104 PURIFICATION OF RESTRICTION ENZYMES [13]

10C

9C

8C

7C

z6c
Q

.5£

3C

2C

1C

0
5 10 15 20
Time (rain)
FIG. 1. Independent cleavage of 2 EcoRI sites of pVH153 DNA. The reaction (0.5 ml)
contained 0.1 M Tris-HC! (pH 7.6), 0.05 M NaCI, 5 mM MgCI2, 0.2 mM EDTA, 50 ~,g/ml
bovine serum albumin, 2.74 nM pVH153 DNA (as molecules), and 0.14 nM EcoRI endonu-
clease as dimer. Incubation was at 3T. Samples (0.04 ml) were removed as indicated, and
reaction terminated by addition of 0.02 ml of 0.03 M NaaEDTA, 0.8% SDS, 30% sucrose,
and 0.005% bromphenol blue. Reactants and products were separated by electrophoresis on
agarose gels and quantitated as described,s'19 Uncut, 7.3 x 10° dalton circles; one cut,
7.3 x 106 dalton linear; two cut, sum of 2.3 x 106 and 5.0 x 10a dalton linear fragments.

Acknowledgments

Work performed in the authors' laboratory was supported by grants from the National
Institutes of Health and the National Science Foundation.

[13] Purification a n d P r o p e r t i e s o f HindII a n d HindIII

E n d o n u c l e a s e s f r o m Haemoptdlus irnquenzae R d
By HAMILTON O . SMITH a n d GARRY M . MARLEY

Haemophilus influenzae Rd is the source for two restriction endonu-

cleases, HindII and HindlII, that may be separated and purified using
conventional ion exchange chromatography mediaJ ,z The basic steps in
the procedure are removal of nucleic acids from the cell extract, separa-
i H. O. Smith and K. W. Wilcox, J. Mol. Biol. 51, 379 (1970).
2 H. O. Smith, Methods Mol. Biol. 9, 71 (1975).

Copyright© 1980by AcademicPress,Inc.

METHODSIN ENZYMOLOGY,VOL.65 All fightsof reproductionin any formreserved.
ISBN 0-12-181965-5
[13] HindlI AND HindlII 105

tion of the two activities by DEAE-cellulose chromatography, and

chromatography of each activity on phosphocellulose as a combined
purification and concentration step.

Assay Method
HindlI. Reaction mixtures (20/zl) contain 1/zg of phage h DNA, 10 mM
Tris-HCl (pH 7.5), 10 mM MgCI2, 7 mM 2-mercaptoethanol, and 1-5/~1 of
enzyme fraction. Incubation is for 60 min at 37°.
HindlII. Reaction mixtures (20/zl) contain 1/xg of phage h DNA, 10
mM Tris-HCl (pH 8.5), 10 mM MgCI2, 60 mM NaC1, 7 mM
2-mercaptoethanol, and 1-5/~1 of enzyme fraction. Incubation is for 60
min at 37°.
Reactions are stopped by addition of 5/.d of dye mixture containing
25% glycerol, 0.1 M Na2EDTA (pH 8), 0.05% bromphenol blue. Samples
are analyzed for DNA cleavage by electrophoresis on a 1% agarose gel.
One unit of endonuclease activity is just sufficient to produce complete
cleavage of 1/~g of phage h DNA in 60 min.

Procedure

Materials
Growth Medium. Brain heart infusion (Difco), 18.5 g; tryptone, 5 g;
yeast extract, 2.5 g; NaCI, 2.5 g; H20, I000 ml; autoclave to sterilize.
Store at 4° .
Hemin Stock Solution. Hemin (Eastman 2203), 0.1 g; histidine, 0.1 gm;
triethanolamine, 4 ml; H20, 96 ml. Incubate at 70° for 15 min to sterilize.
Store at 4° .
N A D Stock Solution. fl-Diphosphopyridine nucleotide (fl-DPN;
/3-NAD; Sigma No. D-5755) 10 mg/ml. Sterilize by filtration through a 0.45
micron Millipore filter. Store at - 2 0 °.
Buffer A: 30 mM Tris-HC1 (pH 8.4), 14 mM 2-mercaptoethanol, 5%
glycerol
Buffer B: 20 mM Tris-HCl (pH 8.4); 14 mM 2-mercaptoethanol; 0.2M
EDTA, 5% glycerol.
Buffer C: 10 mM potassium phosphate (pH 7.4), 14 mM 2-mer-
captoethanol, 5% glycerol.

