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'~

STUDIES ON THE ISOLATION OF PROI‘HRQ‘IBIN FRQI
HUMAN PLASMA FRiCTION III BY PAPEE
ELECTROPMESIS

By

Robert Bastian Foy

A THESIS

Suhaitted to the School of waduate Studios of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

EASIER 0F $IMCE

Department of Chantry

1955

ACKNOYLEDGMENT

The author Iii-hoe to expreu appreciation and gratitude
to Dr. H. A. Lillevik for guidance and counsel.

The cOOporation and nuggeetionl of Honors. 1". A. Bette
and F. E. Hood in connection with apparatus construction are
sincerely appreciated.

He is also mindful of the advice given by other Ital!
member. as well as follow otudento.

The author is also very grateful to his wife for her
underutanding and consideration during the cow-e or this

.tw o

W
mm
mm
we
H
i

VITA

The author was born June 11;, 1928. His secondary education was

completed in 19% at Cass City mgh School, Cass City, Richigan.
In 1950 he was graduated from Central Michigan College of Education,
Ht. Pleasant, Michigan, with the Bachelor of Science Degree. lie was
admitted to the School of Graduate Studies at Hichigan State College
in Septmber 1953 and has been in attendance since.

Mploynent experience consists of positions as: Medical Tech-
nologist m 1950 to 1952 with the Clinical Laboratory at Eula:
Hospital, Flint, Kichigang Graduate Teaching Assistant 1953 to 1955
in the Department of Chemistry, Hiohigan State College; and assistant
director of the Clinical Laboratories since January 1955 at the
Edward W. W1: Hospital, Lansing, Hichigan.

He is a Junior asaber of the Auction Chaioal Society, somber
or the lensing Chipter of the Michigan Medical Technologists Society,
associate member or Sign. Xi and a somber of Alpha Phi Mega.

STUDIES ON THE ISOLATION OF PRUl‘IfiCMBIN FRCM
W PLLS‘L-l FRACTION III BI PAPER
ELECTROPHORESIS

By

Robert Bastian Foy

AH 53 REACT

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

EASTER (F 331%.?
Department of Chemistry

Year 1955

Approved \

iv

ABSTRACT

Blood coagulation studies require the use of several enzyme
preparations which are difficult to obtain in pure form, Prothrombin,
which catalyzes the conversion of Iibrinogcn to fibrin, has been
shown to be concentrated in Fraction III and 111(2-3) by the alcohol
nethods of Cohn md Oncley. This study was undertaken to prepare
purified prothrombin from these human plasma fractions by employing
filter paper electrophoresis.

The horizontal strip technique, with apparatus modifications was
first tested. Effective resolution of the crude plasaa fraction
couponents resulted. Staining with brow-phenol blue aided in the
detection of resolved proteins. Quantitative isolation of prothrombin
by elution of protein zones {rm unstained paper was not effected.

Continuous filter paper electmphoresis for isolation and purifi-
cation of prothronbin was carried out on husnn plasma Fraction
m(2-3). Preliminary treatment of un- fraction gave a salt soluble
extract which appeared to be about 95 percent one~conponent by Tiselius
elec trophoretic analysis. When this solution was treated by continuous
paper electrophoresis and the cuts analyzed it was found that two
principal components appeared. Prothrombin assays demonstrated activity
in all outs collected. There was no concentration of prothrcnbin in
any sample over that of the material in the sodim chloride extract.
The assays moved that the prothrescbin activity resided in these cuts

obtained nearest the anode side of the paper. Comparison with Tiselius
electrophoresis was made on those samples which afforded sufficient
material after dialysis and lyophylizaticn.

Inorganic chemicals have been used as adsorbents for the isolation
and purification of prothronbin. Barium sulfate and barium carbonate
were tried for further purification of one of the samples obtained by
continuous paper electroPhoresis. Under the conditions employed further

purification of prothrombin was not effected.

I.
II.

III.

IV.

TABLE OF COPE? MES

31m emf-I‘m:

00000000000000.0000...sooneoosoosoasosoesososs

m. 3 a?
M‘01"IC'WOO0-00.00000000000000cossacs-soot.OOOOOOOOOOOOOO

JIi. HGCIIEIfi.m Cf 810051 Cletting.assesseeosooesoossIeOOOC
B. I3013u0n0: HananProf-Immunoouu.”an”...
C. TI‘Q MQIDment 01: Paper mectmphoflcaiSOsOseasOses.

Emmm:flIT-1LOUOOOOIOOOIOOOOOOOOOODOOOOOOOOI0.0.0.000000000

Q T‘ s 4. .
.- I', I» 3“
“‘0 lq!"3'glm‘""*u0000.0.00sOOOOCOOOOOOsbolOOOOOOOOIO0.0.0.8.

B. Reagent-3 and Hauez'iala......n“announuououoo

C0 I‘f'stlliyiSOOOI-‘I.O-IOOOCOOOOOIOO.OOOOOOOOOOO.OOOOOOOOOOO

315??“ 3 film!

“" 2{IC0033......0.8.6.0...000000000.I.OOCIOODOOQOQOI

NV". ‘lgfi‘
v. Lj’bifiwflq‘yascoogoegoeseseoesooosooeoeoaOCeeeooooooseOeeOOOOOo

Emum'i?moeoo0.00.0.s000.00.000.0000.00-00.00.0.00.00.00.00.

new"
. ale-a‘hfl.......-........og......n.....n.l....DOOOOOOOOOOOOOQOOOI

vii

O‘NN N H

10
10

13
17

37
38

TABLE P5633

I Yields, Prothrombin Activity and Nitrogen Content of

SJEUFIQS Studied,.,,....,......~.......9......-....-....n-o- 2’4
‘ I"
II Pmthrombin :malysea...................................... 29

III Eata or. Electrophoresis Patterm! Cbtainad in 0.1014 Veronal
Buffer or pH 8.6..OOOOCOOOOOOOOOOOOOOOI0.0.900...0.00.0... 26

LI 5‘}? CF FIGURES

F1 gar-a Page

1. Schematic Representation of the Blood Clotting Romanian
2. Horiflonm-Stnp Appu‘tn'.goooooo9.0.0.0000000000000000
3. Continuous Plow Apparotua...............................

h. Dingrmtio Representation of the Preparation of
Emotion III and Fraction 111(2-3) hoarding to Methods

6 md 9..0.0.0.0...IOOQUOOQOOOCOCCOOIOOOOOCOOQ0....0000.

SO Wuon Fl” &%tOOOOOOOO0.0o-OCocoouOooaocooooOOlofi

6. meetr'ophoreais Patterns Prom Fraction 111(2-3)
Pmmmaw TreammoogoooooclaoOCOOO‘OQOCOOCOOOOOGOOOOOa

7 , Electrophoresis Pattern; of Cuts After Contimoua Flow

- TecmquOOOCQOOI.‘IOOOOOOOC00.0.00...OOOOOOOOOOIO.OOOO

8 . Paper Plectmphm'eais of Fraction 111(2-3) . . . . . . . . . . . . . .

