2-terminal residue Designationof peptidc !. Fraction III Fraction Va - - Fraction Vb Fraction VII Fraction VIII -- 0.223 (5) 0.236 (4-5) 0.151 (&6) 0.030 (1) 0.041 (1) 0.019 (1) 0.071 (2) 0.004 (0) 0.021 (1) 0.276 (6-7) 0.089 (2) 0.031 (1) 0.042 (1) 0.154 (3-4) 0.086 (3) 0.105 (%3) 0.043 (1) 0.028 (1) 0.3'72 (9) 0.052 (1) 0.077 (3) 0.046 (1) 0.053 (1) 0.077 (3) 0.086 (2) 0.057 (1) 0.059 (2) 0.241 (6) 0.119 (2-3) 0.055 (2) o.ooo (0) o.ooo (0) o.ooo (0) 0.081 (4-5) 0.039 (1) (lS22) o.cm (0) o.ooo (0) (3) 0.029 (l-2) o.ooo (0) (3-5) 0.071 (3-4) 0.002 (0) (12-14) 0.020 (1) o.ooo (0) (8-Q) 0.011 (0) 0.002 (0) (4-5) 0.075 (4) 0.052 (1) (18) 0.005 (0) 0.018 (1) (6) 0.075 (4) 0.042 (1) (10) 0.057 (3) o.ooo (0) (13) o.ooo (0) o.ooo (0) (0) 0.146 (3) 0.053 (1) 0.041 (2) 0.038 (2) o.ooo (0) (8) o.ooo (0) o.ooo (0) 0.002 (0) o.ooo (0) o.ooo (0) (0) 0.038 (1) 0.088 (2) 0.012 (1) 0.029 (l-2) o.mo (0) (5-6) 0.198 (5) 0.131 (3) 0.068 (2-3) 0.023 (1) o.ooo (0) (11-12) .o.ocm (2)a 0.005 (0)" 0.001 (l)a o.ooo (3)s 0.031 (1) (7) 0.003 (0) 0.007 (0) 0.034 (l-2) 0.016 (1) o.ooo (0) (2-3) - UP - (0) - (0) - (0) - (0) (1) o.ooo (0) 0.054 (1) 0.032 (1) 0.021 (1) 0.041 (1) (4) Valine Alanine Threonine Valine Tyrosine E A C D B Amino acid content I - T Total number of residues a The values obtained from analysis of samples before purification by paper electrophoresis (see the text) were used to correct for destruction of tyrosine. b Determined by staining with Ehrlich's reagent (see the text). 1.5 I- Varigrad, 35' IO 9 8 7 PH 6 s 1.0 5 % P 4 8 3 2 0.5 A "0 100 200 300 FRACTION NUMBER I I 1.5 Vangrad, 35"&pH9.9,50" P ,. : 9 I : 8 : : 7 d 6 PH c 1.0 5 k? 5: : 0.5 0 0 100 200 300 FRACTION NUMBER I----~ ---- Vortgrod, 35"~-+++ 1.5,- 50%+--pH 7, SO"+pH 9.9,508 IO 9 8 7 6 PH `5 4 3 FRACTION NUMBER FIG. 14. Chromatography of tryptic peptides prepared from cyanogen bromide Fragments III (upper), V (middle), and VII (lower), on Dowex 50-X2. Samples equivalent to approximately 4, 2, and 4 rmoles of Fractions III, V, and VII, respectively, were applied. 4381 4382 Partial Sequence of a Staphylococcal Nuclease. I Vol. 241, No. 19 TABLE XII Summary of amino acid analyses of acid hydrolysates of trypiic peptides prepared from cyanogen bromide fragment The values reported are in micromoles, and the assumed ntlmber of residues is given in parentheses. the values obtained were equal to or less than 0.002 @mole. Where no numbers are given, A positive Ehrlich reaction for tryptophan is shown by a plus sign. The dashes indicate that quantitative analyses were not performed. Peptides derived from Sephadex G-50 Fractions III, V, and VII (see Table X) are summarized in Parts A, B, and C of the table, respectively. VII were essentially homogeneous samples of CNBr Fragments E and D. As described in the text and in Table V, Fractions III and t,o yield CNBr Fragments A and C. Fraction V was further separated by curtain electrophoresis Peptide maps of trypsin digests of these purified fragments were used to assign