Further observations on the purification and properties of autoprothrombin III (Factor X)

THROMBOSIS Printed

RESEARCH in the United

FURTHER

OBSERVATIONS OF

Waiter

Vol.

States

ON

THE

PURIFICATION

AUTOPROTHROMBIN

H. Seegers,

Lowell Genesio

E.

III

McCoy,

Murano,

1972 1, PP. 209-220, Pergamon Press, Inc.

Jan

and B.

AND

(FACTOR

X1

Reuterby,

B. L.

PROPERTIES

Nobuo

Sakuragawa,

Agrawal

Department of Physiology, Thrombosis Specialized Center of Research, Wayne State University School of Medicine, Detroit, Michigan, U.S.A. 4.5.1972.

(Received

Accepted

by

Editor

B.

BlombB'ck)

ABSTRACT III (Factor X1 was separated from purified bovine prothromAutoprothrombin bin complex by chromatography on DEAE-cellulose and filtration through a Sephadex G-l 00 column. It had the same amino acid composition and approximately the same sedimentation characteristics in the ultracentrifuge as the first product obtained in this laboratory. The sedimentation constant was 3.58s. The product was studied by disc gel electrophoresis over a wide pH range. It was free of autoprothrombin C (Factor X,1, was stable, and had no tendency to activate spontaneously. On the basis of 27 Sephadex filtration experiments, the molecular weight was 81,900 + 2,770. For autoprothrombin C, it was 53,500; by another method, it was 58,000. The N-terminal amino acids were alanine and serine, while those for autoprothrombin C were glycine and serine. Peptide maps for the zymogen and enzyme were similar except there were fewer peptides on the map of the latter. Based on a specific activity of 1,200 u/mg and a plasma concentration of 30 u/ml, the concentration of autoprothrombin III in bovine plasma most likely ranges from 2-3 mg”/.

In previous

work

isolated

from purified

physical

chemical

is distinctly

from this

bovine

properties

different

laboratory

prothrombin and amino

from the thrombin

(l-31,

autoprothrombin

(prothrombin

complex).

acid

composition

zymogen.

This investigation was supported by research Heart and Lung Institute, National Institutes

(41,

In another

grant HE-14142-01 of Health, U. S. 209

III (Factor It was

X1 was

clear,

from the

that this enzyme

precursor

study

(5,

Public

61,

isolation

from the National Health Service.

and

210

AUTOPROT~O~IN

work proceeded

characterization

independently

ties of their protein corresponded description

studying

(FACTOR

Vol. l,No;3

x)

and the authors concluded

that the proper-

quite well with those reported from this laboratory.

by Esnouf and Williams

they were evidently

111

(7) was significantly

different

(3,

The

6) even though

the same substance.

The main purpose of the work from which some results are reported in this paper was to revise,

simplify,

and further study parameters

prothrombin III from purified to obtain the autoprothrombin We found that the physical those described

bovine prothrombin

(prothrombin

chemical

properties

Peptide

enzyme which was obtained

follows:

A modification

6 liters of oxalated

bonate at neutral pH. centrifugation

AND

of 3.5%

(w/v) trisodium

fractionation

METHODS

-20’

C or lower.

citrate. the 34-60%

description

Fractions

glycine)

from gel filtration

Analytical

washed with physiologic

7.5%

of saturation

magnesium

fraction),

hydroxide

(9-11).

The apparatus

(w/v) acrylamide,

gels were prepared essentially

The concentration

was

layered

by

ml portions

by ammonium sulfate

dialyzed,

and stored at

III activities procedure

III were examined

and 0.2% according

of the electrode

on the gel and allowed

used was manufactured

buffer (0.025

ranged (8).

A

by polyacrylby Shandon

M Tris and 0.38

M

N,N’-methylenebisacrylamide. to Weber and Osborn (11)

was used in our studies. buffer was one-half

Gels were cast in 7 x 75 mm glass tubes. sucrose

was recovered

in two 600

adsorption

of autoprothrombin

only half the amount of ammonium persulfate omitted.

This was as

saline until free of detectable

The final product was collected

Gels were prepared in Tris-glycine pH 8.5,

in

of the procedure is being written for publication.

amide gel electrophoresis Scientific.

of this procedure was also used.