3
11

12

19

27

28
29

I . EWROLUCT I01?

Prothrombin is a pro-enzyme which is vitally a port of the blood
clotting process. Its complete node of action in this couple):
somxenco of. enzymatic event. has not been entirely elaboreted. A pure
tom of hman prothrombin has not been readily available for ltudioo
on this mohaniu. The initial problem was one of isolating a eun-
ably pure preparation that could be utilised in blood coagulation ‘
undies.

During recent years two nothode oi prothronbin ieolation have
been used. One mployo adeorption from plane or modified plans and
tho other utilize. ioo-olootrio precipitation. Either of those involve
long todione procedures and requiro an ample mpply of hmon blood.
The availability of prothrofiin-rioh plum. Fraction III offered the
tonnage of using this source a o starting mtoriol.

Protein separation: by filter paper omtography cud/or iono-
phoretio loans have been applied to plow. The simple apparatus
required suggested he mutation of filter paper electrophoreeie to
the purification of prothrombin. If. no anticipated find: this tech-
niqne night provide o more easily accessible and reproducible couroo

of this enzyme.

II. HISTORICAL

In connection with this investigation to improve the isolation
of prothronbin it is desirable to review blood clotting, bases for
prior isolation methods and how filter paper electrophoresis has been

develoPod .

A. Mechanism of Blood Clotting

In the early 1900's the emulated lmowlsdge about buxom blood
coagulation was formulated into what is now known as the ”Classical
Theory” or blood clotting (33). This mechanism 1. concerned with
three basic phases but at present it is regarded as being much more
complicated than originally assumed .

Briefly the triphasic mechanism is: 1) in activation of blood
platelets 11th production of thrombOplaetin, 2) The resulting conver-
sion of the synogen, prothronbin, to the active enzyme , thronbin, and
3) The transformation of soluble fibrinogen to insoluble fibrin by
thrombin. In addition to these basic steps there are several activator
and inhibitor systems involved (1). Figure l elaborates the above and
sunrises the presently rowded ensynatic reactions and biochemical
factors concerned with the lumen blood coagulation process.

3. Isolation of Himan Ethrombin

Early prothrwin preparations depended upon either a direct
precipitation of the proonsyme or the prelininary removal of tibrinogen

fro-plane.

Figure 1. Schematic Representation of the Blood Clotting Mechanism

 

 

 

 

 

 

 

 

 

PROFIBRINOLISIN FIBRINOLYSIS
£151nt 4
mmmoum FIBEm-JOLIS‘IN —> 4 m1
(Planinogen) I (Plamin) FIBRINOLISIR
I A
mnmwmnmousm FIBRlN cwr
KIHASE Bermuda
Emmoogg —>FIB::.Ie (mansions)
T mmme
+
PEPTIDE

(Proaccelerin,
Factor V,

 

labile factor)

1

men AM + rancmm+ mnmnmm—(mnm)—vnmnmm

1

51m no-o——> ,

(Aooelsrin ,
Factor VI)

custom Ion—a-

 

1111324131

 

 

 

 

 

 

(Convex-tin)
some
Hermann:
comment
momma—v
PRUT new -
+
PRO-em + rmomormsrm
(Freeman) 1
carom ION 77
"me SURFACE" (Antihelophilio globulin,
thromb splastino gen ,
thrmbooytolysin)
earners + men moron s)

(Woplastinogenase)

Hollanby (31) in 1909 recorded the first preparation of pro-v
thrombin and later offered a modification for its purification (32) .
The maul procedure uployed ice-electric precipitation of the
globulin from diluted plasna brought to pH 5.3 with one percent acetic
acid. The precipitate was resuspended in 0.70 percent sodium chloride
and defibrimted with thrombin to yield a pro dnrombin-rich solution.

Seegsrs and coworkers (33) modified the above method by adsorb-r
ing protic‘ombin from diluted bovine plasma at pH 5.3 upon magnesium
hydroxide and eluting with carbon dioxide . Jitter dialysis and
bnplnrlization the pro thranbin preparation was stored with prolonged
retention of activity. Seegers, at 5.1..- (39) , elaborated their earlicr
directions vith more details of procedure in 191.5. Sec-33ers and oo-
miners (10,141) have recently reported the application 01‘ these pro-
oedurea to the isolation of human prothrombin.

Bordet and Delange (3) used twenty percent sodium chloride to
supress thrombin formation ani then obtained crude prothrombin by div
lution and defib rination of plasma. Howell (21) precipitated plant
proteins with. acetone and dried the fractions for storage. when
prothrorebin we wanted the dry precipitate was extracted with slightly
alkaline water and filtered. Condo (5) removed plam fibrinogen by
heat coagulation and after acetone precipitation dried the prothronbin
product with other. Gratis (19) reported a preparation whereby plans
as inmoulated with £52111»st w and incubated six to twelve
hours. Removal of the resulting fibrin left an enriched prothronbin.
Eagle (15) proposed the precipitation of a prothromin-rich fraction

U1

by bubbling carbon dioxide through cold diluted plazas. When centri-
fuged the active precipitate was peptized in 0.85 percent sodium
chloride at pH 7.0.

Partsntjev (36) and Taylor and Adana (It?) 180th a plasma
globulin fraction with considerable clotting power by repeated pre-
cipitations with monitor sulfate solutions. Orr and moon (35) also
produced a plasma globulin out with potential thrombic activity by
using Butler's (1;) phosphate buffer.l Precipitation of active material
occurred principally between 1.7 and 2 .0 molar.

The urgent need for human plasma protein products during Yorld
War II accelerated develOpment of the alcohol fractionation method as
devised by Colm and coworkers (7,8) . Their process involved the
careful control of pH, temperature, protein and ethanol concentration
and ionic strongmz. The resulting availability of various plasma
protein fractions led Gncley, £3 a}... (31.), to investigate their sub-
fractionation in order to concentrate prefix-01503.11 and. any other active
proteins of minute occurrence. They reported a preparation of human
protmcz’nbin from Fraction III with an activity of 30 units per mg.

A different preparation of human prothronbin produced by Surgenor
and co-worlaere (1435,“) involved adsorption upon barium sulfate from
plasma of blood that was decalcifiod by collection over Borax-SO.

The prothrombin was subsequently eluted with citrate solutions.

 

l Consists of equinoleculnr parts of potassium dihydrogen phos-
phate and potassium nonolvdrogen phosphate, pH 6.5, with the total
ionic strength varying from 0.6 to 3.0 molar.

