Bovine Pancreatic Deoxyribonuclease A

THE JOURNAL OF BIOLOGICAL CHEMISTRY VoL248,No. 4,Issue of February 25.~~. 1480-1488,1973

Printed in U.S.A.

Bovine Pancreatic Deoxyribonuclease A

ISOLATION, COMPOSITION, AND AMINO ACID SEQUENCES OF THE TRYPTIC AND CHYMOTRYPTIC PEPTIDES*

(Received for publication, October 2, 1972)

JOHANN SALNIKOW,~ TA-HSIU LIAO, STANFORD IMOORE,AND WILLIAM H. STEIN

From the Rockefeller University, New Yorlc, New York 10021

SUMMARY

The first phase of the determination of the primary struc- ture of chromatographically homogeneous bovine pancreatic deoxyribonuclease A has been the isolation of 23 principal peptides from tryptic digests of the reduced and alkylated protein and 20 peptides from a chymotryptic hydrolysate. Seven of the latter series provide information on the order- ing of peptides of the tryptic series. The results provide the basis for the derivation of the structural formula of DNase A from the subsequent study of the products of cyanogen bro- mide cleavage reported in the accompanying paper.

The determinations of the sequences of the peptides have been facilitated by a scaling down of the subtractive Edman procedure to the use of 1 to 2 nmoles of peptide per step through the use of amino acid analyzers operating in that range.

The preparation of bovine pancreatic deoxyribonuclease in a sufficiently homogeneous form for structural study is based on the experiments of Kunitz (2), Lindberg (3), Price et al. (4), and Sal- nikow et al. (5). Chromatography on phosphocellulose (5) sepa- rates four naturally occurring forms of the enzyme; the major component, DNase A, has been the starting product for the present studies on the amino acid sequence. The earlier experiments (3,4) have shown that the enzyme is a single polypeptide chain with two disulfide bridges and a molecular weight of about 31,000. Catley et al. (6) have established that the single carbo- hydrate side chain is attached at an asparagine residue. This paper and the accompanying contribution (7) summarize the determination of the structural formula of the enzyme.

EXPERIMENTAL PROCEDURE

Materials-DNase A was prepared from Worthington DP grade DNase by the supplier by chromatography on phosphocellulose according to the procedure of Salnikow et al. (5). Trypsin

* This investigation was supported in part by United States Public Health Service Grant GM 07256. A preliminary report of this work has appeared (1).

$ Present address, Institut fur Biochemie, Technische Univer- sit&t, Franklinstrasse 29, Berlin, 10, Germany.

(n-1-tosylamido-2-phenylethyl chloromethyl ketone-treated), LY- chymotrypsin, and carboxypeptidases A and B (both DFPr- treated) were also from Worthington. The carboxypeptidase from yeast (proteinase C of Hayashi and Hata (8)), was a gift from Dr. Rikimaru Hayashi at the Rockefeller University. Aminopeptidases M and 0 were obtained from Rohm and Haas, Darmstadt, Germany. Streptococcal proteinase (9) was a sample prepared by Dr. Stuart D. Elliott and Dr. Michael C. Lin at the Rockefeller University. Thermolysin was obtained from Daiwa Kasei K. K., Osaka, Japan; Nagarse was purchased from Enzyme Development Corp., New York. DFP was a product of Aldrich. The sulfonated polystyrene resin Dowex 50-X8 was Aminex Q-15s from Bio-Rad; the anion exchange resin Dowex l- X2 was AG l-X2 (200 to 400 mesh) from Bio-Rad. Trifluoro- acetic acid and phenyl isothiocyanate (Sequanal grade) were obtained from Pierce Chemical Company. N-Ethylmorpholine was redistilled; pyridine was redistilled over ninhydrin. Iodo- acetic acid was recrystallized from petroleum ether. All other chemicals were reagent grade.

