Fibrinogen, Globulins, Albumin and Plasma at Interfaces - Advances in

12 Fibrinogen, Globulins, Albumin and Plasma at Interfaces

Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch012

L. VROMAN, A. L. ADAMS, M. KLINGS, and G. FISCHER Veterans Administration Hospital, Brooklyn, New York Changes in plasma protein films preadsorbed and then exposed to plasma or deposited by plasma itself, onto various surfaces were studied using ellipsometry and other techniques to observefilmthickness, antigenicity, and activity of adsorbates. Plasma deposited matter onto 7s gamma-globulins; if intact clotting factor XII was present in film or plasma, some removal followed from oxidized silicon substrate. Fibrinogen films were partially removed and, as did globulin to some extent, lost their antigenicity on exposure to intact plasma even if lacking factor XII. Antigenicity was maintained if the substrate had been non-wettable. Albumin was not adsorbed out of plasma though it competed well against purified proteins. Glass preexposed to proteins adsorbed factor XII out of plasma.

At an air interface, water arranges itself like a hydrophobic film ( 1 ) ; even in very dry air, a metallic surface will obtain such a low energy coat of water (2, 3). Coiled protein molecules also adhere to relatively simple, non-yielding surface (4) to display complex surface properties. Onto a hydrophobic solid they adhere with hydrophilic residues exposed to the aqueous phase while on a hydrophilic one their apolar residues will be exposed (5). However, the forces involved in adhesion to a simple surface such as Lucite may be far from simple (6). On more complex surfaces such as gels and cell membranes (7) and in multilayer adsorption (8), reaction rates will be incomputable. If only 14 residue segments of any protein molecule are involved in adsorption (9), the distortion needed to accomplish such adhesion must have varied and indirect effects (10 11, 12, 13, 14) rendering the adhering shape sometimes more, rather than less, antigenic (15,16) or changing its enzymatic activity which is especially sensitive to orientation (17). Neither a hard 255 In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.

256

APPLIED CHEMISTRY AT PROTEIN INTERFACES

surface s u c h as g e r m a n i u m

(18)

nor a hydrophobic

one

need

(19)

destroy t h e g l o b u l a r s h a p e of the a d s o r b e d p r o t e i n m o l e c u l e .

T h u s the

a n t i g e n i c i t y of a d s o r b e d a n t i g e n c a n be r e t a i n e d sufficiently for use i n i m m u n o a s s a y s ( 20, 21)

o n a v a r i e t y of h a r d , h i g h energy, m a t e r i a l s a n d

w i t h a v a r i e t y of t e c h n i q u e s s u c h as a l l o w i n g w a t e r to condense o n glass e l l i p s o m e t r y o n s i l i c o n crystals (23),

(22),

formation on anodized tantalum


Effects of Adsorption

or interference color p a t t e r n

(24).

on Plasmatic Clotting

Factors

I n b l o o d c l o t t i n g , w h e r e a c t i v i t y r a t h e r t h a n a n t i g e n i c i t y is i m p o r tant, b o t h the adsorbent a n d the adsorbate influence events.

Various

p r o t e o l y t i c enzymes b e c o m e active i n c l o t t i n g i f a l l o w e d to f o r m plexes w i t h the p r o p e r p h o s p h o l i p i d m i c e l l e s (25).

com-

It is the a r r a n g e m e n t

of p h o s p h o l i p i d m o l e c u l e s t h a t i n d u c e s t h e r i g h t p a i r s of

proteinaceous

c l o t t i n g factors, I X w i t h V I I I a n d t h e n X w i t h V , to c o m b i n e forces at the interface.

H e r e the e n z y m e s

(factors I X a n d X ) are h e l d at p o l a r

groups w h i l e t h e i r cofactors ( V I I I a n d V ) are h e l d at a p o l a r ones i n the m i c e l l e (26).

P e r h a p s the i n i t i a t i n g factors X I I a n d X I of this c h a i n c a n

i n t e r a c t at a n y i n t e r f a c e that initiates c l o t t i n g (27),

although purified

f a c t o r X I I , once a c t i v a t e d , activates p u r i f i e d factor X I w i t h o u t f o r m i n g a f u n c t i o n a l c o m p l e x w i t h i t (28).

However, under more physiological

c o n d i t i o n s things m a y b e different.

C o l l a g e n f a i l e d to cover itself w i t h

c l o t - p r o m o t i n g m a t e r i a l w h e n exposed to p l a s m a t h a t l a c k e d either factor X I I or factor X I ; i n a d d i t i o n , the c o l l a g e n m o l e c u l e n e e d e d its n e g a t i v e l y c h a r g e d groups as w e l l as its h e l i c a l structure to activate the X I I and X I

(29).

Ramifications

of Factor XII

Activation

A c t i v a t i o n of factor X I I leads to c o m p l e x s e v e r a l areas of p h y s i o l o g y . times i g n o r e d . X I I (31)

factors

events

(30)

connecting

Interfaces a c c i d e n t a l l y i n t r o d u c e d are some-

F o r e x a m p l e , the surface of b a c t e r i a m a y activate factor

a n d thus be responsible f o r the finding that i m m u n e

complex

activates factor X I I d i r e c t l y . T h e r e is the l a c k of s t r i c t specificity i n the actions of s e v e r a l enzymes.

F o r e x a m p l e , factor X I I is n e e d e d f o r the

c o n v e r s i o n of p r e k a l l i k r e i n ( p e r h a p s i d e n t i c a l w i t h F l e t c h e r f a c t o r ) k a l l i k r e i n b y glass (32).

to

T h e n c o n v e r s i o n of k i n i n o g e n to k i n i n b y k a l l i -

k r e i n f o l l o w s ; b u t s e v e r a l precursors m a y activate themselves as w e l l as others

(33).

T h e p a i n - p r o d u c i n g factor formed b y diluting plasma m a y be i d e n t i c a l w i t h a c t i v a t e d factor X I I ( X l l a ) a n d splits i n t o p r e k a l l i k r e i n a c t i v a t o r w i t h f a c t o r X I I a c t i v i t y a n d some fragments (34).

C o l l a g e n gains k i n i n -

In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.

12.

VROMAN E T AL.

generating

Protein

( kallikrein-like )

257

Interfaces

activity upon

exposure

to

plasma.

Most

significant h o w e v e r is t h a t m a x i m a l a c t i v i t y is g a i n e d o n shortest exposure (15 sec) to p l a s m a ( 3 5 ) .

Y e t , i n most laboratories the r e m o v a l of k i n i n o -

genase f r o m p l a s m a b y q u a r t z (36)

or b y C e l i t e (37)

is c a r r i e d out

w i t h o u t c o n s i d e r i n g that short r a t h e r t h a n l o n g exposure c a n b e effective.

O n some surfaces n o r m a l p l a s m a deposits

fibrinogen

most

i n two

seconds a n d converts it (renders i t u n a b l e a n y longer to attract m a t t e r out of a n t i - h u m a n

fibrinogen

sera) w i t h i n t w e n t y seconds i f t h e p l a s m a

is i n t a c t ( h a d n o t b e e n exposed

to surface t h a t activates factor X I I ) .


F a c t o r X I I seems to h a v e n o r o l e i n this c o n v e r t i n g a c t i v i t y , b u t o n l y i n the presence

of i n t a c t factor X I I d i d p l a s m a r e m o v e

converted matter

some of

the

(39).

A n t i b o d y / a n t i g e n c o m p l e x itself m a y not a d s o r b a n d a c t i v a t e factor X I I (31),

b u t its f o r m a t i o n leads to a c t i v a t i o n of c o m p l e m e n t factor C

w h i c h i n t u r n m a y a c t i v a t e c l o t t i n g (40, 41). appear poorly

s o l u b l e a n d m a y offer

6

H e r e too, some p r o d u c t s

p h y s i c a l interfaces

rather than

chemical activity. F o r m a t i o n of the p r o t e o l y t i c p l a s m a e n z y m e , p l a s m i n , out of its precursor,

plasminogen,

r e q u i r e s factor X I I (42) m a y w e l l create

by

adding

k a o l i n or

chloroform

as w e l l as at least one cofactor.

an insoluble protein

film

at the

to

plasma

Chloroform

plasma/chloroform

interface, a g a i n i n t e r p o s i n g m o r e p h y s i c a l events. A t least one H a g e m a n factor cof actor has b e e n p u r i f i e d (43). activating powders

I t is r e m o v e d f r o m p l a s m a b y

s u c h as glass a n d is p e r h a p s

responsible

l y s i n e esterase a c t i v i t y present i n u n a c t i v a t e d p l a s m a (44).

for

the

Thus, par-

t i c u l a r amounts of c e r t a i n interfaces m a y s i m u l t a n e o u s l y a d d a n d r e m o v e e n z y m e activities. Relationships

between Adsorption

of "Plasma Proteins and

Adhesion of Platelets Activation of Clotting and Adhesion of Platelets. p o r t e d (45)

It has b e e n r e -

that a l l u n c h a r g e d h y d r o p h o b i c surfaces a d s o r b the

proteins f r o m p l a s m a i n the same u n d e s t r u c t i v e w a y , b u t the

same

adsorbed

proteins are yet u n i d e n t i f i e d . A n e x p e r i m e n t i n w h i c h a test t u b e first receives h e m o g l o b i n

and

t h e n b l o o d has l e d to the p r o p e r c o n c l u s i o n t h a t h e m o g l o b i n i n h i b i t s surface a c t i v a t i o n of c l o t t i n g (46),

b u t a w i d e r a n g e of other p a r t i a l l y

p o s i t i v e l y c h a r g e d substances w i l l c o m p e t e just as successfully for the n e g a t i v e l y c h a r g e d a c t i v a t i n g sites o n a s o l i d (47)—especially

if given

a chance to get there before f a c t o r X I I does. B l o o d p l a s m a contains some components a b l e to a t t a c h themselves to p o s i t i v e sites o n c o l l a g e n , w h i c h t h e n b e c o m e s u n a b l e to aggregate platelets (48).

