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The adsorption of gaseous hydrogen chloride and ammonia on egg albumin surfaces

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The adsorption of gaseous hydrogen chloride and ammonia on egg albumin surfaces

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Content THE ADSORPTION OF GASEOUS HYDROGEN CHLORIDE AND AMMONIA
ON EGG ALBUMIN SURFACES
. A Thesis;
P r e s e n t e d 1 to
t h e F a c u l t y o f t h e D e p a rtm e n t o f C h e m is tr y
T h e U n i v e r s i t y o f S o u th e r n C a l i f o r n i .a
I n P a r t i a l F u l f i l l m e n t
o f th e- R e q u ire m e n ts f o r 1 t h e D e g re e
M a s te r o f S c ie n c e i n C h e m is try
by
J e r r o l d M o rto n S e e h o f
J a n u a r y 19 50
U M I Number: EP41580
All rights reserved
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This thesis, written by
jm RQ LD . MORTON. SEEHOF.....................
under the guidance of h%Jk... Faculty Committee,
and approved by all its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fulfill
ment of the requirements for the degree of
M a s te r o f S c ie n c e
Faculty Com m ittee
A c k n o w led g em en t s
I s h o u ld l i k e t o t a k e t h i s : o p p o r t u n i t y t o th a n k
D r. S . W. B e n so n f o r h i s ; m any h e l p f u l s u g g e s tio n s : o n ex
p e r i m e n t a l p r o c e d u r e ,; D r. D . A . E l l i s ; f o r h i s ; n e v e r - e n d
i n g s u p p ly o f p a t i e n c e an d u n d e r s t a n d i n g ,, a n d m any o f
my a s s o c i a t e s : ■w ho -were e v e r - p r e s e n t w ith new a n d s t a r t
l i n g t h e o r i e s o n p r o t e i n s t r u c t u r e .
ta b l e: of contents
CHAPTER PAGE
I . INTRODUCTION .. , . . . .. . ., . . . ... . * 1
I I . HISTROICAL SURVEY CONCERNING ACID-BASE
BINDING PROPERTIES OF PROTEINS . . . . . . . 3
I I I . EXPERIMENTAL PROCEDURE . . . ..................... . . 10
A. P r e p a r a t i o n o f s a m p le s • . . . . • . . . 10
B. P u r i t y o f s a m p le s ......................... 1 2
C. S p ra y f r e e z i n g an d d e n a t u r a t i o n . . . . 13
D. P u r i f i c a t i o n o f g a s e s . . ............................. 1 ?
E . D e s c r i p t i o n an d o p e r a t i o n o f
a p p a r a t u s . . . . . . . . . . . . . . 20
F . A d s o r p tio n r u n s an d c a l c u l a t i o n s . . . . 25
G. T e s t r u n s ................................................... 3 ^
IV . DATA ............................... 35
A. D e s c r i p t i o n o f a d s o r p t i o n r u n s .. .. . 35
B. P r o t e i n d e n a t u r a t i o n . . . . . . . . . . 37
G. I n d i v i d u a l g r a p h s o f a l l r u n s ... .. kO
D. C o m p o site g r a p h s o f a l l r u n s . . . . . . 8 2
V. DISCUSSION OF RESULTS . ............................... 89
BIBLIOGRAPHY ....................................... 10 6
CHAPTER I
INTRODUCTION
T h is t h e s i s i s a n a tt e m p t t o e l u c i d a t e some o f
t h e c o m p l e x i t i e s o f p r o t e i n s t r u c t u r e b y a s t u d y o f t h e
a d s o r p t i o n o f p o l a r g a s e s u p o n t h e s u r f a c e o f a r e p r e
s e n t a t i v e com pound. On t h e b a s i s o f e a s e o f p r e p a r a t i o n
an d p u r i f i c a t i o n , a n d a v a i l a b l e c h e m ic a l a n d p h y s i c a l i n
f o r m a t i o n c o n c e r n in g i t s p r o p e r t i e s , eg g a lb u m in w as s e
l e c t e d .
H arro w ^ I n t r o d u c e d t h e s u b j e c t o f p r o t e i n s i n h i s
t e x t i n t h e f o l lo w in g m a n n e r:
The p r o t e i n s b e lo n g t o a g ro u p o f t h e m o st c o m p le x
o f c h e m ic a l s u b s ta n c e s * They a r e e s s e n t i a l c o n s t i t
u e n t s o f a l l p r o t o p la s m , and t h e y a r e a l s o e s s e n t i a l
fo o d c o n s t i t u e n t s .
The p r o t e i n s a r e c h a r a c t e r i z e d b y t h e f a c t t h a t
o n h y d r o l y s i s th e y y i e l d fro m 20 t o 2 5 d i f f e r e n t -
am ino a c i d s . ( P r o l i n e an d h y d r o x y p r o l i n e , h y d r o l y t i c
p r o d u c t s o f p r o t e i n s , a r e e x c e p t i o n s i n t h a t t h e y
a r e im in o r a t h e r t h a n am ino a c i d s . )
T he d i f f e r e n c e s b e tw e e n p r o t e i n s a r e l a r g e l y a
m a t t e r o f t h e n u m b e r, t h e k in d a n d th e . a rr a n g e m e n t
o f s u c h am ino a c i d s w i t h i n t h e p r o t e i n m o le c u le .
S in c e t h e p r o t e i n m o le c u le i s o f t e n b u i l t up o f h u n
d r e d s , a n d e v e n th u o s a n d s , o f t h e s e am ino a c i d s , t h e
p ro b le m o f p r o t e i n s t r u c t u r e i s o n e o f a lm o s t i n s u p
e r a b l e d i f f i c u l t y .
1 H a rro w , B e n ja m in , T e x tb o o k o f B io c h e m is tr y
( P h i l a d e l p h i a an d L o n d o n : ¥.. B . S a u n d e rs Com pany, 19*+6),
2
The num ber a n d k i n d o f am ino a c i d s i n m any o f t h e common
p r o t e i n s h a v e b e e n a s c e r t a i n e d * U n s o lv e d a s y e t , h o w e v e r,
i s t h e i n t r i c a t e p ro b le m o f t h e o v e r - a l l a r r a n g e m e n t o f
t h e am ino a c i d s w i t h i n t h e p r o t e i n , a n d t h e r e l a t i o n o f
s u c h a r r a n g e m e n t t o p r o t e i n f u n c t i o n . The r e s o l u t i o n o f
t h i s s t r u c t u r a l r i d d l e w i l l p r o v e o f g r e a t v a l u e i n c l e a r
i n g up some o f t h e m y s t e r i e s o f m e d i c i n a l a n d p h y s i o l o g
i c a l c h e m is tr y .
The s tu d y o f t h e a d s o r p t i o n o f p o l a r g a s e s , s u c h
a s HC1 an d NH3 , o n eg g a lb u m in , s h o u ld y i e l d r e s u l t s
w h ic h m ay a i d i n d e te r m i n i n g t h e n u m b er an d n a t u r e o f
t h e v a r i o u s a c i d i c a n d b a s i c g r o u p in g s i n t h i s p r o t e i n .
D a ta t o t h i s en d i s i n c l u d e d an d e v a l u a t e d o n t h e f o l lo w
in g p a g e s .