Certain physioo-chemieal information concerning bovine pro-
titre-bin has been reported (25,26), while the date. on m prepara-
tion. 18 incomplete. Samples of the latter have been reported to
be paladiaperae in the ultracentrifuge (h2,b6) and to have electro-
phoretic nebility'comperdble to that of the‘bovine prothrenbine(h9,n2).
Sane evidence has‘been offered for the presence of tyroeine and
tmtophane (to), while Lanchantin (27) has suggested the preunne of
cytteine but the Absence of cystine in human prothrofibin,

C. The Devnlonmert of Paper Electrovforrsis

 

Initislly the whole field of paper chomato graph; and subsequent
paper electrophoresis received its impetus from Tavett's original (hh)
experiments on the separation.o£ plant pigments'by adsorbents conteinod
in a column. Other workers brought out different eleborations of this
technique, such as solvents for elation (37) and frontal analysis (29).
Sane have applied electrical potentials across the emits of the
colurn (10,I3),

Integration of the previous research led to the development of
zone electroplwresie. Zone electrophoresis involves the migration of
charged components as aspirate zones in a supporting medimn and givee
the advantage of securing more complete separations. However,
‘Iobilities and ire-electric points are not generally determined by
this method. The matter is complicated by adsorption on paper fibers

and by electroosmotic phenomena.

One of the firet reporte ebout elect‘mphoreeie upon paper no
that by Ionig (23) in 1937 . The first application to protein capers-
tione eppeared in 1939 when von nobueitcky end Icnig (22) deecribed
the resolution of e yellow chronoprotein from e snake ”not. During
the decade of 1938 to 191:8 paper chromatography and urine electro-
chroletogrephie method! eppecred to overshadovnaper electrophoreeie
development . The introduction of acting boundary electrophoreeie by
Tieeliue (50) in 1937 elec delayed the deve10pment end ecceptance of
the paper work.

In the late l9b0'e various methode of peper electrOphoreeie rere
independently euggeeted fro. eereral laboratoriee. The eilpleet
eppmtue coneieted of a circuit which use oonpleted by dipping the
end- of en electrolyte-mintened paper into veeeele conteining electro-
lyte end eloctrodee. The problem of eupporting the ptpee' to prevent
buffer 'pooling" end eurfeee enporetion eere two diudmtegee of
thin eetup.

For paper uppert there are basically three eon-traction. One
ie the glue plate nethod (11,214) whereby the noietened peper etrip
1e placed between two glee- pletee winch are reeled to mini-ire mfece
enporetion. A eeoond construction, known co the gallon type (13,16),
elploye linimm contect by eupporting the paper over e glue rod fit):
the end. pulled outward and dipped into electrolyte. Thirdly, the
box-type cpparetul (2e.9) mpe the piper horizontal while giving
linimn contact to the ligretion eurfece. The use of glue rectenglee
or reieed pointed projectione on e pleetic surface provide eupport.

A weighted type or stretching frame niy’also be used to keep the
paper in a taut, level position,

For control of temperature ani surfice evaporation the glaee
plate seen: quite effective, but other setups provide a cover and
refrigeration. Honopolar immieicible liquids such as chlorobenzene (9),
heptane (2a) or cerbon tetrachloride (11) act as selling agents to aid
in heat dissipation. HcDozmld (3o) tried hydrogen and helium as water-
eatnrltod gaeeons sealants.

Hangaard and Kroner (20) were first to express the idea of combin-
ing a flowing'buffer with electrophoretic migration on.paper. This
led to the separetion process known as "continuous electroghoreeis”.
Simultaneously yet independently, Gresemann.and Hennig (17, 8) and
Sveneson and Bratesten (hS) described an apparatus employing the above
rinciples. More recently Darren (lb) and Larson.and Fcinberg (28)
have presented some variations of the continuous electrophoresis
apparatus. A notable difference in this continuous technique and other
paper electrOphoresis is that the slowly'migrating components do not
necessarily pass over the same path as do the feeter moving components.

The principal construction in a filter paper sheet hung over the
edge of a'buffer vessel ouch that buffer flows continuously downward
by capillarity. The protein.mixture to be separated is supplied from
e reservoir dbove to the fig of the paper in a continuous winner by'
the aid of a filter paper'wiok; The lower edge of the paper is serrated

to provide drip points for receiving vessels and the lower corners are

inserted into electrode vessels smash that an electrical potential is
horizontally applied across the paper.

T223 problem of aurfaca distillation has Men met by coating the
edges of the pip-91‘ with paraffin (31) or by clamping mam with glass

plates (LS . Ref'flg-eration has also aiczed in texzxgerature control.

10

III. EXPERM PEETAL

”fl

A. filament

Horizontal Strip Apparetug - The power supply unit (0-300 volts)
was obtained from Ivan Small, Inc., New York, labeled Type 32903-110.
Rods of 1/1; inch stainless steel and 3/16 inch carbon were used es
cathode and anode, respectively. “batman Ho. 1 filter paper we the
supporting nedime. Buffer wells were constructed from 3/8 inch Lucite
sheets. One end of each vessel was provided with e tsp which served to
connect them by e 12 inch length or thin plastic tubing. Paper supports
were either an 8 x 12 x 1/1; inch glass plste or a plastic true with
tension holders .1 The housing unit consisted of en 18 x 30 x 1; inch

box provided with e Incite cover. Figure 2 illustrates the epperatus.

Continuous Flow Amtns .. The power supply unit (0-750 volts)
m Model E-800-2 sold by Research Equipment Corporation, Oakland,

California. Platimne sire of 25 gauge served as electrodes. One
hundred n1. tell-form bonkers were the electrode vessels. These were
provided with a tap near the bottom for connection by e lllll bore
plastic leveling tube . Paraffin was applied (1/2 inch mm) to the
long edges of e 6 x 16 inch sheet of Whetman 3 m4 filter paper
tensininating at five inches from one end. it the untreated end the
paper was out to provide five drip points, each two cm. apart. The

apperetus is chem in Figure 3 .

 

1 The frame was constructed end kindly made evaflsble for this
work by Dr. J. R. Brunner, Dairy Department, Michigan Stste College.

«a mmo> ocouuooam .0
made; nomusm .m
ufio>emmm have: .¢
maneueums aduum ngCOnanom .N easmfim

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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13

Moving Boundary ippamtus - Schliéren diagrams and mobility data
were obtained by use of a Tieclius electrophoresis instrument, the
Perkin-Elmer Model 38. Conductivity measurements were made with a
Model RC-lB Conductivity bridge manufactured by Industrial Instruments,
Inc., Jersey City, New Jersey. The conductivity cell supplied by
Perkin-Elmer had a cell constant of O.h893. All dialysis was performed
with the external rotating liquid dielyzer as constructed by Djang,
Lillevik and an). (12). The equilibration membrane was from Canoe,
catalog nmnber 70160-A-l-l/8. Lyophylizations were carried out with
an all glass apparatus similar in construction to that advertised by
the E. H. Machlett Co., Her Iork. Centmrugations were accomplished
with the Sonall Swedish angle-head, Model 31420, or the Model V Inter-
national Centrifuge manufactured by the International Equipment Co. ,

Boston, Mass .