N o t only the polarity


258


of surface groups (49)

b u t also t h e i r a b i l i t y to act specifically as a s u b -

strate f o r a p l a t e l e t e n z y m e m u s t be essential i n b i n d i n g p l a t e l e t surface to n o n - p l a t e l e t surface (50).

T h e r e f o r e the process w o u l d b e affected

by

specific r a t h e r t h a n b y g e n e r a l p r o p e r t i e s of activators a n d i n h i b i t o r s . F o r e x a m p l e , o n l y a specific g r o u p of l i p o p r o t e i n s , p e r h a p s b y m a s k i n g c e r t a i n sites o n platelets, enhances t h e i r a g g r e g a t i o n (51);

certain basic

p o l y m e r s p r o m o t e a g g r e g a t i o n a n d the release of p l a t e l e t m a t e r i a l b u t d o r e q u i r e adenosine

diphosphate

(ADP)

specifically (52).

It seems

l i k e l y t h a t a l l s u r f a c e - r e l a t e d p r o p e r t i e s of p l a t e l e t s : a d h e s i o n , a g g r e g a -


t i o n , release ( 5 3 ) , a n d clot r e t r a c t i o n (54)

have their o w n requirements.

W h e r e a d h e s i o n or a g g r e g a t i o n of platelets is f o l l o w e d b y release

of

c l o t t i n g f a c t o r m a t e r i a l , n e w p a t h w a y s m a y l e a d to i n i t i a t i o n of c l o t t i n g w h i l e b y p a s s i n g factors X I I a n d X I (55, 56, 57).

W i t h the v i e w to l i n k

c l o t t i n g a n d platelets w i t h t h r o m b o s i s , efforts h a v e b e e n m a d e to s h o w that a c t i v a t i o n of factor X I I leads to p l a t e l e t a d h e s i o n in vivo, b u t e v i d e n c e is s t i l l insufficient (58).

A l s o , o t h e r factors m a y p r e c e d e factor X I I

i n s u c h a c h a i n ; F l e t c h e r factor deficiency is c o r r e c t e d in vitro b y a c t i v a t e d factor X I I (59), the interface. (60),

a n d p e r h a p s this factor precedes factor X I I at

Therefore,

d i a g r a m s of

clotting may

b u t there is always>room at the top.

enzyme

appear

complete

Elsewhere, even a c o m m o n

m a y penetrate the system; for e x a m p l e , t r y p s i n c a n

f a c t o r XI

Adsorption of Other Proteins, and Adhesion vs. Platelets.

activate

(61). Aggregation

of

W e h a v e b e e n interested i n proteins t h a t p l a s m a m a y deposit

a l o n g w i t h factor X I I ( a n d p e r h a p s F l e t c h e r factor a n d factor X I )

on

v a r i o u s surfaces. F i b r i n o g e n seems to p l a y a p e c u l i a r r o l e (38, 39, 62-83). O n t o a glass-like suface, n o r m a l i n t a c t p l a s m a deposits f a c t o r X I I a n d fibrinogen

w i t h i n a f e w seconds at a rate that seems i n d e p e n d e n t of the

p a r t n e r protein's presence i n s o l u t i o n (62).

D o e s either of these

p l a t e l e t a d h e s i o n in vitro or t h r o m b o s i s in vivo?

cause

T h e p r o p e r t i e s of a

surface that d e t e r m i n e its t h r o m b o g e n i c i t y in vivo m a y not be r e l a t e d to its a b i l i t y to m a k e platelets a d h e r e in vitro. T o f o r m a t h r o m b u s , platelets m u s t a d h e r e to platelets (63).

M u c h w o r k has b e e n done to

find

a

specific p r o t e i n t h a t w o u l d act as a g l u e o n either the p l a t e l e t or the s o l i d surface.

Patients l a c k i n g

fibrinogen

c o n g e n i t a l l y (64)

as a r e s u l t of streptokinase injections ( 6 5 )

or p e r h a p s

h a v e platelets w i t h p o o r a d -

h e s i o n to glass, b u t these experiments f a i l e d to d e t e r m i n e w h e t h e r the glue p r o t e i n m u s t b e l o c a t e d o n p l a t e l e t or s o l i d . It m a y b e c o m e active or adhesive u n d e r the i n f l u e n c e of d i s t o r t i n g forces at the i n t e r f a c e 66).

T h e distortions t h a t proteins other t h a n

fibrinogen

may

(19,

undergo

at the site of a w o u n d a p p e a r insufficient to cause p l a t e l e t a d h e s i o n a n d a g g r e g a t i o n : the b l e e d i n g t i m e of a f i b r i n o g e n e m i a patients is p r o l o n g e d a n d c o r r e c t e d b y i n f u s i o n of

fibrinogen

(67).


12.

VROMAN E T AL.

Protein

259

Interfaces

It has b e e n suggested t h a t c a t i o n i c proteins—i.e., XII,

fibrinogen,

factor

a n d g a m m a - g l o b u l i n s — c a n a l l restore t h e a g g r e g a t i n g a b i l i t y of

w a s h e d platelets a n d d o so b y r e d u c i n g t h e n e t n e g a t i v e c h a r g e o n t h e platelet membrane

T h a t platelets w i l l also a d h e r e to n e g a t i v e l y

(68).

c h a r g e d surfaces, s u c h as those c o a t e d w i t h h e p a r i n , is e x p l a i n e d b y a s s u m i n g that the h e p a r i n i z e d surface first adsorbs a p r o t e i n f r o m t h e p l a s m a (69).

O t h e r s h a v e suggested it is

needed for platelet aggregation

fibrinogen

e s p e c i a l l y w h i c h is

(70, 71) b u t f o u n d that at least i n a

s o l u t i o n c o n t a i n i n g g e l a t i n glass coated w i t h g l o b u l i n s r a t h e r t h a n w i t h


fibrinogen nin

c a u s e d platelets n o t o n l y to a d h e r e b u t t h e n to release serotoP o l y s t y r e n e i n t w o configurations a p p e a r e d t o adsorb

(72).

more

g l o b u l i n a n d h e n c e cause m o r e platelet a g g r e g a t i o n i n one c o n f i g u r a t i o n t h a n i n t h e other (73).

S i m i l a r efforts to see i f c o n f o r m a t i o n changes i n

m o r e n a t u r a l substrates, s u c h as c o l l a g e n , fibrin, a n d

fibrin-coated

collagen

(74) h a v e significant consequences a n d h a v e b e e n unsuccessful. Adsorption of Proteins out of Plasma; Relationship of Their Fate Platelets s u s p e n d e d i n s e r u m , the l i q u i d i n

to Adhesion of Platelets.

b l o o d left after c l o t t i n g a n d therefore l a c k i n g fibrinogen, or i n the p l a s m a of a p a t i e n t suffering f r o m a f i b r i n o g e n e m i a a d h e r e d o n l y to glass that had

adsorbed

fibrinogen

on it. T h e

fibrinogen

film

could be deposited

either b y exposure of the glass to fibrinogen s o l u t i o n , or to n o r m a l p l a s m a d u r i n g exposure of less t h a n a b o u t 5 sec (75).

L o n g e r exposure, c a u s i n g

the p l a s m a to convert t h e a d s o r b e d

left a film to w h i c h p l a t e -

lets w o u l d no l o n g e r adhere.

fibrinogen,

I n a range of 0.5 to 10 m g %

v a l u e b e i n g a b o u t 300 m g % ), i n c r e a s e d c o n c e n t r a t i o n of

(a normal

fibrinogen

in

p l a s m a w a s reflected b y i n c r e a s e d n u m b e r s of platelets p e r u n i t surface area of glass f o u n d to adhere (76).

Platelets a d h e r e d m o r e o n c o l l a g e n

i n presence of l o w concentrations of

fibrinogen

t h a n of other p r o t e i n s

tested (77), t h o u g h a l b u m i n at p h y s i o l o g i c a l concentrations times those of necessary f o r for a d h e s i o n

fibrinogen) fibrinogen

(78).

w a s also effective.

( a b o u t 10

W h i l e A D P was found

to cause platelet a g g r e g a t i o n , i t w a s n o t n e e d e d

T h i s is e v i d e n c e t h a t a difference exists

between

platelet-to-platelet a n d p l a t e l e t - t o - s o l i d a d h e s i o n . Could

platelet

surface

a d s o r b e d elsewhere? in vitro.

form

fibrinogen

on the platelet

dimers

fibrinogen;

T h e less dense ones adsorb most

adsorb i n j e c t e d over

fibrinogen

Platelets d o a d s o r b

(79),

with

fibrinogen

b o t h in vivo a n d b u t t h e i r a b i l i t y to

in vivo at a l l w o u l d suggest a r a p i d t u r n -

membrane.

A l s o , t h e amounts

of

fibrinogen

that w i l l a i d a g g r e g a t i o n p r o v o k e d b y other means are v e r y s m a l l 80).