CHAPTER I I
HISTORICAL SURVEY CONCERNING ACID-BASE
BINDING PROPERTIES OF PROTEINS
D u rin g t h e p a s t f i f t y y e a r s , t h e n a t u r e o f p r o
t e i n c o m b in a tio n w ith a c i d s an d b a s e s h a s r e c e i v e d m uch
a t t e n t i o n . N e v e r t h e l e s s , a s a t i s f a c t o r y u n d e r s t a n d i n g
i s s t i l l l a c k i n g . The e n i g m a t i c a l a s p e c t o f t h e s t u d y
w as o b s e r v e d a s e a r l y a s 1 8 6 6 w hen P la tn e r-* - s t a t e d t h a t
o n e a n d t h e sam e p r o t e i n c a n co m b in e w i t h b o th a c i d s an d
b a s e s . R e f e r e n c e s t o t h e l i t e r a t u r e o f 187*+ t o 1 9 0 6
may b e fo u n d i n a n a r t i c l e p u b l i s h e d b y R o b e r ts o n 2 i n
1 9 0 6 , i n w h ic h t h e “a m p h o t e r i c ” p r o p e r t i e s o f p r o t e i n s
w ere a g a i n r e p o r t e d .
I n 1931 a n e x c e l l e n t r e v i e w o n t h e s u b j e c t o f a c i d
an d b a s e b o n d in g b y p r o t e i n s w as p r e s e n t e d b y B e ld e n ^ ,
m any o f w hose i m p r e s s io n s a r e i n c o r p o r a t e d i n t o t h e r e
m a in d e r o f t h i s d i s c u s s i o n .
I n t h e l i g h t o f t h e i n t e r p r e t a t i o n o f a c i d - b a s e
p h en o m en a h e l d i n 1 9 0 0 , o n e m ig h t s u p p o s e t h a t t h i s com
b i n a t i o n o f p r o t e i n s w i t h a c i d s an d b a s e s i s s im p ly o n e
1 P l a t n e r , Z . B i o l . , 2 , ^ 1 7 (1 8 6 6 )
2 R o b e r ts o n , J . P h y s . C h em ., 1 0 , 52k- ( 1 9 0 6 )
3 B e ld e n , J . P h y s . C hem ., 216k (1 9 3 1 )
o f n e u t r a l i z a t i o n ; t h a t i n t h e p r e s e n c e o f a n a c i d a p r o
t e i n w o u ld a c t a s a b a s e , t h e i n t e r a c t i o n y i e l d i n g a n
i o n i c p r o t e i n s a l t , a n d i n t h e p r e s e n c e o f a b a s e , t h e
c o n v e r s e w i l l a p p ly , y i e l d i n g a m e t a l p r o t e i n a t e . The
p r o t e i n , t h e n , may b e c o n s i d e r e d a s a n a m p h o te r ic s u b
s t a n c e , c o n d u c tin g i t s e l f i n a m a n n e r a n a lo g o u s t o i n
o r g a n i c a m p h o te r e s . I n t h i s m a n n e r, t h e n a t u r e o f p r o
t e i n c o m b in a tio n w i t h a c i d s and b a s e s w as p re s u m e d t o
b e s e t t l e d i n t h e e a r l y l i t e r a t u r e .
From a c r i t i c a l p o i n t o f v ie w , h o w e v e r, t h e s i t
u a t i o n i s i n no w is e so s im p le a s i t m u s t h a v e a p p e a re d
i n 1 9 0 0 . On c l o s e r i n s p e c t i o n m any s u b t l e a s p e c t s o f
t h e s u b j e c t w e re e n c o u n t e r e d . I t a p p e a r e d , f o r i n s t a n c e ,
t h a t t h e b in d in g p o w er o f v a r i o u s p r o t e i n s f o r h y d ro c h lo r= -
i c a c i d w as g r e a t e r t h a n t h a t w h ic h c o u ld be e x p e c te d
o n t h e b a s i s j o f t h e know n am ino g ro u p c o n t e n t , t o w h ic h ,
a t t h a t tim e , t h e b a s i c p r o p e r t i e s o f t h e p r o t e i n w e re
a t t r i b u t e d .
The o r i g i n a l v ie w t h a t t h e n a t u r e o f t h e co m b in
a t i o n o f p r o t e i n s w ith a c i d s and b a s e s i s o n e o f chem
i c a l u n i o n h a s , h o w e v e r, p e r s i s t e d an d b e e n c a p a b ly u p
h e l d b y t h e r e s e a r c h e s o f m any w o r k e r s . I n 1 9 1 2 , t h i s
c o n t e n t i o n w as c l e a r l y p r e s e n t e d b y R obertsop**^ W hose
* * R o b e r ts o n , D ie P h y s i k a l i s c h e C hem ie d e r P r o t e i n e .
(1912).
c o n c l u s i o n s m ay b e su m m arize d a s f o l l o w s : t h e p r o t e i n s
fo rin t r u e a q u e o u s s o l u t i o n s an d n o t c o l l o i d a l s u s p e n s io n s
an d t h e y r e a c t i n s t o i c h i o m e t r i c p r o p o r t i o n s w i t h a c i d s
a n d b a s e s t o fo rm c h e m ic a l co m p o u n d s. F u r t h e r , i n s u p p o r t
o f t h i s v ie w o n e c a n f i n d , among o t h e r s , w o rk s b y L o eb ^
a n d W ils o n ^ . A n o th e r v ie w p o in t i s e x p o u n d e d b y B a n c r o f t ?
Q
i n a r e v ie w o f R o b e r ts o n ’ s b o o k i n w h ic h h e a s s e r t s t h a t
p r o t e i n s a r e n o t i n t r u e s o l u t i o n .
T h is l e a d s u s t o t h e c o n s i d e r a t i o n o f a n o p p o s in g
t h e o r y w h ic h h a s b e e n a d v a n c e d t o e l u c i d a t e t h e n a t u r e
o f t h e c o m b in a tio n o f p r o t e i n s w i t h a c i d s an d b a s e s .
R o b e r ts o n h o l d s t h a t p r o t e i n s a r e i n t r u e s o l u t i o n . I f ,
h o w e v e r, I t b e su p p o s e d t h a t p r o t e i n s fo rm c o l l o i d a l s u s
p e n s i o n s i n w a .te r, t h e n i t i s a p r o b a b l e c o n s e q u e n c e ,
th o u g h n o t a n e c e s s a r y o n e , t h a t t h e c o m b in a tio n w ith
a c i d s an d b a s e s i s a d s o r p t i o n a n d t h a t s t o i c h i o m e t r i c
r e l a t i o n s do n o t n e c e s s a r i l y h o l d . I f t h e p r o t e i n c o n
s t i t u t e s a s e c o n d p h a s e , a d s o r p t i o n w i l l b e a l a r g e p a r t ,
i f n o t t h e w h o le , o f t h e s t o r y .
5 * L o e b , P r o t e i n s an d t h e T h e o ry o f C o l l o i d a l Be
h a v i o u r . ( 1 9 2 4 ) .
6 W ils o n , The C h e m is tr y o f L e a th e r M a n u f a c tu r e .
( 1928).
