Partial purification of bovine sulfation factor

Vol. 39, No. 3, 1970

BIOCHEMICAL

PARTIAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PURIFICATION

-OF BOVINE J. P.

SULFA

TION

FACTOR

Liberti

Department of Biochemistry Medical College of Virginia Health Sciences Division Virginia Commonwealth University Richmond, Virginia 232 19

Received --Summa

March

10, 1970

ry

A method for the partial purification of sulfation factor (S. F. ) from bovine serum utilizing the techniques of ultrafiltration and molecular sieve chromatography is presented. The method is simple and approximately 50% recoveries of the initial S. F. activity have A 400-fold purification is obtained when been consistantly obtained, the potency is based on biuret reactive material; on a dry weight basis, a 125-fold purification. From the data presented herein, it appears, that serum S. F. is similar in size to the muscle S. F. It is of Hall but considerably smaller than that reported by others. suggested that freezing and thawing of the samples prior to and during the purification procedures may dissociate S. F. from a large protein or macromolecule which would lead to artificially high molecular weight assignments. Sulfation

factor

the conversion Sulfation

(S. F. ) is a constituent

of chondroitin

activity

to chondroitin

is related

to pituitary

in acromegalics

and depressed

growth

hormone

is the pituitary

vivo,

it apparently

in modulating S. F.

is unknown,

(“active

core”)

In a recent

by cartilage.

homeostasis

since

dwarfs

(1).

it is elevated Although

stimulates

since

growth

hormone

is ineffective

(2).

Although

the nature

it may

be a catabolite

it has been

postulated

communication

stimulates

which

in vitro --

hormone

which

agent

activity

of growth

sulfate

in hypopituitary

is not S. F.

sulfation

of blood

that

sulfation

-in

of

(3).

a scheme

356

yielding

a 65-fold

purification

BIOCHEMICAL

Vol. 39, No. 3, 1970

of S. F.

from

acromegalic

We wish A 400-fold

to report

plasma on our

purification

ultrafiltration

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was

efforts

has been

and molecular

presented

to purify

obtained

sieve

(4).

S. F.

utilizing

from

bovine

serum.

the techniques

of

chromatography.

Methods Bovine Serum

blood

was

-lo0

was

prepared

in volumes

in a Model

by centrifugation

were

in Table

the filtration

chromatography

The

S. F.

activity

incorporation

corrected

biuret

for

cell

rats

(5).

endogenous

G-15

was

chondroitin

activity.

kept

and batches

)

in ice during fraction

subjected

to molecular

equilibrated

and eluted

were

at -10”.

stored using

was

the --in vitro

cartilage

activity

Protein

Mass.

BS-2F

by costal

sulfation

at

of membranes

assayed

sulfate The

slowly

The

filtrates

cartilage)

day and stored

was

and then

All

in ice.

, Lexington,

at 4-6’.

Sephadex

formate.

kept

thawed

Corp.

by lyophilization

of 35 S into

were

a series

(Amicon

collected

(c/m/mg

hypophysectomized

was

ultrafiltration

employing

0. 001 M ammonium

was

The

2-fold

with

serum

through

cell

1.

which

on the same

ultrafiltered

and the filtrate

concentrated

from

The

401 ultrafiltration

as depicted

sieve

in vessels

of 200 mls.

of 200 to 400 mls

was

collected

obtained

of test

determined

samples by the

reaction.

Results A flow activities

obtained

50 membrane the overall activity components

diagram

of the purification are

shown

represents procedure

and about having

in Table

the most representing

a 200-fold a molecular

scheme 1.

employed

Fractionation

effective

single

a recovery

purification. weight 357

and

The

the sulfation

through step

the XM-

purification

of 80% of the initial majority

> 50, 000 are

of serum retained

by the

in

Vol. 39, No. 3, 1970

Table

BIOCHEMICAL

I - Summary

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

of Purification

Procedure

Bovine Serum (lOO%:l. XM-50 membrane; 1 70 psi 502

(lO%:O.

1)

positive more

(BS-SOR) material

viscous

passed

8o)’

o)

approximately

The

water,

resultant

is clear membrane.

