LAB JOURNAL OF OLULOGICAL CHEMISTRY
Vol. 241, No. 19, Issue of October 10, pp. 4366-4385, 1966
Printed 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)

Hirosui Tanrucut{ anp Curistran 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 amino- 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 valine 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 aureus 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 this two-fold function and

* Preliminary reports of this work have appeared (1, 2).
} 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 rotatory properties.

The polypeptide chain of the highly purified nuclease was
subjected to 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 diisopropy!
fluorophosphate as described by Potts e al. (5). Diisopropyl
fluorophosphate-treated 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--H
DNA from Escherichia colt, Kish, 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.

Carboxymethy] cellulose (medium) was obtained from Sigma.
Phosphocellulose (Whatman, column chromedia, p11 fibrous
powder) was obtained from Angel (7.4 meq per g). Sephadex
G-50 (medium) was obtained from Pharmacia (water regain =
5.1 + 0.3 g per g; particle size, 100 to 270 mesh). Dowex AG
50W-X2 (200 to 400 mesh) was obtained from Calbiochem. All
of these ion exchange materials were washed with distilled water,
1 nw NaOH, distilled water, 3 n HCl, and distilled water. Other
chemical reagents were analytical grade.

Cellophane dialysis tubing (Visking) was heated at 80° for 72
hours or boiled in 1% NaHCO; for 10 min! to decrease pore size.

Assay of Enzymic Activity—The methods for the determination
of ribonuclease and deoxyribonuclease activities, and the def-

1R. 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

inition of units, were described previously (4). The concentra-
tion of protein was determined by measuring absorbance at 280
mu. The activity of the nuclease is expressed in RNase units
unless otherwise specified. One absorbance unit at 280 mu was
assumed to be 1 mg of protein per ml for the calculation of specific

H. Taniuchi and C. B. Anfinsen

4367

activities. DNase activity with DNA-*H as substrate was deter-
mined ecccrding to the methed cf Weissbach and Korn (6).
Yeast RNA was used as inhibitor of DNase activity, and calf
thymus DNA was added as unlabeled carrier DNA after the
incubaticn.

 

 

 

 

 

8.0 8000

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a S
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Oo

no 2
a
at

2.0 — 2000

1.0

Oo 0
0 600 1200 1800

EFFLUENT VOLUME, ML

 

] ] I I

3.0/-

T

ABSORBANCY AT 280 mp

7.0

pH
N
°

°

 

 

| I |. 4

 

T i I J T
3000
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z
4
2000 +»
m
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0 200 400 600

800

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1000—-«1200—s:« 1400 1600 1800

EFFLUENT VOLUME, ML

Fig. 1. Chromatographie 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 1) was applied to the column in the first frac-

tionation. In the second fractionation the fraction containing the nuclease (410,000 units) was applied.

mp; O---O, RNase activity; A——-A, pH.

@——@, absorbance at 280
4368

Purification of Nuclease—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 extract ) was applied to a
carboxymethyl] cellulose column. The unadsorbed activity was
7% of the total activity applied. The procedure for the first
fractionation on the carboxymethy] cellulose column (4 x 30 em)
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

Purification of nuclease from S. aureus V8*

 

 

 

 

 

 

Total |Activity| Absor- . :
Enzyme fraction vol cunts bance « 7 area Total i
ml
Crude extracts... ....| 670 | 1,160| 39.7 2.9/776 000°} 1.04
First carboxymethyl]
cellulose fraction. . .| 530 ; 1,650) 1.82) 920 |877,000 | 1.65
Second carboxymethyl
cellulose fraction. ..| 103 | 4,750} 2.37/2,000 |489,000 | 1.70

 

* One hundred grams of the filter cake were used as starting
material.

' 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.

Partial Sequence of a Staphylococcal Nuclease. [

_ summarizes the results of this purification procedure.

Vol. 241, No. 19

60 ml per hour. Fractions were collected every 10 to 15min. A
typical elution diagram is shown in Fig. 14. The fractions giving
specific activities of more than 900 units per mg were pooled.
After adjustment to pH 6 by the addition of phosphoric acid, the
pooled fraction was diluted with distilled water to 2.5 times its
volume (approximate final concentration, 0.05 m phosphate).
The acidified solution was applied to the second carboxymethyl
cellulose column (2.2 x 75 cm) which was cquilibrated with 0.05
M sodium phosphate buffer, pH 6.1, and gradient elution was
carried out as with the first column. Fractions were collected
every 30 min at a flow rate of 16 ml per hour. A typical diagram
of the second fractionation is shown in Fig. 1B. The fractions
with constant specific activity were combined. The combined
solution (approximatley 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
lyophilization was used in the studies described below. Table I
Fig. 2
shows the ultraviolet absorption spectrum of the purified nu-
clease.

Determination of Ultraviolet Absorbance—A sample of the
purified nuclease was dried over P20; under reduced pressure at
25° for 3 days to constant weight. An aqueous solution of the

 

‘dried protein, containing 1.0 mg per ml, showed an absorbance at

280 my of 1.16.

Electrophoresis and Ultracentrifugal Analysis—The procedures
employed were essentially 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 the Gross and Witkop procedure (10) was
utilized. Protein preparations were dissolved in 70% formic acid

 

ABSORBANCY

9°
a
L

 

 

 

i L
260 270

1 !
280 290

mp

Fic. 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

to make a 1% solution. A 30-fold molar excess of CNBr? 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 90% as judged by amino acid
analysis.

Digestion with Trypsin—Nuclease and separated cyanogen
bromide peptides were incubated with trypsin (1% by weight of
substrate) at 37° for 3 hours. The pH during digestion was ad-
justed to 8.0 with NH,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 yl of 0.05 m ammonium bicarbonate, and the
solution was adjusted to pH 8 (using phenol red) by the addition
of 15 wl of 0.01 ma NH,OH if necessary. Subtilisin (Nagarse) (1
mg in 5 ul of 0.05 mM ammonium bicarbonate) 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 Anfinsen (11). A small drop of phenol red
was added at the origin as a reference standard in the chromato-
graphic 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 methionine-containing peptides were located on the map with
Ehrlich, Sakaguchi, Pauli, and platinum iodide reagents, respec-
tively (13).

For the preparation of certain purified peptides, aqueous
solutions of the trypsin digest, containing 0.1 to 0.2 wmole of each
peptide, were applied on Whatman 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.

Polyacrylamide Gel Electrophoresis—Polyacrylamide gel electro-
phoresis was performed at 4-6° with 7.5% “standard gel” and
15% “4.3 gel” as described in the manual supplied by the Canal
Industrial 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 constant current of
3 to 4 ma per tube for 3 to 5 hours.