(77,

F u r t h e r studies are r e q u i r e d to assemble these facts i n t o a coherent

theory. C e r t a i n platelets u n d e r c e r t a i n c o n d i t i o n s w i l l adhere to

fibrino-

gen-coated surfaces w i t h o u t r e l e a s i n g t h e i r a g g r e g a t i n g m a t e r i a l , w h i l e


260


on gamma-globulin-coated f o l l o w (81). fibrinogen

Platelet

surfaces release, a n d t h e n a g g r e g a t i o n

fibrinogen

a n d o c c u r r i n g m o s t l y i n p l a t e l e t granules

(82)

behave unlike the

fibrinogen

will

b e i n g s o m e w h a t different f r o m p l a s m a (83)

may

w e w o r k w i t h o r m a y not b e a v a i l a b l e to

react at a l l . G a m m a - g l o b u l i n s a d s o r b e d onto latex (84)

or glass

(72)

c a u s e d platelet a g g r e g a t i o n , b u t o n glass this was true o n l y i f the platelets were suspended i n an unphysiological m e d i u m . t h e y a d h e r e d , t h o u g h w i t h o u t a g g r e g a t i n g , to

In their o w n plasma,

fibrinogen-coated

surfaces.

S i n c e a l l of these proteins are a v a i l a b l e to the platelets i n t h e i r n a t i v e


m e d i u m ( the p l a s m a ) itself, s u c h a p r o t e i n ( as w e l l as a n y specific p l a t e let e n z y m e substrate) w o u l d h a v e to c h a n g e c o n s i d e r a b l y to b e c o m e a glue.

P e r h a p s the n o r m a l l y d i s s o l v e d m o l e c u l e s s i m p l y b e c o m e a b n o r -

m a l l y c o n c e n t r a t e d , o r i e n t e d , or p a c k e d at the s o l i d i n t e r f a c e , or t h e y undergo

a change

attractive.

i n conformation

f o r c i n g t h e m to expose s o m e t h i n g

F o r e x a m p l e , as c l o t t i n g f o l l o w s surface a c t i v a t i o n of f a c t o r

X I I , so a c h a i n of reactions c a n f o l l o w the f o r m a t i o n of a c o m p l e x tween

antigen

(such

as a f o r e i g n

protein)

and

antibody.

a d h e r e to a surface that has b e e n exposed to s u c h i m m u n e reactions once complement ponent C

6

component

C

3

h a d joined.

W h e n complement

also j o i n e d , the platelets l y s e d (86).

be-

Platelets (85) com-

O n the other h a n d , no

c o m p l e m e n t was n e e d e d for g a m m a - g l o b u l i n s o n latex to m a k e platelets release t h e i r n u c l e o t i d e s

(87);

t h e g l o b u l i n s h a d m e r e l y to be i n the

a d s o r b e d state. W h e t h e r or n o t t h e c o m p l e m e n t components

themselves

are s t i c k y , i t has seemed that o n l y those surfaces a b l e to a c t i v a t e the c h a i n of c o m p l e m e n t release m a t t e r (88,

reactions

c a n cause

platelets to aggregate

and

89).

T h e p l a t e l e t enzymes i n v o l v e d (90), actions a n d of others (91),

the c o m p l e x i t y of t h e i r i n t e r -

and their relationships, both physiologically

a n d p e r h a p s p h i l o g e n e t i c a l l y , w i t h the reactions of c e r t a i n w h i t e b l o o d cells (90, 9 2 ) h e l p us to see platelets as l i v i n g p a r t i c l e s . T h e i r a g g r e g a t i o n is u s e d as a q u a n t i t a t i v e a i d i n i m m u n o a s s a y (93)

i n a technique

that m a y w e l l b e a p p l i c a b l e to q u a n t i t a t i o n of heat aggregatable g l o b u l i n s (94)

a n d of specific i m m u n o g l o b u l i n G

gamma-

(95).

The Search for Surfaces to Which Platelets Will Not

Adhere

I t is d e s i r a b l e t o find a s i m p l e surface p r o p e r t y that w i l l i n d u c e a n e q u a l l y s i m p l e a n d h e n c e p r e d i c t a b l e b e h a v i o r of p l a s m a a n d its p r o t e i n s a n d t h e n of b l o o d a n d its platelets. A r e s i m p l e g u i d e l i n e s for b u i l d i n g non-thrombogenic (96),

flow

m a t e r i a l s a v a i l a b l e or

(97, 98, 99,100),

even

possible?

Wettability

a n d the effects of a i r / l i q u i d interfaces (101 )

a l l seem to b e r e l a t i v e l y s i m p l e , p h y s i c a l factors w i t h a clear effect o n p l a t e l e t a d h e s i o n . P h y s i c a l , h y d r o p h o b i c b o n d i n g , e.g.,

a force i m p o s e d


12.

VROMAN E T AL.

Protein

261

Interfaces

b y t h e c o m p l e x i t i e s of w a t e r , m a y b e i n v o l v e d i n p l a t e l e t a g g r e g a t i o n (102) as w e l l as i n a d h e s i o n . C h a r g e d i s t r i b u t i o n s o n t h e p l a t e l e t surface are p r o b a b l y also r e q u i r e d (103) a n d m a y w e l l b e so specific that t h e y act r a t h e r l i k e c h e m i c a l l y acute a n d d i s c r i m i n a t i n g sense organs w i t h t h e a b i l i t y to r e c e i v e a s h a r p i m a g e o f t h e i r n a t u r a l w o r l d a n d of its abilities to change. F o r platelets n o t to a d h e r e , aggregate, a n d cause t h r o m b o s i s , a n o n - t h r o m b o g e n i c surface m a y h a v e to r e a c t as w e l l as l o o k n a t u r a l H e p a r i n has b e e n t h o u g h t a n a t u r a l a n t i t h r o m b o t i c surface c o n -

(104).

stituent, b u t o n a surface this a n t i c o a g u l a n t m a y cause m o r e r a t h e r t h a n


less a d s o r p t i o n of proteins a n d a c t i v a t i o n of factor X I I (105), d e p e n d i n g o n t h e substrate c a r r y i n g t h e h e p a r i n (106). Albumin.

T h i s p r o t e i n m a y f o r m a m o r e n o n - t h r o m b o g e n i c surface

t h a n does h e p a r i n . I t is t h e most a b u n d a n t p l a s m a p r o t e i n . A l b u m i n s h a v e b e e n s t u d i e d extensively i n surface c h e m i s t r y laboratories. B o v i n e s e r u m w a s f o u n d to b e a d s o r b e d m o s t s t r o n g l y onto h y d r o p h o b i c surfaces (107) a n d i n t u r n b i n d s a r o m a t i c c o m p o u n d s b o n d i n g (108).

mostly b y hydrophobic

U n d e r a v a r i e t y of c o n d i t i o n s , platelets d o n o t a d h e r e

to a l b u m i n - c o a t e d glass (72, 75), b u t t h e h y d r o p h o b i c n a t u r e per se of the a l b u m i n film f a c i n g t h e platelets m a y n o t b e r e s p o n s i b l e .

Albumin

f o r m i n g a p r o t e c t i v e c o a t i n g o n platelets i n c e r t a i n e x p e r i m e n t s

(109)

m a y d o so b y u s i n g h y d r o p h o b i c b o n d i n g p r e c i s e l y to a d h e r e to t h e platelet m e m b r a n e .

C o n s i d e r i n g the h i g h concentration of a l b u m i n i n

p l a s m a , a n d t h e h i g h a v a i l a b i l i t y o f its h y d r o p h o b i c , t h o u g h p e r h a p s often

occupied,

albumin

even

sites, o u r o b s e r v a t i o n onto

hydrophobic

that plasma never

surfaces

(23,

38,

deposited

110, 111, 112,

113, 114), is s u r p r i s i n g . Present Efforts to Unravel

Interactions Among

Plasma

and Its Proteins at an Interface It is clear t h a t : a ) p l a s m a deposits s e v e r a l proteins onto o n e m a t e r i a l ; b ) i t t h e n affects e a c h d e p o s i t e d p r o t e i n d i f f e r e n t l y ; c ) n e i t h e r c o m p e t i t i o n a m o n g these proteins f o r a surface n o r t h e i r subsequent fate i n t h e p l a s m a / s o l i d i n t e r f a c e is s i m p l y p h y s i c a l . T o separate these o v e r l a p p i n g events, e a c h p u r i f i e d p r o t e i n t y p e w a s p r e - a d s o r b e d s i n g l y onto a v e r y s i m p l e surface a n d t h e n exposed to p l a s m a . T h e results w e r e r e c o r d e d i n terms of thickness a n d a n t i g e n i c i t y . T h e sequences w e r e p u t together to m o d e l t h e b e h a v i o r of w h o l e p l a s m a m e e t i n g this s i m p l e surface. O n e s u c h surface w a s that of a n a c i d - t r e a t e d s i l i c o n c r y s t a l slice.

I t also

s e r v e d to a n s w e r questions s u c h as: does a l b u m i n c o m p e t e as p o o r l y against separate proteins as i t does i n w h o l e p l a s m a ? D o h i g h l y c h a r g e d m o l e c u l e s s u c h as h e p a r i n affect t h e fate of p r e - a d s o r b e d p r o t e i n

films?

A s i n t h e past (22, 23, 24, 38, 75, 106, 111, 112, 113, 114), o u r m e t h o d s


262


i n c l u d e e l l i p s o m e t r y , a n o d i z e d t a n t a l u m interference color

observation,

w a t e r v a p o r c o n d e n s a t i o n p a t t e r n a n d C o o m a s s i e B l u e studies, a d h e s i o n of platelets, a n d the a b i l i t y of p l a s m a deposits to correct t h e c l o t t i n g of f a c t o r X I I deficient p l a s m a . T h e latter t w o m e t h o d s m a y give a m e a s u r e of the significance of a deposit to platelets a n d c l o t t i n g u n d e r o u r e x p e r i mental conditions.


