The binding of deoxyribonucleases of Escherichia coli to deoxyribonucleic acid immobilized in agarose

326

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95295

T H E B I N D I N G OF D E O X Y R I B O N U C L E A S E S OF E S C H E R I C H I A

COLI TO

D E O X Y R I B O N U C L E I C ACID IMMOBILIZED IN AGAROSE

J.

E. NABER',

A. M. J. SCHEPMAN

AND

A. RCIRSCH

Medical Biological Laboratory o/the National De/ence Research Organization TNO, Rijswijh, Z.H. (The Netherlands) (Received April 29th, 1965)

SUMMARY

The binding of the enzymes endonuclease-I, exonuclease-I and exonuclease-II of cell-free extracts of Escherichia coli KI2 S to highly polymerized calf thymus deoxyribonucleic acid, immobilized in agarose, has been studied. Columns containing 3 ~o agarose carrying native or denatured DNA were prepared. The three enzymes studied were bound to such columns at low ionic strength. Subsequent elution with I M NaC1 led to a substantial degree of purification of these enzymes. The influence of various conditions on the binding of the nucleases to the immobilized DNA was studied.

INTRODUCTION

In general the purification procedure for an enzyme contains an essential step in which the enzyme is bound more or less specifically to another substance, such as for example Ca~(P04)2, DEAE- or CM-cellulose. Obviously the most specific binder of an enzyme is its own substrate; therefore it seems attractive to use the substrate as means for purification. The application of the substrate however is limited since (a) the enzyme-substrate complex m a y be even more difficult to isolate than the enzyme itself, and (b) the enzyme tends to alter its substrate. With nucleases however the situation is favourable; these enzymes can be bound firmly to their substrates (the nucleic acids), under conditions that prevent a significant breakdown of these highly polymerized compounds. In this way LEHMAN et al. 1 and WEISBACH AND KORN2 separated deoxyribonucleases from cell free extracts of Escherichia coli by concomitant precipitation with nucleic acids by protamine sulphate and streptomycin. The nucleases were recovered from these precipitates by subsequent elution with salt or a concentrated solution of streptomycin. In this paper we describe the binding of deoxyribonucleases to, and the elution * Stationed at the Medical Biological L a b o r a t o r y of the National Defence Research Organization TNO b y of the Royal Air Force.

Biochim. Biophys. Acta, 114 (1966) 326-337

PURIFICATION OF DEOXYRIBONUCLEASES OF

E. coli

327

from, DNA in an immobilized form. The DNA was embedded in an inert carrier which enabled us to prepare a column that simulates a conventional chromatographic column, though here the substrate, instead of an unspecific ion-exchanger or protein adsorbing material, was used to retain the enzymes. In a similar way McCARTHY AND BOLTON~-~ immobilized denatured DNA in agar in order to study D N A - R N A hybridization. Native agar however has the disadvantage that it binds considerable amounts of other proteins that occur in bacterial extracts. Therefore agar as such is unsuitable for immobilizing DNA for our purpose. From agar we prepared its main constituent agarose which has a strongly decreased affinity for proteins 6. Nevertheless it is still able to bind the enzyme endonuclease-I to a great extent 6. We made use of this property to purify this particular enzyme. The binding capacity of agarose itself for the enzyme however prevented its application as an inert material for the study of the interaction of endonuclease-I with embedded DNA. We were able to show that, after washing the agarose with a solution of 5.IO -a M EDTA, the affinity for endonuclease-I is eliminated. The agarose washed in this manner, used as an inert carrier for highly polymerized calf thymus DNA in the native and denatured form, could be used to study the binding of endonuclease-I, exonuclease-I and exonuclease-II at various pH's.

MATERIALS AND METHODS

The following chemicals were used: bovine pancreas deoxyribonuclease (deoxyribonucleate oligonucleotidohydrolase, EC 3.i.4.5) , Sigma; 82PO~-, cartier free, Radiochemical Centre, Amersham; calf thymus deoxyribonucleic acid, highly polymerized, NBC; Sephadex G-75, Pharmacia; Special Agar Noble, Difco; polyethylene glycol, mol. wt. 6000, Shell Nederland Chemie; streptomycin sulphate, K.N.G. and S.F., Delft.