Paper Electrophoresis—Pyridinium acetate buffers, pH 3.6 and
6.5 (11), were used on Whatman No. 83MM 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 bicarbonate, 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 120ma. 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 Peptides—All procedures
were carried out at 25°. A column of Sephadex G-50 (8 x 260
em) was poured from a thin slurry, which was allowed to settle

2 The abbreviations used are: CNBr, cyanogen bromide; DNP-,
2,4-dinitrophenyl-; DFB, 1-fluoro-2,4-dinitrobenzene.

HH. Tantuchi andl C. B. Anfinsen

4369

 

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-HCI 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% ammonium acetate.
The sample, an 8% solution of the peptide mixture in the same
buffer, was applied to the column and washed in with three 1-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 mp. Suitable aliquots (15 to 100 wl) 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 Dewex 50—The procedures described by
Canfield and Anfinsen (12, 16) have been employed. A Dowex
AG 50W-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. C. Craig, Rockefeller University, personal communication.
4370

Further Purification of Peptides—All of the tryptic peptides
that separated and the subtilisin peptides derived from one of
them were examined for purity by two-dimensional mapping as
described above. Larger aliquots of the fractions that contained
more than one tryptic 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 cyenogen bromide pep-
tide mixture were examined by paper electrophoresis (2500 valts)
at pH 6.5 for 90 min. Larger aliquots of each cyanogen bromide

+ Ascending

Partial Sequence of a Staphylococcal Nuclease. I

 

Vol. 241, No. 19

peptide fraction were applied as wide bands. After location by
staining cf a side strip with ninhydrin the components were
eluted with 50% aqueous pyridine and lyophilized.

Amino Acid Analysis—Samples were hydrolyzed in 0.5 ml of
constant boiling HCl in evacuated, sealed tubes at 110° for 20
hours. After removal of HCI by rapid evacuation over NaOH
pellets, amino acid analyses were performed by the method of
Spackman, Moore, and Stein (17) on a Spinco model 120 amino
acid analyzer equipped with an Infctronics model CRA-10A
integrator. The recorder of the analyzer was equipped with a
4- to 5-mv resistor card (Minneapolis-Honeywell, kit part No.

Fig. 4. Moving boundary electrophoresis of the purified nuclease, obtained in the Perkin-Elmer model 238 electrophoresis apparatus:
Protein concentration was 6.1 mg per ml, in glycine-HCl buffer (0.1 ionic strength), pH 3.51 (see ‘“‘Experimental Procedure’’); con-

ductivity, 6.2 mmhos.

The photograph was taken after 115 min at a constant voltage of 100 volts. A similar symmetrical schlieren

pattern was obtained with free electrophoresis of the purified nuclease in 0.05 m citrate, pH 7.5, which was kindly carried out by Dr.

William R. Carroll.
Issue of October 10, 1966

-endcm

365928-999). A series of analyses on the acid hydrolysates of
the nuclease containing between 0.01 and 0.1 umole 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 Cystine—The nuclease, oxidized
with HCOOOH according to the method of Hirs (20), was ana-
lyzed for cysteic acid. Reduction and subsequent alkylation
with iodoacetate-“C (21) was also applied to the nuclease prepa-
ration. Radioactive iodoacetate (specific activity 16,500 cpm
per umole; 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 ul of 2-mercaptoethanol, and
0.1 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 ecntent of tryptophan, with the use of 0.1 n NaOH
solutions of the nuclease. Alkaline hydrolysis of the nuclease
was performed in evacuated, sealed tubes at 110° for various
lengths of time by a modification of the method of Lugg (24).
The hydrolysate was analyzed with the amino acid analyzer.
The tryptic peptide containing tryptophan was detected on two-
dimensional peptide maps with Ehrlich’s reagent. The Ehrlich-

H. Taniuchi and C. B. Anfinsen

 

Fig. 5. Comparison of purity of the crude extracts (Crude), the purified nuclease (2nd CM), 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.

4371

Crude Mix.

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 nuclease was determined according to the method of Dr.
Robert Hill. Dried nuclease, 6.6 mg, was dissolved in 0.60 ml
of constant boiling HCl. Aliquots 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 evacuated, 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 ecmplete hydrolysis by
the method described above and analyzed on the amino acid
analyzer.

NH.-terminal Residues by Dinitrophenylation—The NH.-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 umole, dissolved in 20 yl of
water, were placed in test tubes (1.5 X 15 cm), and 200 ul of 1 m
trimethylamine acetate, pH 9.7, were added. One volume of
1-fluoro-2,4-dinitrobenzene was dissolved in 20 volumes of
anhydrous ethyl aleohol. Aliquots of 5 ul of the fluorodinitro-
benzene solution were mixed with the sample solutions. The

4R. L. Hill, Duke University, personal communication.
4372

reaction mixtures were incubated for 3 hours at 37° and lyophil-
ized. The dried mixtures were put in a vacuum oven over NaOH
pellet, and sublimation was performed at 60° for 4 hours. The
residues were extracted with ether and again placed in a vacuum
oven at 60° for 3 hours. The dried materials were dissolved in
0.5 ml of constant boiling HCl and hydrolyzed in evacuated
sealed tubes at 110° for 16 hours. The hydrolysates were sub-
jected to two-dimensional chromatography by the modified Levy
procedure. All manipulations were performed in a darkened
laboratory. The two-dimensional chromatographic separation
of ether-soluble DN P-amino acids was achieved with the lert-amyl
alcohol-1 » NH.OH (4:1) system in the first direction and 15M
potassium phosphate, pH 6.0, in the second (19). The water-
soluble DNP-amino acids were extracted with n-buty! aleohol
and examined by paper electrophoresis at pH 6.5.

Edman Degradation—The Edman procedure modified by
Margoliash (26) was employed for the determination of the N He
terminal residue cf the nuclease. Phenylthiohydantoins of
amino acids were identified by thin layer chromatograms with
two different solvent systems; chloroform-methanol (9:1) and

+

 

A

Partial Sequence of a Staphylococcal Nuclease. I

 

 B Mix.
Standard gel

Vol. 241, No. 19

chloroform-formic acid (20:1). The chromatographic plates
were made with MN-cellulose powder 300 (Machery, Nagel and
Company). The spots were located with iodine azide spray
(27). The NH;-terminal residues of tryptic peptides and of a
small cyanogen bromide fragment of the nuclease were also de-
termined by the following modified Edman degradation (28-30).
The reaction mixtures consisted of 0.2 to 0.5 umole of peptide in
50 yl of distilled water, 50 ul of 2% phenylisothiocyanate in py-
ridine, and 5 yl of 25% aqueous trimethylamine. The mixture
was placed in a conical 8-ml glass tube fitted with a standard
taper glass plug which had two openings, one stoppered with a
rubber vaccine port and one with a ground glass stopper. After
the tubes were flushed with O.-free nitrogen through a hypoder-
mic needle for 2 min, the vessel was sealed and incubated at 40°
for 4 hours. The mixture was extracted five times with 0.5 ml
of thiophene-free benzene under nitrogen. The upper layers
were discarded following each extraction after centrifugation in
an International clinical centrifuge. The residual solutions were
rapidly taken to dryness under reduced pressure. Cyclization
of the phenylthiocarbamy] derivatives was performed by adding

Origin

 

4.3
gel

Fic. 6. Electrophoretic pattern of er
crystals; Miz, a mixture of both samp]
gel.’’

ystalline nuclease on acrylamide gel electrophoresis (standard gel).

es.