the tryptic pep- tides obtained from Fraction V to either CNBr Fragment, A or C. Dowex 50 Fractions TIII-10, TIII-11, TV-3, TV-4, TV-14, TV-16, TV-18, TVII-A, TVII-9, and TVII-12 corresponded to minor spots on peptide maps, and their compositions are not reported here. A sample of Peptide TIII-2 was digested with subtilisin. scribed in "Experimental Procedure." The two fragments formed were separated by paper chromatography as de- These fragments were designat,ed TIII-2-8-l and TIII-2-S-2. TIII-4c Table XZZA I ' TIII-4e TIII-Sb ' TIII-5c 0.009 (lj; 0.012 (Ij 0.012 (1 - ) 111-2-s. 17 1.119 (3 1 1.064 (2 ) ).086 (2 )1 1.040 (1 j.043 (1 ,' Swine E -d `III-Z-S- 0.024 (1 I.024 (1 3.023 (1 + (1) Leucine E -d TIII-8b TIII-9c TIII-12 I.016 (1 j.070 (2 0.023 (I I.071 (2 0.029 (1) 0.020 (1) 0.026 (1) 0.042 (2) 0.018 (1) 0.048 (2) 0.033 (I) 0.003 (0) 0.010 (0) 3.031 (1) 3.003 (0) 1.050 (2) I.026 (1) - - Lysioe Lysine Valine E + E + E + TIII-6 `l-111.7a TIII-71 .2' ) I I 1 ) ( TIII-tc TIII-2 0.021 (1) T 1 ( ; : 1 ( ) ( 1 i 3 1) ( I 11, ( l)l 1) ( 1) ic 0.017 (1 0.029 ( 0.031 ( 0.028 ( 0.034 ( 0.038 (' - Glutam acid E + LYS His, Thr ser Glu ., Pro Gly. Ah.. _' .; :. CYS.. I Val. Met :: ..:: xte. Leu... TY~ Phe. 1.` :: :., Trp Homoserine NHz-terminal residw 0.072 (4 0.032 (1 0.025 (1 0.030 (1 0.003 (0 0.030 (1 0.057 (2 0.032 (1 - Vatine E + 0.006 (Oj~ 0.007 (1) 0.016 (2) 0.014 (1 0.003 (0 0.016 (2) 0.031 (2 0.027 (1) 0.044 (2 0.019 (1 0.017 (1 0.019 (1 0.010 (1' - Glutamk acid E -c 0.025 (lj 0.022 (1 0.026 (1) 0.022 (1 I.004 (1 I.003 (1 1.003 (1 - Valine E + E -c + UY Leucine E + Swine E -e - Glutamk acid E -I - Lysine E -B Assigned to cyanogen bromide peptide Inclusion in summa. tion (Table XIII: indicated by + Table XIZB TV-l TV-2 TV-5b TV-6 TV-ib TV-8a TV-10 TV-13 TV-lSa 0.011 (1) 0.010 (1) 0.012 (1) 0.012 (1) 0.013 (1) 0.007 (1) 0.035 (3) - Leucine C + 0.029 (2) 0.015 (1) 0.013 (1) 0.016 (1) - Lysine C + 0.010 (1) 0.007 (1) 0.016 (1) - Threonine C + 0.015 (1) 0.030 (2) 0.016 (1) 0.013 (1) 0.014 (1) 0.016 (1) 0.014 (1) Alanine A + 0.010 (1) 0.004 (1) 0.018 (1) 0.028 (2) 0.009 (1) 0.012 (1) 0.010 (1) 0.011 (1) 0.021 (2) 0.006 (1) 0.011 (1) 0.006 (1) 0.011 (1) 0.031 (2) 0.013 (1) 0.014 (1) 0.013 (1) 0.003 (0) 0.013 (1) 0.010 (1) 0.006 (1) 0.008 (1) 0.010 (1) - - Tyrosine Glutamicaci< Alanine Threonine C + A +" A + C +" Lys His. Arg.. ..,. ksp Tbr. ser.. : Glu Pro. Gly Ala CYS I vat Met. Ile Leu Tyr. Pbe.. _. .: :: TIP. Homwerine. NIIrterminal residue. Assigned to cysnogen bromide peptide. Inclusion in summation 0.017 (1) 0.017 (1) Leucine A + Issue of October 10, 1966 H. Taniuchi and C. B. Anfinsen 4383 Lys His Arg Asp. Thr.. ser Glu Pro Gly Ala eye I Val. Met. He. Leu.. Tyr Phe Trp Homceerine. 