Stability of isoproterenol bound to cyanogen bromide-activated agarose

Vol. 64, No. 3, 1975

BIOCHEMICAL

STABILITY

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF ISOPROTERENOL BOUND TO CYANOGEN BROMIDE-ACTIVATED

G. VAUQUELIN*,

M.-L.

LACOMBE**,

J.

AGAROSE. HANOUNE** and A.D.

STROSBERG'.

*Laboratorium Chemie der Proteinen, V.U.B., St. Genesius Rode 1640, Belgium. **Unite

Received

April

de Recherches Mondor, 94010

INSERM U-99, H8pital CrGteil, France.

Henri

24,1975

SUMMARY. The chemical stability and release of isoproterenol bound by diazotation to an insoluble agarose matrix has been investigated. It is demonstrated, by a double labeling procedure ([3H]isoproterenol and [14C]spacer arm), that the bound ligand is readily released in a soluble form. This occurs primarily through a chemical hydrolysis of the arm-linked ligand from the cyanogen bromide-activated agarose, and can be observed under extensive washing procedures as well as under more "physiological" incubation conditions. The instability of the agarose-arm linkage should lead to a critical analysis of physiological effects obtained with agarose bound hormones in vitro.

Peptides spacer

arm,

and small

to a solid

reportedly

released

physiological light

hormones

were

in vitro.

from

upon used

This

of free

matrix

(4),

et al

of this the

coupled we report that

link

strongly

at pH above

incubation to glass here

the release

to the instability

various

has been

Tesser

explained

this

5. Finally,

beads

(12).

Using

from

the agarose

of the agarose-arm

C&right D I9 75 b.v Aradenzic Press. Irx All rights of reproduction in ar?y form resewed.

for

from

coupling

spacer

the

arm and

Experiments

suggesting

by

a tlsolvolyse"

the

labeled

ligand

[3H]catecholamines arm and isoproterenol,

together,

in our conditions,

linkage. 1076

11).

labeled

are released matrix,

(6,

exchange

reported

desorption

used for

between

hypothesis,

differently

arm and hormone

immobilized systems

by the simple

suggested

tritium

under

whereby

azo-bond

of linkage

was also

supported

or incubation

are

has shed a new and (8-10)

either

a single

beads,

or subcellular

of the

Cleavage

or through

or agarose

storage

cellular

medium was recently

that

glass

approaches

or by the cleavage

to the spacer arm (11). (glass beads or agarose)

with

during

technical

ligand matrix

(6)

directly,

Such a phenomenon

to stimulate

release

ligand

matrix

(l-7).

several

coupled

such as porous

this

conditions

critical

molecules

which is

suggests mostly

due

Vol. 64, No. 3, 1975

EXPERIMENTAL

BIOCHEMICAL

PROCEDURE. MATERIALS.

dl-isoproterenol

was from Aldrich.

were

p-Nitrobenzylazide

from Merck.

and solvents (2.11

were

Ci/mmole)

was obtained HCl (7.95

by the

Sepharose

4B was suspended

dissolved

in a minimal stirred

from New England

in

from

for

prepared were

buffer

24 h at 4".

prepared

by the

dark

at 4" for unreacted

2-3

0.1 M phosphate at 4" for

buffer

groups

with

with

was determined

spectrometer.

The release

and incubation After

Data

conditions

washing,

containing from

with

stored (v/v).

the literature

report

water.

75 mg glycine,

was

obtained

in

stirred water.

in

the

In order

to

IO ml of in the dark

Labeled

gels

and [ 14C] -ethylenediamine of the gel preparation.

a Packard

0.5 % n-butanol

suspension

was stirred

50 ml distilled

Tri-Carb

ligand

liquid

under

in the legends

at 4" in

the dark,

the coupling

to the

scintillation

was followed

as depicted

gelswere

solution

100 ml distilled

of the bound

This

pH 4, to the previously

obtained by adding [3~] -isoproterenol compounds at the corresponding steps

Radioactivity

to 50 mg of

the gel was suspended

(pH 8) containing

24 h and then washed

with

Isoproterenol-agarose

This (14),

of

funnel

of 18.1 mg of dl-iosproterenol

buffer,

days and washed

diazonium

to the

p-aminobenzamidoethyl-agarose (13).

addition

added

on a glass

pH 10, and added

The diazotized

of washed

pH 11 by addition

washed

to Cuatrecasas

p-aminobenzamidoethyl-agarose.

block

around

was

bromide,

were

in 5 ml of the same buffer.

according

weight)

250 mg cyanogen

the gel was rapidly

in 20 ml of 0.1 M acetate

RESULTS.