The yields of prothrombin and autoprothrombin

much higher than with our original more detailed

from plasma by methods developed

of the prothrombin complex was accomplished

(retaining

precursor.

with adsorbed prothrombin complex,

and was repeatedly

Elution

for other research.

bovine plasma were mixed with saline washed barium car-

The salt,

protein.

Another goal was

maps were studied and compared

from its purified

Bovine prothrombin complex was isolated (8).

auto-

of the products now obtained are similar

reports (l-3).

MATERIALS

this laboratory

complex).

III and leave all the prothrombin available

in the previous

to those of the active

in our procedure for obtaining

The

protein

to electrophorese

except

The detergent was

that of the gel buffer. solution

in 10%

w/v

for 20 min at constant

to

~~TOPR~T~OMEXN

Vol.l,No.3

current (3 ma/tube;

v/v methanol 454

Blue (Coomassie

Blue 1.2

g, glacial

follows:

pH 2.8

pH 6.8

III was also examined

(0.02

(0.005

4.8,

6.8,

and 8.6.

M NaH2P04,

M NaH2P04,

by polyacrylamide

of 7.5% v/v

0.02

0.005

M H3P04);

M Na2HP04);

pH 4.8

pH 8.6

The concentration

one-half

of electrodes

that of the gel buffer.

The electrophoresis

ode at the bottom). longer

time

destaining

(at pH 2.8,

4.8,

DEAE-cellulose

was Whatman

of Andrews

(12).

sis by chromatography be satisfactory); as described

microgranular,

reduction by Smyth amino

solution

of purified

protein/ml.

The material

Buffers:

Tris

(in previous

analyses

soybean

2.0

2.0

M Tris-HCI

buffer

of 1 .O M NaCl

and 10

M NaCI, to 500

to 1 liter.

mix 150 ml.

inhibitor

0.150

Dissolve

M NaCI,

and dilute

(31,

Staining

and

to 500

ml of 2.0

M Tris-HCI

ml of 1.0

M NaCl

size

were

24

based amino

of Hirs

(13);

acid

analy-

proved

to

trypsinolysis et al

of Edman

was made to contain

Sepha-

on the

by Iwanaga

by the methods

0.1

(16). mg

Biochemical.

242.28

g of Tris water,

mix 75

ml of 1.0

buffer

mu.

hr hydrolysis

in distilled

ml.

cellulose.

and the column efflu-

as described

by Worthington

(Eastman)

(stock)

(cath-

for slightly

recording at 280

procedure mapping

M (stock).

For

buffers was

min).

quantitative

work

performed

trypsin

2-(hydroxymethyl)-1,3-propanediol) and dilute

were

was manufactured

Buffer

HCI,

was as follows:

and then peptide

acid

30

Fine Chemicals,

and carboxymethylation

(14);

of electrode

preswollen

studies for particle

analysis

instrumentation

N-terminal

A stock

DE-52

filtration

Protein

M Na2HP04).

was reversed at pH 2.8

with an LKB Uvicord II,

“chromatography”

M NaH2P04);

(0.0133

min; at pH 8.6,

products were obtained from Pharmacia

dex thin-layer

(0.02

above.

ents were monitored automatically

(15).

50

in

of buffers was as

was carried out as before except

and pH 6.8,

done as described

were

Sephadex

The polarity

gel electrophoresis

The composition

The figures represent final concentrations.

dilute

acetic acid 46 ml and 50%

acid - 5% v/v methanol by washing during a 24 hr period.

phosphate buffers at pH 2.8,

0.3

Gels were

ml) for 2 hr. The excess dye was removed with a solution

Autoprothrombin

work

211

24 ma total; the voltage was normally about 270).

stained with Coomassie

acetic

III (FACTOR x)

For 0.175 (stock)

and 10 ml of 2.0

base

pH to 8.0

(2-aminowith

2.0

M

M NaCl and 10 ml of M NaCI,

and dilute M Tris

mix 87.5

to 500

buffer

ml.