Electrophoretic behavior of the purified nuclease at pH 4.3 (see

A, needles; B, hexagonal
the text) is labeled “4.3
Issue of October 10, 1966

0.3 ml of trifluoracetic acid anincubating the resulting solution
at 40° for 15 min under nitrogen. After having been dried under
reduced pressure, the residue was extracted four times with 0.5 ml
of ethylene chloride. The solvent was removed by Pasteur
pipette. The extracts and the residual material were taken to
dryness under reduced pressure over NaQH pellets at 25°. The
dried extracts were dissolved in 0.5 ml of 30% aqueous alcohol,
adjusted to pH 1 by the addition of concentrated HCl, and in-
cubated at 80° for t 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 phenylthichydantoin derivatives. The
dry residual material, containing the protein or tryptides in which
the original NH, terminals had been removed, was dissolved in
200 ul of 50% aqueous pyridine. Two aliquots of 10 wl were
removed and placed in a hydrolysis tube. The aliquots and the
residual solutions were lyophilized. The aliquots were then
hydrolyzed in evacuated, sealed tubes with 0.5 ml of constant
ting HC! at 110° for 16 hours. Amino acid analyses of the
hydrolysates were performed with the automatic amino acid
analyzer, and by paper electrophoresis by the method of Dreyer
“and Bynum as 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 umole 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 w 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 ef al. (31) was used to. examine the nucle-
ase for carbohydrate, with p-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-H was found to be 14 x 104 cpm.

RESULTS

Purity of Nuclease Preparation—The purified nuclease de-
scribed above appeared to be homogeneous on the basis of ultra-
centrifugation,® free boundary electrophoresis,’ 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

5 These experiments were performed with the cooperation of
Dr. R. Suriano.

H. Baniuchi and C. B. Anfinsen

4373

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 preparative 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), peptide 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 signifi¢ant differences. 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 II). 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

 

 

 

] I T T T ]
32.0 | 7
€
°
@o
N
7
>
Oo
a
< 1.0- 4
ac.
oO
n
ao
<
+ _-
0 1 } | J

 

O 10 20 30 40 50

FRACTION NUMBER

Fig. 7. Preparative electrophoresis 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.”
 

A B Mi
: IX.
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 separated by preparative electrophoresis. A and B show the mobilities on standard gel, of
samples obtained from tubes 12 and 24, respectively (see Fig. 7); and Miz represents the pattern obtained from a mixture of the two.

4374
Issue of October 10, 1966

TaBLe II

Comparison of amino acid compositions and sugar contents of two
components obtained electrophoretically from nuclease preparation

 

t

 

 

 

Component I? Component II*
pmole pmole % pmote umole %
Lys. 0.270 15.7 0.303 15.6
His... ow... eee 0.0380 1.7 0.040 2.0
Ar ook 0.055 3.2 0.065 3.4
Asp. eee eee 0.166 9.7 0.187 9.6
Thr. ooo... eee eee 0.122 7.1 0.127 6.5
Ser... ... 20.0022 0.062 3.6 0.062 3.2
Glu... eee 0.197 11.5 0.240 12.4
Pro... oi... eee ee 0.071 4.1 0.087 4.5
Gly. eee eee 0.113 6.6 0.138 7.1
Ala. oo... eee eee ee 0.173 10.1 0.185 9.5
Cys... eee 0.000 0.0 0.000 0.0
Val... eee 0.116 6.7 0.121 6.2
Met.............005- 0.026 1.5 0.036 1.9
Tle. oo. eee 0.070 4.1 0.065 3.4
Leu... ..... ee eee 0.132 7.7 0.155 8.0
0 0.076 4.4 0.088 4.5
Phe... .............- 0.040 2.3 0.041 2.1
mole/mole protein mole/mole protein
Glucose equivalents?. 0.238¢ 0.66¢

 

 

 

¢ 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.

> See ‘Experimental Procedure.”’

¢ The amounts of protein used for analysis were 0.080 and 0.12
umole for Components I and II, respectively.

 

 

 

 

T T
03h 4.5
i >
4 &
E oS
© @
“ 0.2b 410 8
a 7
>
S ° zi
a ®
< oO
” 3
2 otk dos F
4 |
\ !
%5 0.5 1.0

ENZYME CONCENTRATION

Fig. 9. RNase and DNase activities of the nuclease as a func-
tion of enzyme concentration. The methods of assay are described
in “Experimental Procedure.” O-——O, RNase; @-—@6@,
DNase.

H. Taniuchi and C. B. Anfinsen

4375

. TaB_e III
Effect of phosphate and citrate ions on RNase and DNase activity

 

Activity
Addition

 

| DNase RNase

 

% of control % of control

None*®. 0... eee eee 100 100
Phosphate, 5 X 10-4 M.......... 117 105
Phosphate, 5 X 10-9 M.......... 228

Citrate, 5 XK 10 M............. 337 239
Citrate, 5 X 10-3 M.........-.e. 542 184

 

* The concentrations of contaminating phosphate ions from the
original enzyme solution were 1.2 X 10-' m and 5 X 10-5 M in the
reaction mixtures for the DNase and RNase assays, respectively.

 

 

 

 

 

3.0 T T T T 3000
a
é A
8 6
a 2.0- 1b ~2000

1
q 1 S
3 4
2 v
o m
iid a
zB z
ao
a 1000
o=} #
900 1200 | 1500 1800

EFFLUENT VOLUME, ML

Fig. 10. RNase and DNase activities of chromatographic frac-
tions of the nuclease from carboxymethyl cellulose. @——®,
absorbance at 280 mu; O- --O, 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 mp were diluted 100-fold with 0.05 m sodium phos-
phate buffer, pH 6.1. From each diluted solution, 5- and 10-ul
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 results 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-
4376

TasBLe IV

Effect of RNA on DNase activity

|
Concentration of RNA | Concentration of DNA-3H

 

DNase activity

absorbance units/ml epm

0 0.25 3535
0 0.75 9700
90 0.25 1387
90 0.75 4222

 

Partial Sequence of a Staphylococcal Nuclease. I

Vol. 241, No. 19

value of 7 residues of tyrosine per molecule (Table VII). This
was checked further by staining peptide maps of trypsin and
eyanogen bromide digests with Ehrlich’s reagent. Both of the
digests showed only a single Ehrlich-positive component. The
Ehrlich-positive tryptic peptide was isolated and subjected to
further digestion with subtilisin as described above, giving two
fragments. Only one fragment, with a high Rp value on the pa-
per chromatogram, showed a positive reaction with Ehrlich rea-
gent. These results indicate that 1 residue of tryptophan is
present per molecule of the nuclease.