The preparation o/cell-]ree extracts [rom E. coli K 12 S Cell free extracts were prepared from E. coli K I2 S as described previously 6. Cells grown in M 9 medium, supplemented with casaminoacids, were broken in the Raytheon sonic oscillator. Unbroken cells and cell wall material were removed b y centrifugation. The supernatant was brought to IO % streptomycin sulphate in order to precipitate the nucleic acids. Under these conditions no nuclease activity is coprecipitated. Next the solution was saturated with ammonium sulphate, the precipitate collected and redissolved in the appropriate buffer. This preparation will be referred to as the nucleic acid free extract.

Preparation o/ agarose Agarose and agarose columns were prepared as described previously 6. In contrast with our preceding procedure all agarose columns were washed with a solution of 5.1o -8 M E D T A before equilibration with the buffer used for elution. Biochirn. Biophys. Acta, 114 (1966) 326-337

328

J. E. NABER, A. M. J. SCHEPMAN, A. RORSCH

Preparation o/ DNA-agarose columns Highly polymerized calf thymus DNA was embedded in agarose by mixing a 6 % agarose solution with an equal amount of a DNA solution (2 mg/ml) at 9 o°, followed by rapid chilling, as described b y McCARTHY AND BOLTON~,a. For the preparation of agarose columns containing denatured DNA the DNA solution in water was heated for IO min at IOO° before mixing it with agarose. Agarose containing native DNA was prepared from a DNA solution in I M NaC1. This high ionic strength prevents denaturation during the mixing of the DNA solution with agarose at 9 o°. DNA-agarose columns were prepared in the same way as agarose columns n. Before equilibration with suitable buffer the columns were washed with 5-1o -3 M EDTA. The DNA content of the columns was calculated from the DNA washed out from the columns and the total amount of DNA used for their preparation. The results were checked with the determination of the DNA concentration in parallel columns b y incubation with pancreatic DNAase or by boiling with 5 M NaCIO, as described b y McCARTHY AND BOLTON5.

Preparation o~ R N A and DNA sRNA was prepared from E. coli B as described b y Zt~BAY7. 3zp-labelled DNA was prepared as described previously 6.

Assay o/ E. coli endonuclease activity Endonuclease-I activity was measured b y incubation of enzyme preparations with asP-labelled, native E. coli DNA at p H 8.o s. The incubation mixture contained in a total volume of 0.45 ml 3o/*mole Tris p H 8.0, 3/*mole MgC12, approx, o.02 A,6om#-units Es*P]DNA (2"Io4-6"1o4 counts/ min) and an appropriate amount of enzyme. After 30 min of incubation at 37 ° 0.5 ml cold 0.5 M HC104 was added followed by 0.2 ml solution of bovine serum albumin (1 mg/ml). After centrifugation 0.25 ml of the supernatant was taken to dryness and the radioactivity counted in a Nuclear Chicago Ultrascaler Gas flow counter model 192 or in a Nuclear Chicago sample Changer model lO41 with GeigerMiiller end window counter. Specific enzyme activities were calculated from assays in the proportional range as described previously 6. A unit of enzyme activity is defined as a/*mole 32p made acid soluble b y 30 rain of incubation at 37 °. Protein concentrations were determined from the absorbance at 260 and 280 m/* (ref. 9) and by the method of LOWRY et al. x°.

Assay o/ E. coli exonuclease-I activity Exonuclease-I activity was measured by incubation of enzyme preparations with [3zP]labelled denatured E. coli DNA at p H 9.5 s. The denatured single stranded DNA was prepared from native E. coli DNA by heating for IO rain at IO0 ° followed b y rapid chilling in ice. The incubation mixture contained 2o/,moles glycine buffer p H 9.5 instead of Tris buffer. For the rest the same technique was used as described for endonuclease-I. Biochim. Biophys. Acta, 114 (1966) 326-337

PURIFICATION OF DEOXYRIBONUCLEASES OF

E. coli

329

Assay o/E. coli exonuclease-II activity Exonuclease-II activity was measured also by a procedure similar to that described for endonuclease-I. The incubation mixture contained 20/~moles glycine buffer p H 9.2 and in addition 5/,moles of E. coli sRNA s.