(stock)

ml For

and

212

AUTOPROTHROMBIN

111 (FACTOR x)

EXPERIMENTAL General Outline

activation

prothrombin complex was passed through a DEAE-cellulose

thrombin zymogen from autoprothrombin of the latter,

After separation

some soybean trypsin

of autoprothrombin

from the frozen state. filtered

III,

G-l

as the final product.

but there was always

inhibitor

To minimize

column

autocatalytic

was added to the protein solution,

IIIwas then dissolved

00 column to separate

III. The latter separation

autoprothrombin

III (17).

the salts were removed by dialysis

The autoprothrombin

through a Sephadex

selected

RESULTS

of Procedure

The purified to separate

Vol.l,No.3

before drying

in a small volume and

inert material

from the desired

was sharp and tubes with no overlap could be

A second filtration

on Sephadex was tried several

times,

loss of activity.

FIG.

1

Chromatography of purified prothrombin complex on DEAE-cellulose in 0.04 M Tris-HCI pH 8.0 buffer. Elution of fractions occurred with increments of NaCl concentration in the same Tris-HCI buffer. 1) 0.15 M NaCI; 2) 0.175 M NaCI; and

TUBE NUMBER (20 ML ) Seoaration

of Autonrothrombin

Approximately

420,000

were brought to 0.04 cellulose

III u of purified

M Tris-HCI

column was 2.5

by adding 0.41

The prothrombin complex was applied

buffer.

Flow rate was 2-3 M Tris-HCI.

Then an additional

0.3

thrombin III(Fig.

ml/min.

to 0.04

0.175

1).

M Tris-HCI.

in 20 ml

The DEAE-

M Tris buffer and used at by 0.04

M Tris

After about 60 min, the buffer was changed to 0.15

This eluted the main protein material

M NaCl in 0.04

M NaCl in 0.04

complex)

to the column and followed

fraction was removed to remove residual

line was just reached, was removed,

ml 2.0

x 25 cm and conditioned

4’ C.

M NaCl in 0.04

prothrombin (prothrombin

M Tris-HCI

M Tris-HCI

(thrombin zymogen).

prothrombin. was applied.

After the base After this

buffer was used to elute the autopro-

About 67 products were chromatographed

in this manner with a

Vol.

mTomom~o~~I~

l,No.3

recovery averaging

83% of that applied

preserve the activity

at -20’

111

(FACTOR

to the column.

trypsin

inhibitor,

an efficient cation work,

Our distilled

sodium carbonate dialysis,

activation

described

by the addition against

by Seegers

solution was generally resistance

zen and dried from the frozen state. III activity

to

III in prepa-

III, in a volume of 60-80

ml, was

of about a half ml of stock soybean

repeated changes of distilled

water with

(8) and used commonly in prothrombin purifi-

water tended to become acidic and a small amount of saturated

and if the specific

autoprothrombin

the autoprothrombin

The autoprothrombin

It was then dialyzed

dialyzer

it was possible

C for at least a month.

column.

protected from spontaneous

213

At this point,

The purpose of the next step was to concentrate ration for the Sephadex

x)

added to the cold dialysis was at least 2,000

Recovery

water.

After 3 hr of

ohms, the product was fro-

from this procedure was near 8 0% of the

obtained from the DEAE-cellulose

column.

The main loss

occurred during dialysis.

AUTO-E

0.40 0.30 0.22 0. 16 0. IO 0.05

A

z

:

0.00

I

loo

I

300

200

EFFLUENT

VOLUME

200

I

400

EFFLUENT

(m I 1 FIG.

I

300

VOLUME (ml

1

2

Sephadex gel filtration of the autoprothrombin III product obtained from DEAEcellulose chromatography. Left: Sephadex G-100 superfine (2.5 x 95 cm column). Right: Sephadex G-100 (2.5 x 183 cm column).

Purification

of Autoprothrombin

The G-100

Sephadex

III

column was 2.5

with upward flow at an approximate nium bicarbonate

pH 7.8-8.0,

x 183

rate of 25 ml/hr.

cm and was used at 20-25’ The buffer used was 0.1

and the sample volume was 3-4

ml.

