Immobilization of enzymes and affinity ligands to various hydroxyl group carrying supports using highly reactive sulfonyl chlorides

Vol.

102,

No.

BIOCHEMICAL

1,198l

September

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

16, 1981

IMMOBILIZATION

OF

CARRYING

ENZYMES

SUPPORTS

AND

AFFINITY

LIGANDS

USING

HIGHLY

REACTIVE

Kurt Department

of

Pure

and

Nilsson

Applied

and

July

SULFONYL

07

GROUP

CHLORIDES

Chemical

Lund,

HYDROXYL

Mosbach

Biochemistry,

S-220 Received

Klaus

TO VARIOUS

449-457

Centre,

P.O.B.

740,

Sweden.

28,1981

SUMMARY A facile method for the activation of hydroxyl group carrying supports such as agarose, cellulose, diol-silica, glycophase-glass or hydroxyethyl methacrylate gels with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride) is described. On reaction of the resulting tresylate containing supports with different enzymes or affinity 1 igands at neutral pH, overnight and at 4 oC, coup1 ing yields and retained specific activities of up to 80 and 50 %, respectively were obtained. Thus, Nb- (6- aminohexyl)The bound ligands showed excellent affinity properties. AMP coupled to diol-silica completely separated a mixture of albumin, lactate dehydrogenase and alcohol dehydrogenase in less than 15 min when the technique of high performance liquid affinity chromatography was employed.

INTRODUCTION Recently

a new

method

p-toluenesulfonyl affinity over

of

activating

chloride ligands

presently

was used

of

the

support

In

the

literature

to

be more

allowing

described ones

and

no

side

than

In

this

report

derivatives

these

tosylates

compound number

as of

chromatography.

it

allows

different

to efficient supports

The

reactions

carrying

supports

immobilization

This

method

was

are

bound

directly

occur

of

and we wish

ligands

reactions

trifluoromethanesulfonate

group

subsequent (l-3).

since

a number

reactive

hydroxyl

during of

of

shown to

activation

sulfonic

including

esters

an

including involved

under those in

have

and

advantages

the

carbon

atoms

and

coupling.

have

been

reported

(tresylate)

immobilization

coupling

proteins

p-nitrobenzenesulfonate,

2,2,2-trifluoroethanesulfonate

describe

to

using

method very

used immobilization

mild for

based

on

conditions

high

performance of

biomolecules

(4). the and

latter to

a

liquid using

0006-291X/81/170449-09$01.00/0 449

Copyright G 1981 b-v Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 102, No. 1,198l

tresyl

chloride

BIOCHEMICAL

or

tosyl

AND

chloride

are

BIOPHYSICAL

believed

to

RESEARCH COMMUNICATIONS

be

as

given

in

the

scheme

below:

Activation:

+

H2OH

ClS02CH2CF3

+

H,OSO,CH2CF3

b tresyl

+

H20H

chloride

tresylate

ClSO2C6H4CH3

+

H20S02C6H4CH3

b tosyl

chloride

tosylate

Coupling:

H20S02R

+

H2N-L

-

H20S02R

+

HS-L

-

+

HOS02R

H2-S-L

+

HOS02R

room

temperature,

4-c

ti (R

H2-NH-L

=

CH2CF3

MATERIALS

or

C6H4CH3;

AND

Activation

L

=

ligand

or

enzyme)

METHODS

with

tresyl

chloride

was

performed

at

principally

as described for activation with tosyl chloride (1,2). Sepharose (48, CL-48 or CL-6B, Pharmacia) was transferred to dry acetone via the following washing steps on a glassfilter funnel: 10 gel volumes of dist. water, 30:70, 60:40 and 80~20 of acetone p.a.: water (V/V), 2x10 volumes of acetone and finally 3x5 volumes dried acetone (dried over molecular sieve, 100 g/3 1 acetone). The gel (32 g) was then transferred to a beaker containing 0.1 gel volume dry acetone and pyridine (2 ml/ml chloride reaction acetone, finally entire Cellulose ing in

tresyl (>$I7 was 10 with

chloride). %, Fluka) continued volumes 2x10

of volumes

Under vigorous was added dropwise for 10 min. The gel

magnetic stirring for 1 min to the was then washed

30:70,

and stored

of

50:50, 70:30 1 mM HCI and

activation procedure can be completed (Sigmacell, type 100) was activated 1 M NaOH for 1 h. To facilitate stirring