8. Reagents and Materials

Crude StartingLMaterials - Frac-tion III end Fraction 111(2-3)
were received as frozen pastes after the alcohol fractionation of pooled
human plasma. This process is simmer-iced in Figure h. For this in-
vestigation the crude fractions were provided by the Plasma Products
Division of the Michigan Department or Health} These frcctions were
prepared according to Heth 9 (3t) and Method 6 (7). Fraction III
contained approximately 38 .14 percent solids , of which about 37 .l

 

l Supplied through the kind generousity of Drs. H. D. index-con
m K. B. “com.

Figure 1.1.. Ding-emetic Representation of the Preparation of Fraction
III and Fraction 111(2-3) According to Methods 6 and 9.

 

 

 

 

 

 

 

 

Plasma
E ' 16
u 0.11;
pH 7.2
Temp. -3°C.
Protlein 5.1%
f i
fiemstant Fraction I
. we 2
u 0.09
pH 6.8
Tm. -500.
Protein 3%
L 7
Supernatant Erection 11-!- III
1:3th 20
u 0.005
pH 7 2
Tempo - 0
Protein 13
F W
cMmstant Fraction II+III
If. one
u 0.015
562
Temp. «- C.
Protein 1.2%
r
:2 rustmt Fraction III
" no
u om
pH m
Temp. «0°C.
Projein 2.1%
r V
deer-name W

 

percent was protein. Fraction 111(2-3) consistcc‘i of about 28.0 per-

cent solids, of which 27.0 percent was protein.

Buffers .. Barbitsl, U.S.P., and sodium Ivdronde from Fisher
Scientific Co. were used. In the continuous flow experiments glycerol,
Baker, U.S.P., was added to the buffer to the extent of twenty percent
(v/V). The glycinate buffer was prepared according to Oncley, and co-
uorkers (3b). In sane instances Baker, C.P. sodium chloride was added
to increase the ionic strength.

Electrode Bridges -- Agar aga', U.S.P. from Sargent , and the
Verona]. buffer sent into filling the bridges connecting the electrode

vessels to the buffer wells.

Sealant .. Chlorobensene rm Eastman Kodak-Distillation Products,
catalo; mmber 70, was used in the horizontal strip studies.

Staining - The dye solution for protein identification was pre-
pared by dissolving one gs. bros-phenol blue (Nutritional Biochesiosls
Corporation) in one liter 95 percent ethanol satm-ated with mercuric
chloride (Baker, C.P.). Acetic acid and sodium acetate in the rinse
solutions were Baker, 0.P. chemicals. The drying oven was Model 0-1500,
fro- Research Equiplent Corporation, Oakland, California.

Adsorbent for Prothronbin --- Mallinckrodt analytical reagent

barium sulfate and barium carbonate were used.

Smi—miom Fijeldem Lea-dyadic -- Procedures 213‘ reagents were

followed according to Clark (6) . Digestion and distillation equip-ant

for these analyses was that as modified and used in this laboratory.

frothrombin Assay -- The reagents were all prepared by the analyst
at the Michigan Department of health Laboratories} The tImmnboplastin
was of human placental origin. The prothrombin conversion mixture con-
tained thie tln'omboplastin, gum acacia,2 inidazole buffer, sodiml
chloride and calcium chloride. A one percent (it/V) saline dispersion
of tumor: fibrinOgen was used as substrate in the essay. The accelerator
globulin (Lo-G) diluent was sorely fresh bovine serum diluted 1:75 in
0.85 percent (Ir/v) sodium chloride. The standard thrombin curve
(clotting time versus activity units) sue prepared beforehand using the
above reagents and a sample of tin-ombin supplied by the National
Institute of Health (11.1.3), Biologics Control Division, Bethesds, Md.
One unit of 1.13022sz activity is considered to be derived from one unit
of prcthrombin. A unit of thrombin has been defined as that amount of
thrombin which will clot one m1. of fibrinogen in 15 seconds under
standard conditions as given in detail in the Know Requirements of
Dried Thrombin, Second Revision, September 1916, issued by H.I.H.

 

1 The analyses were performed through the fine cooperation and
courtesy of Dr. L. A. madman end Mr. H. Gellick.

2 This provided 0.68 percent (w/v) calciu.

C . Metro-13

Horieontafl, Strip Techniwggg -- The 7 x 12 inch filter paper sleet

 

13 planed in the frame with the ends dipping 1313.0 the buffer wells.
One hour is allowed for equilibration with buffer as it 11101:! tens the
paper by canilL-zry action. The samples for paper electrophoresis are
prepared to contain one percent (w/v) protein from fraction III or
Fraction III(2—3) in 0.05 molar veronal buffer of pH 8.6. A 200‘!-
eample in applied at a point six on. from the cathode end and one on.
in mm each edge by streaking a filled capillary pipe .159 across the
and of the paper. Alternn‘mly one may place tmnty A]. on each of ten
Spots 1.5 cm. 31m across the end of the p’lpal‘.

One-half hour is allowed for equilibration with buffer after
application of sample. Current in then applied and adjusted to ten
nfllianperes with the mung. rousing from 250 to 300 volts. Running
tin. varies from 12 to 21; hours. After each run the paper is dried
and stained u subsequently described. The elation of protein zones
from the unstained paper it accmnliahad after cutting the damp paper
cross-vise into one on. strips. The cuts start at one cm. on the
cathode side of the amp].- origin and proceed toward the anode until
ten one on. strips on had. Each chip is placed into a. beaker con-
tnining two 111. of 0.85 percent (If/Y) sodium chloride and allowed to
soak in tln refrigorotor one to two hours. The “line extract is
than “sued for prothrombin nativity (vide infra) .

The none separation! achieved with this apparatus may be observed
in Figure 8. 5.1.1 samples are in 0.05 H. veronel of pH 8.6 with an

18

applied potential gradient of 9.8 volts/cm. The only difference in

conditions for the samples. shown is the length of running time.

Staining - Tho method is a modification of the procedure of
ancks (26). The papers are dried for one-half hour in the chromatog-
raphy oven at 100°C. The dyo is sprayed on in Just sufficient quantity
to cover the paper without dripping. After air-drying overnight,
amass dye is removed by two five-minute rinses in two percent (v/v)
acetic acid and one rinse in 0.05 M. sodium acetate contained in two

percent acetic acid. The paper is finally allowed to air-dry.

Continuous Flow Technique -- All Operations from the beginning
through dialysis and preliminary to lyOphylisstion are carried out in
the cold room at O to 5°C .' The separation described as follows is
schematically shown in Figure 5.

Four hundred and forty :11. of cold distilled water is added to
15 p. of. crude Fraction III(2-3) and allowed to stand for two hours.
The dispersion is slowly agitated with a mechanical stirrer for five
more hours. The pH is adjusted to 6.8 with a 0.5 H. glycinato buffer
of pH 9.8 and about 0.65 ml. is required. The molarity with respect
to sodim glycinats in this protein solution is about 9.1: x 10".