[U-14C]Centre

Agarose

One gram (wet

and adjusted

diazotized

figures.

bromide reagents

Co. and

conjugates.

method.

dissolved

pH 4.8,

Nuclear

other

dZ-' lsoproterenol

[7-3H]

of dimethylformamide,

at 5-10"

dissolved

stirred

washing

All

the Radiochemical

10 ml water.

amount

0.5 M carbonate

ethylenediamine

conjugates

bromide

20 min,

25 ml of cold,

were cold

available.

Ci/mmole),

cyanogen

slurry

1 N NaOH. After

gently

grade

from Pharmacia.

and cyanogen

was from Eastman.

of isoproterenol-agarose

activated

was then

4B was obtained

England).

METHODS. Preparation

gently

Sepharose

Dimethylformamide

of the purest

ethylenediamine, (Amersham,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

various

to the in 100 mM EDTA,

of catecholamines,

such as epinephrine and isoproterenol, to diazonium agarose and diazonium glass beads at pH 7.5 (2,9,15). However, under these conditions, the coupling

is

the formation whereas

accompanied

of pyrocatechol

the highest

pH 4 (data

by several,

yield

to be published).

unexpected

azoderivative of isoproterenol Thus

in order

1077

side-reactions as well

azoderivative to obtain

leading

as other is

to

by-products, observed

a maximal

amount

at of

Vol. 64, No. 3, 1975

the expected

BIOCHEMICAL

conjugate

(9),

AND BIOPHYSICAL

the coupling

RESEARCH COMMUNICATIONS

step was routinely

performed

at

pH 4. The release conditions. double

To elucidate

labeling

mine

of free

for

and bound

the "true"

procedure

hormones

mechanism

to prepare

the gels

the

spacer arm and [3H] isoproterenol both [ 14 C] labeled arm and tritiated

contained agarose.

Under

activity

was not

this

bound,

condition,

Structure agarose

of the conjugate

it

but

only

of release, (Table for

I):

[14c]

ethylenediaThus gel

that

bound

part

Content (pmoles/g

t

Gel II

2

Gel III Symbols

"A-B-N2-*IPR (agarose

*A

0.03

III

I3113 1.5

0.35

used: [14C]-ethylenediamine;

-*A

I I:

I .a5

and gel

in labeled products gel) before washing

*IPR

$A-B-NH~

*A:

Table

alone)

*A + "IPR

to the

of the radio-

Gel II

t4c1 Gel I

various

the hormone.

on the gel.

Labeled products adsorbed

under we used a

hormone

was possible adsorded

was studied

Structure

"IPX:

[3H]-isoproterenol.

i)H -Band labeling

’

“IPR -

of the gels.

Gel I was prepared as described under "Methods". Gel II was prepared by the treatment of inert agarose (not activated by cyanogen bromide) with [14C]-ethylenediamine, followed by the other steps described under of p-aminobenzamidoethyl "Methods". Gel III was prepared b treatment agarose (not diazotized) with [ 37H -1PR and by the subsequent steps described under "Methods". The specific activit 7 of L3Hl isoproterenol was 2.7 Ci/mole for gel I and III and that of [ 4C] ethylenediamine was 240 and 40 mCi/mole for gel I and II respectively.

1078

I

Vol. 64, No. 3, 1975

were

prepared

native

BIOCHEMICAL

to correct

[14c]

ethylenediamine

to be only

adsorded.

of unbound

material

subtract calculate

for

it

from

This

from

gel

with

10 mM HCl.

from gel

II.

allowed

release

As shown in Fig.

observed

of bound during

Almost

When the release

contained which

were

us to measure

washing

gel

I.

considered

the "desorption"

and incubation from

respectively

steps

and to

Thus we could

material.

1, appreciable

I and III,

they

isoproterenol

the various

release

released

possibility:

procedure

the total

and then

this

and [3H]

