Reprinted from the ProcerviInGs oF THE NATIONAL ACADEMY OF SCIENCES
Vol. 58, No. 3, pp. 1235-1242. September, 1967.

NUCLEASE-T: AN ACTIVE DERIVATIVE OF
STAPHYLOCOCCAL NUCLEASE COMPOSED OF TWO
NONCOVALENTLY BONDED PEPTIDE FRAGMENTS

By Hirosui Tantrucui, CuristiAn B. ANFINSEN, AND ANN SopJa

LABORATORY OF CHEMICAL BIOLOGY, NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES,
NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND

Communicated July 26, 1967

An extracellular nuclease produced by Staphylococcus aureus!—* has been shown
to contain 149 amino acid residues (mol wt, 16,807) and to lack both sulfhydryl
groups and disulfide bonds (Fig. 1).2-5 The enzyme catalyzes the cleavage of
both DNA and RNA to yield 3’-nucleotides.2 ? The absolute requirement for
Cat+1,2,5 may be explained by the interdependency of Cat+ and nucleotide
binding to the protein, and the conformation of the enzyme is stabilized by this
reversible ligand interaction.7,* The present communication summarizes experi-
ments on the cleavage of the polypeptide chain by limited digestion with trypsin in
the presence of 3’,5’-deoxythymidine-diphosphate to yield three peptide fragments:
Nase-T-p,° (residues 1-5), Nase-T-p. (residues 6-49), and Nase-T-ps (residues 50-
149). The latter two fragments associate reversibly to form an active complex
designated nuclease-T (Nase-T) which possesses approximately 8 per cent of the
enzyme activity of the native enzyme.

“ GOGSOOHSGHOSSOSIOSSOSY
SSOSOOOSESSSOOTOSOGOE

4g + 50

OOOO OU OOO OOOO ITS
CNTR EESPHIRTC ENE IN HOEIEEIVIOS OSA ENICIEY
SOGBOSOSOOSS S COOUEVES
G COCCOOOOGOWOOE CIO NTLS

149

AIAG BOA OVEQAQAHOQGOQALE-com

Fic. 1.—The proposed amino acid sequence of staphylococeal nuclease Vs.5 The arrows indi-
cate the bonds cleaved by trypsin in the presence of 3',5’-dTDP and Ca** (see the text). The
amino acid sequences of nucleases produced by strains V; and Foggi are very similar, if not iden-
tical, with respect to NH.- and COOH-termini and peptide maps of the tryptic digests (unpub-
lished results by H. Taniuchi, C. Cusumano, and C. %. Anfinsen). Accordingly, the designations
of the spots on the peptide map are the same as described previously.4

1235
1236 BIOCHEMISTRY: TANIUCHI ET AL. Proc. N. A. 8.

Methods.—Staphylococcal nuclease (Foggi strain), obtained from Worthington Biochemical
Corp., was further purified on a phosphocellulose column. Other materials and methods have
been reported elsewhere,?—® unless otherwise specified. The results of amino acid analyses!) are
presented in pzmoles.

Preparation of nuclease-T: Four ml of a solution at pH 8.0 containing 50 mg nuclease, 5 mg
deoxythymidine-3’,5’-diphosphate (3’5’-dTDP) (Calbiochem), CaClk (0.01 M@), NHsHCOs; (0.05
M), and 0.5 mg trypsin (Worthington, DFP-treated), were incubated at 25° for 2 hr. The reac-
tion was stopped by the addition of 1.5 mg soybean trypsin inhibitor (Worthington, crystallized)
and the mixture was lyophilized. The dried material was dissolved in 0.5 ml 0.01 N acetic acid—
0.1% ammonium acetate and applied to a Sephadex G-50 (medium, Pharmacia) column (1.2
22 em) equilibrated with the same buffer. The column was developed with the buffer at 5-6 ml
per hour at 4°. The absorbancies of the fractions were read at 280 mz. Two peaks (effluent
volumes 10-19 mi and 21-39 ml, respectively) were detected. Fractions comprising the peaks were
pooled and lyophilized. The second fraction contained Nase-T-p,, free lysine, and 3’,5’-dTDP
as determined by absorbancy measurements at 280 and 260 myz and by peptide mapping (see
below). Nase-T-p,: and lysine were purified by paper electrophoresis.‘ The material in the first
peak from the Sephadex column was dissolved in 4 ml 0.3 M ammonium acetate, pH 6.0, and
applied to a phosphocellulose column (1.2 x 10 cm), equilibrated with the same solution. A
gradient elution was performed at 70-80 ml per hour using a Varigrad containing, in the first and
second chambers, respectively, 100 ml of 0.3 M@ ammonium acetate, pH 6.0, and 100 ml 1.0 M
ammonium acetate, pH 8.0. The gradient was followed by additional elution with 50 ml of the
second solution. The absorbancies of the fractions, collected every 5 min, were read at 280 my,
and each fraction was assayed’ for enzymic activity. Only one major peak appeared, at the same
position as previously observed with native nuclease. Essentially constant specific activities
(8-9% of the native value) with both DNA and RNA were observed throughout the fractions
constituting this peak. The over-all recovery of Nase-T, based on absorbancy at 280 my, was
45-50%. The active fractions were pooled and lyophilized, dissolved in H,O, and lyophilized a
second time.