At this point a fifty s1. aliguot is dialysed saltu-trao against dis-
tilled water, lyophylisod and the residue stored in tho deep-freeze
labeled as Sanpls I. The remaining solution of about [:00 n1. is
treated sith 100 m1. of 2.9 percent (w/v) sodium chloride. Since the
glycinato concentration is negligible the addition of salt makes the

19
Figure 5. Separation Flow Sheet

Crude Fraction III(2-3)

1. Suspended in Inter
2. pH adjusted to 6.8

 

F
Supernatant Preolpitate
(Rot removed)

1. Aliquot dialysed and
. dried as S . lo I
2. Made 0.1 me with
sodium chloride
3. Stirred and centrifuged

 

r ‘

 

 

 

Supernatant Precipitate
. _ (Dialyzed, dried)

1. Aliquot dialned and Sagple III

dried as 5.212%“ II
2 . Equilibrated buffer
3. Subjected to oontimous

paper electrOphoresis

F i - t i 1

Residual Cut 1 Out 2 Cut 3 Cut 2; Cut 5

Solutren

Dialysis and lyoprwlization

 

 

 

 

1r t
Sample I? 88111235"? m“ le VI M9 VII mle VIII flu IX

 

20

total ionic strength practically 0.10 molar and the protein concen-
tration approaches 0.9 percent (ll/V). Following two hours of stirring
the suspension is centrifuged 15 minutes at 1500 RPM to give a pre-
cipitate that is resuspended in twenty to thirty ml. distilled water,
dialysed, dried, stored and identified as Smple III.

A 100 nl. aliquot of the above supernatant is dialysed, dried,
stored and referred to as Sample II. The remaining supernatant
(about 250 II.) is dialyzed against four one-half hour changes of. 0.05
H. veronal buffer, pH 8.6, which contains twenty percent by voltme of ‘
glycerol. Il'he equilibrated solution is then placed into the reservoir
for continuous paper electrophoresis. To attain equilibrium the paper
is kapt in contact with buffer for six hours before application of
protein solution and two hours prior to separation the current is
turned on. The protein is admitted to the paper at the rate of 15
drops per minute but it can vary from: 12 to 15 drape per minute. The
full amount of 700 volts from the power unit is applied and throughout
the run the current ranges from three to seven miniamperes.

After 2h hours the current is shut off and those solutions of
this separation referred to as cuts 2 and 3 are dialysed 12 hours .
against two changes of three to four liters of distilled enter for
removal of glycerol and salt. Each is lyophylised, labeled as Samples
VI and VII, respectively, and stored in the deep-freeze. The small
volumes in cuts 1, h and 5 were not dialyzed but were directly dried,
labeled as Samples V, VIII and II, respectively, and stored.

21

All samples are as sayed for prothrcm‘oin activity and analyzed
for nitrogen content. Electrophoretic patterns are obtained sherepthe
anount of sample is sufficient. Information regarding nitrogen content
and enzymatic activity is given in Tables I am II. The Schlieren

diagrams by Tiselius electrophoresis are shown in Figures 6 and 7.

much Experment - Two lumdred mg. f Sample III is sus-
pended in ten ml. distilled water and stirred for twenty minutes at
room temperature. One hundred mg. of sodium chloride in two ml. of
0.10 PI. veronal buffer of pH 5.6 is added and the stirring continued
for one hour.

The mixture is centrifuged in the Swedish angle-head centrifuge
and the precipitate discarded. One ml. or the supernatant is trans-
ferred to a tared bottle for protein estimation. The rest of the
swernatant is placed into a test-tube containing two grams of barium
sulfate and gently stirred ten minutes. The barium sulfate is removed
by centrifugaticn. Again one :1. of the supernatant is placed in a
tsrsd bottle for protein estimation as is subsequently described.

The final supernatant is equilibrated with 0.10 H. veronal of
pH 8.6 with 0.05 l. sodiul chloride and electrophorstically analysed.

ivsisilsr experiment as performed with 200 ng. of Sample III and
two grams of barium carbonate.

Pmthrombin Lesa: - Assays are performed according to the di-
motions of Warner, Brinkhous and anith (53) as modified by Ware and

Seegers (52). The procedure is as follows:

2?.

To 0.9 ml. of ic-G diluent is added 0.1 ml. of a saline diaper-
cion or the cannula to be tested. The resolting 0119 ml. is added to
three ml. or prothmnbin conversion mbzture (P .C .24.) and allowed to
undergo [emotion of throm‘oin at room temperature . At varying time
intervals (about five minutes) a 0.2; ml. aliquot is added to 0.1 :1.
of the fibrinogen substrate which indicates progreu of conversion.
With 1. stop watch the time 0! formation of the first visible fibrin
clot is noted as the clotting time. in activity factor is obtained
from the standard thrombin curve (clotting time versus thronbin units)
using the clotting time of e thii-ty minute conversion aliquot. Thirty
minutes is considered to give maxim conversion of prothrwxbin.

The units of protlu‘ombin in the original undiluted sample are then
calculated as shown in Appendix I with Sample I as an eminple.

Protein Estimation - An estimation of solids as protein is
performed by placing : Imam volume of test solution in a tax-ed weigh-
ing bottle and obtaining its total weight. The sample is dried at
103°C. for eight hours. Extraction. are made to follow: 1-2 ml. of
(Ii-tilled later is added to the bottle and then heated for 15 minutes
at 103°C . The supernatant in then carefully removed by a. capillary
tube saving the imluble denatured protein. This procedure is re-
peeted until an almost constant '31th on the dried couple is obtained.

Preparation formthe Tisoliue in sis .. Samples are dissolved

to one poment (It/V) in five ml. buffer. Dialysis is for one to two

hours with 101‘) ml. buff-er at room toppemture, then five to seven
hours against a 260 ml. charge of buffer at O to 5°C. and a. final
overnight equilibration versus 30C ml. fresh buffer at 0 to 5°C. The
equili‘f rated sample it clear is now ready for analysis; otnerwisa

it is centrifuged encmgh to clarify. ill subsequent operational pro-
cedures are according to the flannel of Instruction; as supplied by

the instrument manufacturer.

TABLE I

HMS, PROTERCMBIH ACTIVITY RID nrruocm CONTENT OF

Sample!» I

Volume of

Sample
141:

LyOplwlized

50
in

220

Weight in

Mg! .

Prothrombin

lho

Activity in
Units per :11.

8:11:10

figs. Nitrogen

per :1.

Saline

Prothmbin

135

Activity 1::
Units per mg .
Ritz-03m:

i Nitrogen on

lyophililed
residues

1.130

10.1 8.90 10.0

SAMPLES SPUDIED

II III I? V

100 30 ‘ 10
335
180

111. 3801114

1.78

101 111;

1 Refer to Figure 5.

" These are not glycerol-colt free weights.

b Weight m not obtainable.

