Isolation of a monodisperse protein fraction from cottonseeds

Vol. 15, No. 2, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ISOLATION OF A MONODISPERSEPROTEIN FRACTION FROhl COTTONSEEDS*

Rossi-Fanelli, E. Antonini, M. Brunori, A. Caputo and F. Satrisni

A.

Institute

Received

January

Little

of Biological Chemistry, Rome, Italy

is still

To a great

properties

heterogeneous

this

parations

seed proteins 1963).

is due to the fact

a pure protein

fraction

by modern fractionation

as cottonseed

that

these

and often

is found to be

techniques

proteins

are concerned,

out in the past,

were found to be heterogeneous

or in electrophoresis

(Altschul,

We wish to describe

*

physicochemi-

from the seeds in complex mixtures

have been carried

perties

of Rome,

(Altschul,

l

As far tions

of individual

1954; Altschul,

extent,

what was considered

1963)

known about the chemical,

and Sandegren,

are extracted

University

6, 1964

cal and biological (Brohult

M.R. Bruzzesi,

of a monodisperse

Supported by a grant of Agriculture.

but all

fractionathe pre-

in the centrifuge

Lyman, and Thurber,

here the isolation

1958).

and some pro-

protein

component which accounts

FG-It-125

from the U.S. Department

110

BIOCHEMICAL

Vol. 15, No. 2, 1964

for

a large

analogy called

fraction

with this

AND BIOPHYSICAL

of the proteins

similar

proteins

protein

t'Acalin

RESEARCH COMMUNICATIONS

in cottonseeds.

from other

In

seeds, we have

A."

EXFERIMERTAL AIVDRESULTS Acala glandless the Southern

cottonseeds

Regional

were kindly

Laboratory,

supplied

U.S. Department

by

of Agri-

culture. The seeds were dehulled, volumes

(W/V) of acetone

suction

on a filter.

flaked,

cooled

extracted

at -20°,

The acetone powder was then stored of the protein

present

can be extracted the removal solution

with

in this

extract

the ionic tion

experiments,

unusually

large

the solubility fore

devised

effect

in the cold.

Most

After

the pale yellow the presence

Most of the proteins

of

which are present

precipitated

by lowering

of the solution.

In preliminary

fractiona-

it was noted that

the temperature

had an

effect,

at a given

of a protein

salt

the extract concentrations

of the various

have so been obtained,

ionic

fraction.

to fractionate

of different

the solubility

different

material,

can be reversibly

strength

by

2 M NaCl at room temperature.

shows, in the ultracentrifuge, components.

and dried

5

in the acetone powder of cottonseeds

of the insoluble

several

with

proteins.

strength

and pR, on

A procedure

was there-

making use of the and temperatures

A number of fractions

which show , in the ultracentrifuge,

amounts of individual

components. 111

on

The procedure,

Vol. 15, No. 2, 1964

which will protein

BIOCHEMICAL

be described

component,

affected

below,

(Acalin

water

a heavy precipitate

appears

soluble

proteins

cipitate

A). at pH 7, is dialyzed During

24 hours. in the dialysis

with

is discarded

temperature.

and the pre-

NaCl 0.05 M in the cold. and the precipitate

The material

at a temperature

is then centrifuged

The precipitate

is extracted

at the same again with

of 0.3 M NaCl at 30' C, and the clear

obtained

from the extractions precipitate

in the cold.

appears

cooled

The

is then sus-

portions

a white

The water

sack.

pended in 0.3 M NaCl, at pH 7, and extracted near 30' C.

the dialysis

are removed by ceatrifugation

is extracted

supernatant

for

of the

of which is particularly

in 2 M NaCl, buffered

in the cold against

RESEARCH COMMUNICATIONS

leads to the isolation

the solubility

by temperature

The extract

AND BIOPHYSICAL

supernatants

at O-2' C.

and is collected

The bulk of the precipitate

smaller

On cooling,

by centrifugation is dissolved

in a

minimum amount of 0.3 M NaCl at 30' C and the insoluble al removed by centrifugation. again at O-2' C. dissolved

The precipitate

is insoluble

in water;

is precipitated

The solubility solutions,

is then cooled

which forms is collected

end

in 0.3 M NaCl at 20' C.