Separation of Nase-T-p, and Nase-T-p3;: Nase-T (25 mg), in 1.0 ml 50% CHsCOOH contain-
ing phenol red, was applied to a Bio-Gel P-20 (50-150 mesh, Bio-Rad Laboratories) column (2 X
200 cm), equilibrated with 50% CHsCOOH for 48 hr at 4°. The filtration was performed at 5.6
ml per hour at 4° with 50% CH;COOH. The absorbancies of fractions, collected every 30 min,
were read at 280 my. Aliquots of fractions, dissolved in H,O after lyophilization, were assayed

 

       
      

 

 

Tye
15 Lok g 4 2.0
> i >
L2 6 B
av ‘ 8]
> 3 tl @
& 2: 4 2
N Za \ 5
ba | x
~ ZFA5E 410%
3 9?) on
<t a g Phenol red a

a
5S 2
A oO =
aw °
<t <J 4 | 4
O 1 1 mene tg om A 1 ‘ 4 ! ~

 

to
0 50 100 i50
FRACTION NUMBER

Fig. 2.—Separation of Nase-T-p. and ps3. Details are given in the text. Enzymatic activity
was determined against both DNA (4) and RNA (A). The immunological reactivity with anti-
nuclease rabbit serum (presented as +, -+:, and —) was carried out with the cooperation of Dr. 8.
Fuchs. The second activity peak appeared between peptide fractions containing Nase-T-p, and
Ps, respectively, and may be due to reassociation of the two peptides. The presence of the two
peptide components in this overlapping region was shown by polyacrylamide gel electrophoresis
at pH 2.3 and was also consistent with the immunological findings.
VoL. 58, 1967 BIOCHEMISTRY: TANIUCHI ET AL. 1237

for enzymic activities. Total amino acid content was estimated by reaction with ninhydrin after
alkaline hydrolysis (Fig. 2). The peak fractions (42-49 and 55-61), constituting Nase-T-p;
and ps, respectively, were pooled and lyophilized.

Isolation of reconstituted Nase-T (Nase-T’): A solution at pH 8.0 containing, in 1.5 ml, 0.32
umoles Nase-T-ps, 1.2 pmoles Nase-T-p:, 1.5 mg 3’,5’-dTDP, CaCl, (0.01 17) and NH,HCO;
(0.05 ) was incubated with 0.1 mg trypsin at 25° for 3 hr. The subsequent isolation of Nase-T’
by chromatography was the same as described above for the isolation of Nase-T. Approximately
0.2 umole of reconstituted Nase-T’ was recovered.