1820 72a 53‘

VI

60

75

85

1.00 2.39 1.72 0.99

159 65.5 86

9 .90

100

1024

115

L9

1.13 0.88

102

55.5

10

2h

. .d. 2.0 3900 .. .000 £380
b.5503. a .09 909.33 00.5.9.2. 3033.3 mamfimm I 590. “0090000.. .059. 3300.8 I so 33.952 .05.“. 833009 I 9x

 

 

 

 

 

 

 

 

 

 

mfl m9..." 009 0.3 on

09 9.0 009 0:90 on m: 2.0 009 0. am 0m 1. I 009 0.3 00

I .... 009 .1. 99 00 ..... I... 009 «.mm 00. a- .3 00.9 0.09 m9

5. I. 009 .2. 09 .3 2. 009 0.3 09 m; 0...... 009 m.m~ 09

.... 1. 009 .2. m 2. I 009 N. .909 m I. ..... 009 0.0m m
99 «emu. MM.» 093...... M90 0905....

mm 3.0 009 79 mm .99 0.9.9 000 0.90 on can mad 0% m.m9 m.0~

:0 0......0 009 0:9 mm mm 2.0 000 0.0.0 00 I. I. 0.3 m.09 00

90 9......0 .89 flow m9 mm. 00.0 009 m5” m9 2. .2. 0.04 0.2 m9

0m 0.....0 0.0.9 0.3 09 .2. I. 009 0.0.... 09 z. 2.. 009 méa m

1. E. 009 9.00 m I ..... 009 .38 m I 2. 009 0.00 n
HP @.NQETM PH ”MN“. m

0.99 00.0 9.0m 0.09 0m 0.: 09.0 3m 0.09 on

0.9 00.0 000 0.09 0a 009 .330 0.00 0.00 00

099 09.9 00.9 0.09 on 0.9 00.0 00... «.09 m9 009 00.0 00.9 05 m9

00 0.0.0 009 on: 00 t. 2.0 009 0.9.9 09 m. 40.9 0... 0.99 09

«0 «0.0 009 4.00 9 3 «5.0 00 0.00 0 9w 3.9 am. 0.00 m

on 00.0 009 0:9 0 I. 2. 009 0.2. m .I I 00 9.0.0 m
999 finanm... E 099...... H 09mg...

0.50 m .5901 9.01.90 .30 m $9.8 .6 .E 9E m 14.9.90 .8 E

 

 

Ema: figasmm
HH 39.9 .

TABLE III

DATA 0!! IEIIITROPHORESIS PETE-ENS 0371137120 IN
0.1024 VEONAL BUFFERQF pH 8.6

0 m - 0 4
a

 

 

 

 

 

. ‘ Ionica The Potential Mobilitfi Concentration
Sample Stren h ( Sec 3) Gradient omponent Co . one nt
(3018:; (Volta/cm) a b a 5%) L

I 0.15 52.00 7.51 7.13 2.29 81 19

II 0.10 Shoo 0.91. 5.02. -- 93 7

m 0.15 5100 1.2.; 7.69 2.00 76 21.

111.1 0.15 51.00 6.89 7.11 1.32 86 12.
(3.150.)

71 0.10 5060 7.97 7.35 2.96 83 ‘ 17

VII 0.10 1.500 0.91. 7 .09 1.8:. 03 17

3- Protein concentrations are one percent.

I Values our 0.10 H. include 0.05}! N301.

I liabilities are calculnted from: descending patterns and are
”pruned 1n Tia-1171: units, cm/uc. per volt/m. x 10'.

27

N. , c I, .. ‘5 .l.‘ '_ “ _ (f . ' -_. \ . ...._- -
11911 c o. 1.100... 01.01 0.01.; 13.... 00:. n3 prom ii‘actlon 111(2-3)
Pr 011. 1:11.? ;,' ‘11'0a‘mont.

“ULRHJIQG
m

UQQJIUiAOU

 

 

 

 

 

 

 

 

 

     

L
r—

4 J.
151.1 in 0.10 vsroml 01’ 9.1 0.0, see Table III for conditions
,01' 0.1011 sagplc.
“nor 13111.5 am; all other 301.1105 rater to blame h.
alter 0.1111. 0.11000 ausorg'oion.

Figm‘e 7o flectrOphorcsis rattcrns ci‘ Unis Arml-
Continuous blow ‘i‘ecimiquc.

QCENDING DESCENDING

 

 

 

 

 

 

 

111.11 in 0.10 M Vux‘OllLl 01' pH 8.0, 30.. 1'00]..- .111. 1'0: (302.111.010.13

,-oi' each surplc.
(Tor ”CELLS 8410 all 01.1101 15;-z_)1;:; 11:11:; 1.0 1151:: 1;.

29

 

Chang
.3

non-m

ounce..-

nouaoduh Ho 300.34—

9

.Anu~009

I \ u a o
a

N

Bow 0

 

30

IV . DIS3USSIOH

This discussion with reference to prothromhin isolation will
attenpt to evaluate the results and significance of experiments con-
coming: (1) Apparatus testing and modification, (2) The separation
of proteins in Fraction III by horizontal-stflp teclmiques, (3) The
use of continuous paper electrophoresis to produce separation, and

(h) the utilization of adsorption nothods.

Horizontal-Strip Tochniguc .. lies was made of the box-typo appara—
tus to establish sons of tho procedures inhcrsnt in the isolation or
separation of proteins by loans of paper eloctrophcrcsis. In decoup-
ing surface cvaporation control and Iaintonancc of paper tension
Fraction III served as test natsrial.

The initial runs were node with a glass plate as paper support
and chlorobcmono as sealant. 1 "clear-typo" migration on tho result.
The components did not separate sharply but appeared to migrate as a
ma. 0mm. tho glass-plats-shlorobsnsens combustion was dis-
cardcd. 1 plastic franc for holding the paper was substituted into
the setup. This proved to be more satisfactory when using either strips
or a single paper shoot.

The center compartment which had held ths glass plate was filled
nth water to provide s saturated shosphsm tithin the closed box.
Migration was of such better quality. The application of the protein

31

solution to the paper by the spot method was found to give much
sharper resolution than by the streaking technique.

Considerable electrolysis occurred in the buffer wells when they
also served as the electrode vessels. This led to loss of buffer
ability. This problem was improved by replacing the steel anode with
a carbon rod. Furthermore , the electrodes were moved to containers
separate from the buffer sells but connected with an agar~bridge.

The final construction is shown in Figure 2.

Several runs were made with no attempt to remove the resolved
proteins from the paper. The sheets were merely stained in order to
observe the effects of various apparatus modifications. Reproducible
results, similar to such as shown by Figure 8, were obtainable when
the apparatus and techniques as described in the experimental section
were followed. These developments were then directed toward definite
isolation of prothrombin from Fraction III(2-3) . The four runs of
Figure 8 indicate that the longer time gives a better separation. Two
components are distinguishable after brainphenol blue staining. This
observation was confirmed by Tieelius electrophore tic analysis and
the Schlioren diagram can be seen in Figure 6, Sample I.