Reduction and AZkyZa ion-Prior to reduction, DNase A was incubated at room temperature at pH 8.6 in the presence of approximately 0.1 y. (v/v) of DFP for 30 min. RCM-DNase was prepared according to Crestfield et al. (lo), but without need for urea (Price et al. (4)). Approximately 300 mg of DNase A were reduced in 1% mercaptoethanol (v/v) in pH 8.6 Tris-HCl buffer (0.36 M) and, after 3 to 4 hours at room temperature, carboxymethylated with iodoacetic acid (equivalent to the molarity of added sulfhydryl groups). For RAE-DNase A, instead of iodoacetic acid, three 0.5-ml portions of ethylenimine (11) were added. The reduced and alkylated DNase A was separated from excess reagents by the passage through a column of Sephadex G-75 (2.5 x 100 cm), with the pH 8.6 buffer as eluent. The modified enzyme was dialyzed against distilled water and lyophilized.

Digestion of Derivatized DNase A with Proteolytic Enzymes Conditions for the digestions are given in Table I. Tryptic action was terminated by bringing the pH to 11 with 0.5 N NaOH, and the whole clear solution was applied directly to a Sephadex column, G-25 for the small peptides and G-50 for the large peptides.

1 The abbreviations used are: IIFP, diisopropylphosphofluori- date; RCM-, reduced and S-carboxymethylated; RAE-, reduced and S-aminoethylated; dansyl, l-dimethylaminonaphthalene-5- sulfonyl; the peptides of the tryptic series are prefixed by T and those of the chymotryptic series by C.

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The chymotryptic digestion was carried out with RAE-DNase A, with the aim of obtaining aminoethylcysteine-containing peptides that could be further hydrolyzed by trypsin, but the need for further cleavage did not arise. The digestion was stopped by DFP (a few drops), and the cloudy solution was centrifuged at 48,000 x g. Only the clear supernatant solution was gel-filtered and studied.

Methods of Isolation of Peptides-In many instances the peptides isolated by the combination of gel filtration (at room temperature) and ion exchange chromatography (at the indicated temperature) were pure enough for sequence studies, as judged by amino acid analysis and paper electrophoresis. When neces- sary, further purification was accomplished by one-dimensional paper electrophoresis or paper chromatography. The load on paper was about 20 miioles per cm along the line of application. Two chromatographic systems were used: System I (l-butanol- pyridine-acetic acid-water = 15:10:3:12) and System II (l- butanol-acetic acid-water = 200 :30 : 75). Chromatograms were developed in all glass tanks for 12 to 16 hours by the descending technique. Electrophoresis was carried out at 60 to 75 volts per cm in tanks (Savant) containing Varsol as coolant. Two buffer systems were used: pH 6.5 (pyridine-acetic acid-water = 100:4:900) and pH 1.9 (formic acid-acetic acid-water = 25:lOO: 875). Samples were applied as a narrow band on Whatman No. 3MM paper. Guide strips (5 mm) were cut and stained with ninhydrin-cadmium reagent (12). Ninhydrin-negative peptides were visualized with hypochlorite-starch-iodide stain (13)) and tryptophan-containing peptides were located with Ehrlich’s reagent (14) on separate guide strips. Neutral or basic peptides were eluted from the paper with 300/, acetic acid, and acidic peptides with 1 ye pyridine. When a large peptide was subjected to further proteolysis, the hgdrolysate was usually applied directly to paper and the resulting peptides were separated by electrophoresis or chromatography or both.

When gel-filtered fractions containing some of the large tryptic peptides were lyophilized, the residues were not soluble in many aqueous solvents but did dissolve in 50 y0 acetic acid. Therefore, these peptides were further fractionated by gel filtration in 50% acetic acid.

It was not always practical to include thiodiglycol to protect methionine residues from oxidation (15) ; if the peptide, as isolated, containing methionine sulfoxide, the amino acid analysis will show methionine since the sulfoxide reverts to methionine during acid hydrolysis (16).