A l t h o u g h the d a t a s h o w a r e l a t i o n s h i p b e t w e e n the a b i l i t y of a sur face to a d s o r b

fibrinogen

this

fibrinogen,

a n d the t e n d e n c y of platelets to a d h e r e , i t does n o t c o n

out of p l a s m a , the plasma's i n a b i l i t y to convert

firm

that a d s o r p t i o n of

fibrinogen

m u s t p r e c e d e a d h e s i o n of platelets to

a l l k i n d s of surface, o r t h a t surfaces w h i c h a d s o r b

fibrinogen

u n d e r these

c o n d i t i o n s w i l l be b a d b i o m a t e r i a l s in vivo as h e a r t valves, b l o o d vessels, or c a n u l a e , or ex vivo as a r t i f i c i a l k i d n e y m e m b r a n e s — e v e n

t h o u g h the

latter are most l i k e l y to b e i m p e d e d b y a n y adsorbate. D a t a presented supported

here represent m o s t l y single observations

i n subsequent

modifications

of

each

experiment.

further Because

e l l i p s o m e t r y r e c o r d i n g s are t i m e c o n s u m i n g , a n u m b e r of v a r i a b l e s , s u c h as p H a n d c o m p o s i t i o n of buffer s o l u t i o n , h a v e b e e n k e p t constant. Experimental M a t e r i a l s . T h e m a t e r i a l s u s e d i n these experiments are l i s t e d w i t h t h e i r source a n d p r e p a r a t o r y details. S i l i c o n c r y s t a l slices, η-type, o x i d i z e d ( S i O ) , are u s e d as d e s c r i b e d ( 2 3 ) . A n o d i z e d t a n t a l u m s p u t t e r e d glass ( T a O ) was o b t a i n e d f r o m M i l l i s R e s e a r c h , M i l l i s , M a s s . B u f f e r ( V S ) , isotonic V e r o n a l s o l u t i o n w a s a d j u s t e d to p H 7.4 a n d d i l u t e d 1:4 w i t h 0 . 8 5 % N a C l . 15 m l w e r e u s e d p e r e l l i p s o m e t e r cuvet. A l b u m i n , h u m a n , c r y s t a l l i n e , 1 0 0 % ( M a n n R e search L a b s . , N e w Y o r k , Ν. Y . ) , was p r e p a r e d i n s o l u t i o n of 2 m g / m l V S . 0.2 m l w e r e u s e d p e r e x p e r i m e n t . F i b r i n o g e n , h u m a n , l y o p h i l i z e d , p l a s minogen-free, protein 9 6 % clottable containing 0.4% sodium citrate and 0.9% N a C l (from A l a n J . Johnson: N e w Y o r k U n i v e r s i t y ) was prepared as 3 m g / m l V S . 0.4 m l w a s u s e d p e r e x p e r i m e n t unless o t h e r w i s e i n d i cated. F r a c t i o n I (fibrinogen), h u m a n ( M a n n Research L a b s . ) was pre p a r e d i n s o l u t i o n of 3 m g / m l V S a n d 4 m l w a s a d d e d p e r experiment. 7s gamma-globulins, h u m a n , chromatographically isolated 1 0 0 % ( M a n n R e s e a r c h L a b s . ) i n c o n c e n t r a t i o n of 8 m g / m l V S ; 0.2 m l was a d d e d p e r e x p e r i m e n t . F r a c t i o n I I I - I , h u m a n ( H y l a n d - d i v . of T r a v e n o l , C a l i f . ) i n c o n c e n t r a t i o n of 3 m g / m l V S ; 0.2 m l was a d d e d per experiment., E p s i l o n - A m i n o - n - C a p r o i c A c i d ( E A C A ) , C P . , homogeneous ( M a n n R e s e a r c h L a b s . ) i n c o n c e n t r a t i o n of 0 . 1 M ; 0.4 m l p e r e x p e r i m e n t w a s added. Plasminogen, human, purified ( M a n n Research Labs. ) i n concen t r a t i o n of 1 m g / m l V S (15 R P M I u n i t s / m g ) ; 0.4 m l was a d d e d p e r experiment. A p p r o x i m a t e l y 4 4 - 4 5 u n i t amounts ( d i s s o l v e d i n V S ) of S t r e p t o k i n a s e ( S K ) , 100,000 C h r i s t e n s e n u n i t s / v i a l . 0.1 m g = 400 units ( L e d e r l e Laboratories) was used per experiment. T r y p s i n Inhibitor, S o y b e a n ( S B T I ) , c h r o m a t o g r a p h i c ( c o m p o n e n t V I , salt free, l y o p h . ) , 10,000 B A E E u n i t s of i n h i b i t i o n p e r m g ( M a n n R e s e a r c h L a b s . ) , w a s



12.

VROMAN ET A L .

Protein

Interfaces

263

p r e p a r e d as 2 m g / m l V S . P l a t e l i n ( G e n e r a l D i a g n o s t i c s D i v . of W a r n e r C h i l c o t t , M o r r i s P l a i n s , N . J . ) was u s e d as d i r e c t e d b y the m a n u f a c t u r e r . Stock s o l u t i o n of C o o m a s s i e B r i l l i a n t B l u e R ( S i g m a C h e m i c a l C o . , St. L o u i s ) w a s p r e p a r e d b y d i s s o l v i n g 2.5 m g i n 50 v o l % m e t h a n o l a n d 10 v o l % acetic a c i d q.s. to 100 cc. F o r s t a i n i n g , the stock w a s d i l u t e d 2 0 - f o l d w i t h a s o l u t i o n of 40 v o l % m e t h a n o l , 5 v o l % acetic a c i d , a n d 2.5 v o l % g l y c e r o l . H e p a r i n , N a , 100,000 i n t e r n a t i o n a l u n i t s / g m ( K & K L a b s . , N e w Y o r k , Ν. Y . ) i n c o n c e n t r a t i o n of 14 m g / m l V S ; 0.1 m l was a d d e d per experiment. P r o t a m i n e ( S a l m i n e sulfate) ( P S ) ( G e n e r a l B i o c h e m i c a l s , C h a g r i n F a l l s , O h i o ) i n c o n c e n t r a t i o n of 2 m g / m l V S ; 0.4 m l was a d d e d p e r experiment. N o r m a l i n t a c t p l a s m a , c o l l e c t e d i n 0.1 v o l . A C D , r e n d e r e d p l a t e l e t p o o r b y c e n t r i f u g a t i o n , a n d stored i n p o l y s t y r e n e tubes ( F a l c o n Plastics D i v . , B e c t o n , D i c k i n s o n , & C o . , O x n a r d , C a l i f . ) at — 4 0 ° C w a s u s e d w i t h i n 4 hrs after t h a w i n g . A c t i v a t e d p l a s m a was p r e p a r e d b y exposing n o r m a l i n t a c t p l a s m a to 60 m g S p e e d e x ( G r e a t L a k e s C a r b o n C o r p . , L o s A n g e l e s ) p e r m l p l a s m a for 10 m i n , c e n t r i f u g i n g , a n d c o l l e c t i n g the supernatant. F a c t o r X I I deficient p l a s m a was c o l l e c t e d i n a 3 % s o d i u m citrate f r o m a p a t i e n t w i t h severe factor X I I deficiency. N o r m a l s e r u m was p o o l e d f r o m n o r m a l donors, stored for several hours at 3 7 ° C , a n d f r o z e n i n glass tubes. R a b b i t antisera to the f o l l o w i n g h u m a n p r o t e i n s : F r o m H y l a n d , antisera to a l b u m i n ( l i s t # 0 7 1 - 1 0 7 ) , fibrinogen (list # 0 7 1 - 1 0 8 ) , h u m a n f r a c t i o n I I I - I ( l i s t # 0 7 1 - 1 0 3 ) , β-lipoproteins ( l i s t # 0 7 1 - 1 1 3 ) , h u m a n s e r u m ( l i s t # 0 7 1 - 1 2 1 ) , F a b ( 0 7 1 - 2 5 8 ) , F c ( 0 7 1 - 2 5 9 ) . F r o m M a n n R e s e a r c h L a b s . , antisera to 7s g a m m a - g l o b u l i n s (cat. # 2 3 1 ) a n d t o t a l g a m m a - g l o b u l i n s (cat. # 8 0 9 0 ) . F r o m H o e c h s t , W o o d b u r y , Ν. Y . : a n t i s e r u m to p r e - a l b u m i n ( c o d e #8506). Techniques. E l l i p s o m e t r y was c a r r i e d out as f o l l o w s : a n S i O c r y s t a l slice c o v e r e d w i t h test m a t e r i a l is p l a c e d i n 15 m l V S w h i c h is s t i r r e d b y v e r t i c a l m o t i o n a n d k e p t at 37 ° C . R e a d i n g s are t a k e n , a n d the r e c o r d i n g was started. O f t e n , 0.1 m l n o r m a l i n t a c t p l a s m a is a d d e d to the V S , a n d the m i x t u r e is r e p l a c e d b y fresh V S t w i c e , after 2 m i n , a n d i n other series after 45 m i n . R e a d i n g s are r e p e a t e d , a n d the differences b e t w e e n first ( b l a n k ) a n d s e c o n d r e a d i n g s of m i n i m u m l i g h t t r a n s m i t t i n g positions of the a n a l y z e r (for the e l l i p s o m e t e r w i t h q u a r t e r w a v e p l a t e p l a c e d after reflecting s u r f a c e ) or p o l a r i z e r (for t h e ellipsometer w i t h q u a r t e r w a v e p l a t e p l a c e d before reflecting s u r f a c e ) is r e p o r t e d . 0.1 m l of a n t i - h u m a n fibrinogen, a n t i - h u m a n 7s g a m m a - g l o b u l i n s or a n t i - h u m a n a l b u m i n s e r u m is a d d e d , f o l l o w e d 45 m i n later b y r e p l a c i n g c u v e t contents w i t h fresh V S , a n d readings are t a k e n . V a r i o u s sequences of the antisera a d d i tions w e r e u s e d since w e f o u n d a n t i g e n i c i t y of a p r o t e i n film m a y be d e s t r o y e d b y e x p o s i n g i t to a n o n - m a t c h i n g a n t i s e r u m . T h e final a n t i s e r u m was one against t o t a l h u m a n s e r u m . T a O interference colors are o b s e r v e d on a 1 X 3- or 2 X 3 - i n c h T a O slide c o v e r e d w i t h test m a t e r i a l . S o m e V S is d r o p p e d o n the slide a n d some n o r m a l i n t a c t p l a s m a is p l a c e d i n t o this d r o p . A f t e r 10 m i n , another d r o p of V S is p l a c e d elsewhere o n the slide a n d p e r m i t t e d to r u n i n t o the V S - p l a s m a m i x t u r e . A s soon as the d r o p s flow together, the entire slide