Resulis—The native nuclease is highly susceptible to digestion by proteases.?—*
However, a potent inhibitor, deoxythymidine-3’,5’-diphosphate, protected the
nuclease from such extensive digestion!? (Table 1). The progress of limited diges-
tion by trypsin is summarized in Figure 4. A peptide map of the trypsin digest
(Fig. 3) showed only two spots and a streak, the latter suggesting the presence of
large peptides. One of the two discrete spots was identified as lysine by amino
acid analysis (0.3 moles/mole nuclease after 2 hr), and the second as a pentapeptide,
Ala-Thr-Ser-Thr-Lys (Nase-T-p,). Amino acid analysis of an acid hydrolyzate
gave the following results: Lys, 0.03 (1); Thr, 0.047 (2); Ser, 0.022 (1); Ala, 0.021
(1). Leucine amino peptidase digestion for five hours released Ala, 0.006; Thr,
0.004; Ser, Lys, <0.002. Amino acid analysis of a fragment produced by CPase-B
digestion and purified by peptide mapping yielded Ala, 0.013 (1); Thr, 0.036 (2);

TABLE 1

Errect or DEOxYTHYMIDINE-3’,5'-DIPHOSPHATE AND Cat+ ON THE SUSCEPTIBILITY
or NucLEasE To ProTEotytic Digestion

Presence or absence of Per cent of Initial Activity against DNA

Proteases 3’,5’"-dTDP and Catt (30 min) (60 min) {120 min)
Trypsin + 11.7 4.1 6.4
_ 9.8 2.4 0.1
a-Chymotrypsin 4.5% w/w of + 75.2 85.8 85.4
nuclease - 38.6 20.2 4.3
Subtilisin 4% w/w of nuclease + 89.3 90.8 70.2
- 34.7 22.7 9.1
Pronase 3% w/w of nuclease + 8.1 5.6 6.7

— 17.4 4.3 4.

The components in the incubation (25°) mixtures (0.1 mi total volume) were as described in the section above
describing the preparation of Nase-T. Aliquots (5 ul) were removed and mixed with 5 ul 5 N acetic acid. In
the case of trypsin, soybean trypsin inhibitor was added instead of acetic acid. The enzymie activities were
examined with suitably diluted aliquots. The presence of both 3’,5’-dTDP and Ca*t+ was necessary to protect
the nuclease from complete digestion by trypsin, but had no effect on the tryptic digestion (1 hr) of apomyo-

globin (horse heart), as judged by peptide maps. The relative values with RNA as substrate were parallel to
those with DNA.

Ser, 0.018 (1). CPase-A digestion of this fragment for three hours yielded Thr,
67 per cent. Hydrazinolysis of the fragment yielded threonine only.
Characterization of Nase-T: The isolated Nase-T showed a single symmetrical
peak upon ultracentrifugation (kindly performed by Dr. E. Steers). In these ex-
periments, samples, dissolved in a solution containing 0.1 Mf Tris, 0.001 4f EDTA,
and 0.05 M NaCl at pH 7.5, were centrifuged at 56,000 rpm, 25°. The standard
cell contained 1.1 per cent native nuclease and the wedge cell, 0.63 per cent Nase-T.
The sedimentation velocity of Nase-T was essentially the same as that of the nu-
clease.6 Nase-T was also immunologically identical with the nuclease as ex-
amined by the method of Ouchterlony using rabbit antinuclease serum.” However,
the pattern on polyacrylamide gel electrophoresis was different from that shown by
native nuclease (Fig. 5). Two discrete bands with different intensities appeared
1238 BIOCHEMISTRY: TANIUCHI ET AL. Proc. N. A. 8.