85:15 at within by

the during

4

0.2-1.0 gel-suspension. with 2x10

ml gel

1 mM HCl:acetone 'C until used.

1.5 h. same procedure activation

tresyl The volumes

and The after 0.5

swellgel

volumes of acetone was added. Diol-silica (1,2-dihydroxy-3-propoxypropylsilyl-silica), prepared from LiChrospher Si 500 (Merck) as described reviously (5), LiChrosorbR Diol (5 urn, Merck) ? M/CPG-40, and glycophase-glass (Glycophase-G 5-10 urn, Corning) were initially washed with go:10 acetone:water (V/V), then washed with dried acetone and treated with tresyl chloride as above, except for using 12 ml of dry acetone/g dry gel to facilitate stirring. After washing of the gels as described above they could be washed with acetone and they were ready for use. Alternatively, sucked dry for storage. Hydroxyethyl methacrylate (Spheron R P 100,000, Koch-Light) was treated as the silica gels, however 1 gel volume acetone was added to the settled gel to allow proper stirring during activation. Coupling. type l-S,

Trypsin Sigma),

(bovine albumin

pancreas, (bovine,

type fraction

450

III, V,

Sigma), Sigma)

tryps'n inhibitor b and N -(6-aminohexyl)-AMP

(soybean,

Vol. 102, No. 1,198l

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

(Sigma) were dissolved in cold coupling buffer (either 0.2 M sodium bicarbonate or 0.2 M sodium phosphate, + 0.5 M sodium chloride). One volume of settled activated gel was briefly washed with the cold coupling buffer and added to one volume of ligand solution. Coupling proceeded at 4 ‘C with gentle agitation. Coupling was terminated by washing with 3x10 gel volumes of coupling buffer, 0.2 M sodium acetate, pH 3, containing 0.5 M sodium chloride, 0.5 M sodium chloride, dist. water and coupling buffer. Hexokinase (yeast, type III, Sigma) was coupled in 0.2 M Hepes buffer, pH 7.0 containing 15 mM MgC12, and washed as above omitting the acetate buffer. Concanavalin A solution (14.5 mg in 0.5 ml saturated sodium chloride, Miles), was added at 4 oC to 1.1 g wet tresyl Sepharose 48, which had been rapidly washed with buffer (0.2 M sodium phosphate + 0.5 M sodium chloride pH 7.5). The gel was washed as recommended for CNBr coupled Concanaval ;n A (6). Protein content was determined by amino acid analysis and bound AMP-analogue was determined by uv-measurements (1). Determination of free and immobilized enzyme activities was done spectrophotometrically as described previously (1). The assay system for hexokinase was as given by the Worthington Manual (7). Activation with CNBr of Sepharose Cyanate groups were determined by Coupling of trypsin inhibitor (10 to CNBr and tresyl chloride activated 4 OC as described above. Coupling by measuring c251]activity of the

CL-be was done according to March et al (8). the method described by Kohn and Wilchek (9). mg/g gel) andu25galbumin (5 mg/g gel) to Sepharose CL-4B were done at pH 7.5 and yield and leakage of albumin were determined gel and the collected washings.