TABLE V

Amino acid composition of nuclease

 

 

 

 

 

 

 

 

Amino acid content after acid hydrolysis for® Calculated moles
Residue Average per 3 moles of
17 hrs 34 hrs 57 hrs phenylalanine?
umole pmole pmole Bmole
Lys.......2.0.... 0.148 0.144 0.148 0.148 0.138 0.150 0.147 18.4 (21.4*)
His.............. 0.024 0.022 0.024 0.022 0.023 0.025 0.024 3.0
Arg... 0.20.00... 0.039 0.038 0.039 0.036 0.038 0.039 0.038 4.8
ASp............. 0.113 0.113 0.112 0.112 0.105 0.108 0.117 14.6
Thr. wo... ee... 0.077 0.075 0.078 - 0.076 0.073 0.075 0.0774 9.6
Ser.............. 0.040 0.037 0.038 0.039 0.032 0.033 0.0414 5.1
Glu. ....... 22... 0.145 0.148 0.154 0.144 0.144 0.148 0.146 18.3
Pro.............. 0.041 0.041 0.040 0.036 0.039 0.043 0.041 “*8.]
Gly..... 022... 0.064 0.064 0.064 0.062 0.063 0.063 0.063 7.9
Ala... 2.02. .2... 0.113 0.116 0.112 0.112 0.110 0.114 0.115 14.4
Cys. .........0.. 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.0
|
Val... eee. 0.069 0.068 0.076 0.071 0.071 0.077 0.074 9.2
Met............. 0.026 0.028 0.029 0.027 0.026 0.028 0.028 3.5
lle. ............. 0.038 0.038 0.042 0.038 0.040 0.040 0.040 - 5.0
Leu............. 0.091 0.091 0.098 0.088 0.090 0.094 0.093 11.6
Tyr. ............ 0.049 0.049 0.054 0.049 0.052 0.054 0.053 6.6
Phe. ............ 0.023 0.018 0.025 0.021 0.025 0.023 0.024 (3.0)
Trp. ........ 2... 0.000 0.000 0.000 0.000 0.000 0.000 0.000 le

 

 

 

 

* The results of duplicate analyses are given (see the text).

> Three tryptic peptides containing phenylalanine were obtained from tryptic digests of cyanogen bromide fragments by separation

on Dowex 50 columns (see the text).

¢ 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.
4 Corrected for slight destruction during hydrolysis.
* See the text.

ysis for cysteic acid after performic acid oxidation and by
radioactive measurements after alkylation with “C-iodoacetic
acid. Cysteic acid levels were less than 0.08 mole per mole of
nuclease (Table VI). Although approximately 1 mole of iodo-
acetic acid-1-“C 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 fragments® 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.

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

§ To be published.

The amide-NH; content, determined as described above, was
shown to be 11 moles per mole of nuclease, based on an assumed
value of 3 residues of phenylalanine per mole (Table V).

The amino acid analyses permit the calculation of a minimum
molecular weight for the protein of approximately 17,000.
Preliminary studies on the amino acid content of the separated
tryptic peptides are in good agreement with this figure, as de-
scribed below.

NH:-terminal Residue—The DFB method yielded alanine ex-
clusively as the NH.-terminal residue in a yield of 80 to 90%.
The. phenylthichydantoin 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 the ultracen-
trifuge, together with determinations of partial specifie volume in
density gradients, yield a molecular weight value in agreement
with this number within experimental error; J. Heins, R. Suriano,
H. Taniuchi, and C. Anfinsen, unpublished results.
Issue of Gictober 10, 1966

ferent solvent systems on thin layer chromatograms. Alkaline
hydrolysis of the intermediate thiazolinone, formed by eyclization
of the original phenylthiocarbamy! nuclease, yielded alanine,
with a trace amount of glycine upon amino acid analysis.

COOH-terminal Residue—Treatment with carboxypeptidase A
liberated glutamine in a yield of approximately 80% after incuba-
tion at both 25° and 37° for 24 hours. Glutamine was identified
qualitatively on ascending chromatograms with 80% pyridine as
solvent (Ry, 0.21) (aspargine, Ry, 0.14; serine, 0.35). No other
amino acids were detected either on the automatic amino acid
analyzer or on paper chromatograms. After 24 hours of incuba-
tion with the enzyme at 25° and 37°, respectively, 85 and 78%
of the specific enzymic activity of the nuclease remained.

Peptide Maps—Fig. 11 shows a peptide map obtained with a
mixture 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 preparation. Only F25
gave a positive Ehrlich reaction for tryptophan. Components
F15, F17, F18, and F19 were clearly bleached by the platinum
iodide test for methionine. F17 and F19 also gave a positive
Sakaguchi test for arginine. 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 peptide 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
with 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 method con-
tained methionine as expected and account for the 4 residues of
this amino acid indicated 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 the same method as above indicated the
compositions (His, Pro, Lys) and (Lys), respectively.

Cyanogen Bromide Fragments—If the nuclease contains 4
residues of methionine, five fragments should be formed after
cleavage with cyanogen bromide. End group analysis by the
DFB method of CNBr digests showed the presence of 2 moles of
NH,-terminal 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 mu) was 65%. An aliquot 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, VI, and
VIII, which were relatively homogeneous on the basis of these

A. Taniuchi and C. B. Anfinsen

Taste VI

Amino acid analysis of nuclease after perfermic acid oxidation

 

 

 

 

 

mine sett concent_ |, Ciluate moles per
umole
Lys. 0... eee eee eee 0.825 22.2
His... ....... cece 0.100 2.7
APB. eee eee 0.175 4.7
Cysteic acid............ 0.003 0.1
Methionine sulfone..... 0.135 3.6
ASP. occ ccc cece cece ees 0.502 13.6
Thr... ooo. ee eae 0.341 9.2
Ser... eee ee eee 0.166 4.5
Glu... eee eee 0.664 17.9
Pro... oo... ee eee 0.217 5.9
Gly ooo. 0.360 9.7
7 6 rr 0.511 13.8
CYS. cece eee 0.000 0.0
|
Valo cece eee 0.325 8.8
Met.................... 0.600 0.0
He... 0.175 4.7
Leu... oo... eee 0.426 11.5
Os 0.196 5.3
Phe... ........-2 000000 0.111 (3)
TaBre VII

Spectrophotometric analysis of mole ratio of tyrosine to tryptophan

 

Ratio of tyrosine

Method of analysis to tryptophan

Method of Beneze and Schmid (23)............... 5
Method of Goodwin and Morton (22)............. 6.6
Amino acid analysis after alkaline hydrolysis.....