After locating the proteins by staining, an attwpt was made to
elute then from unstained paper. Assays upon saline extracts showed
presence of prothronbin activity. However, clotting times were not of
sufficient magnitude to warrant designation of maximal activity for
any particular region along the strip. Although this deveIOpment

produced clear sons formation with sharp resolution , it may be concluded

to

that sufficient material can not be applied to the paper for a
preparative concentration of active prothrombin by the horizontal-
strip technique.

Ccntinumsflow TPChllij‘Re -- ie a conseguence of the above dis-
cussed experiments, the centimetre-flow technique was next explored
as a possible way of preparing prothrombin from Fraction HIM-3) .

The presence of the two components in the starting material suggested
the desirability of preliminary separation. A scheme as shown in
Figure 5 was devised before attempting continuous paper electrOphoresis.
Principally the procedure of Oncley and coworkers (3b) was followed.
The supernatant (which should have been essentially prothroubin) was
found by the Tiselius method to contain about ninety percent of the
total eaterisl sigrating as one component. Figure 6, Senple II
demonstrates this result. The precipitate, presumably planinogen in
part, was found by similar analysis to contain two principal components
as canbe seen in Figure 6, Suple III. Since this pattern is very
sinilar to cm. of Fraction 111(2-3) it in quite probable that the
precipitate also consisted of undiseolved starting saterial.

Glycerol in the buffer served to increase the viscosity of the
protein solution, thereby decreasing its flow rate on the paper.
Concentration changes across the paper due to surface distillation were
kept at a minim by paraffin treatnsnt of the edges. As Valset and
Svensson (51) suggested, electrical equilibrium was establihhed by
turning the current on two hours before application of sample .

33

Heating of the paper occurs when the current is first applied and as
the current rises there is produced a swelling of the paper fibers.

It evidently causes flow of liquid from the electrode vessels produc-
ing an even greater rise in current. Hence, pro-equilibration lessens
subsequent complications after application of the protein solution.

After the continuous-flow apparatus was put into Operation, five
cuts of Sanple II sore collected. Data concerning value and lyophyl-
ised residue weight are given in Table I. In three of the five cute
such slall volume were collected that glycerol and salt-free weights
were not obtained upon the lyopkwlised residues. However , all samples
were subjected to nitrogen analysis and prothronbin assay. Only
Samples VI and VII provided adequate residue for Tiselius analysis.
Tables I, II and III report the results of these analyses.

Suples which gave sufficient glycerol and salt-free neighable
residue were found to contain approximately ten percent nitrogen.
This nay suggest uniform drying but if 11; to 16 percent nitrOgen in
prothronbin is asmed there is evidence of incomplete dehydration.

The prothronbin assay-1 demonstrated no enrichment of activity
in any particular out. There is no simificant activity difference
between Sample II (uncut) and Sample VII (the principal fraction of
the cuts). The latter is obtained fun the drip point directly
beneath the point of couple application. Perhaps the gravitational
force on the flowing protein solution was [rector than the horisontal

_._

1 The samples were also checked for the possible presence of
thrombin and found to be essentially free.

electrical vector. Although tho experiment we»: of relatively short
duration it is possible that channeling of the protein solution
occurred.

Prothraubin activity is present throughout all the cuts indicat-
ing the difficulty of completely segregating it from its co-proteins
in Fraction 111(2-3).

Although no out showed any activity greater than that of Sample II
(see Table I) there is quantitative evidence that the prothrombin
Ioiety was most ottractod towards the anodio side of the paper and
that very little activity resided in the cuts nearest the cathode.

This agrees with results of previous emerinento whore the component:
at Fraction 111(2-3) nigrntod medically in the horizontal-strip
apporotus.

Hoot workable was the finding of greatest protln‘ombin activity
in tho rosidufl. solution, Smple IV. One would expect activity loss
if any change at all. The activity units in this solution approximate
the values of prothronbin concentrates reported by Oncley and co-
vorkorl (3’4) 38 about 187 units per mg. nitrogen.

Schlie’ron diagrams were obtained on the two largest cute, Samples
VI and VII. Information concerning the composition by electmphoreeis
as well as conditions of_analysio may be had in Figaro 7 and Table III.
According to the patternshoth out: possess two components, 2 and 9.
Component 3 in both Samplo VI and VII has essentially the me mobility
tnd caprioes the some percent or the total material. This observo-

tion in also two for omnponent g. Tho mobilities of component-

g and g in Samples VI and VII (allowing for difference in effect of
ionic strength) sre comparable to those of crude Fraction III(2~3).
No outstanding difference in percentage composition can be noted in
the three cases.

Eloctrophoretic analysis of Sample II, as seen in Figure 6 ,
(mtorid prepared for continuous electrophoresis) reveals a principle
component that mounts to about 93 percent of the total. Its mobility
is loss, by 2 Tiselius units, than the fastest component of any other
simple so analyzed. This material was the soluble portion of an
aqueous extract of Fraction III(2-3) after treatment with sodium
chloride. Yet when it was subjected to continuous electrOphoresis the
staples obtained showed the presence of two components again. It may
be surmised that the sodium chloride precipitation left in solution
I. complex or materifls that under conditions of moving boumiary electro-
phoresis migrated singly whereas upon paper there was resolution into
two fractions. The similarity of mobilities of the components in
Samples I, VI and VII leads one to suspect that Sample II could

represent such a complex.

Adsorption Emeriments -- Prothrombin has been reported to be
adsorbed from whole plasma upon barium sulfate (14,7). Sample III ,
available in substantial mount, we tested in this regard. The
results with barimn sulfate indicated partial revision of the propor-
tions of components 3. and _b_ by electrophoretic enflysis, as seen in
Figure :3, Simple III-.31. Apparently a ten percent loss of component

b with a correSponding increase in 5 occurred. Protein estimation.

36

revealed about a four percent weight loss after barium sulfate
treatment.

Barium carbonate has been used to purify preparations of bovine
prothrombin (h2). An attempt vestmade to purify Sample III with this
compound. The result use an Opaque solution which upon electro-
phoretic analysis gave only partial patterns. These, however, indi-
cated no particular change in composition as did the protein esti-
mation.

Hence the proteins of Sample III and presumably Fraction III(2-3)
under the conditions employed and with the adsorbents need do not

sea to be conducive to separation by this technique.

37

7. SUMMARY

1) The best resolution of Fraction 111(2-3) by horizontal strip paper
electrOphoreeie wan obtained when: a) eurface evaporation was
controlled by a saturated aqueous atmosphere et room teMperature,
b) the paper was supported by a tension holder, and c) the protein
was applied by the spot method.

2) Fraction 111(2-3) separated into tucprincipal couponente, but gave
no distinct resolution of prothrombin activity.