RESULTS

Agarose column chromatography o/ nucleic acid [ree extract Prior to the investigation of the binding of the deoxyribonucleases to DNA immobilized in agarose, the binding of these enzymes to agarose itself was studied. In contrast to the procedure described previously 6 the agarose column was washed with a solution of 5.1o -3 M EDTA. 7 ml of a nucleic acid free extract--containing approx. IOO mg protein--was dialysed against 0.02 M Tris buffer p H 7.5 with 5.1o -3 M MgC12. This preparation was applied to a IOO ml 3 % agarose column equilibrated with the same buffer. The column was eluted with that buffer and subsequently with the buffer supplemented with I M salt. At an elution velocity of 50 ml/h, fractions of IO ml each were collected. The fractions were dialysed for 2.5 h against 50 times their total volume of 0.02 M Tris buffer (pH 7.4) and subsequently assayed for enzyme activity. The results are presented in Fig. I and Table I. We observed that not only all the protein but also all the enzyme activity that could be recovered was washed through the column with the first effluent, and no enzyme activity could be detected in the salt eluate. The specific enzyme activity and the enzyme units recovered for the various enzymes are presented in Table I. The enzyme recovery gives the percentage of enzyme units recovered in terms of the material applied to the column. This seems more reasonable than to relate the recovery to the total activity of the crude extract since this figure is meaningless owing to the presence of inhibitory nucleic acids. Apparently 55.8 % of the endonuclease-I activity, 35.0 % of the exonuclease-I activity and 77.4 % of the exonuclease-II passed through the agarose column and were recovered without having been bound. Moreover, since no increase in specific activity for any of these enzymes was observed we assumed that agarose, previously washed with E D T A and subsequently equilibrated with MgCI~, m a y be considered sufficiently inert to serve as a carrier for DNA in our studies. 1400-

~

-2. 8

1MiaCl

E

%1ooc

2.0 c

600-

1.2

o~ 2000

-0.4 0 ]00

200

300 Effluent

(ml)

400

Fig. I. Agarose chromatography of nucleic acid free extract. 0 - 0 , O - O , endonuclease-I activity; x - X , absorbance at 28o m/~.

exonuclease-I activity;

Biochirn. Biophys. Acta, 114 (1966) 326-337

&

Ox

4~

tz

OF

Abbreviations: EN-I,

I;

p H 7.5

379

305

119

EX-II

155

278

EN-I

24

31

EX-II

55 .8

EN-I

(%)

35 .0

EX-I

77-4

EX-II

E n z y m e recovery

exonuclease-II.

420

12o2

EX-I

Total enzyme units x 10 -3

exonuclease-I; E X - I I ,

6769

II958

3590

EX-I

EX-I,

2570

542

EN-I

F i r s t effluent agarose c o l u m n

Salt eluate agarose c o l u m n

AT

Specific enzyme activity (units/rag protein)

E. coli K I 2 S

2778

endonuclease

OF EXTRACTS

Nucleic acid free e x t r a c t

Crude e x t r a c t

AGAROSE CHROMATOGRAPHY

TABLE I

I00

EN-I

I00

EX-I

I00

EX-II

Distribution o/ recovered enzyme units over /irst effluent and salt eluate

ota

xl ©:

~z

m t~ ~0

>

>

t~

©

PURIFICATION OF DEOXYRIBONUCLEASES OF

E. colt

331

Chromatography on agarose containing denatured DNA Columns of IOO ml containing 3 % agarose and 7 ° mg denatured D N A were prepared as described under methods. The retention of the various nucleases that occur in the nucleic acid free bacterial extracts was studied on these columns at pH 7.5 and pH 9.5. One column was equilibrated with 0.02 M Tris buffer pH 7.5 and 5.1o -3 M MgC12, and another with 0.05 M glycine buffer (pH 9.5) and 5.1o -3 M MgC12. Samples of 15 ml nucleic acid free extracts, containing approx, zoo mg protein, were dialysed against the corresponding buffers and applied to these columns. After elution with the same buffers the protein retained on the column was eluted with I M salt. The result of the experiment at pH 7.5 is illustrated in Fig. 2 and Table II, and the result of the experiment at pH 9.5 is presented in Table III. In the first three columns of Tables II and III the specific activities of endonuclease-I, exonuclease I and exonuclease-II in the crude extract, in the nucleic acid free extract, the first effluent in 0.05 M buffer and in the salt eluate are listed. The next three columns show the total amount of enzyme units in each stage. The enzyme recovery was calculated as the total amount of enzyme units that appeared in the first effluent and the salt eluate together in proportion to the number of enzyme units applied to the agaroseD N A columns. Lastly the distribution of the recovered enzyme units over the first effluent and the salt eluate are presented. For the enzyme endonuclease-I the proportion of enzyme units, initially 700 -