7 B a n c r o f t , J . P h y s . C h em ., l £ , 7 0 4 (1 9 1 2 )
8 R o b e r ts o n , l o c . c i t .
6
R o b e r ts o n ^ s u g g e s te d t h a t i t i s a m a t t e r o f d e f i n
i t i o n i f we a r e r e l u c t a n t t o a d m it a p u r e l y c h e m ic a l m ech
a n ism o f n e u t r a l i z a t i o n a n d p r e f e r to in v o k e a p h y s i c a l
o n e , su c h a s t h e m o le c u l a r a t t r a c t i o n s among t h e c o l l o i d
a l p a r t i c l e s o f p r o t e i n a n d t h e m o le c u le s o f a c i d o r b a s e
w h ic h a r e n e u t r a l i z e d . The a d s o r p t i o n o f d i s s o l v e d su b
s t a n c e s by s u r f a c e s i s i t s e l f a n e x p r e s s i o n o f c h e m ic a l
a f f i n i t i e s . T ru e a s t h i s l a s t s t a te m e n t p r o b a b l y i s ,
a d s o r p t i o n c a n b e v a r i e d c o n t i n u o u s l y w i t h i n l i m i t s .
T h is i s n o t w h a t we u s u a l l y m ean by c h e m ic a l r e a c t i o n
i n s t o i c h i o m e t r i c p r o p o r t i o n s .
T h a t t h e s t r u c t u r e o f t h e p r o t e i n m o le c u le c a n n o t
b e i n t e r p r e t e d e n t i r e l y a s t h e l i n k i n g t o g e t h e r o f t h e
c a r b o x y l an d am ino g r o u p s , a f t e r t h e m a n n e r o f E m il F i s c h
e r ' s s y n t h e s i s o f p o l y p e p t i d e s , h a s b e e n know n f o r some
t im e . One r e v i e w b y K L a rm a n ^ o f t h e v a r i o u s p r o p o s a l s
w i t h r e g a r d to p r o t e i n s t r u c t u r e l i s t s 1 26 r e f e r e n c e s
t o t h e c h e m ic a l l i t e r a t u r e , w h ic h s e r v e s to i n d i c a t e t h e
i n t r i c a t e n a t u r e o f t h e s u b j e c t .
9 R o b e r ts o n , C o l l o i d C h e m is tr y , 2 , 25 5 ( 1 9 2 8 ) .
10 K L arm an, Chem. R e v s ., it, 51 (1 9 2 7 )
7
One p o s s i b l e m ove i s to e l i m i n a t e t h e s o l u t i o n
p h a s e e n t i r e l y an d t o w ork w ith a s o l i d (p o w d e re d ) p r o
t e i n a n d a v o l a t i l e a c i d o r b a se * I n so d o in g , t h e d i f
f i c u l t i e s i n h e r e n t i n t r y i n g t o m ake a n a n a l y s i s o f th e
a d s o r p t i o n co&Eplex w hen t h e s o l i d p h a s e i s p e p t i z e d i n a
s o l u t i o n p h a s e w i l l h a v e b e e n e l i m i n a t e d .
S e v e r a l I n v e s t i g a t i o n s u t i l i z i n g t h i s g e n e r a l p r o
c e d u r e w e re u n d e r t a k e n d u r in g t h e p a s t tw e n ty y e a r s , t h e
f i r s t o f t h e s e b e in g m ade by B a n c r o f t a n d Barnett-*--*-.
U pon a d d i t i o n o f HC1 an d NH3 g a s e s t o p r o t e i n s o l i d s ,
t h e y d e te r m in e d t h e am o u n t o f a c i d o r b a s e a d s o r b e d p e r
u n i t m ass o f t h e s o l i d a t v a r i o u s e q u i l i b r i u m p r e s s u r e s .
A n a l y s i s o f t h e i r d a t a y i e l d e d t h e i n f o r m a t i o n t h a t f o r
HC1 a d d i t i o n , g l i a d i n fo rm e d t h r e e c h e m ic a l c o m p o u n d s,
z e i n n o n e , w h ile a r a c h i n , c a s e i n , e d e s t i n , an d f i b r i n
a l l fo rm e d d e f i n i t e co m p o u n d s. Compound f o r m a t i o n w as
n o t o b s e r v e d w i t h a n y o f t h e a b o v e -m e n tio n e d p r o t e i n s
u p o n a d d i t i o n o f N I^ . H ie c r i t e r i o n f o r com pound f o r m a t io n
w as t h e a p p e a ra n c e o f f l a t p o r t i o n s o n t h e a d s o r p t i o n
r u n 's g r a p h i c p l o t .
O th e r i n v e s t i g a t i o n s b y B a n c r o f t a n d B arn ett-* -^ ,
1 1 B a n c r o f t a n d B a r n e t t , P r o c . N a t l . A c a d . S c i . ,
1 6 , 1 1 8 ( 1 9 3 0 ) ; 1 6 , 1 3 ? ( I 93O)
1 2 B a n c r o f t a n d B a r n e t t , J .. P h y s . C hem ., ||? |, W ?
( 1 9 3 0 ) ; -2 ^ 3 3 .(1 9 3 0 )
8
B e ld e n * ^ , P a r k s a n d M e la v e n 1 -1 *, a n d B e e k - 5 - ^ g a v e r e s u l t s
i n a c c o r d w ith t h e i n i t i a l s tu d y o f B a n c r o f t an d B a r n e t t .
1 6
T s a i , h o w e v e r, w o rk in g .w ith h i d e p r o t e i n , r e p o r t e d t h a t
no p r o t e i n com pounds x^ere fo rm e d w i t h e i t h e r HC1 o r NH3 .
I n p a s s i n g , i t w o u ld be i n t e r e s t i n g t o n o t e t h a t
T a f t 1 '7’ 1 ^ , M cC hesney a n d M i l l e r 1 ^ , B i l t z . and R a h l f s 2 0 ’
M cC hesney an d R o b e r t s 2-*-, a n d M i l l e r a n d R o b e r t s 22 h a v e
d o n e some w o rk o n t h e p r o p e r t i e s o f s o l u t i o n s o f p r o t e i n s
i n l i q u i d am m onia. I t w as fo u n d t h a t g e l a t i n a n d eg g
a lb u m in s w e ll s l i g h t l y . G l u t e n i n fo rm s a f a i r d i s p e r -
_ 13 B e ld e n , I 0 6 . c i t .
l^f P a r k s a n d M e la v e n , J . P h y s . C hem ., IjO, *+71
( 1 9 3 6 ) ; l a , 1101 (1 9 3 7 )
15 B e e k , B u r. S ta n d . J . R e s . , 8 , 5^9 (1 9 3 2 )
1 6 T s a i a n d H s ia o , J . C h in . Chem. S o c ., 2 , 87
(1 9 3 ^ )
1 7 T a f t , T r a n s . K a n sa s A c a d . S c i . , 3 2 . 3 8 (1 9 2 9 )
18 T a f t , J . P h y s . C hem ., 2792 ( 1 9 3 0 ) ; 3 5 ,
578 (1 9 3 1 )
19 M cC hesney a n d M i l l e r , J . A m er. Chem. S o c .,
5 3 , 3 8 8 8 (1 9 3 1 )
20 B i l t z an d R a h l f s , Z . A n o rg . A llg e m . C h em ., 1B 7,
313 (1 9 3 1 )
21 M cC hesney an d R o b e r t s , J . A m er. Chem. S o c .,
6 0 , 1 9 3 5 (1 9 3 9 )
22 M i l l e r a n d R o b e r t s , J.. A m er. Chem. S o c ., 5 6 .
9 3 5 (1 9 3 ^ )5 5 8 , 309 ( 1 9 3 6 ) ; 6 1 , 355*+ (1 9 3 9 )
9
s i o n an d g l i a d i n i s r e a d i l y d i s p e r s e d ., No s w e l l i n g o r
d i s p e r s i o n i s fo u n d f o r t h e o t h e r p r o t e i n s .