Amino Acid Analysis-Peptides were hydrolyzed (17, 18) in 200 ~1 of 6 N HCl containing 0.2% (w/v) phenol and 0.1% (v/v) mercaptoacetic acid in evacuated, sealed tubes (10 x 75 mm Pyrex No. 9820) at 110” for 22 hours (unless otherwise stated) ;

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the additions, which ensure good recoveries of tyrosine and car- boxymethylcysteine when peptides have been eluted from paper, cause no complications when half-cystine or cysteine has been derivatized. The HCl was removed at 25” in a desiccator by an oil pump protected by a trap containing pellets of NaOH and a trap immersed in solid Cot-acetone. When 10 nmoles or more could be utilized for amino acid analysis, the chromatography was performed according to Spackman et al. (19) with an acceler- ated system (20), Beckman M 82 and PA 35 resins, and a 0.1 absorbance range on the recorder. For 1 to 10 nmoles, an analyzer of the above type was modified for use with 2%mm bore columns instead of g-mm columns. Several commercial models of analyzers reach this range by a combination of optical and electrical amplification and, in some instances, through narrower columns. The yields of the peptides were based upon the amino acid analyses.

The values for serine and threonine are not corrected for de- struction during hydrolysis, unless so stated. Tryptophan was detectable either through the recovery of about 50% under the conditions of acid hydrolysis or by a positive Ehrlich reaction on paper. Accurate analyses for tryptophan were obtained after alkaline hydrolysis (21).

When peptides are eluted from paper, there is the problem of the blank from the paper. By keeping the load at 20 nmoles per cm at the application line and using for analysis about one-tenth of the sample eluted per 1 to 2 cm2, the contribution from the paper (after hydrolysis of the eluate) generally amounted to no more than 0.1 nmole per amino acid, which was tolerable when 1 residue corresponded to 1 nmole or more.

Sequential Degradation of Peptides-In the initial studies on DNase, the Edman degradation was carried out under conditions similar to those employed by Dopheide et al. (22). In order to be able to perform subtractive Edman degradations on the amounts of peptides eluted from paper, amino acid analyzers operating in the 1 to 10 nmole range were used and the procedure for the Edman degradation was scaled down. Approximately 10 to 20 nmoles of peptide in a 3-ml test tube (10 x 75 mm) were dissolved in 100 ~1 of N-ethylmorpholine-pyridine-acetate buffer, pH 8.6, and 5 ~1 of phenylisothiocyanate were added. The tube was thoroughly purged with nitrogen and covered with Parafilm. The reaction mixture was incubated at 45” and after 30 min, dried in a vacuum desiccator (Precision Scientific No. 63351) at the same temperature for 20 to 30 min. As soon as the drying was complete, 2 to 3 drops (-100 ~1) of trifluoroacetic acid were added and the tubes were again purged with nitrogen, incubated at 45” for 10 min, and dried in vacua at room temperature for 20 to 30 min. The residue was dissolved in 200 ~1 of glass-distilled water and extracted with benzene three times (1 ml each). A

To obtain:

Small tryptic peptides (< 10 residues)

Large tryptic pept ides (> 10 residues)

Chymotrypt,ic peptides

TABLE I Conditions for digestion of derivatized DNase A with proteolytic en2

I /

% (w/w) % Wu) hrs

Trypsin RCM-DNase A 1.5 1.3 11.5

Trypsin RCM-DNase A 5.0 3.0 2.0

Chymotrypsin RAE-DNase A 1.5a 3.0 4.5

nes at W

Bllffer

None (lm~ CaCl2)

1% NIL- HCO,

None

PH

8.0

ca 8

8.0

PH adjustments

pH-stat with 0.5 N NaOH

None

pa-stat with 0.5 N NaOH

a At 3.5 hours, 1% (w/w) of additional chymotrypsin was added to make a total of 2.5%.


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portion (usually one-fifth to one-tenth) of the aqueous phase was taken for amino acid analysis after acid hydrolysis and the re- maining portion was dried in vucuo for the next degradation cycle. Occasionally, if relatively hydrophobic peptides were degraded, the portion for amino acid analysis of these peptides was taken from the buffer, pH 8.6, after the residue had been dissolved for the next step. For the confirmation of sequences, many of the chymotryptic peptides were sequenced by the dansyl procedure of Gray (23) combined with the thin layer chromatography of the derivatives according to Woods and Wang (24).