 

¢ 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 68-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 NH:-terminal threonine and
alanine, was subjected to preparative electrophoresis in the
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 NH2-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
fraction 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, VI, and
VIII (Peptides A, C, D, and B, respectively). Fraction HI
(Peptide E), lacking this residue, must be COOH-terminal in the
nuclease; and Fraction Va (Peptide A), which contains an NH:
4378

> ELECTROPHORESIS

 

 

Fig. 11. Peptide map obtained from a trypsin digest of the nuclease.
Electrophoresis was carried out at 2500 volts for 80 min.

man No. 3MM paper for two-dimensional separation.

Partial Sequence of a Staphylococcal Nuclease. I

 

Vol. 241, No. 19

—> CHROMATOGRAPHY

The digest, containing 1 mg of protein, was applied to What-
The peptides giving

a positive platinum iodide reaction are indicated as MET. The spots indicated by Y gave yellow color upon staining with ninhydrin-

aleohol solution.

terminal alanine residue, must be the NH,-terminal peptide.
Only Fraction IIT (Peptide E) gave a positive test for tryptophan
and, as discussed below, yielded a fragment upon trypsin diges-
tion which corresponded to the single Ehrlich-positive compo-
nents on the peptide map. The sum of the residues determined
for the individual CNBr fragments corresponds well with the
total amino acid content presented in Table V above.

Ehrlich indicates the spot with a positive Ehrlich reaction.
only spots clearly identified on the original map are shown (as Arg).

Among those spots giving a positive Sakaguchi reaction,

Trypsin Digestion of CNBr Fragments—Samples of cyanogen
bromide peptide Fractions III, V, and VII were digested with
trypsin, and the resulting mixtures of peptides were subjected to
separation on Dowex 50-X2 columns as described under “Experi-
mental Procedure.” The chromatographic patterns are shown
in Fig. 14. The purity of the separated peptides was examined
by two-dimensional peptide mapping, amino acid analysis, and
Issue of October 10, 1966

end group determinations by both the DFB and the Edman
procedures. Further purification, when necessary, was performed
by either paper ¢électrophoresis or paper chromatography as
described in “Experimental Procedure.”’ Table XII summarizes

Tas ie 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 umole.

 

 

 

 

 

 

Designation on peptide map
F-15 F-17 F-18 F-19

Lys... 0.2.02. ee. 0.012 (1) | 0.025 (1) | 0.003 (0)
His. .......-.... 0.003 (0)
ATE. oo... eee 0.004 (0) | 0.015 (1) | 0.015 (1)
Asp.. 0.023 (1) | 0.005 (0) | 0.020 (1)
Thr............. 0.003 (0) | 0.016 (1)
Ser.....0.....00. 0.003 (0) | 0.009 (0) | 0.004 (0) | 0.007 (0)
Glu... 2.2... 0.028 (1) | 0.015 (1) | 0.017 @)
Pro 0.007 (0) | 0.015 (1)
Gly. oo... eee. 0.004 (0) | 0.011 (0) | 0.013 (1) | 0.006 (0)
Ala. oo... ...00.. 0.023 (1) | 0.004 (0) | 0.018 (1)
Cys... eee ee
Val... : 0.021 (1) 0.029 (2)
Met............. 0.010 (1) | 0.013 (1) | 0.012 (1) | 0.007 (2)
5 te
Leu............. 0.009 (1) 0.017 (1)
Tyr... eee eee 0.007 (i)
Phe... .......... 0.004 (0) | 0.015 (1)
NH_.-terminal

residue*.......| Leucine Methionine, Glycine {Methionine

 

H. Taniuchi and C. B. Anfinsen

 

* The analysis was qualitative, but only a single ether-soluble
DNP-amino acid was found for each peptide.

4379

the data obtained on these tryptie 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

Taste IX ;
Amino terminal analysis of cyanogen bromide fragments
of nuclease with DN P-method
Sample 1, intact nuclease; Sample 2, nuclease treated with 70%
HCOOH (without CNBr); Sample 3, nuclease treated with CNBr
in 70% HCOOH. Each sample contained 0.12 umole of the nu-
clease.

 

DNP-amino acid found*

 

 

Sample
Alanine | Valine | Threonine Tyrosine
pmole umole umole umole
1 0.10
2 0.11
3 0.12 0.24 0.01% 0.02°

 

 

¢No water-soluble DNP-amino acids, other than «-DNP-
lysine, could be detected by paper electrophoresis in pyridinium
acetate, pH 6.5.

» The low recovery of these end groups in the present experiment
is diseussed in the text.

 

a
E
2
v1.0
a
~~
Oo
2
aq
oO
a
D
co 0.5
oi

 

 

AwOss LY ADNVaHOSSYV

 

 

0
Oo 100

300

TUBE NUMBER

Fig. 12. Gel filtration pattern of cyanogen bromide peptides prepared from the nuclease.
cyanogen bromide digest. Other details are given in ‘“‘“Experimental Procedure.” @-——®, absorbance at 280 my;

sorbance at 570 mu.

The column was loaded with 360 mg of
O-- -O; ab-
4380

appear in the peptide maps of Peptide C. Cyanogen bromide
Freement B (obtained from Fraction VIII of the Sephadex G-50
separstion) did not fiagment further upon trypsin as tested by
peptide mapping. The composition of the CNBr fragments is
compared with the sum. of the amino acid compositions of the
trypsin fragments assigned to them in Table XIII. The total
amino acid analysis of the original nuclease is included in this
table for over-all comparison.

TABLE X
Qualitative summary of several sets of quantitative analyses for
amino-terminal residues of cyanogen bromide fragments
prepared by Sephadex G-50 gel filtration

 

DNP-amino acid found®

 

 

 

Fraction Pooled tubes®

Alanine Valine |Threonine | Tyrosine
I 128-145 + + + +
II 151-168 _ +++ _ _
IV 169-173 + ++ | i+ +
Vv 174-191 +++ | Trace | +++ | Trace
VI 192-195 - + ++ + _
VII 196-213 - +++ _ _
Vir 272-295 _ _ - +++

@ See Fig. 10.

> Only ether-soluble DNP-amino acids were analyzed (see Ta-
ble IX). The relative intensities of the DNP-amino acids are
qualitatively indicated by plus signs. Ether-soluble DNP-amino
acids, other than those shown in this table, were not found.