Affinity experiments were done with the gels packed in Whatman columns of 0.3 cm diameter. The flow rate was controlled with a peristaltic pump. The absorbance of the eluate was recorded with a Uvicord (Pharmacia). The experiments were done at 4 OC except for the purification of peroxidase which was done at room temperature. Gels used for affinity chromatography were prepared by coupling at pH 7.5 and 4 ‘C. Soybean trypsin inhibitor (15 mg) was coupled to 1.5 g Sepharose 4B (0.8 mmol tresyl groups/g dry weight) as described under Coupling. To remove any interfering tresyl groups, the coupled gel was washed with cold 0.1 M Tris-HCl, pH 7.5, for 30 min on a glass filter. A mixture of 4 mg albumin (bovine,type V,Sigma) 2 mg trypsin (bovine pancreas, type III, Sigma) and 2 mg a-chymotrypsin (bovine pancreas, type l-S, Sigma) in 0.5 ml effluent (0.2 M sodium phosphate, pH 7.2) was applied to 1 g gel containing 7.5 mg inhibitor. The flow rate was 20 ml/h. Peroxidase (horse-radish, grade I I, Boehringer) was applied to 0.5 g Sepharose 48 containing 6.5 mg Concanavalin A. The effluent contained 0.1 M sodium acetate pH 6.0, 1 M sodium chloride and 1 mM MgCl2, MnC12, and CaC12 each. After application of 0.25 ml enzyme solution (containing 1.25 mg protein the flow was stopped for 30 min to allow equilibration. Fractions were then collected at a flow rate of 10 ml/h. Preparation of AMP-silica used for High Performance Liquid Affinity Chromatography (HPLAC). LiChrosorb” Diol (5 Urn, Merck), 2.5 g dry weight, washed with acetone as was suspended in acetone (10 ml) described under Activation with tresyl chloride, Tresyl chloride (0.5 ml) was added dropand pyridine (1 ml) at room temperature. wise (1 min) with stirring. The gel was washed after 20 min as described under Activation. N6-(6-aminohexyl)-AMP (40 mg) was dissolved in 2.5 ml 0.7 M sodium bicarbonate and added to the wet tresyl silica (7 g). Coupling proceeded for 20 h at 20 oC at pH 7.5. After washing with coupling buffer the gel was treated with pH 7.5, for 4 h at 20 OC (treatment with 0.1 M Tris-HCl 0.1 M mercaptoethanol, is believed to be a good alternative as was found for Sepharose buffer, pH 7.5, After washing of the gel as described under Coupling it bound affinity ligands). was washed with acetone, sucked dry and analyzed for nitrogen. It was found that 13.2 mg AMP-analogue/g dry gel had coupled (i.e. 79 % yield).

451

Vol. 102, No. 1,198l

BIOCHEMICAL

Table

1.

Activation

Type of Sepharose

of

mmol/g

wet

RESULTS

AND

The of

studied

of ratio

well

with

only

rather

life

of

about

gels

of

fluorine

30

can

be

chloride.

tregyl

dry

for as

weight

10 minutes

as described

determined

by elemental

on

For been

for lyophilized

storage

at

least

three or

kept

nitrogen

by

at

4 ‘C

overnight

with

CNBr

the

recommended coupling

months in

The

acetone.

452

elemental

to

of 0.1

these

settled

corresponded that

both

S and

pH

the

gels

enzymes

conditions.

pH

where is

no

F decreased

M phosphate,

neutral

for

the

indicating

agarose

keep

swelling

analysis,

amount

at

capacity under

The

groups

water.

activated

phase

the

tresyl

since

to

in

for

of

product).

introduced

ion.

is

amount

a

was

obtained

activation

was

aqueous

high

as

activation

was

back

react

the

during

dry

the

the

it that

mmol/g

determined

rasts in

the

transferred

No

storage

groups

is

during

as

cant

found

used

preparations

pattern

(>l

when

during

Sepharose

substitution

change

sulphur,

stability

(9).

not

numbers.