A c r o s s s e c t i o n o f r e f e r e n c e s f o r s o l u t i o n r e a c t i o n s
i s i n c l u d e d a n d n u m b ere d fro m t w e n t y - t h r e e t o tw e n ty -
n i n e .
23 B r a c e w e l l , J . A m er. Chem. S o c ., O c t. (1 9 1 9 )
2*+ S a n d s tro m , J . P h y s . C hem ., 3*+. 1 0 7 1 (1 9 3 0 )
25" H i t c h c o c k , J . Gen., P h y s i o l . , 1 ^ , 99 (1930)-;:
1 6 , 3 57 ( 1 9 3 2 ) .
26 L lo y d , J ., I n t e r n . S o c . L e a th e r T r a d e s C hem .,
1 Z , 2*+5 (1 9 3 3 )
27 S c h u lz an d E t t i s e h , Z . P h y s i k . C h em ., A l6* fr. 9 7
( 1 9 3 3 ) ; KL6kr h i7 (1 9 3 3 )
28 C a n n a n , Chem. R e v ., 3 0 . 3 9 5 ( 1 9 l+2)
29 C o n r a t an d C o o p e r, J . B i o l . C h em ., I5*f. 239 (19%ig)
CHAPTER I I I
EXPERIMENTAL PROCEDURE
A . PREPARATION OF SAMPLES
The s a m p le s o f eg g a lb u m in u s e d i n t h i s r e s e a r c h
w e re p r e p a r e d by D., A., E l l i s u n d e r t h e g u id a n c e o f D o c to r
J . W. M ehl o f t h e D e p a rtm e n t o f B i o c h e m i s tr y o f The U n iv
e r s i t y o f S o u th e r n C a l i f o r n i a . The f o l l o w i n g d i s c u s s i o n
o n t h e p r e p a r a t i o n o f t h e s a m p le s w as a b s t r a c t e d fro m
E l l i s t h e s i s o n The M e a su re m e n t o f S u r f a c e A r e a s o f
P r o t e i n s .
The p r o t e i n s o l u t i o n s w e re d r i e d i n t h e a p p a r a t
u s show n i n F i g u r e 1 . T h is i s a v a r i a t i o n o f t h e l y o -
p
p h i l i z i n g a p p a r a t u s o f F l o s d o r f a n d M udd.
The s o l u t i o n c o n t a i n i n g t h e s a m p le i s f i r s t f r o z e n
by r o t a t i n g t h e f l a s k i n w h ic h i t i s c o n t a i n e d i n a b a t h
o f e t h e r a n d d r y i c e . The f l a s k i s t h e n a t t a c h e d t o t h e
vacuum l i n e , an d t h e t r a p i s s u i t a b l y c o o le d by im m e rs io n
i n a d e w a r f l a s k c o n t a i n i n g a l o w - t e m p e r a tu r e b a t h .
The s a m p le r e m a in s f r o z e n u n t i l i t i s d r i e d t o a lo w
v a p o r p r e s s u r e o f w a t e r ,d u e t o t h e c o l l i n g e f f e c t o f
e v a p o r a t i o n . The p r o c e s s r e q u i r e s fro m e i g h t t o tw e lv e
h o u r s f o r c o m p le tio n .
1 E l l i s , “The M e a su re m e n t o f S u r f a c e A re a s o f
P r o t e i n s , ” ( u n p u b l is h e d M a s t e r ’ s t h e s i s , The U n i v e r s i t y
o f S o u th e r n C a l i f o r n i a , L os. A n g e le s , l ^ S ) •
11
F i g u r e 1
LYOPHILIZING APPARATUS
t o pump
sa m p le f l& s k
f l a s k
t r a p
" V
The a lb u m in w as i n i t i a l l y o b t a i n e d fro m e g g s an d
w as t h e n c r y s t a l l i z e d fro m ammonium s u l f a t e a f t e r t h e
3 h
m eth o d o f H o p k in s , A d a ir a n d R o b in s o n , a n d T a y l o r ,
A d a i r , an d A d a ir.-* S e v e n c r y s t a l l i z a t i o n s w e re m ade t o
b e s u r e o f a p u r e p r o d u c t . The sa m p le w as t h e n d i a l i z e d
a g a i n s t d e - i o n i z e d w a te r u n t i l i t w as s a l t - f r e e , an d t h e n
l y o p h i l i z e d .
B . PURITY OF SAMPLES
E l l i s ^ h a s t h e f o l l o w i n g t o s a y a b o u t t h e p u r i t y
o f t h e egg a lb u m in p r e p a r e d i n t h i s m an n e rs
1 . X - r a y D i f f r a c t i o n . X - r a y d i f f r a c t i o n p a t t e r n s
w e re t a k e n o n s a m p le s o f b o v in e p la s m a f r a c t i o n V,
c r u d e ; *. • . egg a lb u m in l y o p h i l i z e d fro m a 2% s o l u
t i o n ; « • » .
T h is w o rk w as d o n e th r o u g h t h e c o u r t e s y o f D r.
R . D. V o id an d M r. J . D. G ra n d in e o f t h e D e p a rtm e n t
o f C h e m is tr y , o f t h e U n i v e r s i t y o f S o u th e r n C a l i f
o r n i a
2 F l o s d o r f an d M udd, J . Im m u n o lo g y , 2 9 , 389 (1 9 3 5 )
3 H o p k in s , J . P h y s i o l . , ;2£, 306 (1 9 0 0 )
J+ A d a ir a n d R o b in s o n , B io ch em . J . , 2*K 993 (1 9 3 0 )
5 T a y l o r , A d a i r , a n d A d a i r , J . H y g ., 3 2 , 3^5 (1 9 3 2 )
6 E l l i s , ’’The M e a su re m e n t o f S u r f a c e A r e a s o f
P r o t e i n s , ” ( u n p u b l is h e d D o c t o r 's t h e s i s . The U n i v e r s i t y
o f S o u th e r n C a l i f o r n i a , L o s A n g e le s , 1 9 % ) , p* 7»
13
No p e a k s w e re fo u n d e x c e p t f o r f a i n t p e a k s c o r
r e s p o n d in g t o 1 a n d 2 A s p a c i n g . T h e re w as no e v
i d e n c e o f c r y s t a l l i n e s t r u c t u r e i n a n y o f t h e s a m p le s
e x a m in e d . I d e n t i c a l r e s u l t s w e re o b t a i n e d f o r s a m p le s
a t e q u i l i b r i u m w i t h w a t e r v a p o r i n t h e a i r an d f o r
d r i e d s a m p le s .
2 . E l e c t r o p h o r e s i s p a t t e r n s .
E l e c t r o p h o r e s i s r u n s w e re m ade o n a sa m p le o f egg-
a lb u m in l y o p h i l i z e d fro m 5 *6$ s o l u t i o n and o n . . . .
The r u n s w ere m ade fro m a p e r i o d o f 3 h o u r s a t
pH 8 .I f . T h is pH w as c h o s e n b e c a u s e ^ t h e g ra p h g i v e n
by L o n g s w o rth , c a n n e n , a n d M a c ln n e s ' o f m o b i l i t i e s
v s pH f o r t h e v a r i o u s c o m p o n e n ts o f eg g w h ite show s
t h a t t h e c o m p o n e n ts a r e s e p a r a t e d i n t h i s r e g i o n a s
m uch a s p o s s i b l e .