Essentially

(BS-ZOF).

Subsequent

through The

a UM-2

majority

based

(85-fold

of the initial obtained

positive

upon

material

purification

activity.

filtrate

activity

The

chromatography

for

of the total

tacky

resided

280 rnp are

on dry

358

was

through

a UM-10

was

retained.

(BS-2F),

and

purification

and the S. F. The

having

passed

and contained

1.

found

activity

finally

a 400-fold

in Fig.

contained

activity

substance

weight)

was

of the remaining

S. F.

was

absorbance shown

BS-50F

in the filtrate

represented

based

most

(BS-1OF)

although

(BS-20R)

ultrafiltration

and a white,

of the S. F.

The

all the S. F.

of l/5

The

membrane

on biuret

of S. F.

of 80.

BS-SOF,

retentate

and accounted

in retention

activity

filtrate,

The

material.

a specific

activity

95% of the biuret

and colorless.

biuret

resulted

1000

Chromatography G-15 Sephadex

S. F.

membrane

jyT-mqG;

F=filtrate

no measurable

in the filtrate

lo,ooo

vated

a UM-20

positive

q;.;

0:

of serum.

through

20,000

0:

2~(oq

and represent

than

Factor

O)a 50, OOOb

0:

a Values in parentheses represent % Recovery and Relative Specific A ctivity/mg biuret positive material b A pproximate mol. wt. cutoff of membrane Abbreviations: Rzretentate;

Sulfation

50F ;80%:210) 1 UM-20 membrane; 50 psi

2°2;;~;;oq

membrane

of Bovine

45% activity

fractions

con-

BIOCHEMICAL

Vol. 39, No. 3, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

GEL FILTRATION OF SULFATION FACTOR ON SEPHACEX G-15

A280 hp)

---

A2w SuIfulion

05 Activity

0.4

0.3

0.2

0.1

0 EFFLUENT

Legend

to Fig.

(ml)

1

Molecular sieve chromatography of S. F. 95 mls of twice concentrated BS-2F was applied to a Sephadex G-15 column (2.0 x 40 cm) and eluted with 0. 001 M ammonium formate. The flow rate was 10 ml per hr and 5 ml fractions were collected. All operations were done at 4”. Each fraction was lyophilized, dissolved in Hz0 and aliquots were assayed for S. F. activity.

taining

S. F.

after

the void

activity volume.

positive

tests

of S. F.

on the basis

although

small

fold

were

confined

The

to ninhydrin.

purification

which

No increase

of biuret

molecular was

eluates

to a single

obtained

possessed

was

contaminants

on a dry

which

was

S. F.

activity

in the relative

reaction

weight

peak

noted were

weight

basis

eluted

specific

in these

gave activity

fractions

removed.

A 125-

however.

Discussion The

technique

of ultrafiltration

offers 359

a method

for

the relatively

BIOCHEMICAL

Vol. 39, No. 3,197O

crude

separation

methods ease

of fractionation.

which

S. F.

of serum

is of extreme

which

effects

that

can arise

is the slow

rate

membrane ml/min leakage

after

continuous

tration

steps

in the BS-2F

molecular

weight for

no similar

reported’that

based

65-fold relative

on a dry

weight

of the initial

and

represents

membrane

S. F.

basis,

concentration activity.

observed

subsequent flow

very

even ultrafil-

rates

were

>30% It would

on biuret

that

(6).

on the dry

yields

in the assay be of great

physiologic 360

Calculated

on the UM-2

In mixing serum of serum

results

experiments, was

inhibited

to a final

in a reduction

importance

(4)

of the isolated

retained

addition

system

weight

since

Wyk et al,

purification.

activity.

that

Van

material.

and of unfractionated

It is known

material

a 125-fold

the substance S. F.

recovered This

positive

of the starting

procedure

was

purification.

is proteinaceous

weight

inhibited

of BS-2F

material. of

our

to note

completely

by this

to

However,

activity

a 400-fold

of S. F.

to the dry

activity

decreased

rapid

S. F.

based

purification

It is of interest

i. e. using

was The

are

). one-half

material

as

In addition

increased.