C

M ammo-

To this we added

214

0.1

AUTOPROT~~~IN

ml soybean trypsin

tration varying from l-2/0

activity

found a molecular

bin C.

by the autoprothrombin

From the filtration

weight of 81,900

it was 78,000.

The average

1

We also used Sephadex

2,770.

mg/liter

G-100

superfine

Russell’s

used for the purification

The specific

purified autoprothrombin

of autoprothrombin

III.

activity

of

The

and properties

III to autoprothrombin

This gave good results.

advantage

(3,

The Sephadex

filtration

data (4 products)

On thin-layer

filtration

(3 products) according

53,500.

analysis

x 95 cm.

The active enzyme was purified on the Sephadex

procedure has no special 17).

“filtration”

of

C

times we converted

viper venom.

The

Almost all of the applied

and a column 2.5

for both procedures.

Several

2, right).

plasma.

the autoprothrombin

of Autoprothrombin

1) came

III product was free of autoprothrom-

2, left).

Purification

(Fraction

III (Fig.

By thin-layer

flow rate was 15 ml/hr or less (Fig. III were identical

A protein concen-

data we took the average for 21 runs and

The autoprothrombin

yield was 2.5

Vol.l,No.3

Inert material

amounted to 65% of the total protein applied.

was recovered.

4 products,

followed

x)

we combined two lots.

proved to be satisfactory.

through with the void volume, latter usually

Sometimes

inhibitor.

(FACTOR

III

over the DEAE-cellulose

G-100

C with column

However,

the

used in previous work

indicated

a molecular

to Andrews

weight of

(121,

we found

58,000. Miscellaneous

Observations

Our amino acid analysis values

(3,

4).

A single component was found by microzonal

electrophoresis.

In previ-

studies,

to those of Jackson

and Hanahan (6) than our first ones (3,

the autoprothrombin

III was found to be 1,200

fresh bovine plasma contained

30 u/ml.

bovine plasma ranges from 2-3

mg%.

zymogen range from 15-19 neighborhood

published

= 3.4. Now we found 3.58, but the concentration So 20,w slope was the same as in previous work. These results are thus even closer

ous ultracentrifuge dependence

results were in accord with our previously

mg% (8,

u/mg protein.

4).

The specific

18).

of

By the same analysis,

This implies that the concentration It is interesting

activity

that estimates

in normal

for the thrombin

The ratio of the two zymogens is thus in th e

of 1~8 in bovine plasma.

Repeatedly tubes containing

we determined the fractions.

the specific Generally,

activity

the material

of the autoprothrombin corresponding

III in all the

to the descending

Vol.l,No.3

AUTOPROT~OMBIN

had the highest

limb of the recording with

the ascending

disc

parts which

phosphate

buffers

were

as well

range where

the activity

8.6,

consistent

migration

subsequent

activity

preparation

215

x)

by approximately

as Tris

together

buffer.

is preserved

20% as compared

might

The

(Fig.

a single

3).

It migrated point

anionically

for on the basis

into

We used

was observed

in the pH

at pH 4.8,

earlier

(3).

generally

in poly-

to separate

not be certain.

reported

material

component

band appeared

best homogeneity

Freeze-dried

be accounted

a single

that one could

its low isoelectric

the cathode.

which

gave essentially

Sometimes

so close

with

was toward

electrophoresis

III

gel electrophoresis.

two equal

etc.,

specific

(FACTOR

limb of the curve.

The autoprothrombin acrylamide

111

At pH 2.8,

gave extra

of molecular

6.8,

bands

association

on

and

aggregation,

FIG.

3

Polyacrylamide disc gel electrophoresis. Tubes 1-4: pH 2.8, 4.8, 6.8, and 8.6 in phosphate buffer. Tubes 5 and 6: In Tris buffer at pH 8.5 and approximately 3 and 6 pg protein respectively. At pH 2.8, polarity was reversed.

The thrombin

protein

fraction

III was found

demonstrate

somewhat studies, N-terminal

Amino

to those

satisfied Acid

We analyzed

10

column

no Factor

VII

or anticoagulant

the gel,

Amino

acid

for the autoprothrombin

III.

is not related

before

autopro-

activity

nor could

activity.