Partial Sequence of a Staphylococcal Nuclease. I

Vou. 241, No. 19

Reconstruction of Tryptie Peptide Map- The map pusitiens of
separated tryptic peptides of each cyanogen bromide pet!

   
 

 

 

 

 

 

0.5 I rT
+ —
+p +
0.4b 4
=
oO
@o
“ o3b _
i 0
>
Oo
Z 02 —
© PHENOL RED
°
o
@ Oo)
Oo
0 10 20° 30 40 50

TUBE NUMBER

Fic. 13. Preparative electrophoresis of cyanogen bromide frag-
ment Fraction V, obtained by gel filtration. Approximately 10
umoles of peptides, dissolved in 0.5 ml of 0.05 M NH,HCO;, were
applied through the entry port above tube 30. Other details of
the procedures are described in “Experimental Procedure.’
Pooled fractions are indicated by horizontal arrows.

TasLe XI
Approximate amino acid composition of cyanogen bromide fragments
Values are reported in micromoles. The values in parentheses represent assumed number of residues.

 

 

 

Amino acid content
Total number
of residues
Fraction III Fraction Va Fraction Vb Fraction VII Fraction VIII
Lys... 2. eee. 0.223 (5) 0.236 (4-5) 0.151 (6-6) 0.081 (4-5) 0.039 (1) (19-22)
His.................. 0.080 (1) 0.041 (1) 0.019 (1) 0.000 (0) 0.000 (0) (3)
ATg. oo. 0.071 (2) 0.064 (0) 0.021 (1) 0.029 (1-2) 0.000 (0) (3-5)
ASP... 2. ee 0.276 (6-7) 0.089 (2) 0.031 (1) 0.071 (38-4) 0.002 (0) (12-14)
SY 0.042 (1) 0.154 (3-4) 0.086 (3) 0.020 (1) 0.000 (0) (8-9)
Ser... 2... 0.105 (2-3) 0.043 (1) 0.028 (1) 0.011 (0) 0.002 (0) (4-5)
Glu... ee, 0.372 (9) 0.052 (1) 0.077 (3) 0.075 (4) 0.052 (1) (18)
Pro... 0.6... eee 0.046 (1) 0.053 (1) 0.077 (3) 0.005 (0) 0.018 (1) (6)
Gly... ooo cece cee 0.086 (2) 0.057 (1) 0.059 (2) 0.075 (4) 0.042 (1) (10)
Ala. oo... 0.241 (6) 0.119 (2-3) 0.055 (2) 0.057 (3) 0.000 (0) (13)
Cys... ccc. 0.000 (0) 0.000 (0) 0.000 (0) 0.000 (0) 0.000 (0) (0)
vel Leet ee 0.146 (3) 0.053 (1) 0.041 (2) 0.038 (2) 0.000 (0) (8)
Met...........0..000. 0.000 (0) 0.000 (0) 0.002 (0) 0.000 (0) 0.000 (0) (0)
He... ee, 0.088 (1) 0.088 (2) 0.012 (1) 0.029 (1-2) 0.000 (0) (5-6)
Leu... 0... 0... eee 0.198 (5) 0.131 (3) 0.068 (2-3) 0.023 (1) 0.000 (0) (11-12)
Tyr... cece eee 0.000 (2) 0.005 (0) 0.001 (1) 0.000 (3) 0.031 (1) (7)
Phe.................. 0.003 (0) 0.007 (0) 0.034 (1-2) 0.016 (1) 0.000 (0) (2-3)
Trp..............00.. — (1) — (0) — (0) — (0) — (0) (1)
Homoserine. Pees 0.000 (0) 0.054 (1) 0.082 (1) 0.021 (1) 0.041 (1) (4)
NH.-termina! residue. Valine Alanine Threonine Valine Tyrosine
Designation of peptide. E A Cc D B

 

 

 

 

 

 

 

¢ The values obtained from analysis of samples before purification by paper electrophoresis (see the text) were used to correct for

destruction of tyrosine.

* Determined by staining with Ehrlich’s reagent (see the text).
 

   

 

 

 

 

 

 

 

 

 

 

  

 

 

50° .
st Voriarad, 35° _—_—_——_»le-ole-pH 9.9,50°—> io
yore 9
i
I 718 H
p
} 47
' 6
a < io oo”
L o> OO 4
<x poo +4
o J
5 TH2—> 43
a .
a
at
0.5
TI-|
t
Oo I
0 100 200 300
FRACTION NUMBER
, 50°
+ Varigrad, 35° ———-*#e_ +4 pH 9.9, 50°
15 eo 10
i Z 9
: 48
i od,
aa
! 6
je yy 4
> 1,0 pg PPO POOLS OO OO LOLS 5
2 poag ree 7 4
a TV-2—> 3
9°
a
o
<_
0.5}
TV-|—>
0 L l
0 100 200 300
FRACTION NUMBER
Le Vorigrod, 35°-———————-—>#@- 50° pH_7, 50° PH pH 9.950)
1.5 gt lO
i 49
i,
t
(oA7
ptr 0070 008 6 Pt
Ppoow!
S 1.0} woads 99g OTS TOPO HOO 5
9 DO--o poorer e a4
Zz Cow
a 343
ao
a
9
vy
a
t
O.5}-
TH yy ig
TEN-2 TUS
TYI-!>
ok
° 100 300 400

FRACTION NUMBER

Fre. 14. Chromatography of tryptic peptides prepared from cyanogen bromide Fragments IT (upper), V (middle), and VII (lower),
on Dowex 50-X2. Samples equivalent to approximately 4, 2, and 4 zmoles of Fractions III, V, and VIT, 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 tryptic peptides prepared from cyanogen bromide fragment

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 umole. 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. As described in the text and in Table V, Fractions ITI and
VII were essentially homogeneous samples of CNBr Fragments Eand D. Fraction V was further separated by curtain electrophoresis
to yield CNBr Fragments A and C. 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 TIIL-10, THTi-11, TV-8, TV-4, TV-14, TV-16,
TV-18, TVII-6, TVIL-9, and TVII-12 corresponded to minor spots on peptide maps, and their compositions are not reported here. A
sample of Peptide TIITI-2 was digested with subtilisin. The two fragments formed were separated by paper chromatography as de-
scribed in ‘‘Experimental Procedure.’’ These fragments were designated TIII-2-S-1 and TIII-2-8-2.