3) irony: of protein eluatee from unstained papers showed no prepara-
tive concentration of prothronbin'by the horizontal-strip technique.

1.) A cult-treated extract of Fraction 111(2-3) with prothrombin
activity ehoued eeeentially'e singly'nigrating component by Tiseliue
electrOphoretic Analytil.

5) Continuous paper electrophoresis of the ebove material gave five
cute, of which two comprieed dbout eighty percent of the total
volume. .

6) Prothronbin.uee present in.dll cuts with no enrichment of activity
in eny one couple. Thronbin use found to be abeent.

7) The highest prothroibin activity its located in the residual solu-
tion from continuoue electrophoresis.

8) The use of‘beriul eultote and barium cerbonate ee adsorbents was
not found to be Adaptdblo to tho purification of prothronbin in

the manner tried.

1.

2.

3.

9.

10.

11.

38

BIBLIOGRAPH!

Alexander, 8. Blood Cells and Plasma Proteins, Their State in
Nature, Harvard Memoirs Ho. 2, page 75. Academic Press, Inc.
new York, 1953.

Block, R. J., Durrun, E. L. and Zweig, a. A Hamel of Paper
Chromatography and Paper flectrophoresis, 2a-page 359 and 2b-
Chapter 18. Academic Press, Inc., New Iorrk, 1955.

Bordet, J. and Delange, 1.. Blood Coagulation and the Formation
of Thronbin, Ann. de 1' Inst. Pastuer, 23, 657 (1912).

Butler, 1. U. and Montgomery, H. The Solubility of the Plasma
Proteins, J. Biol. cm... 22, 173 (1932).

Gouda, E. The preparation and Properties of Prethronbin, Am. J.
Physiol., 8, 512 (1926).

Clark, E. P. Seninicre Quantitative Organic Analysis, Chapter
III, Acadenio Press, Inc. , New York, 19143.

Cehn, E. J., Strong, 1.. 3., mghes, W. L., Jr., Huli‘ord, D. J.,
Lshworth, J. 11., Helin, H. and Taylor, R. L. Preparation and
Properties of Seru- and Plasna Proteins. IV a systen tor the
Separation into Fractions of the Protein and Lipoprotein Gen--
5 entag Biological Tissues and Fluids, J. An. Chem. Soc., 28.,
9 1 .

0011!, E. L, Gard, F. B. 3., fill-gen”, D. 21., Barnes, 3.1.,
Bron, R. 1., Deronaun 0., Gillespie, J. H... Kalirt, I. m,
Lever, H. F., Lin, C. i, Xittlnan, 1)., Houlton, R. 1.,

Sclnid, I. and Urenia, E. i Systen for the Separation of the
Caponents of has Bleed! Quantitative Procedures for the
Separation er the Protein Oenpenents or Runes Plans. J. in. Chen.
Soc., 1;, has (1950).

Consden, R. and Stanier, U. H. i Sinple Paper Electrophoresis
ipparatus , Nature, 110. 1069 (1952).

Coolidge, '1’. B A Sinpls Catapheresis Apparatus, J. Biol. Chem"
.131. 551 (19395.

Cremer, E. D. and fiselius A. flee sis of Egg lemin
on Filter Paper, Biechen. L. :20. 273 1950).

39

12. Djang, 8. S. T. The Isolation fractionation and electrophoretic
characterisation of the Globulins of Mung Bean, ”Unpublished
Ph. D. Thesis" Hichigan State College, 1951, 16!; Numb. Leaves.

13. Dumas, E. L. A MicroelectrOphoretic and Hicroionophoretic
Technique J. Am. Chem. 800., L2, 2912.3 (1950).

111. Durrun, E. 1.. Continuous nectrOphoresis and Ion0phoresis on
Filter Paper, J. Am. Chen. Soc., 12, L875 (1951).

15. Eagle, H. L. Studies on Blood Coagulation, J. Gen. Physiol., E
531 (1935).

16. Flynn, F. V. and Deli-aye, P. Microelectrophoresis of Protein on
Filter Paper. Lancet, 261, 235 (1951).

17. Grassnann, W. and Hannig, I. A Simple Method for the Continuous
Separation of a Mixture of. Compounds by Filter Paper Electrcphoreeis ,
Natureiss, 21, 397 (1950). -

18. Graemann, V. and Hannig, K. A Simple Method for the Analysis of
Serum Proteins and other Protein Mixtures, 1mm, 31, 397 (1950).

19. Gratia, 1. Studies on the Hechanien of Anti-Coagulant Action,
Ann. de 1' Inst. Pasteur, 11, 513 (1921).

20. Haugaard, G. and Kroner, I. D. Partition Chromatography of Amino
Acids with Applied Voltage, J. An. Chen. Soc., 19, 2135 (1918).

21. Howell, W. H. Prothrombin, Am. J. Physio)“, 5, 11711 (19111).

22. von Klobusitsky, D. and Konig, P. Venom of the Genus BothrOps VI,
Arch. Enptl. Path. Pharnakol" 192, 271 (1939). 'Seen in abstract
only" G. 1., 35, 1081 (1910).

23. Konig, P. Actas e Trabalhos do Terceiro Congreesc Sud-Americana
de Chinica, Rio ds Janeiro e 380 Paulo, g, 3311 (1937), Quoted from
Block, R. J., DuPrum, E. L. and Zweig, (1., page 333, A Innual of
Paper Chronatography and Paper Beetrophoresis , Academic Press,
Inc., Rev Iork, 1955.

211. Kunkel, H. G. and Tiselius, i. Electrophoresis of Proteins on
Filter Paper. J. Gen. Physiol., 31, 89 (1951).

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APPE'S'IDIX I
Sanple Cello? tion of Pro thrombin Activity

1) Ten :15. of 38mph I is dissol'md in one 111. 0.9 psrcant 30:13.31;
chloride.

2) To 0.1 ml. of solution (1) is added 0.9 ml. of Soc-G diluent.
his gives o 1:10 dilution of solution (1).

3) Tim one :11. of solution (2) is combinod with three ml. of
protilronbin conversion mifluro to yield four ml. of reaction
mixture. The dilution of solution (1) is now 11110.

b) To 0.1 111. of fibrinogen is adiez‘. 0.1. ml. of reaction mixture
and the time of clotting obtained. (If clotting times are loos
than 20 seconds before 15 minutes of rooction time has passed
than a dilution of solution (3) is made with 56-0 diluent rash
that at 30 minu+323 of relation time the clottillr the will be
12 to 20 seconds.) Total dilution of aolution (l) 1: now 1:50.
In this case an ad-Ltional dilution was made to give a final
value for solution (1) of 1:200.

S) The clotting time is referred to the staniord tlmozlbill curve.
This gives thrombin units per ml. of tile diluted oanplo. For
Salnple I the activity factor for an 18.9 second clotting time
after 30 mitmtos of reg-381011 is 0.70. This protizrmnbin activity
is (0.70 times 200) 11.9 units/m1. or solution (1).

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