cent

various

chloride

reaction

unchanged to

ester

has

storage

formed

Noteworthy

did

was

per

min it

of

groups mmol/g

content

tresyl same

of

ncorporated

h gh

of the

10 min

expected

cyanate

4 OC as after

gel

a few

This

Amount

acetone

of

preparations.

the

was

substitution

seen

Sepharose

the

pyridine by

As

only

of

volume

gel

tresyl

COMMUNICATIONS

0.80 1.28 0.79 1.35 0.45 0.98 1.10

amounts

1).

after

properties

with

tresyl useda

were d,one in and Methods. from the sulphur

of

different

Sepharose

obtained

The

degree

(Table

different

Sepharose

RESEARCH

DISCUSSION

Activation. function

BIOPHYSICAL

0.15 0.30 0.15 0.30 0.10 0.20 0.30

The reactions in Materials Calculated analysis.

b)

of

Amount chloride

48 4B CL-4B CL-4B CL-6B CL-6B CL-6B

a)

AND

the

half-

reported in

did

to

1 mM HCI not

Alternatively,

7.5.

be

at

decrease the

Vol. 102, No. 1,1981

Table activated

2.

BIOCHEMICAL

Coupling with

Supporta

of

AND

enzymes chloride.

tresyl

and

BIOPHYSICAL

affinity

L i gandd

RESEARCH

ligands

to

different

Yield

Bound ligand v/g dry support

48 4B

Concanavalin A Soybean trypsin inhibitor Sepharose CL-6B Hexok i nase ________________________________________--------------------------------------

360 195 94

53

26

Sepharose CL-6B Trypsin 3, Cellulose II Diol-sil icab ______-____---____----------------------------------------------------

124 61 24

87; 80

33e 56 63

Sepharose

CL-6B

Cellulose Diol-silica Glycophaseglassb Hydroxyethyl methacrylateC

a)

b) c) d)

e)

b

II

hydroxyl

chloride

group

followed

conditions

by

(Table

Coupling

of

As

seen

neutral

optimal

whereas Table

2

87

32

49 18 15

44 36 45

10

12c

- - - - - -- -

2).

carrying

supports

efficient

were

coupling

of

conveniently

enzymes

or

activated affinity

with

ligands

tresy

under

mild

2).

enzymes.

studied.

relatively

also

N6- (6-aminohexy 1) -AMP II II II

activity to enzyme

Sepharose 48 and CL-6B and cellulose contained 1.28, 1.1 and 0.5 mmol tresyl groups/g dry weight, respectively. Activation of all polymers lasted for 10 minutes and 1.0 ml pyridine and 0.5 ml tresyl chloride were added to 17 g gel-suspension (i.e. support suspended in acetone, see Materials and Methods). 1,2-dihydroxy-3-propoxypropylsilyl-silica and Glycophase-GTM/CPG-40 were used. Spheron P 100,000 (this preparation has a low surface area, which may explain the relatively low coupling yield). Amount of ligand applied/g wet gel during coupling: 13 mg Concanavalin A; 10 mg soybean trypsin inhibitor; 10 mg trypsin; 10 mg hexokinase and 16 mg N6-(6-aminohexyl)-AMP. Concanavalin A and trypsin inhibitor were coupled for 15 h at 4 ‘C and at pH 7.5 and hexokinase at pH 7.0. The comparative studies involving trypsin and the AMP-analogue were carried out with the preparations coupled at pH 8.2. The affinity experiments described in the text however were made on gels coupled at pH 7.5. On coupling for shorter time (4 h) 55 % was bound but the specific activity was higher (50 %).

Different

was

supports

Specific relative soluble %

%

Sepharose Sepharose

COMMUNICATIONS

The

coupling

from

Table

of

3,

conditions trypsin The

bound stability

more of

high

coupling

7.5).