. . .. The p a t t e r n s o b t a i n e d a r e i d e n t i c a l t o t h o s e
show n b y L o n g s w o rth , C a n n a n , a n d M a c ln n e s f o r eg g
a lb u m in . T h is may b e c o n s i d e r e d a s e v id e n c e t h a t
t h e c o m p o s itio n o f t h e p r o t e i n i s n o t a f f e c t e d b y
t h e d r y i n g p r o c e s s e s u s e d h e r e .
C. SPRAY FREEZING AND DENATURATION
A tw o p e r c e n t s o l u t i o n o f t h i s p r o t e i n w as p r e p a r e d
an d s u b s e q u e n t ly f r o z e n b y s p r a y in g i n t o l i q u i d n i t r o
g e n . The s a m p le f l a s k o f t h e a p p a r a t u s d e p i c t e d i n F i g
u r e 1 w as f i r s t h a l f - f i l l e d w i t h l i q u i d n i t r o g e n . The
s o l u t i o n w as t h e n s p r a y e d i n t o t h e f l a s k w i t h a s m a ll a i r
s p r a y g u n . T h is w as a n a s p i r a t o r w i t h a n o r i f i c e o f
a p p r o x i m a t e ly o n e m .m . d ia m e te r ., The f i n e m i s t o f i s
s u in g p a r t i c l e s w as f r o z e n b y c o o li n g w i t h t h e l i q u i d
7 L o n g s w o rth , C a n n a n , an d M a c ln n e s , J . A m er. Chem.
S o c ., 6 2 , 2580 (1 9 ^ 3 )
lb
n i t r o g e n i n t o a n i r r e g u l a r l y s h a p e d m a s s . The n i t r o g e n
w as t h e n a llo w e d to e v a p o r a t e , an d t h e f l a s k w as c o n n e c te d
t o t h e l y o p h i l i z i n g a p p a r a t u s an d d r i e d i n v a c u o , t h e t r a p
b e in g k e p t i n a b a th o f d r y i c e an d e t h e r . The r e s u l t i n g
sa m p le w as a f i n e w h ite p o w d e r. The w a t e r c o l l e c t e d i n
t h e t r a p show ed o n ly a s l i g h t fo a m in g a c t i o n , i n d i c a t
in g t h a t t h e am ount o f p r o t e i n w h ic h h a d b e e n c a r r i e d
o v e r i n t h e d r y in g p r o c e s s w as v e r y s m a l l .
The egg a lb u m in s a m p le s w e re k e p t i n t i g h t l y c l o s e d ,
s c re w - c a p p e d b o t t l e s a t room t e m p e r a t u r e , w i t h o u t a n y
o t h e r s p e c i a l p r e c a u t i o n s b e in g t a k e n . T h e se w e re c h e c k
ed f o r v a r i a t i o n i n w e ig h t o v e r a p e r i o d o f tw o w e e k s .
T h e re w as a n i n c r e a s e i n w e ig h t o f a b o u t tw o p e r c e n t ,
w i t h o u t d o u b t c a u s e d b y w a te r a d s o r p t i o n fro m t h e a i r .
The p e r c e n t i l e i n c r e a s e i n w e ig h t w as c a l c u l a t e d o n t h e
b a s i s o f w e ig h t o f p r o t e i n r a t h e r t h a n t o t a l w e ig h t o f
s a m p le .
The t o t a l am ount o f w a t e r i n t h e l y o p h i l i z e d sam
p l e s Was d e te r m in e d fro m t h e w e ig h t b e f o r e a n d a f t e r a
_
d a y ’ s p e r i o d o f e v a c u a t i o n a t p r e s s u r e s o f 10 ' m .m . o f
H g. I t h a s b e e n show n t h a t t h e am o u n t o f w a t e r r e m a in in g
o
a f t e r t h i s p e r i o d i s v e r y s m a l l 0 . The p e r c e n t d e c r e a s e
8R .Z w anzig, U n p u b lis h e d w o rk , ( M a s t e r ’ s t h e s i s ,
The U n i v e r s i t y o f S o u th e r n C a l i f o r n i a , L o s A n g e le s , 1 9 5 0 ) .
1 5
was? fo u n d t o b e a b o u t s i x p e r c e n t .
One c r i t e r i o n u s e d t o d e te r m in e w h e th e r o r n o t a
p r o t e i n w as d e n a t u r e d i n t h e s e p r o c e s s e s w as w a te r s o l
u b i l i t y . T h is w as c o n v e n i e n t , s i n c e t h e s a m p le s w e re
a l l w a te r s o l u b l e i n t h e i r n a t u r a l s t a t e , an d becom e
i n s o l u b l e w hen d e n a t u r e d . T h u s, s i n c e t h e f i n a l s a m p le s
o b t a i n e d w e re s t i l l w a te r s o l u b l e , i t w as assu m ed t h a t t h e y
w e re n o t d e n a t u r e d b y t h e a f o r e m e n tio n e d p r o c e s s e s .
D. PURIFICATION OF GASES
The h e liu m a n d n i t r o g e n u s e d w e re p u r i f i e d b y
p a s s a g e th r o u g h th e a d s o r p t i o n t r a i n p i c t u r e d i n F i g u r e
2 . The g a s e s w e re f i r s t d r i e d by p a s s i n g them th r o u g h
a tu b e c o n t a i n i n g b u q e e e d in g l a y e r s o f c a lc iu m c h l o r i d e
an d p h o s p h o r u s p e n t o x i d e . The b l o w - o f f tu b e w as u s e d a s
a s a f e t y v a l v e t o p r e v e n t p r e s s u r e b u i l d - u p i n t h e s y s
tem .
H e liu m w as p u r i f i e d . o f i t s n i t r o g e n an d o x y g e n
c o n t e n t b y s u c c e s s i v e p a s s a g e o v e r a c t i v a t e d c h a r c o a l ,
i n a l i q u i d n i t r o g e n b a t h , an d t h e n o v e r c o p p e r h e a t e d '
t o **00°C.
N i t r o g e n w as r e n d e r e d p u r e b y p a s s a g e o v e r h e a t e d
c o p p e r .
The c o m m e rc ia l g r a d e h y d ro g e n c h l o r i d e w as p u r i f
i e d i n a t r a i n s i m i l a r t o t h a t d e p i c t e d i n F i g u r e 3»
16
F i g u r e 2
GAS PURIFICATION TRAIN
t o s t o r a g e b u lb
fro m t a n k
CaCl-
PoO
2U5
■€r
1 U
d r y in g
tu b e
1
a c t i v a t e d
c h a r c o a l
c o p p e r
b l o w - o f f
tu b e
17
F ig u re 3
POLAR GASES PURIFICATION TRAIN
to vacuum l i n e
and b u lb s
in ta k e
t r a p s
e t h e r _
d r y - i c e
n i t r o g e n ( l )
The g a s w as a llo w e d t o e n t e r t h e s y s te m th r o u g h t h e b lo w -
o f f tu b e a t a p r e s s u r e o f o n e a tm o s p h e r e . Any w a t e r
p r e s e n t w as f r o z e n i n t h e d r y i c e , e t h e r b a t h . A l l
d i f f i c u l t l y c o n d e n s a b le g a s e s , s u c h a s n i t r o g e n a n d o x y
g e n , w e re e l i m i n a t e d by f r e e z i n g t h e HC1 i n l i q u i d n i t r o
g e n a n d s u b s e q u e n t ly e v a c u a t in g t h e s y s te m . A t a l l t i m e s ,
a t r a p n e a r t h e vacuum pum ps w as c o o le d by a d e w a r f i l l e d
w i t h l i q u i d n i t r o g e n t o g u a rd a g a i n s t a n y p o s s i b l e m is
h a p s . The HC1 w as d i s t i l l e d a fe w tim e s fro m t r a p to
t r a p w i t h s u b s e q u e n t e v a c u a t i o n s t o m ake c e r t a i n t h a t
a l l i m p u r i t i e s w e re re m o v e d .