since

such

in ultrafiltration

of 2 ml/min

proteins

problem

relative

and salts

fractionation,

72 hours.

with

and the deleterious

solvents

of the retentate

other

amounts.

encountered

rate

over

material

we encountered

flow

ultrafiltration

it has been

S. F.

initial

is an approximation

their

isolating

the use of organic

The

fraction

value

are

in the initial

posed

can be handled

in minute

of filtration

Approximately

100%

is present

drawback

of large

advantages

when

One definite

( >5 ml/min.

the S. F.

volumes

as the viscosity

little

latter

Large importance

with

XM-50.

obtained

has definite

of pH and temperature

circumvented.

0.3

which

in all likelihood

no extremes

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

if the in-

BIOCHEMICAL

Vol. 39, No. 3,197O

hibition

by BS-ZR

The

serum

and thawed serum

employed

at least

which

once

distributed

respectively 30,000

to 50, 000.

frozen

and thawed

that

the molecule

that

several

would

may

This

data

7) which

indicated

(3,

( (20,

have

000 molecular

and a molecule interesting

that

may

group

have

isolated

In both zone

those

et al.

a skeletal studies,

of the high-voltage We are

to characterize

presently S. F.

S. F.

S. F.

bound.

(unpublished muscle

It is known

as thyroxine

and

An explanation

for

a small

sulfation activity

by repeated the conflicting

molecule

a dissociable In this

molecule

connection

it is

assigned

the procedures were

cited

in reference

substance

which

resided

a molecular employed similar 4) who

is a ‘peptide’.

in the identical

electrophoretogram. processing and to study

larger further

substance, 361

quantities

of serum

the sulfation

these

to a transport

for

chromatography results

the sulfation

weight).

(4) tentatively

although

to Sephadex

indicate

account

molecule,

from

Sephadex

bound

as being

of

of S. F.

can be dissociated could

Van Wyk et al.

by this

of Hall

which

be protein

of -> 20, 000 to plasma subsequent

in blood

a large

weight

such

conjugates,

explanation

weight),

which

to note

to those

hormones

or as an aggregate

and thawing.

patterns

molecular

exists

the S. F.

in the range

on G-15

(15000

S. F.

freezing

size

separation

as protein

be that

sera,

in the 50R and 50F fractions,

a molecular

weight

in blood

macromolecule

and 800/o-15%

of

and unpredictable

unfrozen

and its behavior

molecular

frozen

Ultrafiltration

utilizing

small

has been

in a different

The ultrafiltration

low

discrepancies

results

indicate

serum

scheme

to ultrafiltration.

studies

is quite

occur

purification

prior

607’0~40%

which

S. F.

in this

In several

cortisone

for

is not so treated

fractionation. activity

is specific

AND BlOPHYSlCAL RESEARCH COMMUNICATIONS

in order

inhibitory

Vol. 39, No. 3,197O

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Acknowledgements --Expert technical assistance was provided by Mrs. Dorothy E. This study has been supported by research grant AM 12892 Wood. from the National Institutes of Arthritis and Metabolic Diseases.

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Ellis, Exptl,

2. Salmon, Clin.

S. , Huble, Biol. Med.

J. and Simpson, 84, 603.

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and Doughaday,

3.

Engel, Pincus,

F. L. and Kostyo, G. and Thiman,

4.

Van Wyk, J. J. , Hall, K., Biophys. Acta 192, 560.

5.

Salmon, (1963)

J.

W. D., Jr., Lab. Clin.

Kouman, J. and Doughaday, Phys. 5, 152.

7.

Doughaday, Ed. Res.,

E.

J. L. (1964) K. V. Academic

and Kipmis, G. 22, 49.

H.

D.

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Proc.

(1957)

R. P.

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and Thompson

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M.

J.

Sot.

Lab.

The Hormones, Press, V, 83.

and Weaver,

W.

(1953)

W. H.

Bower, P. H., Med. -’61 120.

6.

W. H. Pincus,

M.

(1966)

Trans.

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Y.

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