Purification Procedure
Growth of Cells. Six 2-liter flasks, each with 1 liter of growth medium
containing l0 ml of hemin stock solution and 0.2 ml of NAD stock solu-
106 PURIFICATION OF RESTRICTION ENZYMES [13]

tion, are inoculated with 50 ml of frozen log phase H. influenzae Rd corn

10- (exonuclease III-) cells and incubated at 37° with vigorous shaking
until about 3 hr beyond the beginning of stationary phase. Cells are har-
vested in a Sorvall GSA rotor at 8000 rpm. The pellets are quick frozen in
the plastic centrifuge bottles, knocked loose as frozen chips, pooled to-
gether, weighed, and then stored at - 7 0 ° until use. Yield is about 3 gm of
cell pellet per liter.
Preparation of Extract. Cells (18 gm) are suspended in 120 ml of buffer
A in a 250 ml stainless steel beaker and disrupted at maximum intensity on
a Branson Sonifier. Temperature is maintained at around 5° by cooling in
an ice-salt water bath. Sonication is continued for about 20 min until the
turbidity of 50-fold diluted samples show a decrease of 70 to 80%. Cell
debris is removed by centrifugation at 20,000 g for 10 min. The superna-
tant is recovered and adjusted to 4 mM MgCI2 and 0.3 M NaC1 in a final
volume of about 140 ml. This is centrifuged at 100,000 g for 5 hr at 4° to
remove ribosomes and fine debris. The supernatant (130 ml) is recovered
(fraction I).
Ammonium Sulfate Precipitation. Ammonium sulfate is added slowly
with stirring to achieve 70% of saturation (0.472 g/ml of fraction I). After
standing 10 min on ice, the precipitate is collected by centrifugation at
10,000 g for 10 min. The precipitate is then dissolved in buffer B and
adjusted to a final volume that gives a conductivity equal to 0.16 M am-
monium sulfate in buffer B (about 15.5 mmhos at 0°).
First DEAE-Cellulose Chromatography. Nucleic acids are removed by
passage through a DE52 column. The column (2.5 × 30 cm) is equilib-
rated with buffer B. The ammonium sulfate fraction is loaded at approxi-
mately 150 ml/hr and washed through with one column volume of buffer B
containing 0.16 M ammonium sulfate. Amber colored breakthrough and
early wash fractions containing the bulk of the unadsorbed protein are
identified by inspection (or A2a0 measurements) and pooled (fraction II,
approximately 200 ml).
Ammonium Sulfate Precipitation. Fraction II is adjusted to 0.3 M NaCI
by addition of 5 M NaC1. Ammonium sulfate (0.313 g/ml of fraction II) is
added slowly with stirring to give 50% of saturation. The precipitate is
removed by centrifugation at 10,000 g for 10 min. The supernatant is
adjusted to 70% saturation by addition of 0.159 g of ammonium sulfate per
milliliter of fraction II, and the precipitate is collected by centrifugation.
The precipitate representing the 50-70% ammonium sulfate cut is dis-
solved in a minimum volume of buffer C (about 50 ml usually).
Sephadex G-25 Chromatography. A Sephadex G-25 (medium grade)
column of approximately 300 ml bed volume is equilibrated with buffer C.
The 50-70% ammonium sulfate fraction is loaded and eluted with buffer
[13] HindII AND HindlII 107

C. The void volume fractions, containing the bulk of the protein, are
easily identified by their yellow color. These are pooled.
Separation of HindlI and HindllI Activities by DEAE-Cellulose
Chromatography. A DE52 column (1.75 × 8 cm) is equilibrated with buf-
fer C. The Sephadex G-25 pooled fractions are loaded at a flow rate of
about 1 bed volume per hour and the column is washed with 5 bed vol-
umes of buffer C. Fractions of approximately 10 ml are collected. The
breakthrough and wash fractions that contain restriction enzyme (HindlII)
activity are pooled (fraction III, about 100 ml). HindlI restriction endonu-
clease should be quantitatively retained on the column.
Phosphocellulose Chromatography of HindlIL A phosphocellulose
(Whatman P l l ) column (1 × 8 cm) is equilibrated with buffer C. Fraction
III is then loaded at about 1 bed volume/hr. This is conveniently done
overnight using an inlet tubing loop that drops below the column outlet so
that the bed does not run dry. After loading, the column is washed with 3
to 4 bed volumes of buffer C. The HindlII endonuclease is then eluted
with buffer C containing 0.5 M NaCI. Fractions (2 ml) are collected and
assayed for activity. Activity begins to emerge after about 1 bed volume
and is mostly eluted by 2.5 bed volumes. The pooled active fractions
(about 11 ml) are mixed with an equal volume of glycerol and stored at
- 2 0 ° (fraction IV).
Comment. Fraction IV should contain around 10,000 units/ml of
HindlII endonuclease activity with a specific activity of 20,000 units/mg
protein and should be free of detectable exonucleases. A DNA binding
activity is usually present which interferes with DNA cleavage and gel elec-
trophoresis when large excesses of enzyme (greater than 10 units//zg of
DNA) are used. Stepwise or gradient elution may be used to obtain
slightly better purity. Hin dlII endonuclease elutes at approximately 0.3 M
NaC1.
Elution of HindlI Endonuclease from DE-52. A 200-ml linear gradient
from 0 to 0.3 M NaC1 in buffer C is applied to the HindlI activity retained
on the 1.75 x 8 cm DE-52 column. HindII activity elutes in a broad peak
centering at 0.1 M NaC1. Active fractions are pooled (fraction V, about
80 ml).
Phosphocellulose Chromatography ofHindlL A 1 × 10 cm phosphocel-
lulose column, equilibrated with buffer C, is loaded with HindlI fraction V
at 10 ml/hr. Elution is stepwise with 30 ml of 0.1 M NaC1, 30 ml of 0.2M
NaCI, and 50 ml of 0.3 M NaC1 in buffer C. HindlI activity elutes between
1 and 4 column bed volumes during the 0.3 M NaC1 elution. Fractions
containing activity are pooled (25 to 30 ml), adjusted to 50 mM Tris-HCl
(pH 8.5) with a 1 M stock solution, and placed in a dialysis bag. The
enzyme is osmotically concentrated about fivefold against dry
 108 P U R I F I C A T I O N OF RESTRICTION E N Z Y M E S [13]