A f i n a l c h e c k o f t h e p u r i t y o f t h e HC1 w as o b t a i n
ed b y c o m p a rin g t h e v a p o r p r e s s u r e o f t h i s g a s w i t h t h a t
o f a n HG1 th e r m o m e te r , b y i n s e r t i o n o f b o th i n t o t h e
sam e CS2 s l u s h . The v a p o r p r e s s u r e r e a d i n g s fro m b o th
c h e c k e d t o w i t h i n o n e m .m . Hg.
The am m onia w as p u r i f i e d i n a n a n a lo g o u s m a n n e r.
E v e ry fe w w e e k s , t h e s t o r a g e b u l b s c o n t a i n i n g t h e s e
g a s e s w e re e v a c u a t e d , w i t h t h e g a s e s f r o z e n i n t h e s i d e -
arm s ( F i g . *+ ) b y l i q u i d n i t r o g e n , t o a s s u r e t h e e l i m i n
a t i o n o f a n y a i r t h a t m ig h t h a v e l e a k e d i n t o t h e c o n t a i n
e r s d u r in g t h i s p e r i o d .
19
g as
s to r a g e
b u lb s
ADSORPTION
APPARATUS
6 .
—=_*.to g a s
p u r i f i c a t i o n t r a i n
pumps
gauge
and
t r a p s
sam ple tu b e d e t a i l
b u r e t t e
manome-
t e r
c a p i l l a r y
ground j o i n t
sam ple^-
tu b e
le v a r i
temp
v a p o r p r e s s
u re therm o
m e te r b u lb
g l a s s TT
b u lb U
sam plaTA
tu b e —
g l a s s
p o i n t
v a p o r p r e s s u r e
th erm p m eter
to a i r l i n e
to vacuum l i n e
20
E*. DESCRIPTION AND OPERATION OF APPARATUS
F i g u r e ^ i s a r e p r e s e n t a t i o n o f t h e a d s o r p t i o n
a p p a r a t u s u t i l i z e d d u r i n g t h i s e x p e r im e n ta tio n * I t i s
Q IQ
a m o d i f i c a t i o n o f o n e by P e a s e * ’
The g a s e s a r e k e p t i n t h e b u l b s , a n d w hen i t i s
d e s i r e d t o e l i m i n a t e a n y p o s s i b l e a i r i n f i l t r a t i o n , t h e
g a s e s a r e f r o z e n o u t i n t h e s id e - a r m t r a p s w ith l i q u i d
n i t r o g e n , an d s to p c o c k s o n e a n d f o u r a r e t h e n o p e n e d .
T h is p r o c e s s i s c o n ti n u e d u n t i l t h e p r e s s u r e i n t h e vacuum
l i n e d e s c e n d s t o 1 0 “ - * m .m . H g. The g a s i s t h e n r e a d y f o r
u s e .
The b u r e tte - m a n o m e te r i s a d e v i c e d e s ig n e d t o d e
te r m in e t h e am ount o f g a s a d m itte d t o t h e s y s te m , an d
t h e p r e s s u r e o f g a s i n c o n t a c t w i t h t h e s a m p le . The v o l
um es b e tw e e n t h e g l a s s p o i n t s w e re a c c u r a t e l y c a l i b r a t e d
b y m ean s o f d i s p l a c e m e n t o f m e r c u r y . The v o lu m e fro m
p o i n t o n e th r o u g h t h e s to p c o c k b o r e a l s o w as a s c e r t a i n
e d . The p o i n t s w e re p l a c e d a t a p p r o x i m a t e ly 1 , 2 , 3 ,
5 , 1 0 , 2 0 , an d 30 c . c . fro m t h e s t o p c o c k b o re * The c o r
r e c t i o n s fo u n d t o be a p p l i c a b l e a r e i n c l u d e d i n T a b le I*
9 P e a s e , J . A m er. Chem. S o c ., 1 1 7 6 (1 9 2 3 )
10 B r u n a u e r , The A d s o r p ti o n o f G a se s an d V a p o rs
( P r i n c e t o n : P r i n c e t o n U n i v e r s i t y P r e s s , ' 1 9 4 3 ) , P - 32*
21
TABLE I
CALIBRATION OF BURETTE
B u r e t t e
P o i n t ( c . c . )
C o r r e c t i o n
( c . c . )
B u r e t t e
P o i n t ( c . c . )
C o r r e c t i o n
( c . c . )
1 + 0 .2 0 10 +0 .0 3
2 + 0 .0 6 20 + 0 .1 0
3
- 0 .0 9 30 + 0 .1 2
*
+ 0 .1 7
I n o r d e r t o b e a b s o l u t e l y c e r t a i n o f t h e a c c u r a c y
o f t h e s e r e s u l t s , t h e i n t e r n a l c o n s i s t e n c y o f t h e a p p a r
a t u s w as t e s t e d . A d e f i n i t e am o u n t o f h e liu m w as a d m itte d
t o t h e s y s te m b y o p e n in g s to p c o c k s tw o an d n i n e . S to p
c o c k s e v e n i s o p e n t o t h e vacuum l i n e . S to p c o c k n i n e - ,
i s t h e n c l o s e d , a n d m e a s u re m e n ts o f p r e s s u r e , b y m eans
o f a c a t h e t o m e t e r , a r e m ade a t e a c h o f t h e v o lu m e p o i n t s .
A c o r r e c t i o n i s m ade f o r t h e b o r e v o lu m e . A ssu m in g h e l
ium t o a c t a s a p e r f e c t g a s , s e v e r a l s e t s o f PV v a l u e s
w e re o b t a i n e d . A c o n s t a n t r e s u l t w o u ld a s s u r e a c c u r a t e
v o ljp ie m e a s u re m e n ts . T h e se d a t a may^ be fo u n d i n T a b le I I .
W ith t h i s a p p a r a t u s , a n am o u n t o f g a s m ay b e a d
m i t t e d t o t h e l e v e l o f a n y o n e o f t h e g l a s s p o i n t s , an d
t h e p r e s s u r e m e a s u r e d . K now ledge o f t h e PVT r e l a t i o n s
o f t h e g a s i n q u e s t i o n w i l l a ll o w a c a l c u l a t i o n o f t h e
am o u n t a d m i t te d . The b u r e t t e t e m p e r a t u r e i s m a i n t a i n e d
22
TABLE II
INTERNAL CONSISTENCY
P o i n t PVa v .
PV fa D e v i a t io n
fro m PVav#
1 190 190 0 .0
2 19*+
2 .1
3 1 9 3
1 .6
5
190> 0 .0
5 189 0 . 5
10
1 8 7
1 .6
5 1 89
0 . 5
3 191 0 . 5
2 190 0 . 0
2 191 0 . 5
1 190 0 .0
1
2
3 ^ 7 351
3 ^ 5
1 .1
0 . 5
3 0 .9
5
3^8
0 .3
10
3 ^ 5 0 . 5
5 3*+5 0 . 5
3
3 ^8
0 .3
2
3 ^ 7
0 .0 .