-2.8

1MNaCI

"~500 c

-2.0

sc

-2

300 u

-1.2 ~

100-

-0.4

0 _o_ 0

t

100

200

300 Effluent (ml)

0

400

Fig. 2. Chromatography at pH 7.5 of extract on agarose containing denatured DNA. O - O , exonuclease-I activity; 0 - O , endolluclease-I activity; X - x , absorbance at 280 mff. 1 4 0 0J /

2.8

1M NaCI

10004

I ~

-2.0

}

5 000~

=

/ ~/ J

~.2,I

"

I

2 t 2/;! 200 J 0

/

g 100

_ k/

.<

h O . 4 200 300 Effluent (ml)

400

Fig. 3. Chromatography at pH 9.5 of extract on agarose containing native DNA. 0 - 0 , clease-I activity; O - O , endonuclease-I activity; x - x , absorbance at 280 mff.

exonu-

Biochim. Biophys. Acta, 114 (1966) 326-337

uo

t~

OF EXTRACT OF E.

AGAROSE CONTAINING

65 029

Most active fractions of salt eluate

Abbreviations: see Table I.

15 35

Salt eluate a g a r o s e - D N A c o l u m n

167

23

651

EN-1

5572

175

95 °

7o 8o027 265

34 466

2715

944 °

475

EX-II

14 709

EX-I

Specific enzyme activity (units/rag protein)

coli K I 2 S oN

F i r s t effluent a g a r o s e - D N A c o l u m n

Nucleic acid free e x t r a c t

Crude e x t r a c t

CHROMATOGRAPHY

TABLE II AT

lO3

19o

io

319

EN-I

113

142

209

1265

EX-I

33

57

io

I27

EX-II

Total enzyme units × lO -3

DENATUREDDNA,

62. 7

EN-I

27.8

EX-I

52.8

EX-1I

E n z y m e recovery (%)

p H 7.5

95.o

5 .o

EN-I

4o.5

59.5

EX-I

85.1

14.9

EX-II

Distribution o/ recovered enzyme units over [irst e]]luent and salt eluate

Oe~

©:

>

t~

m

(3

Z

Z,O

t~

v

4:,.

TABLE

III

Most

fractions

Abbreviations:

active

of salt I.

eluate

column

see T a b l e

Salt eluate agarose-DNA 1i 914

8287

--

16 9 7 °

32

--

72

34

1274

51

124

9 2 7 4 i i 609

1391

EX-I

7

516

IOO

475

EN-I

58

77

2

139

EX-II

Total enzyme units × I o -~

68

12 7 3 8

1241

column

Nucleic acid tree extract

First effluent agarose-DNA

I4 709

EX-II

651

EX-I

Crude extract

EN-I

Specific enzyme activity (units/rag protein)

46.8

EN-I

(%)

8.3

EX-I

57.0

EX-II

E n z y m e recovery

CHROMATOGRAPHY OF EXTRACT OF E. coli K I 2 S ON AGAROSE CONTAINING DENATURED I ) N A AT p H 9-5

87.9

i2.i

EN-I

67.9

32.1

EX-I

97.4

z.6

EX-II

Distribution o] Yecovered Bn2ym8 units over first e]/luent and salt eluate

~o

©

> ~q

© X

©

0

,o

334

j.E.

NABER, A. M. J. SCHEPMAN, A. RORSCH

bound to the column at low ionic strength and subsequently eluted with I M salt, amounted to 95.0 O/,oat p H 7-5 and to the somewhat lower value of 87.90~/o at p H 9.5. The exonuclease-II was also found to be bound considerably under both conditions though for this enzyme a higher retention was observed at p H 9.5 (97-4 o/ /o) than at p H 7.5 (85.1%). The retention for the enzyme exonuclease-I was considerably less; at p H 7.5 40.5 % of its activity were found in the salt eluate, at pH 9.5 67.9 % • The binding of these enzymes to the immobilized DNA at low ionic strength followed by their elution with I M salt led to a substantial purification of these enzymes. In comparison with the nucleic acid free extract the specific enzyme activity of the salt eluate of the p H 7.5 column had increased 6.5-fold for endonuclease-I, 3.5-fold for exonuclease-I and 6.o-fold for exonuclease-II. When only the most active fractions of the salt eluate were considered, purification ratios of respectively 27 ×, 7.5 × and 29 × were observed. Similar results were obtained at pH 9.5 for endonuclease-I and exonuclease-II; at this p H however the salt eluate showed no increased specific activity for exonuclease-I in spite of the fact that this enzyme is more firmly bound at p H 9.5 (67.9 %) than at p H 7-5 (4o.5 °/o)- Obviously this must be due to the low recovery of exonuclease-I at p H 9.5 (8.3 }L) in comparison with the yield at p H 7.5 (27 .8 %). Lastly we have to draw attention to the fact that under the conditions described here no significant breakdown of the immobilized DNA was observed during the elution since the ratio of the absorbance at 280 m# and 260 me, corresponded for all fractions remarkably well with their protein content.