on gel electrophoresis at pH 2.3 (“2.3 gel’’)
(Fig. 5). The DNase and RNase activities of
Nase-T did not change during incubation for
two hours with trypsin in the presence of 3’,
5’-dTDP and Ca++. When both the nuclease
and Nase-T (2.5 times excess over the nuclease)
were mixed in a solution containing 3’,5’-dTDP
and Catt, the enzymic activity of the mixture
at various times after addition of trypsin was
the sum of the control incubations with trypsin
of the nuclease and Nase-T alone. In the ab-
sence of 3’,5’-dTDP and Cat+, however, com-
plete digestion occurred.
All major tryptic fragments of nuclease ex-
cept Fi. (Fig. 3) were present in trypsin digests
Fig. 3.—Peptide map of a limited of Nase-T. Amino acid analysis of Nase-T
tryptic digest (2 hr) of the nuclease (1 showed a lower content of threonine and serine
mg) in the presence of 3’,5’-dTDP and coo.
Catt, than the nuclease (Table 2), and application
of Sanger’s DNFB method indicated NH.-
terminal lysine and glycine. The yield of bis-DNP-lysine was 32 per cent, and of
DNP-glycine, much less. No other ether-soluble DNP amino acids were found.
Amino end-group analysis of BrCN-treated nuclease and Nase-T produced, quali-
tatively, DNP-Ala, -Val, -Thr, and -Tyr,* and bis-DNP-Lys, DNP-Gly, -Val,
-Thr, and -Tyr, respectively. The first stage of Edman degradation of Nase-T
produced, qualitatively, lysine and glycine, and the second, leucine and valine.
Carboxypeptidase-A released only glutamine (70%, 9 hr) and carboxypeptidase B,
only lysine (80%, 9 hr). To prevent digestion by endopeptidase activity contam-
inating the CPase A and CPase B preparations, 3’,5’-ATDP and Catt were added
to the mixture. Nuclease, digested under the same conditions, served as the con-
trol and yielded COOH-terminal Gln only as reported previously.4 The enzymic
activities did not change significantly during CPase-B digestion (7 hr) in the

ELECTROPHORESIS

 

CHROMATOGRAPHY

 

Fig. 4.—-The kinetics of limited tryp-
tic digestion of nuclease in the presence
of 3’,5’-dTDP and Catt (conditions as
described in text). The following data
are summarized: activities with DNA
(@) and RNA (9); determination of
alkali (0.1 M@ NaOH) consumption (@)
at pH 8 as followed in the pH stat
(radiometer); determination of re
leased Nase-T-p; (4) and lysine (A),
using purified Nase-T-p: as the stand-
ard during paper electrophoretic sep-
aration at pH 3.6 and estimation using
a Beckman analytrol.4 NH,HCO, was
omitted in the digestion carried out in
the pH stat. Further addition of tryp-
sin after 60 min of incubation Cindi-
eated by an arrow) did not cause fur-

80

1
wa

D
°

L
°
(%-9 ‘wy ‘ea ) LN TVAINO’ 37I0W

Ye ACTIVITY (@—@, O--0)
bh
°°

{
o
a

20 ‘

 

 

 

ob ee | 6 ther change in enzymic activities.
6 120 180
TIME IN MINUTES
Vou. 58, 1967 BIOCHEMISTRY: TANIUCHI ET AL. 1239

TABLE 2

AmINo AcipD Compositions oF NUCLEASE-T, AND NUCLEASE-T’ AND
Component Pepripes or Nass-T

Nuclease’ Nase-Te Nase-T-p2 Nase-T-px Nase-T’s
Lys 21.4 19.0 7.3 (8) 15.1 (414) €
His 3.7 3.3 2.7 (2) 1.3 (1) €
Arg 4.6 4.6 1.3 (1) 4.0 (4) 4.2
Asp 14.2 14.1 2.9 (3) 10.9 (11) 14.1
Thr 9.4 7.1 4.9 (5) 3.1 (3) 7.5
Ser 6.1 4.4 0.2 (0) 3.6 (4) 4.5
Glu 17.5 16.9 2.5 (3) 14.9 (15) 16.0
Pro 6.5 6.0 4.3 (4) 2.2 (2) 6.2
Gly 10.2 9.6 2.0 (2) 8.0 (8) 8.6
Ala 13.1 12.8 2.0 (2) 11.6 (11) 12.7
Cys- 0.0 0.0 0.0 (0) 0.0 (0) 0.0
Val 8.6 9.3 2.0 (2) 6.4 (7) 8.7
Met 3.6 3.3 1.7 (2) 2.1 (2) 3.0
Tle 5,2 4.6 1.9 (2) 2.8 (3) 4.6
Leu 10.3 10.6 6.2 (6) 5.1 (6) 11.2
Tyr 6.9 6.3 1.0(1) 6.2 (6) 6.1
Phe (3) (3) (1) (1) (2) (2) (3)
Trp? 1 1 e (0) e (1) e

Values are expressed as moles/mole of phenylalanine.‘
a palues are obtained from analyses of aliquots (0.008 wmole) hydrolyzed for 26, 52, and 92 hr, respec-
tively. .

