Binding of calcium by cellulose membranes and Sephadex

Vol. 50, No. 4, 1973

BINDING

BIOCHEMICAL

OF CALCIUM

AND BIOPHYSICAL

BY CELLULOSE K.C.

Department

RESEARCH COMMUNICATIONS

MEMBRANES

AND SEPHADEX

Reed

of Biochemistry, School of General Australian National University, Canberra, A.C.T., Australia.

Received

January

Studies,

15,1973

Summary: Calcium ions bind to polyglucose matrices (dialysis membranes and Sephadex) with low affinity, but in amounts sufficient to markedly influence the rate of dialysis of Cat+. In applications where such interaction is critical binding studies by flow dialysis or ultrafiltration), Ca I ' binding can be largely prevented by acetylation of the membrane and/or high salt concentration.

INTRODUCTION Numerous lation and

and

reports

have

characterisation

proteins

of

derived

from

(5,6),

kidney

(7),

cartilage

retinal

discs

(lo),

brain

chondria

(14-16).

fractions

have

techniques, the lysis

in

either

more

rapid

and,

technique

of

lose

membranes

in

errors

due

binding

to

point

have

some

out such

that

the

indicated

more

of

these by

the

use

can

that

all

It

in

or

flow

unmodified

result

membrane

various

dialysis

(17). of

mito-

dialysis

accurate

Womack

studies by

and

determined

ways,

(9),

(13)

equilibrium

and

quite

is

diapercelluserious

Additional

itself. polyglucose

matrices

calcium. MATERIALS The

ments

been

iso-

mucosa

tissue

serum

conventional

in

intestinal

properties

Colowick

timely

experiments

to

cases

the

fractions

connective

(11,12),

some

on

binding

(l-4),

ana

binding

recently

calcium

muscle

The

by

haps

bind

appeared

had

flow a lower

dialysis chamber

Copyright 0 1973 by Academic Press, Inc. AII rights of reproduction in any form reserved.

AND METHODS apparatus volume 1136

used of

0.25

in ml

these and

experia maximum

Vol. 50, No. 4, 1973

volume

of

was

0.64

250

ul

BIOCHEMICAL

15 ml cm2.

in

were The

binding

by

incubating

45ca++

of

tilled

to

stirring

were

blotted

membranes

was

After removed, and

results

tubing

tested.

measured

membrane

in

directly

beakers

a specified rinsed by

with time

three

counted

cel-

Scientific

similar

dialysis

dry

and The

Thomas

of

of

area

collected

although

gently.

membranes

water,

from

grades

45Caff

were

membrane

scintillation.

obtained

all

ul

liquid

1 cm2 pieces

the

290

(#4465-A2),

with

and

val,

were

Company

obtained

by

The

chamber.

of

counted

membranes

Instrument

upper

Fractions

aliquots

lulose

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inter-

times

liquid

in

dis-

scintilla-

tion. AND DISCUSSION

RESULTS The cellulose

are

flow

dialysis

dicts

(17)

the

peak

of of

the

to

a second

about

plateau

the

much

Ca

direct

tt

the

binding

measurements

1A.

causes the

rate

Theory

of

is

lower

the

The

in

a sharp

followed

by

initial

decrease

dialysis

this

a

value;

a further of

in

reached

results

the

pre-

chamber).

dialysate,

4oca++

"control"

appearing

plateau of

than

the

45Ca++

4oca++

lower

binding

of

to

5 x (volume

concentration,

phenomena of

the

Since

for

The terms

of

value.

unsatisfactory

x (volume of

adsorption

Figure of

practice,

in

a level

increasing

in

within

excess

Cat+

results

plateau

15

a large

addition

the

concentration

radioactivity

decline

ly,

shown

In

at

addition

in

experiment

should

later

from by

chamber).

much

with

resulting

illustrated

that

dialysate

lower

the

artefacts

in

decreases

system

is

totally

analyses.

apparent

in

Fig.

properties showed

1137

of the

1A are the

binding

explicable

membrane. to

be

Firsttime

de-

Vol. 50, No. 4, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

c

'1;

40.

_

cpmxW

cpmxW

cpm x lo+

/;

41

.

30

,o[ n2030LO lo20P 0203040

20.

Fig.

1

1.

Flow dialysis profiles with 45Ca++. All experiments were done at 4". The flow buffer was 5mM tris-chloride, pH 7.4, and the fraction volume 0.29 ml. The ordinate is cpm/250 ul aliquot of the appropriate fraction. 15 ml of 5mM tris-chloride, A. Upper chamber: pH 7.4. F1 ow r~S~a~i2~~0m~~71nwasA~d~~~. fi ;-;arrow 5 nmoles sequent arrows show the addition of 10 uioles CaC12. The dashed line represents the theoretical profile. El. Upper chamber: l 5 ml of 0.25M sucrose, 1OmM Hepes, pH 7.4 n 5 ml of O.lM NaCl, 1OmM Hepes, pH 7.4 ‘I 5 ml of 0.1 M KCl, 10 mM Hepes, pH 7.4 In each case the flow rate was 0.673 ml/min. At the first arrow 2.5 nmoles 45Catt (5 uCi) was added; the second arrow showsthe addition of 100 umoles CaC12. C. Upper chamber: 5 ml of 5mM tris-chloride, pH 7.4. Acetylated membrane. Flow rate 0.619 ml/min. At the first arrow 2.5 nmoles 45Catt (5 uCi) was added; subsequent arrows show the addition of 100 umoles CaC12.

requiring

pendent, of

45Ca++.

about

Similarly,a

bound

at

These

time-dependent

taken

for

brane. presence in

that

this

time

Ca++

large

to

exchanged

but

a very

approximately

diffuse the large

maximal

slowly

of

internal

binding

was

number the

1138

adsorption

the

with

probably

to

1% of

for

proportion

properties

Secondly, of

20 minutes

45Ca++

40Caft.

reflect sites

in

the the

characteristic of

low

added

affinity 45Ca++

time mem-

of

the

sites, was

bound/cm2

Vol. 50, No. 4, 1973

membrane

BIOCHEMICAL

over

a wide

This

is

further

NaCl

or

KC1

slow

attainment

of

thus

represents

the

with

membrane

binding

displaces ates

all

charge the

ar

the

dialysis

plateau

that

in

with

to

the

presence

45Catt

in

bound.

property

of

certainly

the

due

charge

90%.

dialysis

profile

excess

and

virtually net

satur-

positive a decrease

(cf.