Chromatography on agarose containing native DNA Columns containing IOO ml of 3 % agarose and 7 ° mg native DNA were prepared as described under methods. IO ml of a nucleic acid free extract, containing approx. IOO mg protein, was dialysed against 0.05 M glycine buffer (pH 9.5) supplemented with 5.1o -3 M MgCl 2 and was applied to a column containing native DNA and previously equilibrated against the same buffer. Elution was performed as described for the chromatography on denatured DNA. The results are shown in Fig. 3 and Table IV. We expected that exonuclease-I, that has a pronounced specificity for single stranded DNA, would be less firmly bound to native than to denatured DNA. On the other hand, we expected that endonuclease-I and exonuclease-II would be bound at least as strongly b y double stranded as b y single stranded DNA. We observed that in fact the salt eluate of the column containing native DNA showed less exonuclease-I activity (11.6 °/o) than the salt eluate of the column containing denatured DNA (67. 9 °/o, c/. Table I I I and IV). However, the column containing native DNA at p H 9-5 did not retain as much of the enzymes endonuclease-I (41.8 %) and exonuclease-II (54.4 %) as the column containing denatured DNA (87.9 % and 97.4 % respectively). In contrast to the first effluent and the salt eluate of the column containing denatured DNA, the fractions of the column containing native DNA showed a much higher absorbance at 260 m# than could be accounted for b y their protein contents. This indicates t h a t the native DNA was broken down during the elution in spite of the fact that the experiments were performed throughout at low temperature (4°). Therefore we ascribe the weak retention of endonuelease-I and ]?iochim. Biophys..4cta, 1i 4 (1966) 326 337

~o

?

v

4~

19 932

M o s t a c t i v e f r a c t i o n s of s a l t e l u a t e

A b b r e v i a t i o n s : see T a b l e I.

15 230

Salt eluate agarose-DNA column

800

132o

N u c l e i c ac id free e x t r a c t

F i r s t ef fl uen t a g a r o s e - D N A c o l u m n

651

EN-I

54 °

144o

-

-

21 744

22 50o 17 02o

6520

19 37 °

475

EX-II

I 4 709

EX-I

NATIVE

DNA

26

38

53

132

--

57

433

1937

EX-I

AT

37

43

36

144

EX-II

Total enzyme units × lO -~

EN-I

ON A G A E O S E C O N T A I N I N G

Specific enzyme activity (unitslmg protein)

colt 1{I2 ~

Cr ud e e x t r a c t

CHROMATOGRAPHY OF EXTRACT OF E.

TABLE IV

69 .0

EN-I

25-3

EX-I

54-9

EX-II

E n z y m e recovery {%)

p H 9.5

41.8

58.2

EN-1

ii.6

88. 4

EX-I

54.4

45.6

EX-II

Distribution o/ recovered enzyme units over ]irst effluent and salt eluate

t_o ~o

©

BI

o

© z

t2 ©

©

;>

*v

336

J.E.

NABER, A. M. J. SCHEPMAN, A. RORSCH

exonuclease-II b y native DNA to the observed breakdown of the substrate. Nevertheless the specific endonuclease-I activity and the specific exonuclease-II activity of the most active fractions of the salt eluate was 15 times higher than the corresponding specific activities of the nucleic acid free extract; thus in spite of the breakdown of the DNA a substantial degree of purification was achieved.