264

APPLIED CHEMISTRY A T PROTEIN INTERFACES

Table I.

Behavior of N o r m a l Intact Plasma at 7s

Normal

Intact

Plasma

Top


Exp. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 α 6 c

Globulin 2.30 2.85 2.91 2.93 2.81 2.25 2.04 2.48 2.23 3.09 2.33 2.39 2.94 2.46 1.83

time min 10 15 20 10 10 20

Bottom meas. 0.97 1.18 1.10 1.60 2.28 0.87* 1.21* 1.44 1.67 1.13 0.38 1.24 1.36 1.43' 0.26

—

25 60 10 45 10 20 20 2

c

c

d

time min

meas.

— — —

— — —

180 210 40 150

-1.86 -1.93 -1.02 -0.73

120 220

-0.39 -1.24

20 140 180

-0.09 -1.08 -0.89

—

—

—

—

—

—

e

EACA

SK

— — — — — — — — — — —

— — — — — — — — — —

-0.54

— — —

0.15

— — — —

Plasma premixed 1:1 with S K . Plasma premixed 1:2 with E A C A . Plasma premixed 1:1 with PS (1 mg/ml VS).

30

60

90

MINUTES

Figure 1. Tracing of ellipsometer recording, starting when 0.1 ml normal intact plasma had been added to the 15 ml buffer containing a slice of oxidized silicon on which 7s gamma-globulins had been adsorbed. Curve shows adsorption of about 60 A , followed by desorption. Between R and R, curve was reversed by reversing analyzer deviation from minimal light transmitting position.


12.

VROMAN E T AL.

Protein

265

Interfaces

G a m m a - G l o b u l i n - C o a t e d O x i d i z e d Silicon Surfaces Normal

Intact

Top time min

Plasma

Antiserum

meas.

time min

meas.

SK

Globulin

— — Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch012

To

Bottom Fr. llli

Fibrinogen

3.92 2.81

-0.29 10 10 10 12 10

0.50 1.01 0.62 1.04 0.25

840 35 840 100 60

-0.66 -0.09 -0.84 -0.07 -0.41

— -1.66

1.31 —

-0.07

—

Plasminogen

—

0.21 d e /

Plasma premixed with antiplasminogen. Cuvet contents changed 10 min after the start of removal. Plasma premixed 1:1 with soybean trypsin inhibitor (2 mg/ml VS).

is r i n s e d w i t h m o r e V S a n d t h e n w i t h d i s t i l l e d w a t e r after w h i c h i t is a l l o w e d to d r i p d r y . T h e s l i d e thus consists of 2 areas, one area w h e r e p l a s m a o n l y r e s i d e d a f e w seconds or less a n d a n o t h e r w h e r e i t r e s i d e d for 10 m i n . O n b o t h areas, d r o p s of v a r i o u s antisera are p l a c e d , r i n s e d off w i t h V S , a n d t h e n w i t h w a t e r after 1 or 2 m i n . O n b a r e T a O surfaces, the interference colors of n a t u r a l , v e r t i c a l l y ( n o r m a l ) reflected l i g h t a r e : b r o n z e for the u n t r e a t e d surface, r e d d i s h p u r p l e for s u r f a c e exposed to p l a s m a or p r o t e i n , a n d d e e p v i o l e t for sites w h e r e m a t c h i n g antisera h a d r e s i d e d . O n T a O surfaces p r e t r e a t e d w i t h a b i o m a t e r i a l w h i c h itself causes a shift i n color, a shift t o w a r d b l u e w i l l b e n o t e d i n subsequent deposits. G l a s s surfaces w e r e u s e d i n the same m a n n e r as w e r e T a O surfaces. T h e s e surfaces w e r e o b s e r v e d for a n t i g e n / a n t i b o d y r e a c t i o n sites either b y v a p o r p a t t e r n t e c h n i q u e or b y s t a i n i n g . W a t e r v a p o r p a t t e r n a l l o w s d e t e c t i o n of w e t t a b l e r e a c t i o n sites, as r e p o r t e d ( 2 2 ) . F o r s t a i n i n g , slides are c o v e r e d w i t h C o o m a s s i e B r i l l i a n t B l u e R s o l u t i o n w h i l e w e t a n d r i n s e d w i t h w a t e r 5 m i n later. D r i e d surfaces w e r e p l a c e d face d o w n o n y e l l o w p a p e r for o b s e r v a t i o n . Results and

Discussion

Ellipsometry Data.

M o s t experiments w e r e c a r r i e d out i n 15 m l of

V S at 3 7 ° C o n S i O surfaces. V a l u e s i n the tables u s u a l l y represent single e x p e r i m e n t s ; exceptions are n o t e d . I n a l l tables values are g i v e n i n c h r o n o l o g i c a l s e q u e n c e of a d d i t i v e s f r o m left to r i g h t . T h e r e a d i n g s represent


266


Table II.

Behavior of Factor XII Deficient and of Activated

Normal Intact Plasma

XII-Deficient

Top

Top

Bottom

Globulin

time min

meas.

time min

meas.

time min

meas.

1 2 3 4

2.83 2.90 2.60 2.36

10 5

0.84 1.12

10

0.06

220

-0.81

—

—

10 10 10

1.38 1.42* 2.39

—

120 110 110

-0.17 -2.80 -0.15

5 6

2.34 2.93

— —

— —

— —

Exp. No.


Plasma

—

— — — — —

— — — — —

— —

6

—

° Streptokinase (44-45 u) added to the plasma without rinsing after the curve leveled off. a p e a k or e q u i l i b r i u m p o i n t .

W h e n recordings showed a

down-slope,

suggesting d e s o r p t i o n , the p o l a r i z e r or a n a l y z e r ( d e p e n d i n g o n the e l l i p someter u s e d ) was t u r n e d b e y o n d its m i n i m u m l i g h t t r a n s m i t t i n g p o s i t i o n to a p o i n t w h e r e the r e c o r d e r p e n h a d b e e n i m m e d i a t e l y before. T h e angle of the e l e m e n t w a s n o t e d a l l o w i n g a d d i t i o n a l checks of o p t i c a l thickness change as w e l l as r e v e r s a l of the c u r v e i f t r u e d e s o r p t i o n w a s t a k i n g p l a c e . C u r v e s are not r e v e r s e d b y this m a n i p u l a t i o n b u t c o n t i n u e to d r o p i f a n a i r b u b b l e or a n increase i n t u r b i d i t y of the s o l u t i o n w e r e t h e cause. T y p e s of antisera w e r e a d d e d i n different sequences f o l l o w i n g otherw i s e i d e n t i c a l e x p e r i m e n t s , e.g. i n T a b l e V I , E x p . N o . 1, a n t i - f i b r i n o g e n p r e c e d e d a n t i - g l o b u l i n s e r u m , a n d i n E x p . N o . 2 the sequence w a s r e versed. (110),

O n the basis of p r e v i o u s c a l i b r a t i o n s w i t h step-coated

surfaces

the entries expressed i n degrees c a n be m u l t i p l i e d b y a b o u t 30 to

o b t a i n thickness i n angstroms for a r e f r a c t i v e i n d e x of about 1.60. B E H A V I O R O F P L A S M A A T 7s G A M M A - G L O B U L I N - C O A T E D O X I D I Z E D S I L I -

C O N . F i l m s of 7s g a m m a - g l o b u l i n s w e r e p r e p a r e d i n the e l l i p s o m e t e r c u v e t w h i l e r e c o r d i n g , b y a d d i t i o n of 0.4 m l of 8 m g g l o b u l i n p e r m l V S to the 15 m l V S i n the c u v e t c o n t a i n i n g the S i O slice. A f t e r 90 m i n t h e s o l u t i o n was r e p l a c e d three times b y fresh V S , a n d r e a d i n g s w e r e d o n e w h e n the t e m p e r a t u r e ( 3 7 ° C ) h a d c o m e to e q u i l i b r i u m . F i l m s w e r e a b o u t 2 ° to 3 ° t h i c k (see

T a b l e s I - I V ) . O n t o these, 0.1 m l n o r m a l i n t a c t p l a s m a depos-

i t e d a b o u t 1° to 2 ° of m a t t e r w i t h i n a b o u t 10 m i n after i t h a d b e e n a d d e d to the V S ( T a b l e I , E x p . N o . 1 - 7 ) ,

and then removed nearly all

a n d sometimes m o r e t h a n i t h a d d e p o s i t e d ( E x p . N o . 4 - 7 ) . a n a c t u a l r e c o r d i n g is s h o w n ( F i g u r e 1 ) .