» Theoretical values (from the sequence of nuclease Vs*) are given in parentheses. The amounts of
.. Nase-T-pe and ps analyzed were 0.022 and 0.010 zmole, respectively.

¢ Duplicate samples containing 0.003 nmoles.

@ Estimated as described in ref. 4.

e Not determined.

‘presence of 3’,5’-dATDP and Cat+. Hydrazinolysis produced only lysine (24%).

If the amino acid sequences of the nucleases produced by the V; and Foggi strains
are the same (Fig. 1), these results would suggest the following. Trypsin cleaves
bonds between residues 5 and 6 and residues 49 and 50, releasing Nase-T-p, (residues
1-5). Nase-T is composed of Nase-T-p2 (6-49) and Nase-T-p; (50-149), which do
not dissociate under the conditions of cleavage. The origin of the fractional amount
of lysine released during the limited digestion is unknown. Partial removal of
residue 49 by trypsin seems a likely explanation. The release of Nase-T-p; is com-
plete in 10 minutes while nuclease activity continues to fall for at least 30 minutes
(Fig. 4). Therefore, the cleavage of the bond between residues 49 and 50 appears to
be responsible for the decrease in activity to 8 per cent of the native level.

The correctness of this interpretation was established by the following observa-
tions.

Nase-T-peptides 2 and 3: Polyacrylamide electrophoresis of Nase-T on “2.3” gel
yielded bands at the positions occupied by separated Nase-T-p. and ps; (Figs. 5 and
6). The NH,-terminal sequences of Nase-T-p, and p; were, qualitatively, Lys-Leu
and Gly-Val, respectively, as shown by successive Edman degradation. The
COOH-terminal residue of Nase-T-p. was lysine by both CPase-B digestion (6 hr,
90%) and hydrazinolysis (19%). No amino acids were released by CPase-A. The
COOH-terminal residue of Nase-T-p; was glutamine (11%) as shown by CPase-A
digestion (6 hr). No free amino acids were found upon hydrazinolysis of Nase-T-ps.
The amino acid compositions of both peptides accounted for the assigned portions of
the sequence of native nuclease (Table 2). The tryptic peptide maps of the frag-
ments did not overlap, and the sum of the components on the maps accounted for all
major spots of Nase-T. The locations of major spots on either map were consistent
with the peptides derived from the assigned portion of the nuclease sequence.*: * 13
Identification of major spots in digests of Nase-T-p2 was confirmed by amino acid
analyses of eluted samples.14
1240 BIOCHEMISTRY: TANIUCHI ET AL. Proc. N. A. 8.

       

Bt Origin
Zea Se.lhlUCtélmM UO EU
Nem 3 New Tose Miko T-Nom Pep 3 Pep 2. Mix
Fig. 5.—Polyacrylamide gel electrophoresis Fig. 6.—Polyacrylamide gel
patterns of Nase-T (in this figure and Fig. 6 electrophoresis (‘‘2.3” gel, 3 ma,
labeled T-Nase). ‘Mix’ represents the mix- 4 hr) of Nase-T-p; (Pep 3), Nase-
ture of nuclease and Nase-T. Standard gel, T-p: (Pep 2), and the mixture of

3 ma, 8 hr; ‘2.3’ gel, 2 ma, 2 hr. Ps and pe.

‘The recovery of original enzymic activity was less than 10 per cent after Bio-Gel
nltration, while the recovery of protein, as judged by measurements of absorbancy
at 280 mu, was 64 per cent (Fig. 2). Studies of regeneration of activity upon mixing
separated Nase-T-p, and p; (Fig. 7) indicate that neither of the component peptides
of Nase-T exhibits enzymic activity alone.

Properties of Nase-T: The relative instability of Nase-T to heat, compared with
nuclease, was indicated by measurement of the temperature dependence of the re-
duced mean residue rotation at 233 my (Fig. 8), and by Arrhenius plots of the enzy-
mic activities (Fig. 9). Addition of 3’,5’-dTDP and Ca++ stabilized Nase-T to a
greater extent than native nuclease. The optimal concentrations of Ca++ in the
activity assays of Nase-T and nuclease are the same (10-? /).