By disc

analysis

gave

However,

to autoprothrombin

different

serine

for the one good quality of serine

products

and found alanine

we found valine

and serine.

autoprothrombin

C product

made quantitative

and serine We found

which

from other III.

work difficult.

for autoprothromonly glycine

we analyzed. Tentatively,

we

results

Analysis

In one other analysis,

the presence

detected

did not enter obtained

the Sephadex

procoagulant

that the protein

bin III.

nately,

Our tests

of any other

the protein

comparable we were

came through

to be inert.

the presence

gel electrophoresis,

which

and

Unfortuwe can

216

AUTOPROTHF~OMBIN 111

advance the working hypothesis linked by a disulfide

bridge.

bin I11 to autoprothrombin

Sephadex

that autoprothrombin In some experiments,

C and filtered

nine was found as N-terminal

(FMTOR

we converted

their Factor X preparation In some additional the autoprothrombin the Sephadex

C.

It is interesting

found alanine

Ala-

from the

and serine for autoprothrombin

Esnouf and Williams

to note III (Fac-

and glycine

for

(7). exploratory

observations,

we found only N-terminal

III taken from the DEAE-cellulose

column (Fig.

purified autoprothrom-

weight fraction

column which appeared after the autoprothrombin

tor X1 and also for Factor VII (19).

l,No.3

mixture through Sephadex.

amino acid in a low molecular

glycine

Vol.

III has at least two peptide chains

the activation

that HSgenauer et al reported N-terminal

x)

2) contained

named by HGgenauer et al (19)

glycine

column.

and serine,

for their autoprothrombin

FIG.

serine for

The first protein from

which corresponds

to the two

III.

4

Peptide maps for purified autoprothrombin III (Auto-1111 and purified autoprothrombin C (Auto-C). The two proteins were handled alike. Dotted circles indicate light areas of staining.

Peptide

Maps Samples

of autoprothrombin

methylated

and subjected

(Fig.

The number of spots was far below that expected

4).

content of the protein. precursor.

to tryptic

III and autoprothrombin digestion

There were close similarities

These peptide maps are distinctly

technic

in this laboratory

purified

thrombin (4).

in preparation

with purified

for obtaining

peptide maps

from the lysine and arginine

between the active enzyme and the

different

prothrombin

C were reduced and carboxy-

from those obtained by the same

complex,

purified

prothrombin,

or

Vol.l,No.3

AUTOPROTHROMBIN

III

(FACTOR

x)

217

DISCUSSION The method outlined

for obtaining

complex is more convenient

autoprothrombin

III from purified

and refined than the first one described,

but yields a protein

If carried through as described,

4).

repro-

with the same properties

found previously

ducibility

The procedure is integrated with the production of prothrombin

is excellent.

(3,

prothrombin

complex which is required for many purposes, obtaining

prothrombin as well as autoprothrombin

autoprothrombin thrombin C. directly

II-A

(a competitive

Additionally,

inhibitor

thrombin,

from the purified

of autoprothrombin

autoprothrombin

needed.

For autoprothrombin

of Sephadex

filtration

data.

III,

tations of the methods we employed.

zymogens.

-+ 2,770

68,900

has two chains.

on the basis

They found 52,000,

but were troubled with We appreciate

autoprothrombin

the limi-

III filtered

as if it

and we are quite certain of the molecular

(20).

We realize

that such a comparison

work,

multichain

molecules

weight

must also

is a single chain molecule while autoprothrombin

In filtration

C

of Jackson and Hanahan (6) were based

Nevertheless,

larger than prothrombin,

take into account that prothrombin as isolated,

purified

III and autoprothrombin

we found 81,900

The measurements

and related methods.

namely,

II,

autoprothrombin

C, and prethrombin can be derived

problems which could have a bearing on their results.

of prothrombin,

for

C), as well as autopro-

weight of autoprothrombin

is evidently

might be a little

material

with respect to time and material.

Further work on the molecular

on ultracentrifugation

III and, in addition,

prothrombin complex or from the respective

This approach is economical

stability

which then serves as starting

III,

behave differently

than single chain molecules. For comparative

purposes,

thrombin C, as determined vs 53,500).