A t r a c i n g of

A n o t h e r a d d i t i o n of p l a s m a

r e c r e a t e d to some degree this a d s o r p t i o n a n d d e s o r p t i o n sequence


(Exp.

12.

VROMAN ET AL.

Protein

267

Interfaces

P l a s m a a t 7s G a m m a - G l o b u l i n - C o a t e d O x i d i z e d S i l i c o n S u r f a c e s Normal

Activated Plasma


Τον

Normal

Bottom

Τον

time min

meas.

time min

meas.

time min

10 — — — 120 10

0.61 — — — 1.42 1.57

180 — — — — 120

-0.94 — — — 0.00 -0.25

— 10 — — — 15

b

Intact

Plasma Bottom

meas. — 0.44 — 0.00 — 0.44

time min

meas.

Anti-7s GammaGlobulin

— 840 — — — 120

— -1.93 — — — -0.77

— — 0.27 — — 1.97

Plasma premixed 1:1 with S K .

Figure 2. Tracing of ellipsometer recording; uneventful sections omitted. To buffer containing oxidized silicon slice on which 7s gamma-globulins had been adsorbed, 0.1 ml of normal activated plasma was added at A, causing adsorption. After 135 min, removal still being minimal, cuvet contents were replaced twice by fresh buffer, and at B, 0.1 ml intact plasma was added, resulting in removal. R: reversal (see Figure 1).

No. 10-14). event (exp.

N e i t h e r streptokinase nor 2 A C A a p p e a r e d no. 6 a n d 7 ) ;

therefore,

to affect this

i t seems n e i t h e r a c t i v a t i o n

i n h i b i t i o n of p l a s m i n i n f l u e n c e d this d e s o r p t i o n .

nor

P l a s m i n per se m a y n o t

be i n v o l v e d i n i t at a l l . P r o t a m i n e sulfate d i d i n h i b i t i t ( E x p . N o . 9; also, see b e l o w a n d T a b l e I V ). Factor XII-deficient plasma, l i k e normal plasma, d i d deposit matter onto the g a m m a - g l o b u l i n films b u t was u n a b l e to remoVe i t ( T a b l e I I ,


268


Table III.

Behavior of Variously Anticoagulated Plasmas at Normal

Intact

Plasma

Citrated Bottom

Top


Exp. No.

Globulin

1 2 3 4 δ 6

2.75 2.94 3.09 2.81 3.12 26.3

Exp. No. 3).

time min

meas.

time min

20 10

1.63 1.36 1.13

240 220

— — —

meas.

—

— — —

-0.98 -1.08 -1.24

— — —

— — —

H o w e v e r , i t d i d r e m o v e m a t t e r left b y n o r m a l p l a s m a o n

b r i e f c o n t a c t w i t h a g l o b u l i n film ( T a b l e I I , E x p . N o . 1 ) .

I n this aspect,

n o r m a l a c t i v a t e d p l a s m a b e h a v e d l i k e factor X I I deficient p l a s m a ( E x p . N o . 5 a n d 6 vs. E x p . N o . 2 ) (see

Figure 2).

Streptokinase premixed w i t h

f a c t o r ΧΙΙ-deficient p l a s m a c a u s e d i t to r e m o v e a v e r y large a m o u n t of m a t t e r after d e p o s i t i n g some ( E x p . N o . 4 ) , p r o b a b l y b y a c t i v a t i n g p l a s m i n o g e n i n the p l a s m a . T h e a n t i g e n i c i t y of the u n d e r l y i n g g l o b u l i n was d e s t r o y e d b y this a c t i v i t y ( c o m p a r e E x p . N o . 4 a n d 6 ) . T h u s i t appears t h a t s o m e t h i n g i n t a c t is n e e d e d either w h i l e p l a s m a is f o r m i n g its d e p o s i t or later w h i l e the d e p o s i t is e x p o s e d to p l a s m a , to allow

removal

other

than by

plasminogen

of a c t i v a t e d p l a s m a to r e m o v e its o w n

activation.

The

inability

deposit on the g l o b u l i n

films

suggests that p r o t e o l y t i c a c t i v i t y , c r e a t e d b y the a c t i v a t i o n , was not the d e s o r b i n g agent.

It also suggests t h a t a cofactor of factor X I I m a y h a v e

b e e n r e m o v e d b y a c t i v a t i o n a n d is at least c o - r e s p o n s i b l e for the d e s o r b i n g a c t i v i t y of i n t a c t p l a s m a

Table IV.

Exp. No. 1 2 3 4 5 6 7

(42).

Effects of Protamine Sulfate and of Heparin

Globulin

PS

2.77 2.34 2.65 2.41 2.43 2.85 2.68

0.07 0.14 0.04 0.14 0.21 0.10 0.06

Heparin

— — — — —

-0.25 0.02

Globulin

— —

0.76 0.66 0.91 0.54 0.25


12.

Protein

VROMAN ET A L .

269

Interfaces

7s Gamma-Globulin-Coated Oxidized Silicon Surfaces Normal

Intact

Plasma EDTA

Heparinized


Top

Bottom

time min

meas.

time min

10

1.72

240

Bottom

Top

meas.

time min

meas.

time min

meas.

1.18 — —

— 2-10 10

0.32 0.44

120 60

-0.17 -0.77

N e i t h e r the presence of h e p a r i n nor t h a t of E D T A i n s o l u t i o n i n hibited desorption b y intact plasma ( T a b l e I I I ) .

O n the other h a n d ,

w h e n a p r e f o r m e d g l o b u l i n film w a s first exposed to p r o t a m i n e sulfate, t h o u g h this exposure affected film thickness v e r y l i t t l e ( T a b l e I V ) , n o r m a l i n t a c t p l a s m a c o u l d no l o n g e r r e m o v e

m a t t e r b u t o n l y deposit i t

(Table IV, Exp. No. 2). T h e m a t t e r d e p o s i t e d b y n o r m a l p l a s m a onto g l o b u l i n films that h a d ( T a b l e I V , E x p . N o . 1 ) or h a d not b e e n p r e - e x p o s e d to p r o t a m i n e sulfate ( n o t l i s t e d ) d i d not adsorb m a t t e r out of a n t i - h u m a n a n d therefore was p r o b a b l y not

fibrinogen

serum

fibrinogen.

W h e n exposure of a g l o b u l i n film to p r o t a m i n e sulfate w a s f o l l o w e d b y r e n e w e d exposure to g l o b u l i n , m o r e of the latter w a s d e p o s i t e d after w h i c h n o r m a l i n t a c t p l a s m a w o u l d deposit m o r e matter b u t r e m o v e l i t t l e (Table IV, Exp. No. 4 and 5).

If, h o w e v e r , exposure of the g l o b u l i n film

to p r o t a m i n e sulfate w a s f o l l o w e d b y exposure to h e p a r i n ( E x p . N o . 6 a n d 7 ) a g a i n c a u s i n g m i n i m a l changes i n film thickness, the r e s u l t i n g surface

on 7s Gamma Globulin and its Interaction with Plasma Normal

Intact

Plasma

Top time min

Antiserum

Bottom

to

meas.

time min

meas.

2 10

0.15 1.84

—

—

0.20

-0.09

—

2.30

180

10 10 10 10

1.64 1.83 1.28 1.56

90 180 130 840

-0.23 -0.29 -1.49 -1.34

— — — —

— — — —

Fibrinogen

Globulin


—

270

APPLIED CHEMISTRY AT PROTEIN

INTERFACES

w o u l d a d s o r b o n l y l i t t l e a d d i t i o n a l g l o b u l i n . I n contrast, n o r m a l p l a s m a w a s n o w a b l e to d e p o s i t a n d t h e n to r e m o v e l a r g e a m o u n t s of m a t t e r , suggesting t h a t the i n h i b i t o r y a c t i o n of t h e p r o t a m i n e sulfate o n this a c t i v i t y h a d b e e n n e u t r a l i z e d b y h e p a r i n at the interface. I n most e x p e r i ments successive

a d d i t i v e s d o n o t meet i n s o l u t i o n ; after e a c h

added

a n t i s e r u m c u v e t contents w e r e r e p l a c e d b y f r e s h V S once, a n d after e a c h of the other a d d i t i v e s , at least t w i c e .


Table V . Effects of Substrate and of D r y i n g on Conversion of Pre-adsorbed Fibrinogen and on Desorption by Plasma Fibrinogen Wettable

Exp. No.