during

the actual

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

amounts

washing,

first

no release

of isoproterenol with

80 percent

of ethylenediamine

of chemically

bound

were methanol

was observed

isoproterenol

and spacer

1234123456

1

2

3

4 WASHING

....*.-.*.*. ..“(-I ....*.*.*.*.* .*. ..*. 1...*-*.**** .n....*..... *. 1

2

3

4

5

6

!sTEP!s

Fig.. The washing steps were performed as follows: one g of agarose conjugate (prepared as depicted in Table I) was suspended in 40 ml methanol-water (4:1, v/v), and shaken for 48 h at 4" in the dark. The gel was filtered on a Millipore filter disc (0.45 n pore size). The filtrate was collected and counted. The washing procedure was repeated in similar conditions for several times with either 40 ml methanol-water 4:1, v/v) or 10 mM Xl. Total amount of[l4C] arm (O--W* for gel I) and b H] isoproterenol (O -0 for gel I, and O---Ofor gel III) present in each washing solution is given as percent of their molar concentration in the original gel before washing. These concentrations were calculated from the specific activity data given in the legend to Table I. No release of [14C] arm adsorded to gel II was observed under this washing procedure. Insert: Release of bound[3H] isoproterenol (ol ) and bound [14C]arm was calculated from data in Fig. 1 by subtracting the amount (0 ---0) due to desorption (gel III) from the total amount of release (gel I).

1079

Vol. 64, No. 3, 1975

BIOCHEMICAL

arm was corrected original

molar

and

solutions:

0.6

10 mM HCl,

than

that

[14C]

the

step

was used

washing

for

an important

percent

level

for

each washing

after

five

essentially

It

step.

I percent

3 times

release

higher

the first

seems therefore

arm and matrix

of the hormone

Upon washing for

that

when 80 % methanol

catecholamine-agarose

arm and bound

[3H]

washing

was about

from

steps.

between

the innnobilized part

released

decreased

of the of

in the methanol

for

Furthermore,

this

release

of labeled

materials

(10 min at 37')

used

As shown in Table

renol

were

This

suggests

II,

released

(0.17

that,

during

Radioactivity from

gel

complex,

whereas

was due to the cleavage

isoproterenol

gel

III

13H]from

gel

I minus

from gel

II:

double

labeling

method also

for

when

10 mM HCl was

indicated

occurred

that

during

the adenylate

the

cyclase

similar

amounts

of [ 14C] arm and ['H]

and 0.18

percent

of the initial

incubation

under

released

I (total

[3H]from

Table

release which

between

incubations

[14C]

released

as percent proportions

washing.

appreciable

[3H]

similar

of isoproterenol

additional

of the azo-linkage

17).

-arm were

to 0.1 percent occurred

and expressed

1, insert),

of ethylenediamine

the cleavage

time

(Fig.

to 0.7

with

used

the desorption

amount

isoproterenol

washing

for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

molar

"physiologicalnconditions,

Percent

of the original amount

short

assay

(16,

isoproteamount). as

molar

0.23

release)

0.06

(desorption) L3H] from

gel

III

0.17

I

Ligand

release

during

incubation.

After the washing steps described in Fig. 1, 60 mg aliquots of gel and III (prepared as described in the legend to Table I) were suspended in 1 ml of incubation medium containing 50 mM Tris-HCl buffer pH 7.4, 3 mM MgC12 and 0.5 mM ATP. The suspensions were shaken for 10 min at 37" and the supernatants were removed after centrifugation and counted. The total amount of labeled material present in gel I before incubation was 1.4 pmole/g gel for the [14C] arm, 0.9 pmole/g gel for [3H] to as 100 percent. Values depicted isoproterenol, and was referred are means of four experiments.

1080

I

BIOCHEMICAL

Vol. 64, No. $1975

well

as during

washing

arm and matrix. amount

with

of arm-bound

ligand

to give

a final

represents

a contamination

system

(16,

methanol,

in Table

released

procedure

were

also

described

settled

unbound

isoproterenol

release

of bound

DISCUSSION.

Several

mation

have

the

process,

we observed.

of the hormone

should

of "bound"

physiological be exercized

ligand

of membrane detrimental

is

it

adenylate to affinity

of ligand-binding

for

et a2 (6)

release lead

authors,

of the radio-

to a slight

obviously

. An by

by these

explain

conditions(Table

when interpreting in biological

proteins binding

could

bond,

overesti-

the

from agarose-bound hormones by free, and In fact, since this "chemical" release

cyclase.Release chromatography,

receptor methods

cannot

desorption of arm-bound

reported

described

arm

of ligand

of the arm-agarose

of the total

arm

labeled part

whereas

has been

was due

spacer

release

to by Tesser

incubation

studied

Thus,

the

release

between

washing,

hormone

phenomenon but

from to assess

this

the major

the conditions

of the supernatant active, material. under

that

instability

5 percent This

that

arm itself.

alluded

under

release

of ligand designed

linkages

by extensive

the tritiated

at most

caution

spacer

already

from

of adsorded

by the use of differently evidence

due to the

represent

does occur

study,

observed.

material.

a release

was suggested

of

whereas

presently the level

especially

It

one

desorption

percent)

of the bound

reported

now direct

occur,

(0.02

II,

to that

and iminocarbonate

However,

that

release

purpose,

a continuous flow of 12 1 the subsequent incubation

diminished

incubations

et aZ (12).

contamination physiologically

Alternative

the

with

of tritium

would

procedure

be eliminated

by a "solvolytic"

it

cyclase

to the extensive

For this

to Table

comparable

In this

could

can still

activity

was This

shown)

(4).

with During

was minimal

washing

provide

NaCl.