(pH

the

inhibitor

trypsin

For

yields

at enzyme

453

pH a

various

(72

hexokinase

efficiently labile

under

somewhat

hexokinase

%) 7.0

conditions

were

obtained

was

shown

higher

pH

was

found

at to

be

(8.0,

see

to

very

be

Vol. 102, No. 1,198l

Table (1.28

high

3. Coupling mmol tresyl

6.5 87::

10 10

15 1.5

9.5 10.5

immobilized

of

atom

the

occurs

affinity

for

the

to 7-8

is

as and

of

to

fluorine

of

at

obtain

223 195 200

67: 70

decrease

in

while

such

a clearcut

recommended,

(pH

> 8.5) CF,

for

the

after

a few

tresyl

it

immobi

might

of

d d not

the

car-

be

pass

ible

the

pH

biomo Nlecu

enzymes

adversely

d splacement, is

days.

at

the

ization

range

free

group

with

group

pH values

anyhow

storage

to

nucleophilic

which

detected

displacement

terminal

high

was

activity

Although these

activity

biomolecules

itions

the

a side-reaction. out

%

nucleophilic

cond

48

Yield

2 74(64)

of

alkaline

Sepharose at 4 OC.

inhibitor

enzymic

involve

tresyl was done

dry

4 ‘C,

all

to

70 211(366) 195

no

immobilization

At

carried

performance, pH-values

loss

as

ligands

their

in

be1 ieved

of

question

ted

matrix.

substitution

agarose pH 7.0

t he

Bound mdg support

a.5

tresyl

RESEARCH COMMUNICATIONS

inhibitor Coupling

pH

at

in is

for ing

month

resul

reaction

bon

e

or affect

immobilizat of

choice,

on

notably

enzymes.

Affinity

chromatography.

were

tested

application appeared

in

trypsin

and

acetate

of

11 mg The

one

solution

supports

at

to

for

carrying

in

trypsin weight).

15 15

normal

that

dry

10 li(20)

in

The

of soybean groups/g

BIOPHYSICAL

Time coup1 h

storage

enzyme

AND

Amount inhibitor added mg/g wet gel

when

after

BIOCHEMICAL

by

applying

at

pH

the

7,

void

trypsin pH 4.8

inhibitor/g

specific

The

binding

affinity

properties

a mixture albumin volume could

and as

albumin,

inactive

one

3.0,

wet

polymer

peak. be

respectively.

the

was

to

glycoprotein

The

completely The

shown

capacity be

trypsin and

of

trypsin

bound

and

active

of

a preparation

(from

changing

g of

horse-radish)

On

o-chymotrypsin

forms

by

per

inhibitor

trypsin.

eluted

9 mg trypsin

peroxidase

454

immobilized

a-chymotrypsin molecules

sharp

subsequently

and

of

of

of

of

a-chymoto

0.2

M

containing sorbent. to

Conca-

Vol. 102, No. 1,198l

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

0 Time

(min)

Figure 1. Separation of bovine serum albumin (25 pg). beef heart lactate dehydrogenase (5 ug) and horse liver alcohol dehydrogenase (25 pg) on N6-(6-aminohexyl)-AMP-silica with HPLAC-technique. The AMP-analogue was coupled to tresyl chloride activated LiChrosorb Diol as described in Methods. The sample (450 pl) was applied on a column (0.5x10 cm) containing 29 umol AMP-analogue/g dry support. The flow rate was 1 ml/min which gave a pressure drop over the column of 7 MPa. The column effluent (0.05 M sodium phosphate, pH 7.5) was monitored at 2&O nm and then mixed on-line with assay reagents (13) for dehydrogenases and monitored at 340 nm. The experiment was done at room temperature.

navalin

A immobilized

commercially ting

of

which To

or 0.11

avoid

peptides

groups 0.1

M Tris

agarose the

the buffer,

column

was

bound

peroxidase

to

the

buffer.