Digestion of Peptides with Proteolytic Enzymes-Some of the tryptic or chymotryptic peptides were submitted to further digestion with thermolysin, Nagarse, streptococcal proteinase, or chymotrypsin. Usually, 0.1 to 0.2 pmole of peptide dissolved in 50 to 100 ~1 of enzyme solution (0.5 to 1 mg per ml of 1% NHIHCOI) was incubated at 37” for 1 to 3 hours. The degree of digestion was judged by the results of paper electrophoresis or paper chromatography.

Hydrolysis of Peptides with Aminopeptidases and Carboxypepti- dasesPeptides (1 to 5 nmoles) were hydrolyzed with 5 to 10 fig of either aminopeptidases (M or 0), or carboxypeptidases (A, B, or both) in 50 ~1 of 0.1 M NH4HC03 at 37” or room temperature. Unless otherwise stated, hydrolyses with aminopeptidases were at pH 7.5 (adjusted with acetic acid) and with carboxypeptidases at pH 8.0. The yeast carboxypeptidase (and occasionally carboxypeptidase A for acidic residues) was used to remove proline and acidic residues in 0.1 M ammonium acetate at pH 5.0. The hydrolysates were lyophilized, dissolved in pH 2.2 citrate buffer (19), and applied directly to the amino acid analyzer.

Determination of Amides of Aspartic and Glutamic Acids--In most cases, amides were assigned by the hydrolysis of peptides with either carboxypeptidases or aminopeptidases and chromatographic measurement of glutamine or asparagine. However, if the amino acids to be assigned could not be released by any one of these exopeptidases, the assignment was based on behavior of the parent peptides on paper electrophoresis at pH 6.5. If more than one possible amide occurred in a given peptide, the mobili- ties relative to aspartic acid of the peptides obtained before and after successive removal of residues by Edman degradation were compared.

RESULTS

Isolation of Tryptic Pepl-ides-By the combination of the procedures summarized in Table II and Figs. 1 and 2, a complete series of small and large tryptic peptides from RCM-DNase A was isolated which could be integrated into the ultimate sequence of the chain derived in the accompanying communication (7). The tryptic series contained six peptides resulting from chymotryptic-like cleavages (7) (Fig. 2) at hydrophobic residues. Dur- ing the preparation of DNase it is difficult to remove all traces of chymotrypsinogen B (4, 27) ; there is the possibility that the chymotryptic-like hydrolyses are effected either by a trace of chymotrypsin or by chymotrypsin-like activity inherent in trypsin (28).

The only residues not accounted for in this series were 1 residue of arginine (subsequently found to be liberated (7) as the free amino acid by cleavage of an -Arg-Arg bond) and 1 residue of leucine (liberated (7) as the free amino acid from a chymotryptic- like cleavage of a -Phe-Leu-Phe- sequence).

Isolation of Chymotryptic Peptides-The digest of RAE-DNase A with chymotrypsin contained a greater number of peptides and a larger insoluble fraction than the one with trypsin. The pattern of separation was therefore more complex. After re-

TABLE II Summary of isolation of tryptic peptides from RCM-DNase A

The method of isolation is defined in terms of the position of elution in Figs. 1 or 2 and the subsequent electrophoresis or chromatography on paper, when required. The peptides are numbered in the order of their occurrence in the chain; Fig. 2 of the accompanying communication (7) defines the amino acid composition and sequence of each of the primary peptides itemized below.