10 210 212 1 .0
210 0 .0
3
210 0 .0
2 211
o . 5
1
209 0 . 5
10
209
0 . 5
5
210 0 .0
3
210 0 . 0
2 210 0 .0
1 211
0 . 5
30
88*t 882 0 . 2
20 8 8 ^ 0 .0
10 88b 0 .0
5
88b 0 .0
30 88b 0 .0
23
c o n s t a n t b y m eans o f a w a te r b a t h . The l e v e l o f t h e m er
c u r y co lu m n i n t h e b u r e t t e m ay b e c o n t r o l l e d b y m eans o f
t h e s to p c o c k s t e n an d e l e v e n . I f e l e v e n i s c o n n e c te d to
t h e vacuum l i n e , t h e l e v e l w i l l d e s c e n d , an d v i c e - v e r s a .
O nce t h e am ount o f g a s a d m itte d h a s b e e n m e a s u re d ,
i t may b e b r o u g h t i n t o c o n t a c t w i t h t h e s a m p le b y t u r n
i n g s to p c o c k n i n e to w a rd s l i n e A.
The te m p e r a t u r e o f t h e sa m p le m ay b e m e a s u re d b y
e i t h e r a l i q u i d th e rm o m e te r im m e rsed i n t h e te m p e r a t u r e
b a t h o r b y a v a p o r p r e s s u r e th e rm o m e te r f i l l e d w i t h a n
a p p r o p r i a t e g a s . T h is p r o c e d u r e n a t u r a l l y a ssu m e s t h a t
t h e t e m p e r a t u r e o f t h e s a m p le i s t h e sam e a s t h a t o f t h e
s u r r o u n d in g b a t h .
. I n o r d e r t o d e te r m in e t h e t e m p e r a t u r e r i s e u p o n
a d d i t i o n o f HC1 an d NH3 g a s e s , a sa m p le tu b e w as e q u ip p e d
w i t h a th e r m o c o u p le s t i c k i n g up i n t o t h e p r o t e i n . The
th e r m o c o u p le w as m ade o f c o p p e r an d c o n s t a n t i n . F i g u r e
5 i s a d ia g r a m o f t h i s p o r t i o n o f t h e a p p a r a t u s .
As w i l l b e d i s c u s s e d i n m ore d e t a i l l a t e r , i t w as
fo u n d t h a t t h e r e w as b o th a l a r g e t e m p e r a t u r e r i s e , an d
a v e r y s h o r t tim e l a g t h e r e a f t e r , t o a t t a i n te m p e r a t u r e
e q u i l i b r i u m w ith t h e s u r r o u n d in g b a t h .
T e s t s w e re m ade t o d e te r m in e w h e th e r a g ro u n d -
24
SAMPLE TUBS WITH THERMOCOUPLE
p r o t e i n .
th e rm o c o u p le
, 25
g l a s s j o i n t , l u b r i c a t e d w ith g r e a s e (A p ie z o n L ) , w o u ld
h o ld a vacuum u n d e r c o n d i t i o n s o f l i q u i d n i t r o g e n tem p
e r a t u r e . The s y s te m w as pum ped u p o n f o r s e v e r a l h o u r s .
The p r e s s u r e w as m e a s u re d , an d t h e n a d e w a r f l a s k o f l i q u i d
n i t r o g e n w as p l a c e d o v e r t h e g r o u n d - j o i n t . A f t e r k e e p in g
t h e j o i n t su b m e rg e d f o r tw o h o u r s , t h e p r e s s u r e w as a -
g a i n m e a s u re d . No d e v i a t i o n w a s' d e t e c t e d . ^
A t y p i c a l a d s o r p t i o n r u n w i l l b e d i s c u s s e d i n t h e
n e x t s e c t i o n .
F . ADSORPTION RUNS AND CALCULATIONS
The f i r s t s t e p i n m a k in g a r u n i s t o w e ig h t h e
sa m p le tu b e a n d g l s s b u lb . The p r o t e i n i s t h e n p l a c e d
i n t h e tu b e an d e v a c u a te d f o r a t l e a s t a d a y , t o e lim
i n a t e an y a d s o r b e d w a te r o r o t h e r g a s e s . The tu b e i s t h e n
w e ig h e d , a n d t h e w e ig h t o f p r o t e i n d e d u c e d fro m t h e s e
d a t a .
B e f o re m a k in g a r u n p r o p e r , t h e s p a c e b e tw e e n
p o i n t o n e an d t h e sa m p le tu b e n o t o c c u p ie d b y p r o t e i n
m u s t b e d e te r m i n e d . I t i s a l s o d e s i r e d t o f i n d t h e . d i
v i s i o n o f t h i s v o lu m e i n t o lo w t e m p e r a t u r e ( t h a t i n t h e
l i q u i d n i t r o g e n o r o t h e r c o f tla n t) a n d room t e m p e r a t u r e
p a r t s . T h is i s a c c o m p lis h e d i n t h e f o l l o w i n g m a n n e r:
a . H e liu m i s a d m i t te d t o t h e s y s te m t h r o u g h s to p c o c k s
1 1 'E l l i s , M a s t e r 's T h e s i s .
26
tw o an d n i n e . N in e i s t h e n c l o s e d , an d t h e am o u n t o f
h e liu m p r e s e n t i s m e a s u r e d .
b . The sa m p le i s t h e n e x p o s e d t o t h e g a s b y t u r n i n g
s to p c o c k n i n e to w a rd s l i n e A, an d t h e m e r c u r y l e v e l i s
r a i s e d t o p o i n t o n e . T h is i s a l l d o n e a t room t e m p e r a t
u r e . The k n o w le d g e o f a m o u n t o f g a s , t e m p e r a t u r e , p r e s
s u r e , fro m m a n o m e te r r e a d i n g , e n a b l e s o n e t o c a l c u l a t e
t h e v o lu m e a s s o c i a t e d .
c . A d e w a r f l a s k o f l i q u i d n i t r o g e n i s p l a c e d o v e r t h e
sa m p le t u b e ; t h e . l e v e l i s a d j u s t e d to t h e o n e c . c . m a rk ,
an d r e a d i n g s a r e t a k e n o f t e m p e r a t u r e an d p r e s s u r e .
From t h e s e o b s e r v a t i o n s , t h e d e a d s p a c e ( t h i s v o l
ume i s so c a l l e d ) a t room t e m p e r a t u r e , a n d t h e d e a d s p a c e
a t lo w t e m p e r a t u r e c a n b e c a l c u l a t e d , a s s u m in g t h a t h e
liu m b e h a v e s a s a n i d e a l g a s . The a c t u a l c a l c u l a t i o n i s
a s f o l l o w s :
P b - o r i g i n a l b u r e t t e p r e s s u r e
- o r i g i n a l b u r e t t e v o lu m e
T^ - b u r e t t e t e m p e r a t u r e
P - — p r e s s u r e a t room t e m p e r a t u r e a f t e r h e liu m i s a d -
^ m i t t e d t o t h e sa m p le
Tr - room t e m p e r a t u r e
T^ - lo w t e m p e r a t u r e
- t o t a l d e a d s p a c e
27
Vd r t - d e a d s p a c e a t room te m p e r a t u r e
V d it “ d e a d s p a c e a t lo w t e m p e r a t u r e
PSl - p r e s s u r e a f t e r a p p l i c a t i o n o f l i q u i d n i t r o g e n b a t h
PbVb » PSr ^ d Vd . VfoPb^r
Tb Tr P'SR^b
PbVb = vdrtpSl + VdltPSl Vd r vdrt * Vdlt
T b ’ Tr TX
PbVb = V d r tP g i ♦ VdP S l - v d r t p S I
Tb Tr Tx TX
vd r t * 2 c £ l [ 2 d * * M b ]
Tr - Tl LT 1 TbPSxJ
A. s a m p le c a l c u l a t i o n s h e e t f o r t h i s t y p e o f w ork
m ay b e fo u n d a s T a b le I I I , P I t a n d P r t i n d i c a t e d i r e c t
r e a d i n g s o f b o t h s i d e s o f t h e m a n o m e te r o n a c a t h e t o m e t e r
s c a l e . C o rr i s t h e c o r r e c t i o n m ade f o r t h e c a t h e t o m e t e r
s c a l e e x p a n s io n . The t a b l e f o r t h e s e c o r r e c t i o n s w as
t a k e n fro m a W e a th e r B u re a u c i r c u l a r . ^
A t y p i c a l HC1 r u n m ay b e d e s c r i b e d a s f o llo w s ';
a . The w e ig h e d sa m p le i s e v a c u a te d f o r a t l e a s t t h r e e
d a y s , a s t h e a d s o r p t i o n c h a r a c t e r i s t i c s a r e s e n s i t i v e
t o e x tr e m e ly s m a ll a m o u n ts o f a d s o r b e d g a s o n t h e p r o
t e i n s u r f a c e .