However,

we can observe that the molecular

under identical this applies

prothrombin III to autoprothrombin

was 2.27s. prothrombin

of 24,000

C with the use of Russell’s

(21).

the molecular

In the ultracentrifuge,

viper venom,

the sedimentation

C (221,

the molecular

weight was 34,300

but even with allowances

forms of autoprothrombin

and Sio

w = 3.34

for that fact: evidence

C as obtained by different

conditions

auto-

When the

weight was estimated

When the enzyme was obtained from its precursor by activation

work has inherent pitfalls, ent molecular

is less than its zymogen (81,900

only to an enzyme obtained by transforming

enzyme was obtained with thromboplastin, the neighborhood

conditions,

weight of autopro-

to be in constant with auto(23).

This

favors differof activation.

218

AUTOPROTHROMBIN

III (FACTOR x)

Vol.l,No.3

REFERENCES

1. SEEGERS,

W.H., of autoprothrombin 42, 229, 1964. =

2. SEEGERS,

prethrombin

COLE, E.R., AOKI, N ., and HARMISON, C.R. Separation III from bovine prothrombin preparations. Canad. J. Biochem .:

W .H. and MARCINIAK, subunit of prothrombin.

E. Some activation Life Sci .: 4, 1721, =

characteristics 1965.

of the

3.

SEEGERS, W .H., MARCINIAK, E., KIPFER, R.K., and YASUNAGA, lation and some properties of prethrombin and autoprothrombin III.Arch. Biophys.: 121, 372, 1967.

4.

SEEGERS, W.H., MURANO, agulation of blood: Preliminary structure. Life Sci.: f& 925,

5.

JACKSON, C.M., JOHNSON, T.F., and HANAHAN, D.J. Studies on bovine factor X. I. Large-scale purification of the bovine plasma protein possessing factor X activity, Biochemistry: z, 4492, 1968.

6.

JACKSON, C.M. and HANAHAN, D.J. Studies on bovine factor X. II. Characterization of purified factor X. Observations on some alterations in zone electrophoretic and chromatographic behavior occurring during purification. Biochemistry: 7, 4506, 1968. =

7.

ESNOUF, M .P, and WILLIAMS, W.J. The isolation and purification of a bovineplasma protein which is a substrate for the coagulation fraction of Russell’s_viper venom. Biochem. J.: 2, 62, 1962.

8.

SEEGERS,

W.H.

9.

ORNSTEIN, Acad. Sci.:

L. Disc electrophoresis 121, 321, 1964. =

10.

12.

13.

G., MCCOY, L., and MARCINIAK, E. The cosurvey of thrombin and autoprothrombin zymogen 1969.

Prothrombin.

Cambridge:

DAVIS, B.J. Disc electrophoresis proteins. Ann. N. Y. Acad. Sci.:

K. and OSBORN, M. 11. WEBER, by dodecyl sulfate-polyacrylamide 4406, 1969. ANDREWS, filtration. HIRS, P. 199.

-

1.

Harvard University Background

Press,

and theory.

- II. Method and application I&, 404, 1964.

1962.

Ann,

N , Y,

to human serum

The reliability of molecular weight determinations gel electrophoresis. J. Biol. Chem.: 244,

P. Estimation of the molecular Biochem. J.: 91, 222, 1964. =

C.H .W,

Enzymology.

K. IsoBiochem.

weight of proteins by Sephadex

gel

Reduction and S-carboxymethylation of proteins, In: Methods in C.H.W. Hirs (Ed.) New York: Academic Press, 1967, Vol. XI,

Vol.l,No.3

AUTOPROTHROMBIN

III (FACTOR X)

D.G. Techniques in enzymic hydrolysis. In: Hirs (Ed.) New York: Academic Press, 1967,

219

14.

SMYTH, C.H.W.

15.

IWANAGA, S., HENSCHEN, A., and BLOMBACK, B. On the primary structure I, Two-dimensional “finger prints” of tryptic digests of sulof human fibrinogen. fitolyzed fibrinogen and fibrin. Acta Chem. Stand.: 20, 1183, 1966. =

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