0.006 (1) 0.006 (1) - - 0.003 (0) 0.033 (1) 0.027 (1) 0.005 (0) - - NHz-terminal residue. Alanine Threonine Glycine Valine Assigned to cyanogen bromide peptide ._ :. Inclusion in summation A -k D + D + D + TVII-1 TVII-Zc TVII-3b TVII-3c TVII-4b TVII-Sa TVII-8a TVII-Sb TVII-lla o.M)s (1) 0.022 (1) 0.004 (0) 0.012 (1) 0.031 (1) 0.020 (2) 0.003 (0) 0.035 (1) 0.050 (l-2)i 0.015 (2) 0.003 (1) 0.023 (1) 0.014 (1) a.005 (0) 0.012 (2) 0.006 (1) 0.035 (1) 0.004 (0) 0.008 (1) 0.005 (1) 0.003 (0) 0.011 (0) 0.021 (1) 0.033 (1) 0.027 (1) 0.033 (1) 0.036 (1) 0.033 (1) 0.033 (1) 0.003 (0) o.om (1) - A -k 0.029 (1) 0.029 (1) 0.012 (1) 0.004 (0) 0.026 (2) 0.005 (0) 0.003 (0) 0.055 (1) 0.036 (1) 0.024 (1) 0.030 (1) 0.034 (1) - 0.004 (0) Isoleucine 0.010 (1) 0.010 (1) 0.014 (I) 0.017 (1) - - - Lysine Glycine Tyrosine D + D + D + D + - a The presence of tryptophan was also amEnned by digestion with leucine sminopeptidas (Worthington) by the method of Canfield (18). Ammonium bicarbonate buffer (0.05 M) containing 0.01 M MgClt was used. Tryptophan was determined quantitatively on the amino acid analyzer. b Aliquota of fractiona obtained by chromatography on Dowex 50 were wed for amino-terminal residue determination by both dinitrophenylation and Edman degrada- tion. When B peptide was contamin&.ed with a meoond peptide, the amino-terminal residue could be assigned on the be& of the amino acid composition of the purified peptide. c TIII-lo and TIII-78 have the @ame amino acid composition ae TIIIJb, preeumably due to the formation of pyrrolidone earboxylie acid from a terminal glutamine resi- due. d TIIIJ-S-l and TIII-2-S-2 are eonstituenta of TIII-2. B Peptide TIII-4e is derived from TIII-Bb, by lhss of a single lysine residue. f PeptIde TIII-Sb is assumed to be derived from TIII-So by lam of the NHt-terminal lysine residue. This relationship is now under reexamination. n Only free lyuine was detected. i The bar& of this akgnment was obtained from qualitative amino acid snslyuis of tryptic Peptide TVII-llb, which seemed to inlcude the amino acid components of TV- 7b. This peptide is preaumahly derived from cyanogen bromide Fragment A which contaminated Fragment D as s traiIing fraction during the gel filtration on Sephadex G-50 (see the text). i Further Btudy with successive Edman degradation suggests that the glutamic scid residue is not part of this peptide. The nature of the contamination is not clear at present. i In the summation in Table XIII, 1 residue of arginine hea tentatively been sssigned to this peptide. The presence of s pcuaible 2nd arginine residue ie under investi- gation. k Amino acid compoeitione of TVII-1 and TVII-4b are the same aa thoee of TV-2 and TV-&a, respecti~y (se Footnote hand the text). TABLE XIII which is liberated glutamine in 60 y0 yield upon Z-hour incubat,ion Amino acid composition of cyanogen bromide fragments based on analysis of tryptic peptides derived from each with carboxypeptidase A at 37" as described under "Experi- mental Procedure." The following 24 tryptic peptides (identified in the bottom line of Table XII), together with CNBr Fragment B (Table XI), ac- The remaining CNBr fragments can be aligned on the basis of count closely for the amino acid composition of the nuclease. the methionine cont.ent of the