DISCUSSION

From the results presented it is clear that the binding at low ionic strength of the enzymes endonuclease-I, exonuclease-I and exonuclease-II to DNA immobilized in agarose and their subsequent elution with I M salt leads to a substantial degree of purification of these enzymes. The best results were obtained with columns containing denatured DNA, eluted at p H 7.5; the specific activity at the most active fractions were for endonuclease-I, exonuclease-I and exonuclease-II respectively 27, 7.5 and 28 times higher than the specific activity of the material applied to the column. The method described here can surely compete with conventional chromatographic techniques since in general a IO fold increase in specific enzyme activity in a single step is considered satisfactory. We expect that the application of immobilized DNA will not be restricted to the purification of nucleases only. We m a y now consider the purification of other proteins that interact with DNA as for example histones and polymerases. Recently we subjected cell-free extracts of Micrococcus lysodeikticus to chromatography on agarose containing DNA. These extracts, which show a remarkably low endonuclease-I activity b y the way, contain at least two enzymes that act on ultraviolet-irradiated DNA specifically; one enzyme is able to restore the biological activity of irradiated DNA (repair enzymel2), the other breaks down ultraviolet-irradiated DNA (ultraviolet-deoxyribonucleaseaS). Both enzymes were bound at low ionic strength by DNA immobilized in agarose; b y subsequent elution with a salt gradient the enzymic repair activity and the ultraviolet-deoxyribonuclease activity could be separated. The results of these experiments will be published in detail elsewhere. Immobilized DNA m a y also be used for the study of the affinity of the nucleases for their substrate under various conditions. Though we are quite well informed on the specificity of DNAase action not much is known about the affinity of DNA for these enzymes. We m a y wonder how closely affinity and enzyme activity are correlated. Our results indicate for example that exonuclease-I is more firmly bound b y denatured DNA than b y native DNA. Since it is well known that exonuclease-I has a great specificity for single stranded DNA our experiences indicate a correlation between enzyme activity and substrate affinity. Moreover enzyme activity depends strongly on the pH; the optimum p H for endonuclease-I activity is near 7.5, for exonuclease-II near p H 9.5. In this respect it is noteworthy that under our conditions endonuclease-I was found to be bound better at p H 7.5 than at p H 9.5 whereas exonuclease-II was retained on the DNA column to a greater extent at p H 9.5 than at p H 7.5- These results again indicate a correlation between activity and affinity. However w.e also noted that endonuclease-I and exonuclease-II are well bound b y denatured DNA, though these enzymes break down double stranded DNA preferentially. In fact this enzyme activity interfered seriously with the purification Biochim. Biophys. Acta, 114 (1966) 326-337

P U R I F I C A T I O N OF D E O X Y R I B O N U C L E A S E S

OF

E. coli

337

of the enzymes on double stranded DNA. Evidently we have to search for conditions that assure a m a x i m u m substrate affinity and a minimum enzyme activity for the development of new purification procedures.

REFERENCES i 2 3 4 5 6 7 8 9 IO II 12 13 14 15

I. R. LEHMAN, G. G. ROUSSOS AND E. A. PRATT, J. Biol. Chem., 237 (1962) 819. A. WEIGSBACH AND ~). KORN, J. Biol. Chem., 238 (1963) 3383 • E. T. BOLTON AND B. J. McCARTHY, Proc. Natl. Acad. Sci. U.S., 48 (1962) 139o. B. J. McCARTHY AND E. T. BOLTON, Proc. Natl. Acad. Sci. U.S., 5o (1963) 156. B. J. McCARTHY AND E. T. BOLTON, J. Mol. Biol., 8 (1964) 156. J. E. NABER, A. M. J. SCHEPMAN AND A. R6RSCH, Biochim. Biophys. Acta, 99 (1965) 3o7 • G. ZUBAY, J. Mol. Biol., 4 (I962) 347. If. SHORTMAN AND I. R. LEHMAN, J. Biol. Chem., 239 (1964) 2964 • E. LAYNE, in S. P. COLOWICK AND N. O. KAPLAN, Methods in gnzymology, Vol. 3, A c a d e m i c Press, N e w York, 1957, P. 453O. H. LOWRY, N. J. ROSEEROUGH, A. L. FARR AND P,. J. RANDALL, 3r. Biol. Chem., 193 (1951) 265. 1. R. LEHMAN, Progress in Nucleic Acid Research, Vol. 2, A c a d e m i c Press, N e w York, 1963, p. 89. A. RORSCH, C. VAN DE IfAMP AND J. ADEMA, Biochim. Biophys. Acta, 80 (1964) 346. ]3. STRAIJS, Proc. Natl. Acad. Sci., 48 (1962) 167o. 1. R. LEHMAN AND A. L. NUSSBAUM, J. Biol. Chem., 239 (1964) 2628. I. R. LEHMAN AND C. C. RICHARDSON, J. Biol. Chem., 239 (1964) 233.

Biochim. Biophys. Acta, 114 (1966) 326-337