Dry

Slide Wet

Nonwettable

Slide Wet

Dry

Normal Intact Plasma

AF

S i l i c o n slide 2.70

1

1.64

2

1.60

3

—

1.99

4

—

1.83

5

—

2.98

6

—

2.18

7

—

1.80

8

—

2.85

2.29

0.29

3.36 0.17

0.04

3.56 2.09

-0.43

3.47 3.89

-0.04

O x i d i z e d t a n t a l u m slide 9

1.62

10

1.77

1.40

11

1.46

12

1.47

1.34

-0.53

0.56

1.58

13

1.67

14

1.60

1.40 1.76

-0.64

15

1.74

16

1.76

BEHAVIOR OF P L A S M A

-0.45

1.48 -0.90

1.19

AT FIBRINOGEN-COATED OXIDIZED A N D U N O X I -

D I Z E D S I L I C O N . I n a large n u m b e r of e x p e r i m e n t s ( 2 2 , 2 3 , 2 4 , 3 8 , 3 9 ) o g e n p r e a d s o r b e d onto S i O , as w e l l as

fibrinogen

fibrin-

deposited b y plasma

itself is affected b y i n t a c t a n d f a c t o r - X I I - d e f i c i e n t p l a s m a i n s u c h a w a y t h a t its a b i l i t y to c o m b i n e w i t h a n t i - f i b r i n o g e n is lost. T h i s c o n v e r s i o n is slower on non-wettable

surfaces a n d also o n e i t h e r w e t t a b l e or

w e t t a b l e a n o d i z e d t a n t a l u m surfaces. it is f o l l o w e d b y some d e s o r p t i o n .

non-

I n presence of i n t a c t f a c t o r X I I ,

T o o b t a i n some i m p r e s s i o n of

effects that c o n f o r m a t i o n changes i m p o s e d o n the a d s o r b e d

the

fibrinogen

c o u l d h a v e o n t h e a b i l i t y of p l a s m a to c o n v e r t a n d desorb i t , the f o l l o w i n g e x p e r i m e n t s w e r e done.

A s o l u t i o n of 3 m g

fibrinogen

per m l V S was

a p p l i e d d i r e c t l y to d r y surfaces of t h e f o l l o w i n g : u n o x i d i z e d , n o n w e t t a b l e


12.

VROMAN

Protein

ET AL.

s i l i c o n (23,112);

271

Interfaces

oxidized, wettable silicon; anodized tantalum-sputtered

glass ( T a O ) ; a n d the same r u b b e d w i t h f e r r i c stéarate ( T a N ) to r e n d e r it n o n w e t t a b l e . A s t u d y to be p u b l i s h e d elsewhere suggests t h a t a

fibrino-

g e n s o l u t i o n , or e v e n p l a s m a itself, forms a fibrinogen film at t h e l i q u i d / a i r interface w h i c h , w h e n c a r r i e d b y a d r o p r o l l i n g over a h y d r o p h o b i c surface, is t r a n s f e r r e d at the a i r / s o l i d / l i q u i d interface i n a f o r m t h a t cannot be converted b y intact plasma. exposure to the

fibrinogen

I n the present series, after 1 m i n . of

s o l u t i o n , the slides a n d slices w e r e r i n s e d w i t h

V S a n d either p l a c e d i n to the 15 m l V S i n the e l l i p s o m e t e r or first rinsed Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch012

w i t h w a t e r , a i r d r i e d , a n d t h e n p l a c e d i n V S i n the ellipsometer.

After

r e a d i n g s w e r e p e r f o r m e d o n the s i l i c o n set, 0.1 m l n o r m a l i n t a c t p l a s m a was a d d e d ; 45 m i n l a t e r the c u v e t contents w e r e r e p l a c e d b y f r e s h V S and

readings were repeated.

T h e a n o d i z e d t a n t a l u m - s p u t t e r e d slides,

a l l o w i n g slower or less c o n v e r s i o n of p r e a d s o r b e d

fibrinogen,

were

ex-

p o s e d to 0.2 m l r a t h e r t h a n to 0.1 m l p l a s m a for 90 m i n r a t h e r t h a n for 45 m i n . I n a l l of these tests, 0.1 m l of a n t i - h u m a n

fibrinogen

serum was

a d d e d next, a n d r e a d i n g s w e r e p e r f o r m e d 45 m i n later. T h e results o n s i l i c o n substrates as l i s t e d ( T a b l e V ) are averages of 2 tests for

each

e x p e r i m e n t , w h i l e those o n a n o d i z e d t a n t a l u m substrates represent single experiments.

T h e data indicate that d r i e d

fibrinogen

was n o t c o n v e r t e d

w e l l o n a n y surface ( E x p . N o . 2, 6, 10, a n d 1 4 ) , a n d less c o n v e r s i o n t o o k p l a c e o n n o n w e t t a b l e t h a n o n w e t t a b l e surfaces ( c o m p a r e E x p . N o . 4 a n d 8 w i t h E x p . N o . 12 a n d 1 6 ) , p e r h a p s because of the air interfaces a l l o w e d during deposition.

C o n v e r s i o n o n w e t t a b l e a n o d i z e d t a n t a l u m was less

t h a n o n o x i d i z e d s i l i c o n , e v e n t h o u g h r e m o v a l w a s greater ( C o l u m n 5 ) i n this p r o l o n g e d exposure to p l a s m a , t h a n i t was o n S i O . T h e s e d a t a fit the q u a l i t a t i v e observations of interference colors r e s u l t i n g f r o m exposure of fibrinogen

a d s o r b e d onto T a O slides a n d exposed w h i l e w e t w i t h V S to

u n d i l u t e d c i t r a t e d or h e p a r i n i z e d p l a s m a . F i b r i n o g e n - c o a t e d areas t h a t had

b e e n exposed to p l a s m a are p a l e r ( t h i n n e r ) b u t t u r n p u r p l e w h e n

exposed to a n t i - f i b r i n o g e n sera as do the areas not exposed to p l a s m a . T h u s , the d a t a o b t a i n e d on T a O i n d i c a t e that r e m o v a l of t h e a d s o r b e d film

b y p l a s m a n e e d not be p r e c e d e d

r e m a i n e d that

fibrinogen

b y conversion.

T h e possibility

itself c o u l d be v a r i a b l y a n t i g e n i c d e p e n d i n g o n

the w e t t a b i l i t y of its substrate a n d o n its o w n state of d r y i n g .

However,

i n a series of six tests ( not l i s t e d ), no significant differences w e r e caused b y these v a r i a b l e s ,

fibrinogen

films

a l l c a u s i n g a deposit of about

2.7°-

3.5° b y a n t i - f i b r i n o g e n s e r u m . THE

B E H A V I O R O F P L A S M A TOWARDS ITS O W N DEPOSITS O F F I B R I N O G E N

A N D 7s G A M M A - G L O B U L I N S .

T h e v a r i o u s fates of p r e a d s o r b e d

o n exposure to p l a s m a a n d of

fibrinogen

fibrinogen

a n d 7s g a m m a - g l o b u l i n s

de-

p o s i t e d b y p l a s m a itself d e p e n d e d o n the state of a c t i v a t i o n i n the p l a s m a . B o t h a n t i - f i b r i n o g e n a n d anti-7s g a m m a - g l o b u l i n sera d e p o s i t e d


much

272

APPLIED CHEMISTRY

Table V I .

AT


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Fibrinogen

— — — — — — — — — 2.19 2.28 1.83 1.77

— —

c

INTERFACES

Effects of Activation of the Ability of Plasma to Convert Intact Plasma

Exp. No.

PROTEIN

Activated

Plasma

90 sec

45 min

90 sec

45 min

1.61 1.53

— —

1.54 1.32 1.34

— — — — — —

— — — — — —

— — — — — — —

—

— — — — — — —

1.61

1.37

— — — — — — —

-0.56

1.38

—

— — — —

2.61 2.40

—

-0.14 0.01 0.26 0.49

—

1.72

0.38

b

° A n t i s e r u m to fibrinogen a d d e d after a n t i s e r u m to g l o b u l i n . 14 m i n exposure, instead of 45 m i n . 6

m a t t e r onto films left b y n o r m a l i n t a c t p l a s m a i n IV2 m i n of contact w i t h o x i d i z e d s i l i c o n ( T a b l e V I , E x p . N o . 1 a n d 2 ) b u t n o t onto films left b y the p l a s m a after 45 m i n of contact ( E x p . N o . 3 ) .

This conversion was

o b t a i n e d w i t h 0.1 m l i n t a c t p l a s m a b e i n g a d d e d to the 15 m l V S . A n e a r l i e r s t u d y ( 3 9 ) s h o w e d that p l a s m a converts its o w n

fibrinogen

deposit

w i t h i n 30 sec i f u n d i l u t e d , e v e n at r o o m t e m p e r a t u r e . I n a p r e l i m i n a r y set of single experiments, w e f o u n d n o c l e a r e v i d e n c e of o r i e n t a t i o n i n the g l o b u l i n films left b y p l a s m a o n IV2 m i n of contact w i t h S i O . R e a c t i v i t y w i t h antisera to g l o b u l i n fragments

was

s l i g h t a n d u n i f o r m . C o n v e r s i o n of g l o b u l i n s d i d not a p p e a r as c o m p l e t e as t h a t of

fibrinogen.

N o . 8 and 9)

A g a i n w e could show that activated plasma ( E x p .

w a s less a b l e to c o n v e r t

fibrinogen,

b u t the

fibrinogen

deposited b y intact plasma could be converted b y activated plasma ( E x p . N o . 14) a n d vice versa ( E x p . N o . 1 5 ) . C o n v e r s i o n of p r e a d s o r b e d

fibrino-

g e n o n S i O b y i n t a c t p l a s m a ( E x p . N o . 10) w a s a l w a y s less c o m p l e t e t h a n t h a t of the

fibrinogen

d e p o s i t e d b y p l a s m a itself ( E x p . N o . 3 ) ,

though

i n b o t h situations t h e r e m a i n i n g film adsorbs m u c h less t h a n the 2 . 7 ° - 3 ° a d s o r b e d out of a n t i - f i b r i n o g e n s e r u m b y

fibrinogen

films

that h a d not

b e e n exposed to i n t a c t p l a s m a . T H E BEHAVIOR OF A L B U M I N IN P L A S M A AT T H E OXIDIZED SILICON SUR-

F A C E . A c h a n c e f o r a l b u m i n to b e a d s o r b e d out of p l a s m a , not yet e n c o u n t e r e d o n a n y of the m a t e r i a l s tested, c o u l d b e p r o v i d e d b y r e m o v i n g

fibrin-

o g e n a n d some g l o b u l i n s f r o m c o m p e t i t i o n . S o m e n o r m a l i n t a c t p l a s m a w a s


12.