nuclease (4 per molecule) and the Peptide A: TV-l, TV-2, TV-`lb, TV-8a; Peptide C: TV-5b, TV-6, composition of the four methionine-containing peptides isolated TV-IO, TV-13, TV-15a, F-2; Peptide D: TVII-2c, TVII-3b, TVII-3c, TVII-5a, TVII-8a, TVII-8b, TVII-lla; Peptide E: TIII-2, TIII-6, TIII-7b, TIII-8b, TIII-Sc, TIII-12. The num- bers in parentheses are taken from Table XI. The amino acid composition of the nuclease is taken from Table V. Where no numbers are given, the values obtained were zero. I Amino acid content in cyanogen bromide fragment sum of mino acid tryptic Bmpositioo of the A F sptides" B ca D E nuclease ~- ~~ Lys ... ...3 (5) (1) 6 (5-6) 5 (4-5) 6 (56 21 21.4 His ...... 1 (1) 1 (1) 1 0) `3 3.0 Arg ...... 1 (1) 2 (l-2) 2 (2) 5 4.8 Asp ... ...2 (2) 1 (1) 4 G--4)7 (6-7, 14 14.6 Thr . 4 (34) 3 (3) 1 (1) 1 (1) 9 9.6 Ser .... ..l (1) 1 (1) 3 (23, 5 5.1 Glu ...... 1 (1) (1) 3 (3) 4 (4) 9 (9) 18 18.3 Pro ...... 1 (1) (1) 3 (3) 1 (1) 6 5.1 Gly ...... 1 (1) (1) 2 (2) 4 (4)' 2 (2) 10 7.9 Ala ... ...3 (2-3) 2 (2) 3 (3) 6 (6) 14 14.4 CYS I Val .... ..l (1) '2 '2 (2) (2) 2 2 (2) (2) 4 4 (4) (4) 9 9.2 Met ...... 3.5 Ile ....... 2 (2) (1) (1) 2 2 (l-2) (l-2) 1 1 (1) (1) 5 5.0 Leu ... ...2 (3) 3 3 (2-3) (2-3) 1 1 (1) (1) 5 5 (5) (5) 11 11.6 0.0 Tyr ...... (1) (1) 1 1 (1) (1) 3 3 (3) (3) 2 2 (2) (2) 7 6.6 t Phe ...... 2 2 (l-2) (l-2) 1 1 (1) (1) 3 3.0 1, I I I I I I I, I I 1 Trp ! I I 1 1 (1) (1) 1 1 0 0.2 0.4 0.6 0.8 1 .o ...... Homo- RF (CHROMATOGRAPHY) serine 1 (1) (1) (1) (1) (1) (1) (1) 2 FIG. 15. Reconstructed tryptic peptide map. Phenol red and 0 Tryptic peptide F-2 (His, Pro, Lys) (see the text) which was lysine served as reference standards in the chromatographic and not recovered among the purified tryptic peptides obtained from electrophoretic dimensions, respectively. The significance of the designations shown in parentheses is described in the text. cyanogen bromide fragments, is assigned to Peptide C on the basis On a qualitative basis, the following additional assignments of of histidine content. peptides can be made on the map. Component F-16 on the "ex- b The estimated number of residues in Peptide B is included. perimental" peptide map (see Fig. 11) contains a mixture of pep- tides, some incompletely cleaved by trypsin, including TVII-2c, and TIII-le. Similarly, F-21 contains TV-6 and TV-IO. F-24 digest of the nuclease preparation was also subjected to mapping, corresponds to the addition of TVII3b and TVII-5a, and F-14 and major spots were cut and eluted as described in "Experi- contains TVII-lla in addition to TIII-7b. mental Procedure." The resulting peptides were subjected to amino acid analysis after acid hydrolysis. By comparison of position on the peptide map and amino acid composition, almost all of the major spots of the nuclease digest could be found on the reconstructed peptide map, as indicated in Fig. 15, with the ex- ception of those peptides containing methionine. Of the major LEU-MET-TYR-LYS-GLY-(GLU, PRO)-MET-THR-PHE-ARG trypsin