Table XITA

 

TH I-te | TIT-2 |TIHI-2-S-1 TIII-2-$-2! THI-4c | TilI-4e | TII-5b | TYI-5sc | TITI-6 TIli-7a | TI-7b | TIII-8b | THI-9c | TII-12

 

 

 

0.021 (1) 0.009 (1): 0.012 (1)! 0.012 Ga) 0.017 (1)! 0.029 (1)) 0.054 (2)] 0.034 (2); 0.029 (1)
0.020 (1)
0.032 (1) 0.026 (1)
0.072 (4)| 0.119 (3)| 0.024 (1) 0.006 (0) 0.025 (1) 0.042 (2)
0.018 (1)
: 0.0381 (2)) 0.064 (2) 0.007 (1) 0.016 (1)
0.027 (1)! 0.044 (2)| 0.086 (2) . 0.016 (2); 0.014 (1) 0.030 (1)/ 0.019 (1)) 0.031 (1); 0.070 (2)) 0.023 (1)| 0.048 (2)
7 ‘ . , 0.033 (1)
0.025 (1)! 0.022 (1)] 0.040 (1) 1 0:003 (0) 0.003 (0)| 0.017 (1)| 0.028 (1) 0.003 (0)
-| 0.026 (1)} 0.022 (1)] 0.043 (1) 0.004 (1)| 0.016 (2) 0.030 (1)] 0.019 (1); 0.034 (1)] 0.071 (2) 0.010 (0)
0.003 (1) : 0.057 (2) 0.031 (1)
0.019 (1) 0.024 (1) | 0.003 (0)
Lew... 0.026 (1)! 0.021 (1) 0.023 (1) 0.032 (1), 0.019 (1}1 0.038 (1) 0.050 (2)
Ty? eee 0.003 (1) ' 0.026 (1)
Phe...) 22... oe,
Trp...... bone e es _— + (1)% _— + (1) _ _ _ _ a _ _ _ _ _
Homoserine..........
NH:-terminal residue? Leucine Serine Leucine Valine Serine | Glutamic! Lysine Valine | Glutamic} Glutamic] Lysine Lysine Valine

acid | acid acid

Assigned to cyanogen
bromide peptide. ... E E E E E E E E E E E E E E
Inclusion in summa-
tion (Table XIII)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

indicated by +..... — + —4 4d + —¢ —f _o 4 we 4 + + +4
Table XITB
TV-1 TV-2 TV-5b TV-6 TV-7b TV-8a TV-10 TV-13 TV-15a
0.015 (1) 0.011 (1) 0.010 (1) 0.018 (1) 0.028 (2) 0.029 (2)
0.004 (1)
0.010 (1)
0.030 (2) 0.010 (1)
0.016 (1) 0.012 (1) 0.006 (1) 0.031 (2) 0.014 (1) 0.007 (1)
0.009 (1) 0.013 (1)
0.012 (1) 0.012 (1) 0.011 (1) 0.013 (1) 0.015 (1)
0.013 (1) 0.010 (1) 0.006 (1)
0.013 (1) 0.011 (1) 0.003 (0) 0.013 (1)
0.014 (1) 0.021 (2) 0.011 (1) 0.013 (1)
0.016 (1) 0.007 (1) 0.016 (1)
0.014 (1) 0.008 (1)
0.017 (1) 0.035 (3) 0.010 (1)
0.010 (1)
0.006 (1) 0.016 (1)
0.017 (1)
Leucine Alanine Leucine Tyrosine ‘ Glutamicacid Alanine Threonine Lysine Threonine
A A Cc c A A Cc. Cc Cc
+ + + + + + + + +

 

 

 

 

 

 

 

 

 

 

 
Issue of October 10, 1966 A. Taniuchi and C. B. Anfinsen 4383

 

 

Table XIIC
TVIL-1 TVII-2c TVIH-3b TVII-3c TVII-4b TVIT-5a TVII-8a TVII-8b TVI-tla

Ly8. 0. ees 0.009 (i) 0.022 (1) 0.004 (0) 0.027 (1) 0.012 (1) 0.031 (1) 0.020 (2) 0.003 (0)
His... eee ee
ATE. coco ccc c cent ees 0.035 (1) 0.050 (1-2)?
ABD... eee 0.015 (2) 0.023 (1) 0.005 (0) 0.033 (1) 0.085 (1) 0.012 (1)
Thr... eee eee 0.003 (1) 0.014 (1) 0.012 (2)
Ser... 0.0... eee eee eee 0.006 (1) 0.004 (0) 0.004 () 0.003 (0)
Gla... eee 0.011 (0) 0.036 (1) 0.028 (2) 0.055 (1)
PLO. eee
Gly... eee 0.008 (1) 0.003 (0) 0.021 (1) 0.003 (0) 0.029 (1) 0.005 (0) 0.036 (1) 0.024 (1)
Ala... lee ee 0.005 (1) 0.033 (1) 0.033 (1) 0.008 (1) 0.029 (1)
CyB... cnc cee

|
Val... ee eee 0.006 (1) 0.033 (1) 0.010 (1)
Met... 0.20.00 eee
Tle. 0. eee 0.008 (1) 0.003 (0) 0.030 (1) 0.010 (1)
Leu... eee eee 0.033 (1)
TYP. eee 0.027 (1) 0.034 (1) 0.014 (1)
Phe. ... 2.0.6. ee eee 0.005 (0) 0.017 (1)
TEP... eee eee _ _— — _ a — —_ aol cal
Homoserine..............2-.-- 0.004 (0)
NH:2-terminal residue, ........ Alanine Threonine Glycine Valine Isoleucine Lysine Glycine Tyrosine
Assigned to cyanogen bromide

peptide..... rrr A D D D A D D D D
Inclusion in summation... .... —t + + + — + + + +

 

 

 

 

 

 

 

 

 

 

® The presence of tryptophan was also confirmed by digestion with leucine aminopeptidase (Worthington) by the method of Canfield (18). Ammonium bicarbonate
buffer (0.05 m) containing 0.01 m MgCl: was used. Tryptophan was determined quantitatively on the amino acid analyzer.

» Aliquots of fractions obtained by chromatography on Dowex 50 were used for amino-terminal residue determination by both dinitrophenylation and Edman degrada-
tion. When a peptide was contaminated with a second peptide, the amino-terminal residue could be assigned on the basis of the amino acid composition of the purified
peptide.

© TIII-te and TIII-7a have the same amino acid composition as TIII-7b, presumably due to the formation of pyrrolidone carboxylic acid from a terminal glutamine resi-
due.

4 TIIT-2-S-1 and TIII-2-S-2 are constituents of TIII-2.

* Peptide TIII-4e is derived from TIII-8b, by loss of a single lysine residue.

! Peptide TIII-5b is assumed to be derived from TIII-9c by loss of the NH:-terminal lysine residue. This relationship is now under reexamination.

? Only free lysine was detected.

4 The basis of this assignment was obtained from qualitative amino acid analysis of tryptic Peptide TVII-ilb, which seemed to inleude the amino acid components of TV-
7b. This peptide is presumably derived from cyanogen bromide Fragment A which contaminated Fragment D as a trailing fraction during the gel filtration on Sephadex
G-50 (see the text). :

* Further study with successive Edman degradation suggests that the glutamic acid 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 bas tentatively been assigned to this peptide. The presence of a possible 2nd arginine residue is under investi-
gation.

