Diffusion of protein molecules through membranes of controlled pore size

BIOCHEMICAL

Vol.5, No.3,1961

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

DIFFUSION OF PROTEIN MOLECULES TRROLJGB HEMBRAN~ OF CONTROLLED PORE SIZE1 Eckhardt Fuchs of Chemistry, Stillwater,

Department

Received

May 22, 1961

Membrane sizes

have

teins

through

ison ing

protein

sions

; the

cules

which become

diffusion

separation that

membrane

sandwiched

definite

area

vals

tally. sizes

small and the

Experiments of 10,

New Bedford,

that

represented

to the

in Fig.

0.1

50 and 450-q

diameter,

rangsize

can

dimendiffusion.

of protein

volumes

protein

solution

as nearly

of protein three

When assembled,

Equal

portions

aliquot

1.

in the

ml.)

done with

and shape

cut

were

were

by free

A window

compartments

concentration

pore

molecular

volume,

A and B, respectively,

(ca.

their

of equal

solution.

and of 0.1-l%

of both

weight,

of appropriate

size

Compar-

apparatus.

attainable

the

of some pro-

molecular

with

pore

mole-

way.

two plates.

membrane

buffer

The contents

compartment,

simple

of two compartments between

in a simple

in accordance than

uniform

The diffusion

membranes

concerning

is

and nearly

of increasing

that

presumably

employed

in compartments

of time,

proteins

shows

in this

of the

of 0.02kJ phosphate

sible.

for

information

consists

definite

commercially.

is much sharper

may be obtained

cell

have

has been measured

to urease,

The apparatus

put

available

rates

molecules,

appears

fusion

supposedly

such membranes

lysozyme

sift

Thus it

filters

recently

of the from

and George Gorin Oklahoma State University Oklahoma

exposes

(usually buffer

were as pos-

At

appropriate

inter-

withdrawn

was determined of filter the

from

each

spectrophotometri-

membrane, Millipore

with Filter

pore Corp.,

Massachusetts.

“Work done under Contract of Naval Research.

Nonr-2595(02)

with

196

the

Biochemistry

a

8 ml.1

same time

were

from

by a filter

plates

in the

dif-

at the

stirred.

types

purchased

separated

the

Branch,

Office

Vol. 5, No. 3,, 1961

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

cm. sq.

Fig.

1.

Partially

Diffusion struction In

cells,

which

necessary

phragm, given

similar

have been’used

these

first

cells

exploded diagram of apparatus. Cell lucite; A is 47-mm. filter membrane.

after

in principle

by other

which

by equation

the (1)

concentrations

in details

and discussed

sintered-glass

a state

(Gosting,

different

investigators

have a thick

to establish

though

is made of

of con-

by Gosting

(1956).

or alundum

diaphragm,

it

diffusion

in the

dia-

substance

in A and B are

of steady-state of diffusible

is

19561,

(1)

where

1 is the

concentration

time

elapsed

from

the

chosen

time,

g is

the

diffusion

at that

constant.

This

constant

accurately

known I), but

is best

starting

determined

can be related

to the 197

time,

coefficient, empirically characteristics

c

is

the

and fi is with

initial a’ cell

a substance of the

cell

of and

BIOCHEMICAL

Vol. 5, No. 3,1961 of

an idealized

of total (Gosting,

For

diaphragm.

crossection

AND BIOPHYSICAL a diaphragm

2 and length

RESEARCH COMMUNICATIONS

consisting

II, & is

given

of thin

by equation

straight

tubes

(21 (VA=

VB = V)

1956):

2a

e==

Owing

to the

of solution

thinness

and the

neglected, filled.

time

Equation

of the“Millipore”membrane,

time

and the

(1)

(2)

necessary interval

then

to attain

,0.0150

steady-state

can be determined

reduces

cm. * the

diffusion

from

the

time

amount’

can be the

cell

to (3):

$J _ 2cA _ - -$Dt

In

is

(3)

cl4

For

low values

of the

In (1 + x) 2’~

holds

fraction within

diffused, lU$,

and one obtains

‘A FA = -G

Thus

the

slope

initial

portion

proportional

to the

time

required

of the

to Q. for,

2 shows representative

seen

that

Table in duplicate

the results

I gives

(4):

_ ZfiDt 1

(4)

1% diffusion:

data

0.2 = -jE

obtained

are in qualitative

some numerical

or triplicate

expression

approximation

of F versus t should be linear and its A same token, D should be inversely proportional

T0.1

Fig.

the

plot

By the say,

FA =*CJCi,

(5)

with

450-w

agreement

data.

The values

experiments,

each with 198

membranes, with

equati.on

of To 1 were A a fresh filter

and it (3).

determined membrane,

is

Vol. 5, No.

BIOCHEMICAL

3, ‘1961

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

0. lI 5 Fig. 2.

I I 10 15 Time in hours

Diffusion (21 serum

through albumin;

I 20

450-w membranes: (11 urease; (31 ovalbumin; (4) lysozyme.

TABLE I VALUES OF To 1 WITH VARIOUS FILTER5 . in hours

T0.1 Substance

M. w.

-7 10

-

D -1 cm2 set

450~mp filter

50-nl)l filter

lo-nql filter

Lysozyme

14,000

10.0

2.5

3.5

4.4

Ovalbumin Serum albumin

45,000 69,000

7.3 6.8

3.9 6.6

5.5 18

7.9 oo+

480,000

3.4

10.2

oD*

-44

Urease

* < 1% in 24 hours

and found ters

to agree

are fairly

within

reproducible;

1%.