Peptide

Tl T2 T2a T2b T3 T4 T4a

T4b T5

T6 T7

Also

T8 T9 TlO Tll T12 T13 T14 T15 T16 T16a T17

T18 Also

T19 T20 T21 T22

Also T22a T23

Also

FXiC- tion

Gel filtration Dowex 50-X8

Fig- “E

Fig- FIX- “E tion

1A 1A IA

1A 1A 2A 2A 1A 2A 2E 1A 2A 2A 20 1A 1A 1A 1A 2A IA IA 1A 2A 2A 2A

2E 1A 1A

1A 1A 2A 2A 2A 1A 2A 2A 20

IV 1E 4

IV 1E 2 III 1D 2 VI II 1c 1 II II V III b I IB 3 II II b III ID 4

III 1D 5 IV 1E 1 VI II I 1B 2 IV 1E 4

IV 1E 4

I II III

L PPt

I(

:V 1E 3 V VI III 1D 1 II I II I 1R 1 I II a

- D IEAE-Sephade:

- Fig- “X

2c 2c

1 2

2c 2c

4 5

c

2c 8

2B 3 2c 6

2c 7 2B 1 2c 3

2B 2c

2

6 c

a Plus paper chromatography, System I.

FKK- tion

-

K Ek- tro-

phoresis

PH 6.5

6.5a

6.5

6.5

6.5 6.5”

6.5

6.5

Yield

% 37

4 18 25

48 28 68

57

17 21

23

26 53

3

9 43 44 16 37

17 20

10

42 58 10 33

32

14

17

moval of the insoluble material from the digest, the supernatant solution was gel-filtered on a Sephadex G-50 column (Fig. 3A). The resulting four groups of peptides were further fractionated on a cation exchange resin (Fig. 3, B through E). The unadsorbed materials from Fractions I and II were further fractionated on the anion exchanger Dowex 1 (Fig. 3, F and G). Most of the fractions thus obtained contained pure peptides. Some of them, however, required further purification by paper electrophoresis or chromatography. The purification steps for each isolated peptide are summarized in Table III.

Sequences of PeptidesThe application of the subtractive


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I.0 - r

m lx P m

E J

'c 450 600 750 900 (u 2.0. 0 r * 2.0

.? B 2 3 Fraclton I C Fraciion II

3 fiT

g LO- 1.0 .- u 2 I

Z

b z +JzD h

100 200 0 5 I.C ci P .F z

0.5

2.c

I.C

5'0 lb0

E 4

Fraction IT k&Jl 2 3

100 150 200 Effluent ml

FIG. 1. Separation of the small tryptic peptides. A, gel filtration of a tryptic digest of 2.1 pmoles of RCM-DNase A on Sepha- dex G-25 (fine). Column, 2.5 X 200 cm; eluent, 0.1 M ammonia containing 0.1% thiodiglycol and 0.01% phenol; flow rate, 30 ml per hour. Fractions of 4 ml were collected and O.l-ml aliquots of each fraction were analyzed with ninhydrin after alkaline hydrolysis (25,26). B to E, chromatography of the tryptic Fractions II to V on Aminex Q-15s. A column (0.9 X 15 cm) was developed with a linear gradient of 0.2 M pyridine acetate, pH 3.1 (5 M in acetic acid), to 2.0 M pyridine acetate, pH 5.0 (2.5 M in acetic acid), 200 ml each, at 50”. Fractions of 2.5 ml were collected and 0.1 ml of each fraction was hydrolyzed in alkali and analyzed with ninhydrin. Bars and numbew show pooled fractions.

Edman procedure can be illustrated by the following example of

the results on the sixth peptide of the tryptic series.

Sequence: Asp-Ser-His-Leu-Val-Ala-Val-Gly-Lys

T6: Asp Ser His Leu Val Ala Gly Lys

Compositi0n:l.l 1.0 0.9 1.1 2.1 1.1 1.1 1.0

Edman Degradation:

Step 1: 0.0 0.8 0.9 0.9 1.8 1.0 1.0 0.5

Step 2: 0.0 0.0 0.9 0.9 1.8 0.9 1.0 0.4

Step 3: 0.0 0.0 0.3 0.9 1.8 1.0 1.0 0.4

Step 4: 0.0 O.ON.D.0.2 1.8 1.0 1.0 N.D.

Step 5: 0.0 O.ON.D.0.2 1.2 0.9 1.0 N.D.

Step 6: 0.0 O.ON.D.0.2 1.2 0.3 1.0 N.D.

Step 7 : 0.0 O.ON.D.0.2 0.4 0.3 1.0 N.D.