At room temperature

it

The solution

materi-

and neutral it

dissolves

by ammonium sulfate in ammonium sulfate,

is markedly

affected

pH the protein in salt

solutions

at a saturation just

as in dilute

by temperature. 112

so isolated at II 7 0.2; over 70s. salt

BIOCHEMICAL

Vol. 15, No. 2, 1964

AND BIOPHYSICAL

Fig. 1 Sedimentation pattern of "Acalin A." Protein concentration: 7 mg/ml. Solvent: 0.5 M NaCl, pH 7.

1

The protein shown in sis

appears

Figure

on cellulose

eluted

step

wise

extinction

contains

9.2

S.

at is

u=O.3,

pH 7);

it

is

columns,

Its

ultraviolet

light

278 q.

At this

wavelength

7.00.

coefficient

The sedimentation

zone electrophore-

at pH 7.

has a maximum ,l$ ~~~~

in

as

on DEAE-sephadex

18% nitrogen.

coefficient

ultracentrifuge,

buffer,

absorption

of NaCl

The sedimentation is

from

the

band

(phosphate

gradient

spectrum

in

shows a single

peak

The protein absorption

homogeneous

acetate

as a single

with

the

It

1.

RESEARCH COMMUNICATIONS

in

0.3

constant

M NaCl,

pH 7.0

shows little

(S$,,w)

dependence

on concentration. The weight scattering

average

measurements

and with

the

Fanelli,

Antonini

in

procedure

weight

a Brice

routinely

and Caputo,

molecular

weight

in

0.3

the

of a value

of

basis

molecular

Phoenix used

1959).

M NaCl,

in

113

ml/g.

by light

Photometer our

about

at A=546

laboratory

The weight

pH 7 is

dn/dc=O.192

was determined

(Rossi-

average

180,000

on

w

Vol. 15, No. 2, 1964

The slope essentially 0.3

of

zero

BIOCHEMICAL

AND BIOPHYSICAL

the

scattering

light

over

a protein

RESEARCH COMMUNICATIONS

plots

HC/T

concentration

is

from

7 to

Wm. DISCUSSION Aqueous

extracts

AND CONCLUSIONS from

protein

components

which

tively

few of them

have

The simple tion

from

a homogeneous

protein dence

components

of

its

other

molecular

Further of

its

in

progress

of

the

in

proteins. the

which

This

A, appears

to be one of It

for

similar seeds

major depen-

property

this

forms

respect

and in

to other (Brohult

as

which

the

shows a marked

In is

prepara-

behaves

protein,

, and this

it

and rela-

"cold

and Sande-

1963).

with our

seed,

allows

fraction,

of different

characterization

properties

to

of different

material.

properties,

Altschul,

above

procedure.

globulins

seed as pure

cottonseeds.

isolation

a number

from

isolated

on temperature

basis

1954;

vary

of a protein

solubility

precipitable"

contain

described

name Acalin

the

gren,

been

monodisperse the

of

also

procedure

cottonseeds

we propose

seeds

of this

those

of other

protein

and comparison

seed proteins

are

now

laboratory.

REFZ33NCES Altschul, A.M. Reactions.

(1963) in Corvallis,

Symposium Oregon. 114

on Food Proteins September 4-6.

and Their

Vol. 15, No. 2, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Altschul, A.M., Lyman, C.M. and Thurber, F.H. (1958) cessed Plant Protein Foodstuffs." A.I!L Altschul, Academic Press, New York. Brohult, rath

in "Pro editor.

S. and Sandegren, E. (1954) in "The Proteins." H. Neuand R. Bailey, editors. Academic Press, New York.

Rossi-Fanelli, J.Biol.Chem.,

A.,

Antonini, E. and Caputo, 234, 2906.

115

A. (1959)

in