polyethylene glycol (Carbowax 6000, Eastman 15415). An additional

threefold concentration is achieved by dialysis for 18 hr against 500 ml of
50% glycerol, 50 mM NaC1, 20 mM Tris-HCl (pH 8.5), and 14 mM
2-mercaptoethanol. Enzyme is stored at - 2 0 ° (fraction VI).
Comment. HindlI endonuclease fraction VI is free of detectable
exonuclease but may contain traces of HindlII activity. Effects from the
latter can be minimized by controlling reaction conditions and avoiding
excessive digestion. Total yield is about 10,000 units and specific activity
is > 10,000 units/mg. Haemophilus influenzae Rc contains the isoschizo-
mer HinclI as its only restriction activity and is a better source of the
HindlI cleavage specificity than the Rd strain)

Properties
Enzyme Stability. HindlII is stable for at least 2 years at - 2 0 °. Under
reaction conditions it retains activity for hours at 37° and for several
minutes at 65 °. Activity is > 20% after 10 min at 65 ° in the absence or
presence of DNA substrate. Some loss of HindII activity has been noted
in preparations stored for 6 months at - 2 0 °. HindII is relatively stable in
37° reactions but is > 95% inactivated after 1 min at 65 °.
Substrate Specificity. Both enzymes require double-stranded DNA for
cleavage. HindlII cleaves the sequence (5') A-~A-G-C-T-T, 4 and HindlI
cleaves the sequence (5') G-T-Py*-Pu-A-C. 5 Cleavage produces 5'-
phosphoryl and 3-hydroxyl termini.
Divalent Cation Requirements. HindII endonuclease requires 5-10 m M
Mg 2+ for activity; Mn 2+, Ca 2+, and Co 2+ cannot substitute for Mga+.
HindIII endonuclease requires 5-10 m M Mg z+ or Mn 2+ for activity; Ca 2+
and Co z+ do not confer activity. Neither enzyme shows any activity in the
presence of excess EDTA. HindlII is reported to have an altered specific-
ity in the presence of MnZ+.e
Ionic Requirements. Ionic and pH requirements have been evaluated
semiquantitatively by gel assays. HindIII is optimally active in 30-60 mM
NaC1, retains some activity at 0.125 M NaC1, but is strongly inhibited in
0.25 M NaC1 or in the absence of NaCI. On the other hand, HindlI is
optimally active in the absence of added NaCI, is noticeably inhibited in
30 mM NaC1, and is strongly inhibited in 0.125 M NaCI.
pH Optimum. HindlI is optimally active in the range of pH 7.5-8.0.
HindlII is optimally active in the range of pH 8.0-9.0. HindlII activity is
still appreciable at pH 10.0, but is strongly inhibited below pH 7.0.
s A. Landy, E. Ruedisueli, L. Robinson, C. Foeller, and W. Ross, Biochemistry 13, 2134
(1974).
4 R. Old, K. Murray, and G. Roizes, J. Mol. Biol. 92, 331 (1975).
s T. J. Kelly, Jr. and H. O. Smith, J. Mol. Biol. 51, 393 (1970).
e M. Hsu and P. Berg, J. Biol. Chem. 17, 131 (1978).