Aminopeptidase M, 25”, 16 hours: Aspartic acid 0.7, Serine 0.8,

Histidine not determined, Leucine 1.0, Valine 2.0, Alanine 1.0.

Carboxypeptidase B, 37”, 1 hour: Lysine 0.8. In the cleavage of the larger peptides into segments of a size

suitable for the manual Edman method, thermolysin was es-

pecially useful. Chymotrypsin and Nagarse provided most of the additionally needed overlaps. In order to avoid the repeti- tion of the writing of the extensive sequences of the DNase mole- cule, the reader is referred to Fig. 2 of the accompanying paper (7) for the sequences of all of the peptides of the tryptic series listed in Table I. Quantitative data to support these results are available as a supplement to this communication.*

2 The quantitative data on the amino acid compositions and sequences of the peptides and the supporting analytical data are published as a miniprint supplement immediately following this

osc B I

.c 250 500 750

E FractlanmPreclpitate

Effluent ml

FIG. 2. Separation of the large size tryptic peptides. A, gel filtration of a tryptic digest of 4.8 pmoles of RCM-DNase A on Sephadex G-50 (superfine). Column, 2.5 X 180 cm; eluent, 0.5yc ammonium bicarbonate. Fractions of 4.1 ml were collected. Flow rate, 13 ml per hour. B and C, chromatography of the tryptic Fractions II and III on DEAE-Sephadex. Column, 0.9 X 150 cm; first gradient, 0.2 M pyridine acetate, pH 6.05 (0.028 M in acetic acid), to 2.0 M pyridine acetate, pH 6.1 (0.28 M in acetic acid), 250 ml each. Second gradient, 2.0 M pyridine acetate, 0.28 M in acetic acid, to 2.0 M pyridine acetate, 2.0 M in acetic acid, 250 ml each. Temperature, 39”. Fractions of 3.4 ml were collected. Flow rate, 23 ml per hour. Aliquots of 0.15 ml were analyzed with ninhydrin after alkaline hydrolysis. D, gel filtration of Fraction II-6 in C on Sephadex G-50 (superfine). Column, 2 X 160 cm; eluent, 0.5% ammonium bicarbonate. Fractions of 2.5 ml were collected. Flow rate, 12 ml per hour. E, gel filtration of the precipitate of Fraction IV in A on Sephadex G-100. Column, 2.5 X 95 cm; eluent, 5Oa/c acetic acid. Fractions, 4 ml, were collected. Flow rate, 16 ml per hour. Bars and numbers show pooled fractions.

paper. Material published in miniprint form can be easily read with the aid of a large-field reading glass of a type readily available at most opticians. For the convenience of those who prefer to obtain supplementary material in the form of a microfiche or full size photocopies, these same data are available as JBC Docu- ment No. 72M-1378. Orders for supplementary material should specify the title, authors, and reference to this paper and the JBC Document Number, the form desired, microfiche or photocopy, and the numbers of copies desired. Orders should be addressed to the Journal of Biological Chemistry, 9650 Rockville Pike, Be- thesda, Maryland 20014, and must be accompanied by a remittance to the order of the Journal in the amount of $2.50 for microfiche, or $3.60 for photocopy.


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460 660

100 360

0.3 G Fraction II- I

0.2 ELI

2 0. I

Summary of isolation of chymotryptic peptides from RAE-DNase A

The method of isolation is defined in terms of the position of elution in Fig. 3 and the subsequent electrophoresis or chromatog-

raphy on paper, when required. The peptides are numbered in the order of their occurrence in the chain. Fig. 2 of the accompanying communication (7) defines the amino acid composition of

each of the primary peptides isolated below, except that space did not permit inclusion of Cl9 (residues 231 to 236) and C29 (residues 237 to 242).a