% Amino acid compositions of TVII-1 and TVII-4b are the same as those of TV-2 and TV-8a, respectively (see Footnote h and the text).
4384

TasLe XIII

Amino acid composition of cyanogen bromide fragments based
on analysis of tryptic peptides derived from each

The following 24 tryptic peptides (identified in the bottom line
of Table XII), together with CNBr Fragment B (Table XI), ac-
count closely for the amino acid composition of the nuclease.
Peptide A: TV-1, TV-2, TV-7b, TV-8a; Peptide C: TV-5b, TV-6,
TV-10, TV-13, TV-15a, F-2; Peptide D: TVII-2e, TVII-3b,
TVII-8c, TVII-5a, TVII-8a, TVII-8b, TVII-lla; Peptide E:
TIII-2, TII-6, THI-7b, TII-8b, TII-9c, 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.

amine rome frames Sum of |Combodtion

peptides? | setsese
A B c# D E
Lys...... 3 (5) | () 6 G6)/5 4-5)|6 646)) 21 21.4
His...... 1 (1) 1d) 1 (1) 3 3.0
Arg...... 1 (1) {2 (1-2)|2 (2) 5 4.8
Asp...... 2 (2) 1 (1) |4 @-4)|7 6-7) 14 14.6
Thr...... 4 (3-4) 3 3) 1 da) |1 dd) 9 9.6
Ser...... 1 (1) 1 (1) 3 (2-3) 5 5.1
Glu...... 1 (1) | GQ) 38 @) {4 4) [9 () 18 18.3
Pro...... 1 (1) | @) j3 @) 1 (1) 6 5.1
Gly...... 1) | (1) 2 (2) [4 (4) [2 @) 10 7.9
Ala...... 3 (2-3) 2 (2) |3 (3) 16 (6) 14 14.4
Cys
|
Val...... 1 (1) 2 (2) {2 (2) |4 (4) 9 9.2
Met...... 3.5
Tle....... 2 (2) (1) 2 @-2)/1 @) 5 5.0
Leu...... 2 (3) 3 (2-3)/1 (1) 15 () 11 11.6
Tyr...... (1) 1 @) 43 (3) [2 @) 7 6.6
Phe...... 2 (i-2))1 (1) 3 3.0
Trp...... 1 (1) 1 1
Homo-
serine..j1 (1) | @) | (Wd (1) 2

 

 

 

 

 

 

 

 

* Tryptic peptide F-2 (His, Pro, Lys) (see the text) which was
not recovered among the purified tryptic peptides obtained from
cyanogen bromide fragments, is assigned to Peptide C on the basis
of histidine content.

> The estimated number of residues in Peptide B is included.

digest of the nuclease preparation was also subjected to mapping,
and major spots were cut and eluted as described in “Experi-
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
trypsin fragments studied, only peptide F2 of the original peptide
map (Fig. 9) was not accounted for by the CNBr fragments.
Linear Arrangement of CN Br Fragmenits—On the basis of NH;-
terminal end group data alone, Peptide A can be assigned to the
NH--terminal 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
TV-2 and TV-8a). Peptide E must occupy the COOH-terminal
position since it lacks homoserine and yields the trypsin Fragment
TIII-2 which contains neither lysine nor arginine and from

Partial Sequence of a Staphylococcal Nuclease. I

Vol. 241, No. 19

which is liberated glutamine in 60%, yield upon 2-hour incubation
with carboxypeptidase A at 37° as described under ‘ ‘Experi-
mental Procedure.”

The remaining CNBr fragments can be aligned on the basis of
the methionine content of the nuclease (4 per molecule) and the
composition of the four methionine-containing peptides isolated

 

T T T TO Too 7

T T T
Ok ev" |
T-I-9C
a (F-5) Ore-10

T-M-43
(F6} T-IE-Sb

T-MIE-8b

B® Z|

T-HI-7b
Te 8) M@ir-m-i2 (F1a) Q)rx-ts0
treo (EN)
(TMA G7) 1 -wr- 1 “war @u:
ars Y) 0 Qyw-t 4
Qrm-x

o
n

 

 

 

2 Tin Or-m-2¢ “Y
nm 4e T-WI - 5a (F 24)
2g 5
te
oO L QO)r-reiF20) 4
=
Z Ores
ira (T-MIL-1) T-IEAC T-Ir3b
FE C.4e (F22) T-II-7a Q)
2 g
w
a
iw T-¥-5b
L F @=» Qn 4
x T-¥-6
T-u-1
0.2 Qr-m-80 4
-mI-2
(F25)
PHENOL, RED
oof oO” -
t j 1 | 1 ! 1 l 1 I a
0 02 0.4 06 08 10

Rr (CHROMATOGRAPHY )

Fic. 15. Reconstructed tryptic peptide map. Phenol red and
lysine served as reference standards in the chromatographic and
electrophoretic dimensions, respectively. The significance of
the designations shown in parentheses is described in the text.
On a qualitative basis, the following additional assignments of
peptides can be made on the map. Component F-16 on the “ex-
perimental’’ peptide map (see Fig. 11) contains a mixture of pep-
tides, some incompletely cleaved by trypsin, including TVII-2c,
and TIII-4e. Similarly, F-21 contains TV-6 and TV-10. F-24
corresponds to the addition of TVII-3b and TVII-5a, and F-14
contains TVII-11a in addition to TIII-7b.

| T-V~1 | | T-V-15a |

LEU —MET-TYR~—LYS-GLY —(GLU, PRO) —-MET ~ THR ~PHE~ ARG

| |
Peptide B
} | J
FI5 | F18 |

Fig. 16. The linear arrangement of cyanogen bromide Frag-
ments A, B, and C. Peptide B corresponds to Fragment B.
 

 

 

 

Issue of October 10, 1966 H. Taniuchi and C. B. Anfinsen 4385
_— A po a B >~< Cc >
NH2-ALA - - ------- X-LEU-MET-TYR-—LYS-GLY-(GLU,PRO)—~MET-THR-PHE-ARG~ - ------- X—-MET-—
|, 15 {- F 18 i L_
_— D >t E >
~VAL-(GLU,ASP,ALA)-LYS —---.------------ X—-MET-VAL-(ASP,GLU,ALA,VAL,LEU)-ARG — ------ GLN-COQH
F 17 ————> ir F19 _»
T-VIH-3¢ —_—p4J +t T-Ill-6 >

 

 

 

 

Fra. 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 studies* have indeed shown that these residues are lysine.

from peptide maps. If we compare the amino acid composition
and NH.-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-1 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--terminal 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.

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