This

indicates

no attempt 199

the

was made,

characteristics in these

of

preliminary

the filex-

Vol. 5, No. 3, 1961

periments, (all

BIOCHEMICAL

to determine

filters

with high precision

were from one lot).

from the literature be exactly

(Edsall,

appropriate

for the present

Equation

1956); while used, their

(3) predicts

that

accuracy should suffice

is a rather

small fraction

but not an unreasonable

The important

free diffusion

of the protein

of the surface

crossectional

area available

protein,

like

This indicates

large enough to pass this

of nearly

serum albumin,

that

protein,

interesting

the same molecular

of D for these proteins

(6.25 cm2>

i.e.

are quite

they would do so at nearly

there are no pores in the membrane that 2 becomes

essentially

and, if

zero.

A

membranes.

between two proteins

allowed to diffuse

the same speed; however, the 10-w 200

out”

is passed in the

ovalbumin and serum albumin.

close,

in the denser

appears to be “sifted

to note the difference

weight,

with the membranes

for free diffusion,

is observed with urease and the 50-w

It is especially

h = 0.015 cm., V = 8 cm3);

l’ike lysozyme, To 1 inA be ascribed to a decrease in

by the 10-w membrane; only a very small amount of protein

effect

From the

molecule,

creases by a small amount, which might reasonably

course of 24 hours.

manner.

exposed to diffusion

are those obtained

to be noted

For a small protein

But a larger

(2);

molecules

value.

results

pore size.

“normal”

0.4 cm2 (equation

24

cmS2), not in

However, the agreement is

these membranes in a nearly

values of 1 one can calculate

similar

the values may not

from 2.5 to 4.5 (X 10e6 X l/3600

agreement with the prediction.

takes place through

filters.

in the Table are taken

that the product TO 1D should be constant for mem& Actually, the experimental data obtained with 450-q

good to indicate

4, the effective

of & values

purpose.

good quantitative

of smaller

the repeatibility

1952; Gosting,

membranes give values ranging

sufficiently

RESEARCH COMMUNICATIONS

The values of & listed

for the conditions

branes of the same type.

this

AND BIOPHYSICAL

The values freely,

%illipore”

mem-

BIOCHEMICAL

Vol. 5, No. 3,1961

separates

brane

them sharply.

by no means certain

AND BIOPHYSICAL

It has been suggested (Low, 19521, though it

(Loeb and Scheraga,

albumin molecule might be a prolate and it

is easy to visualize

the membrane. geometric

inter

known particle

alia,

size,

The results

19601.

interactions

these matters;

of polystyrene

“mesh size.”

indicate

it to be a large mole-

weight reported

of molecular

(Sumner -*et al I 1936 ;

through

some dif-

of this

The present

molecular

results

weight would

50- and even lo-rnp membranes, contrary illustrates

of an

to what has been

how diffusion

experiments

may be utilized

to advantage for purposes other than the direct

determination

of molecular

observed.

This result

weight 17,000.

particles

of

to “calibrate”

Several years ago, Hand (1939) reported

subunit

it

latices

on the basis of which he suggested the existence

active

readily

on a

play a part in the diffusion

with respect to their

are not in accord with ‘that report: diffuse

specific

by means of which it might be possible

experiments,

enzymatically

doubtless,

with urease clearly

from passing

of molecules

in progress to clarify

in accordance with the molecular

fusion

sifting

to measure the diffusion

obtained

Creeth and Nichol,

that

involved;

are currently

the membranes empirically

cule,

with a major axis 150 61 long,

and the membrane material

Experiments

is planned,

ellipsoid

is

19571 that the serum

how such a molecule would be prevented

basis is the only effect

process.

1956; Tanford,

It would be naive to believe

between the molecules

RESEARCH COMMUNICATIONS

size and shape.

The work described Craig and co-workers We believe

19601. easily

operated

straightforwardly tage,

bears a close relationship

with dialysis the apparatus

in a precisely related

found it

necessary’to

junction

with proteins

“stretch-

(Goldstein

used in this controlled

to the

however, is the larger

tubing

diffusion

to that recently and Craig,

manner, and the results equations.

tubing

above ca. 30,000 molecular 201

1960; Craig,

work had some advantages;

advan-

whereas Craig et al.

before utilizing weight,

it is

can be,

The principal

range of pore sizes available; dialysis

done by

it

in con-

there is no upper

Vol. 5, No. 3,196l

limit

BIOCHEMICAL

AND BIOPHYSICAL

to the range of the filter

not all desirable

intermediate

RESEARCH COMMUNICATIONS

membranes commercially

available

(even though

sizes may be available).

REFEEENCB Methods of Protein Chemistry” (Alexander, P., L. C., in “Analytical B. J,, eds.1 Vol. I, p; 104-119, Pergamon Press, New York,

Craig,

and Block, (1960).

Creeth,

J. M., and Nichol,

C. W., Biochem. J.,

77, 230 (1960).

Edsall, J. T., in “The Proteins” (Neurath, il., and Bailey, p. 634-9, Academic Press, New York, (19531. Goldstein, Gosting,

J.,

and Craig,

I... J.,

L. C., J. Am. Chem. Sot.,

Adv. Protein

P. .B.,

J. Am. Chem. Sot.,

Loeb,

G. I.,

and Scheraga, H. A.,

Low, B. W., J. Am. Chem. Sot.,

61,

74,

3180

I,

82, 1833 (19601.

(1939).

J. Phys. Chem., 60, 1633 (19561. 4830

(1952).

N., and Eriksaon-Quensel,

(1938).

Tanford,

eds.1 Vol.

Chem., 11, 429 (19561.

Hand,

Sumner, J. B., Gralen,

K.,

C., J. Phys. Chem., 61,

1023 (1957).

202

I.,

J. Biol.

Chem., 125, 37