VROMAN E T AL.

Protein

273

Interfaces

Fibrinogen and 7s Gamma-Globulins at the Oxidized Silicon Surface Antiserum Fab Frag.

Fc Frag.

IgG H&Lch

To Fibrinogen 1.25 0.80 0.12


0.41 — —

— 0.93 —

Human Serum

Globulin

— —

1.50 1.28 0.32

a

— — 0.80

— —

1.40 0.64 0.42 0.76

0.95* 0.40 0.42* 0.73 2.81 2.01 1.90 0.27 0.04

— —

1.13

—

—

1.92 2.06

1.96 2.59 0.54

— —

—

* Activated plasma was added before intact plasma. h e a t e d to 5 6 ° C , a l l o w e d to c o o l to r o o m t e m p e r a t u r e ( a b o u t 2 5 ° C ) after 1 h r , a n d c e n t r i f u g e d to r e m o v e the p r e c i p i t a t e d t r e a t e d d i d not deposit detectable

fibrinogen

fibrinogen.

P l a s m a thus

onto o x i d i z e d s i l i c o n ( T a b l e

V I I , E x p . N o . 1) b u t d i d d e p o s i t some g l o b u l i n s w h i c h o n

prolonged

exposure p e r h a p s d i d u n d e r g o some c o n v e r s i o n ( c o m p a r e E x p . N o . 1 a n d 3).

S o m e other, single experiments suggest t h a t

fibrinogen

preadsorbed

out of its s o l u t i o n ( E x p . N o . 4 a n d 5) or out of n o r m a l p l a s m a ( E x p . N o . 6) w a s at least p a r t i a l l y c o n v e r t e d b y h e a t e d p l a s m a . T h e a b i l i t y of h e a t e d p l a s m a to d e p o s i t a l b u m i n ( T a b l e V I I I ) onto S i O was e n h a n c e d Table VII.

Effects of Heating on Intact Plasma Intact Unheated

Exp. No. 1 2 3 4 5 6

Fibrinogen

— — — 2.19 2.38

—

and

Plasma Antiserum

Heated

a

90 sec

45 min

90 sec

45 min

— — — — —

— — —

1.26 1.31

— —

0.86

-0.56

— —

— — — —

2.40

—

-0.04 1.21

Fibrinogen 0.32 -0.02 0.00 0.73 0.94 0.02

° Plasma maintained at 56°C for 1 hr and centrifuged. Antiserum to globulin added before antiserum to fibrinogen. 6


to Globulin 1.05 1.19* 0.58 0.76 0.73 0.48

274


Table VIII.


Exp. No.

Effects of Heating on Subsequent Behavior of Plasma A l b u m i n on Oxidized Silicon

Intact 90 sec

Heated Plasma 1 1.73 2 1.61 3 — 4 — 5 — 6 — 7 — 8 — Unheated plasma 9 1.46 10 —

Antiserum

Activated

45 min

90 sec

45 min

— —

— — — —

— — — — — —

2.56 2.77

Albumin

Globulin 1.12 1.34 1.07 0.77

2.65 2.82* 2.54 2.03* 3.20

— — — —

1.41 1.40

— —

1.99 1.99

2.74

—

— —

— —

0.54 0.11

1.60

to

—

—

1.32

—

1.18

—

— —

° A n t i s e r u m to a l b u m i n a d d e d after a n t i s e r u m to g l o b u l i n .

r e m a i n e d so e v e n w i t h a d d i t i o n of

fibrinogen

to the h e a t e d p l a s m a . I n 9

tests, one v o l u m e of n o r m a l a c t i v a t e d or n o r m a l i n t a c t p l a s m a w a s d i l u t e d w i t h a n e q u a l v o l u m e of V S , k e p t at 65 ° C for 2 hrs, a n d c e n t r i f u g e d . one v o l u m e of the s u p e r n a t a n t , a n e q u a l v o l u m e of 3 m g / m l V S Table IX.

Exp. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 α

% wt/wt in Albumin 100» 100 100 98.2 93.0 93.0 92.8 88.9 80.0 80.0 57.1 57.1 40.0 25.0 10.0

— —

Soin

Fibrinogen

— — — 1.8 7.0 7.0 7.2 11.1 20.0 20.0 42.9 42.9 60.0 75.0 90.0 100 100

Film Meas. 1.09 0.69 0.94 1.15 1.13 1.49 0.83 0.89 1.00 1.08 1.07 1.43 1.53 1.94 2.05 1.71 1.67

To

fibrinogen

Competition between Antiserum Fibrinogen 0.16 0.66 0.77" 0.52 1.41 0.93 " 1.69 1.73 1.59 0.94 2.15 1.26 2.23" 3.16 3.28 3.18 1.51 6

6

6

To: Albumin 3.41 1.39 3.85 3.32 2.65 2.10 2.26 2.55 1.74 0.78 1.86 0.51 0.26 1.35 0.77 0.29 -0.17

I n this case o n l y , 40 m g a l b u m i n per m l V S was used.


12.

VROMAN E T AL.

Protein

275

Interfaces

was a d d e d a n d t h e n 0.1 m l of this m i x t u r e to t h e 15 m l V S at 37 ° C i n t h e ellipsometer.

Measurements, followed

c a r r i e d o u t as d e s c r i b e d . mixtures of

fibrinogen

b y exposure

to antisera, w e r e

T h e y s h o w e d that t h e films d e p o s i t e d b y t h e

a n d h e a t e d p l a s m a w e r e 1.30°-2.27° t h i c k , a d -

s o r b e d 0 . 1 4 ° - 1 . 3 5 ° ( a v e r a g i n g 0 . 6 1 ° ) o u t of a n t i - f i b r i n o g e n s e r u m , a n d t h e n 1.27°-2.75° ( a v e r a g i n g 2 . 1 2 ° ) o u t of a n t i - a l b u m i n s e r u m . H u m a n s e r u m , another m i x t u r e that lacks t h e c o m p e t i n g

fibrinogen,

d e p o s i t e d films u n d e r t h e same e x p e r i m e n t a l c o n d i t i o n s . T h e films w e r e less a b l e to a d s o r b m a t t e r o u t of a n t i - a l b u m i n s e r u m t h a n w a s h e a t e d Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch012

plasma; neither heated convert

fibrinogen

nor unheated

C O M P E T I T I O N B E T W E E N PROTEINS.

globulins a n d

serum showed

clear a b i l i t y to

(data not listed).

fibrinogen,

C o m p e t i t i o n b e t w e e n 7s

albumin and

fibrinogen,

gamma-

a n d a l b u m i n a n d 7s

g a m m a - g l o b u l i n s f o r t h e S i O surface w a s o b s e r v e d b y p l a c i n g 0.4 m l of a m i x t u r e i n t o t h e 15 m l V S of t h e e l l i p s o m e t e r at 37 ° C a l l o w i n g a d s o r p t i o n f o r 9 0 m i n , r e p l a c i n g t h e s o l u t i o n 3 times w i t h fresh V S , a n d e x p o s i n g to successive antisera as u s u a l , e a c h for 4 5 m i n . 60 tests w e r e m a d e ; i n m a n y , t h e s e q u e n c e of a d d i t i o n of antisera w a s r e v e r s e d . O n e set of d a t a is g i v e n here as a n e x a m p l e ( T a b l e I X ) . G r a p h s are p r e s e n t e d

(Figures

3 a n d 4 ) i n w h i c h t h e p e r c e n t of m a t t e r a d s o r b e d o u t of t h e first a n t i s e r u m a d d e d p e r t o t a l a m o u n t a d s o r b e d o u t of b o t h antisera, is p l o t t e d against r e l a t i v e c o n c e n t r a t i o n , w t / w t , of t h e t w o constituents i n s o l u t i o n . A l b u m i n and Fibrinogen

Total Antisera 3.57 2.05 4.62 3.84 4.06 3.03 3.95 4.28 3.33 1.72 4.01 1.77 2.49 4.51 4.05 3.47 1.34 6

1 Antiserum, % of Total Sera Fibrinogen 4.5 30.7 16.7" 13.5 34.7 30.7 42.8 40.4 47.7 54.6» 53.6 71.9" 89.2» 70.0 89.0 91.6 100 b

b

Albumin 95.5 69.3 83.3 86.5 65.3 69.3 57.2 59.6 52.3 45.4 46.4 28.1 10.8 30.0 11.0 8.4 0.0

Antiserum to fibrinogen added after antiserum to albumin.


276

APPLIED CHEMISTRY I


Figure 3. Amount (degrees, ellipsom eter readings) adsorbed out of antialbumin serum, in % of total adsorbed out of it and out of anti-7s gamma globulin serum (curve G) or anti-fibrin ogen serum (curve F), plotted against % of albumin (wt/wt) in 7s gamma globulin (curve G) or fibrinogen solu tion (curve F). Intercept of curve G with Y axis shows some matter was adsorbed out of anti-albumin serum onto albumin-free globulin film.

00

A T PROTEIN

INTERFACES

r

Fibrinogen, Globulins, Albumin and Plasma at Interfaces - Advances in

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