fragments studied, pnly peptide F2 of the original peptide map (Fig. 9) was not accounted for by the CNBr fragments. Linear Arrangement of CNBr Fragments-On the basis of NHr terminal end group data alone, Peptide A can be assigned to the Peptide 6 1 NH&erminal position of the chain. Furthermore, both of the alanine-terminal peptides isolated from trypsin digests may be assigned to this cyanogen bromide fragment (tryptic Peptides I TV-2 and TV-8a). Peptide E must occupy the COOH-terminal F15 F18 I position since it lacks homoserine and yields the trypsin Fragment TIII-2 which contains neither lvsine nor arzinine and from FIG. 16. The linear arrangement of cyanogen bromide Frag- . - ments A, B, and C. Peptide B corresponds to P'ragment B. 4384 Partial Sequence of a Staphylococcal Nuclease. I Vol. 241, No. 19 Issue of October 10, 1966 H. Tanivxhi and C. B. Anjinsen f A r--B-: c- NH?-ALA - - - - - -- - - X-LEU-hiET-TYR-LYS-GLY-(GLU,PRO)-MET-THR-PHE-ARG- - - - - - - - - X-MET- 4385 + D E + -VAL-(GLU,ASP,ALA)-LYS---.------------X-MET-VAL-(ASP,GLU,ALA,VAL,LEU)-ARG-------GLN-COOH I F 17 c A F 19 + T-VII-3c k T-Ill-6 _T FIG. 17. The linear arrangement of cyanogen bromide fragments derived from the nuclease. The residues indicated as X are pre- sumed to be lysine or arginine. Subsequent studies6 have indeed shown that these residues are lysine. from peptide maps. If we compare the amino acid composition and NH2-terminal residue of Peptide B with those of trypsin Fragments F-15 and F-18 which contain methionine, the arrange- ment shown in Fig. 16 can be assigned. If this arrangement is correct, Leu-homoserine and Thr-Phe-Arg should be found among the trypsin fragments of the neighboring CNBr peptides. Both of these were found only in the trypsin fragments obtained from Fraction V which contains Peptides A and C. Thus, Pep- tide TV-l is derived from Peptide A and TV-15a from Peptide C. These observations serve to establish the order of the five CNBr fragments as A-B-C-D-E. The amino acid compositions of the two NH&erminal methionine-containing trypsin fragments of the nuclease, F-17 and F-19, are the same as the compositions of the terminal peptides of CNBr Fragments D and E when methio- nine is added to each. This consistency gives further confirma- tion to the arrangement shown in Fig. 17. Acknowledgments-We would like to acknowledge the excellent technical assistance of Mr. Clifford Lee and Miss Ann Sodja. 1. 2. 3. 4. 5. 6. 7. REFERENCES TANICCHI, H., ANFINSEN, C. B., HEINS, J. N., .+ND CARROLL, W. R., Federation Proc., 24,288 (1965). ANFINSEN, C. B., TANIUCHI, H., HEINS, J. N., AND SURIANO, J. R., Science, 160,368 (1965). ALEXANDER, M., HEPPEL, L. A., ?LND HURWITZ, J., J. Biol. Chem., !Z36, 3014 (1961). ANFIXSEN, C. B., RUMLEY, M., AND TANIUCHI, H., Acta Ckem. &and ., 17, 270 (1963). POTTS, J'. T., BERGER, A., COOKE, J., AND ANFINSEN, C. B., J. Biol. Ckem.. 237.1851 (1962). WEISSBACH, A., AND KORN, D., j. Biol. 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