Gel filtration Fig. 3A

Dowex 50-X8 Dowex l-X2

Peptide Electro- phoresis

Yield

Fraction Fig- Frac- Fig- FraC- ure tion “W tion

l- %

Cl I 3B 1 3F 20

c2 IV 3E 4 9

c3 II 3c 4 6.5 5

c4 II 3c 1 3G 12 c5 II 3c 2” 11

C6 IV 3E 7 10

c7 IV 3E 8 6 C8 II 3c 5h 22

c9 II 3c 7 6.5 8

C9a II 3c 6 6.5 10 Cl0 II 3c 3 6.5 4

ClOa II 3c 2b 4

Cl1 IV 3E 5 19 Cl2 IV 30 2b 19 Cl3 III 30 3 6.5 22 Cl4 IV 3E 2 6.5 22

Cl5 IV 30 4 6.5 5 Cl6 I 30 1 3F 14

Cl7 IV 3E 9 17

Cl8 II 3c 1 3G 20

Cl9 III 30 1 6.5 20

C19a III 30 1 6.5 19 c20 II 3c 2 20

- 5 In addition to the peptides indicated above, two dipeptides

Asp-Thr, Fig. 30, 2, Leu-Tyr, Fig. 3E, 6, and three free amino

Effluent ml acids (Leu, Fig. 3E, 1, Phe, Fig. 3E, 2, and Tyr, Fig. 3E, 3) were ..-,

FIG. 3. Separation of the chymotryptic peptides. A, gel filtration of a chymotryptic digest of 5.8 pmoles of RAE-DNase A on Sephadex G-25 (superfine). Column, 2 X 190 cm; flow rate, 15 ml per hour; eluent, 0.1 M ammonium bicarbonate. Fractions, 4 ml, were collected. B to E, chromatography of the chymotryptic Fractions I to IV on Aminex Q-15s. Conditions as in Fig. 1C except that the gradient was started after 100 ml of the initial buffer. F and G, chromatography of the unadsorbed peaks from B and C on Dowex l-X2. Column, 0.6 X 60 cm; first gradient, 3qb pyridine to 0.5 M pyridine acetate, pH 5.5,100 ml each; second gradient, 0.5 M pyridine acetate, pH 5.5, to 2 M pyridine acetate, pH 5.1, 100 ml each ; temperature, 35”. Fractions of 2.7 ml were collected. Flow rate, 100 ml per hour. Bars and numbers show pooled fractions.

Peptide T4 and T4a were the only glycopeptides isolated. The results fit the observation of Catley et al. (6) that the carbohy- drate side chain is attached to an asparagine residue in the sequence Ser-Asn-Ala-Thr; Salnikow et al. (5) found the carbo- hydrate moiety of DNase A to contain 2 residues of glucosamine and about 6 of mannose.

The peptides of the chymotryptic series were sequenced only to a sufficient degree to obtain information of value in the order- ing of the peptides of the tryptic series; seven of the peptides of

also isolat.eci. b Plus paper chromatography, System I.

the C-series (1,3,6,8,9, 13, and 15) were particularly important in this respect. The derivation of the complete structural formula required the knowledge of the cleavage at the 4 methionine residues, as summarized in the following paper (7).

Acknowledgments-We wish to express our appreciation to Mrs Dagmar Murphy, Mr. John Ferrette, and Miss Lynne Jacobson for their excellent technical assistance. We are also indebted to Dr. George Robinson for modifications of the amino acid analyzer which facilitated ultramicro analyses.

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2. KUNITZ, M. (1950) J. Gen. Physiol. 33,349-377 3. LINDBERQ, U. (1967) Biochemistry 6, 335-342 4. PRICE, P. A., LIU, T.-Y., STEIN, W. H., & MOORE, S. (1969)

J. Biol. Chem. !&I, 917-923 5. SALNIKOW, J., MOORE, S., & STEIN, W. H. (1970) J. Biol.

Chem. 246, 5866-5690


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Johann Salnikow, Ta-Hsiu Liao, Stanford Moore and William H. SteinPEPTIDES

AMINO ACID SEQUENCES OF THE TRYPTIC AND CHYMOTRYPTIC Bovine Pancreatic Deoxyribonuclease A: ISOLATION, COMPOSITION, AND

1973, 248:1480-1488.J. Biol. Chem.

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Bovine Pancreatic Deoxyribonuclease A

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