NaCl

45Ca++

40ca++

of

causes

mM The

of

repulsion of

the

and

of

the

membrane

a slight the

by

KC1

in

Fig.

1B;

are

simil-

boiling

decrease

binding

cellulose to

100

Cat"),

only

Most

than

resultant

membrane

due

of

resulted

flow

45Ca++

The

excess

more

Addition

the

that

equilibration

bound

Pre-treatment KHCOS

by the

sites.

concentrations.

observation

time-dependent

with

rates

the

in

sites.

rate

calcium

binding

previously

associated

to

of by

inhibited

available

dialysis

range

supported

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

material. hydroxyl

in thus

and

the an

is

moieties

in

O.lM

amount

of

intrinsic

almost

of

the

glucose

residues. In dialysis

terms

the

experiments,

ciently

overcome

However,

by

these

similarly

media

whose affinity

use

of

these

most

of

the

using

inhibiting

proteins high

of

have

the

low

affinity

properties binding

NaCl

may

or

membranes difficulties

also

be

(Fig.

of

1B).

least

of

Cat'

studied. at

suffi-

disadvantage

binding being

flow

are

KC1 media

obvious

are

in

to

the

Specific partially

in-

hibited. To membrane for fied

overcome were

17% hours membranes

these

extensively in

25%

were

limitations, acetylated

acetic rinsed

small by

pieces

treatment

anhydridelpyridine. thoroughly

1139

in

of at The

O.lN

acetic

70° modiacid

Vol.50,No.4,

BIOCHEMICAL

1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

B cpmxlO-2

an*,

100 lo

2.0 I

\ 60..6

1.2

20.-z

0.4

LA. lo

20

3l

lo

20

30

lmclii

Fig.

LO

50

60

70

64

90

ml

number

Sephadex elution profiles. A. 6.0 ml aqueous extract of rat liver mitochondria (19) containing 2mM EDTA and 5 uCi 45Catt was applied to a column of coarse Sephadex G-25 (1.6 x 36 cm) and eluted with 5mM tris-chloride (pH 7.4) at 4O.

2.

l

cpm/lO

pl

aliquot at 280

n absorbance

of appropriate fraction nm (1 cm lightpath).

8. 4.0 ml aqueous extract of rat liver mitochondria containing 5 uCi 45Cat+ was applied to a column of coarse Sephadex G-25 (1.6 x 47 cm) and eluted with 20mM tris-chloride (pH 7.4) at 40. Fraction volume was 1.73 ml and the flow rate 0.99 ml/min. The first peak of radioactivity is due to Cat+ associated with protein.

l

and

water

they

and

were

tions. this

way

in

low

total

for

only

artefact in

0.02%

unmodified

rate

of

in

amount total

Most

both

membranes

experiment the

of

Before

00.

use, direc-

treated

in Figure

exchangeable.

first of

Fig.

using

such

addition

of

45Ca++

compared

membranes,

apparent. the

rapidly

small

at

fraction.

stretched by

45Catt

following

the

(approx.

and

dialysis

peak

appropriate

50% ethanol

and

flow

indicates

of

water of

very

The

membrane

in

binding

is

membrane.

aliquot

stored

a control

4oca++

ul

rinsed The

shows

change

cpm/lO

lA),

bound with but

significantly, Thus,

dialysis.

1140

acetylated

1 a 20 mM

to

0.4%

the of

this

is

the

there

is

no

cellu-

BIOCHEMICAL

Vol.50,No.4,1973

lose

membranes

barrier

for

Gaff

essential or

provide

in

a far

binding

studies

superior

(18),

studies

They

rates

of

although

would

also

of

Ca++

RESEARCH COMMUNICATIONS

semi-permeable

applications. employing

ultrafiltration

dialysis

AND BIOPHYSICAL

are

virtually

dialysis

(cf.

conventional

be

less

8,16)

equilibrium

ambiguous

with

these

membranes. The

binding

encountered

in

Ca++

from

trace

of

experiments

aqueous 45Ca++

added

to

a column

extract

also

contained

a sharp

peak

corresponding

the

absence

very

broad

(Fig.

28).

caused

a more

hydrate taken

into is

of

to

the

about

the

the

bed

volume label

the

times in

the

of

phenomenon applications

as bed

from

the

Cat+

to

where

a volume

elution

which

in

2A).

the

45Catt

binding

the

(Fig. appeared

three

of

all

and

appeared

the

a general

extract

qa++

displacement

in

of

A

of

glucose

is

mitochondria.

When

of

that

endogenous

G-25.

O.lM

appears

account

likely

at

also

Sephadex

all

most

centred

moieties

of

was

remove

liver

a sample

to

EDTA,

rapid

thus

to

residues to

rat

EDTA,

Inclusion

It

tion

of

peak

of

was

applied

glucose

designed

extracts

mixture

In

to

buffer column. carbo-

should such

be

interac-

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