The levels of enzymic activities of Nase-T respond to increasing concentrations of
DNA and RNA in a manner similar to native nuclease.’

Reconstituted Nase-T (Nase-T’): When Nase-T-p; was mixed with a three to
fourfold excess of Nase-T-pe, the regenerated enzymic acitivities against DNA and
RNA were not affected by two hours of incubation with trypsin in the presence of
3/,5’-ATDP and Ca++. The peptide map of the mixture showed a streak, as shown
in Figure 3, and the small tryptic peptides produced were derived only from Nase-
T-p:. The isolated Nase-T’ was identical with the original Nase-T by several

Fic. 7.—Enzymic activity as a function of the ratio
of Nase-T-p. to Nase-T-p; using DNA (@) and RNA
(©) as substrates. The concentration of Nase-T-p3
was constant (4.8 X 10—§ wmole/ml as determined by
the amino acid analysis) in all assays. The aqueous
solutions of both peptides were mixed before the
| assay. The regenerated maximum specific activities
were 1.8 < 10% and 0.38 X 10? units/uwmole Nase-T-
ps with DNA and RNA, respectively. (Native nu-
clease exhibits approximately 2 < 10‘ and 0.5 « 10+
units/umole with DNA and RNA, respectively.)
J The results shown have been corrected for slight en-
dogenous activity in the Nase-T-p2. preparation em-
ployed (accounting for about 5% when maximum

activity was obtained). Neither Nase-T-p. nor ps
04 4 4 ttt ttt tt 5 enhanced the activities of Nase-T or nuclease when
PEP 2/ PEP 3 added alone at concentrations of 4 X 10-4 and 8 x

10-5 wmole/ml, respectively.

 

 

102 x A260 mz ABSORBANCY / MINUTE

 
Vou. 58, 1967 BIOCHEMISTRY: TANIUCHI ET AL. 1241

 

-o500 7 OOOO TO] T T T T T T T

-3000 |-
-3500 +
1,53

-4000 |-

-4500 +

 

?
d
LOGigV

~5000 - 4

 

 

 

20 30 40 50. 60 70 80
T° CENTIGRADE

(Above) Fic. 8.—Temperature-depen-
dence of reduced mean residue rotations at
233 my, [M']oa3, in 0.05 M Tris, pH 7.0, 0.1
M NaCl of Nase-T (@), Nase-T with 3',5’- r
dTDP and Ca++ (0), nuclease (a), and
nuclease with 3’,5’-dTDP and Ca++ (A).
3’,5’-dTDP was added in fourfold excess out ! i

 

 

 

i 1 i i
over protein; Cat*, 10-? M. The concen- 30 31! 32 33 #34 35 3.6
tration of protein was 0.2 mg per ml or less. 1000 /T

 

(Right) Fie. 9.—Arrhenius plot of the activities of nuclease with DNA (@), nuclease with RNA
(QO), Nase-T with DNA (a), and Nase-T with RNA (A). The assays were carried out at 5°, 15°,
25°, 30°, 40°, 50°, and 60°. 7’, absolute temperature; v, specific activity.? The concentrations
of nuclease and Nase-T were 0.22 and 10.1 yg per ml, respectively, for the assays at 5° and 15°.
At 25° to 60°, concentrations were 0.065 and 1.0 ug per ml, respectively. Temperature was
maintained with a Haake (type F) bath for temperatures above 25° and a Formatemp, Jr.
(Forma Scientific, Inc. mode] 2095) bath below 25°. Reaction mixtures were equilibrated for
10-15 min before addition of enzyme.

criteria, including the chromatographic pattern on phosphocellulose, the specific
activities with DNA and RNA, sedimentation velocity, amino acid composition
(Table 2), electrophoresis on “2.3 gel,”’ and the heat transition of the reduced mean
residue rotation at 233 my.'®