II

of isourea

together

ligand

Venter

between the total

the adenylate not

and Kaplan

was washed

of the gels.

(6,ll).

released

exchange

that

10 min incubation

(data

in the caption

gel

during

activity

and agarose

ligand

the

remained

authors

carriers,

and hormone,we free

from

affecting

to the cleavage

is

described

column

without

biological

was calculated

to stimulate

by Venter

material

extensive

insoluble

occurred

of 0.1 PM isoproterenol.

submitted

in a column,

the conditions

material

a cleavage

17).

gram of gel,

Thus,

it

during

concentration

of 0.1 M HCl and 18 1 of 0.8 percent under

II,

susceptible

Gel I and III washing

80 percent

From the data

sufficient

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

experiments systems

of arm-bound especially are

ligands

1081

II), where

the effect

such as the activation ligand

might

when very

to be selectively to insoluble

great

matrix,

also

small

be amounts

purified. e.g.

the use

of

Vol. 64, No. 3, 1975

BIOCHEMICAL

of macromolecular suitable,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

arms or epoxy-activated

and are now under

Sepharose,

are conceivably

more

investigation.

ACKNOWLEDGMENTS. We are grateful

to Drs. E. Kanarek and P. Berthelot for helpful and valuable discussion of this study. This work was supported by the "Institut National de la Sante et de la Recherche Medicale" and by the "DelBgation G&-&r-ale ?i la Recherche Scientifique et Technique". G.V. is aspirant of the "National Fonds voor WetenschappeliJkOnderzoek".

REFERENCES

I.

TESSER, G.I.,

2. YONG, M.S.

FISCH, (1973)

3. VAUQUELIN, G., Hoppe-Seyler's 4. VENTER, J.C. 5. YONG, M.S.

H.U.

and SCHWYZER, R. (1972)

Science,

182,

3,

(1974)

and RICHARDSON, J.B. FISCH,

H.U.

56-58.

157-158.

HANOUNE, J. and STROSBERG, A.D. LACOMBE, M.-L., Z. Physiol. Chem., 335, 1263. and KAPLAN, N.O.

6. TESSER, G.I., Is, 1718-1730.

FEBS Lett.,

Science,

(1974)

185,

Science,

and SCHWYZER, R.

459-460.

185,

(1974)

(1974)

460-461.

Helvetica

Chimica

7. KOLB, H.J., RENNER, R., HEPP, K.D., WEISS, L. and WIELAND, O.H. Proc. Nat. Acad. Sci. USA, 72, 248-252. 8. CUATRECASAS, P. (1969)

Proc.

Nat.

Acad.

9. VENTER, J.C., DIXON, J.E., MAROKO, P.R. Nat. Acad. Sci. USA, 69, 1141-1145. HABER, E. and O'HARA, 10. LEFKOWITZ, R.J., USA, 2, 2828-2832. 11. CUATRECASAS, P. and PARIKH, 12. VENTER, J.C., 1-9.

ARNOLD, L.J.

13. CUATRECASAS, P. (1970) 14. WELIKY,

N. and WEETALL,

15. VENTER, J.C., ROSS, J., (1973) Proc. Nat. Acad.

I.

D. (1972)

245,

(1975)

17. HANOUNE, J., (in press).

and PECKER, F.

LACOMBE, M.-L.

1082

Nat.

Proc.

Acad.

2291-2299.

Mol.

Pharmacol.

Sci.

fl,

3059-3065.

DIXON, J.E., MAYER, S.E. Sci. USA, 70, 1214-1217. PERRENOUD, M.-L.

(1972)

11,

Immunochemistry,

16. LERAY, F., CHAMBAUT, A.-M., J, Biochem., 3&, 185-192.

(1975)

450-457.

Proc.

Biochemistry,

Chem., (1965)

USA, 63,

and KAPLAN, N.O.

and KAPLAN, N.O.

J. Biol. H.H.

(1972)

Sci.

Acta,

2, 293-322. and KAPLAN, N.O.

and HANOUNE, J. (1975)

J.

Biol.

(1973)

Chem.

Eur.