The

with

support

also

the

the

non-specific on

was

and

M mannose well

possible

tresyl

peroxidase

corresponded

tresyl with

available

proteins

addition

on

literature binding

can

a strong

easily nucleophile,

be

the

On application

easily

free

washed

could

completely

obtained data

to

demonstrated.

for

gel,

removed at

A pure

peroxidase

we have

found

by pH

treating

7.5,

4 ‘C

of

contamina-

be

eluted

ratio

403’A280

and

was

by 3.15,

(10). that

the

of

excess affinity

for

30

gel min.

Vol. 102, No. 1,198l

High

BIOCHEMICAL

Performance

experiment

Liquid

on

shown

in

1.

in

vated

volume

were

recovered

As

to

(11).

Chromatography

and

80

added

short

to

with bound

lactate

pulses

this

NAD+

be

an

of

gel

bound

as

100

and oxalate

are

is

to

well

CNBr

acti-

% appeared

alcohol and

a HPLAC Diol)

ligand

ligand

the

plus

results

genera1

this

to

COMMUNICATIONS

(LiChrosorb

dehydrogenase

of

.The

diol-silica of

findings was

RESEARCH

(HPLAC)

biospecificity

albumin

% of

with

the

previous

No

BIOPHYSICAL

bound

seen

analogy

Sepharose

void

Affinity

N6-(6-aminohexyl)-AMP

Figure

conserved

AND

in

the

dehydrogenase

NAD+

plus

pyrazole,

respectively. CONCLUSION We have

found

for

activation

the

suited

for

of

support

the

with

found

that

rose h,

storage

mmol

tresyl

a few

activated 20

gels The

%. in

buffers

coupling and

treated

gels to

the

of

used

problem

of

trypsin

the

with

protein

0.2

leaked

importance

as

leakage

of

pH 7.5,

proteins

4 ‘C

and

overnight

be

higher

for

to

the %,

or

same

amounts

of

even

in

aqueous loose

possible

leakage

it

activated

pH

7.8

whereas

to

35

CNBr

for to

a loss activated

problem

as

tested

chloride groups

‘C

bound

recognized

appears,

was aga-

showed

bound

tresyl

at

albumin

weight)

a well

active

substitution

rapidly

dry

is

better

supports

M Tris,

proteins

being

Activation

chloride

off,

groups/g

than

(82 and

70

(12).

with for

CNBr

% as

com-

respectively). described

affinity of

in

chloride

of

stored

tresyl

cyanate

containing

in

to

mmol

inhibitor,

60

bound

to

tosyl

proteins.

degrees

when

regards

(0.85

at

high

activated

weight)

amines

and

stable

With

125 I albumin dry

is CNBr

I 1

cent

method

preparation

a major

media.

containing

55 and

activation

proteins

aqueouS

groups/g

and

gel

in

capacity

albumin

pared

is

activated

to

supports,

ligands

predictable

applied

agarose

This

to

widely

of

carrying

pH-sensitive

leads the

alternative

group

the

per

excellent

hydroxyl of

and

on

only

various

rapid,

groups

(0.8

CNBr

The

is

In contrast, active

to

immobilization

groups

their

of

chloride of

the

tresyl

media.

48

tresyl

is

well

chromatography

immunosorbents the

alternative

as

suited

for

and

should

leakage techniques

456

of

coupling

of

be especially

immobilized at

ligands

hand.

useful

antibodies The

and

ease

for can

of

direct

be

BIOCHEMICAL

Vol.102,No.1,1981

activation

of

sorbents

tography

constitutes

AND

applicable an

to

additional

BIOPHYSICAL

high

RESEARCH

performance

COMMUNICATIONS

liquid

affinity

Council

for

chroma-

advantage.

ACKNOWLEDGEMENTS We wish

to

thank

support

and

Or

the P.O.

Swedish Larsson

Natural for

helping

Science us

Research running

the

HPLAC

their

experiment.

REFERENCES 1. 2.

3. 4. 5. 6.

7. 8. 9. 10. 11. 12. 13.

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