Discussion —F. M. Richards: * demonstrated ten years ago that bovine pan-
creatic ribonuclease could be converted by limited digestion with subtilisin to an ac-
tive derivative consisting of two fragments that were held together by noncovalent
interactions. The experimental findings on staphylococcal nuclease described in
this paper provide a second example of such a two-component, enzymically active
derivative. As in the case of RNase-S, Nase-T affords an excellent. test object for
direct examination of relationships between structure and function and of the nature
of the side-chain interactions underlying the formation and stabilization of three-
dimensional structure. Nase-T, devoid of disulfide bond stabilization, offers a par-
ticularly striking example of the spontaneous formation of tertiary structure on the
basis of the genetic information in the primary sequence of the polypeptide chain.
Crystallographic experiments on the three-dimensional structures of nuclease and
Nase-T, in collaboration with Drs. Cotton, Hazen, and Richardson! of the Massa-
chusetts Institute of Technology, and efforts in this laboratory to synthesize part or
all of the sequence of nuclease and Nase-T are now in progress.
1242 BIOCHEMISTRY: TANIUCHI ET AL. Proc. N. A. &.

We wish to acknowledge the excellent technical assistance of Mr. Clifford Lee in amino acid
analyses, and the kindness of Dr. Robert Resnik for use of the Cary model 60 spectropolarimeter.

1Cunningham, L., B. W. Catlin, and M. J. Privat de Garilhe, J. Am. Chem. Soc., 78, 4642
(1956).

2 Alexander, M., L. A. Heppel, and J. J. Hurwitz, J. Biol. Chem., 216, 3014 (1961).

’ Anfinsen, C. B., M. Rumley, and H. Taniuchi, Acta Chem. Scand., 17, 270 (1963).

4Taniuchi, H., and C. B. Anfinsen, J. Biol. Chem., 241, 4366 (1966).

5 Taniuchi, H., C. B. Anfinsen, and A. Sodja, J. Biol. Chem., in press.

6 Heins, J. N., J. R. Suriano, H. Taniuchi, and C. B. Anfinsen, J. Biol. Chem., 242, 1016 (1967).

7 Cuatreeasas, P., 8. Fuchs, and C. B. Anfinsen, J. Biol. Chem., 242, 1541 (1967).

8 [bid., in press.

® Abbreviations used are: Nuclease-I', Nase-T; dinitrophenyl, DNP; nuclease-T-peptides 1,
2, and 3, Nase-T-p, px, and ps, respectively; carboxypeptidase A and B, CPase-A and -B; deoxy-
thymidine-3’,5’ diphosphate, 3’,5’-dTDP; reconstituted nuclease-T, Nase-T’; EDTA, ethylene-
diaminetetraacetate.

10 Fuchs, S., P. Cuatrecasas, and C. B. Anfinsen, J. Biol. Chem., in press.

11 Spackman, D. H., 8. Moore, and W. H. Stein, Anal. Chem., 30, 1190 (1958).

12 This possibility was suggested by Dr. P. Cuatrecasas on the basis of experiments on stabiliza-
tion of the nuclease with 3’,5’-dTDP and Catt.

18 The addition of 3’,5’-ATDP and Ca++ did not protect either peptide, incubated separately,
from complete digestion by trypsin.

14 The following components were analyzed: residues 7-9, 10-16, 17-24, 25-28; 29-32, 33-35,
36-45, and 46-48 (see refs. 4 and 5).

18 Standard cell, Nase-T, 0.4%; wedge cell, Nase-T’, 0.17%; buffer, 0.05 M Tris, pH 7.0,
0.1 M NaCl; 56,000 rpm, 25°. Nase-T-p; had a lower S value than Nase-T under the same
conditions; standard cell, Nase-T, 0.74%, 2.06S (uncorrected); wedge cell, Nase-T-ps, 0.5%,
1.558 (uncorrected ).

16 No changes were observed in the rotation at 283 mz (A[m’] < 190°) during temperature
increase from 25° to 70° with either Nase-T-pz or ps. However, reconstituted Nase-T’ showed
the heat-dependent change in optical rotation at 233 mp (A[m’] = 1,000°) with the mid-point
of the transition similar to that of Nase-T (Fig. 8), indicating the restoration of the secondary
structure upon reconstitution.

17 Richards, F. M., these Procrgpines, 44, 162 (1958).

18 Richards, F. M., and R. J. Vithayathil, J. Biol. Chem., 234, 1459 (1959).

19 Cotton, F. A., E. E. Hazen, Jr., and D. C. Richardson, J. Biol. Chem., 241, 4389 (1966).