b . A s m a ll am o u n t o f HC1 i s l e t i n t o t h e b u r e t t e an d
1 2 U. S . W e a th e r B u re a u C i r c u l a r F,. ( 1 9 * 4 - 1 )
TABLE O X
B. 28
before a fte r Date ___________
Run M o .________________ Wt, S | B _____ ____ _
V b lu b _______ _
Sample ___ ; Wt. B ____________ _
V d _________
P r e p .___________________ Wt. S_______ __________ _ _ _ _ _ _
V d rt _________
Gas _______
d e n sity ___ V d l t __________
Bath _ ____
bur s rt 3 I t bur s r t s I t bur s r t s I t bur s r t s I t
Tc vp
atm
T A
\
'
PTE----------
P r t
A P
eo rr
P
V bur
V b-cor
(1)
PbVb
(2) ~ 1
(1 )/P s r
(3 )
Tr/Tb
U )
¥d 2x3
(5 )
PslTb
(6)
1/5
(7 )
Vd/Tl
(8)
7 -6
(9)
TrTl
(10)
T r-T l
(11)
9 /1 0
(12)
Vdrt
8x11
'
(13)
V dlt
A-12
_ — _________ .. .............................................
J i
29
t h e w e ig h t c a l c u l a t e d fro m know n PVT r e l a t i o n s .
c . The HC1 i s a llo w e d to come i n t o c o n t a c t w ith t h e
s a m p le , an d t h e l e v d l i s b r o u g h t u p t o t h e o n e c . c . m a rk .
S u f f i c i e n t tim e i s a llo w e d f o r e q u i l i b r i u m t o t a k e p l a c e .
I n c e r t a i n c a s e s , t h i s m ay i n v o l v e a p e r i o d o f o v e r tw e lv e
h o u r s . V a lu e s o f P a n d T a r e r e c o r d e d .
W ith t h e s e d a t a , I t i s p o s s i b l e t o c a l c u l a t e t h e
am o u n t o f HC1 a d s o r b e d b y t h e sa m p le u n d e r t h e p a r t i c u l a r
e q u i l i b r i u m p r e s s u r e . S in c e t h e i n i t i a l am o u n t a d d e d i s
\
k n o w n , a lo n g w ith t h e v o lu m e s , p r e s s u r e , an d t e m p e r a t u r e
o f t h e s p a c e s u r r o u n d in g t h e p r o t e i n , t h e w e ig h t o f g a s
i n t h e s p a c e a ro u n d t h e p r o t e i n m ay b e c a l c u l a t e d . I t
i s a s im p le m a t t e r o f s u b t r a c t i o n t o d e te r m in e t h e a d
s o r b e d a m o u n t.
d'. The m e rc u ry c o lu m n i s r a i s e d so a s t o p u s h t h e HC1
t h r o u g h t h e s to p c o c k b o r e . >
e . M ore HC1 i s a d d e d .
The t o t a l HC1 a d d e d i s t h e sum o f t h e s e tw o i n i t i a l
q u a n t i t i e s , an d a n a lo g o u s c a l c u l a t i o n s may b e m ade u n t i l
a s h i g h a p r e s s u r e a s d e s i r e d i s a t t a i n e d .
I n o r d e r t o e f f e c t t h e s e c a l c u l a t i o n s , t h e B e r t h -
e l o t e q u a t i o n w as a ssu m e d t o h o l d f o r t h e g a s e s u s e d .
The e q u a t i o n h a s t h e f o l l o w i n g fo rm s
PV s gRT [ l ♦ 9 PT c ( l - 6 I § \ 1
M [_ 1 2 8 P c T V T 2 /J
30
o r
mg = 1000 (PV) 1 / 1 + 9 PTC
1 2 8 P CT
F o r e a s e o f c a l c u l a t i o n , t h e f o l l o w i n g f a c t o r s
w e re e v a l u a t e d a t v a r i o u s t e m p e r a t u r e s a n d p r e s s u r e s an d
t a b l e s p r e p a r e d .
HCls P c - 6300 cm. Hg ; Tc - 3 2 ^ .7 ° K .
N H y P c - 8500 cm . Hg ; Tc - ^ 5 . 7 ° K .
R uns f o r NH^ ani N2 w e re c a l c u l a t e d i n a n a n a lo g
o u s m a n n e r . A sa m p le c a l c u l a t i o n s h e e t i s t o be fo u n d a s
T a b le IV .
F o r e a c h r u n , a g r a p h o f m ass a d s o r b e d p e r gram
o f p r o t e i n w as p l o t t e d a g a i n s t p r e s s u r e . I t w as fo u n d
13 I n t e r n a t i o n a l C r i t i c a l T a b le s (New Y ork an d
L o n d o n : M c G ra w -H ill B ook Com pany, 1 9 2 6 ) •
l*f L a n d o l t - B o r n s t e i n ( B e r l i n : J . S p r i n g e r , 1 9 2 3 )-
1 5 L a n g e , H an d b o o k o f C h e m is try ( S a n d u s k y , O h io :
H andbook P u b l i s h e r s , I n c . , 19^+6) •
f 2 1 / 1 * 9 P T c f1 “ 6T%\
1 2 8 P CT \ ~T2)
mg = (PV)(fi)Cf2)(L000)
An a v e r a g e o f v a l u e s t a k e n fro m I . C . T l ^ B o r n s te in -
an d L a n g e 1 s H andbook-*^, y i e l d e d t h e f o l l o w i n g r e s u l t s :
x a u u i u , j. v
A.
Run No______ ____U t.____ Vdrt_______ Vdlt_______ 3 1
T bur
P I t
P r t
A P
corr
A Pcorr
atm
P
V bur
V b-cor
PV
f a c t
cc stp
cc to t
T"
T rm
•
vap p
atm
atm vp
T low
P I t
P r t
A P
c o rr
A Pcorr
atm
P
r fa c t
Vr x r f
c cr t
1 fa c t
VI x I f
c e l t
a Inha
c e l t cor
cc ads
cc ads/s:
t
Po
1/P o
P/Po
Po-P
P7(Po-P
1/V
( )/V
l / v 2
1
t h a t a I»angm uir-* * 6 p l o t te n d e d t o c o r r e l a t e t h e d a t a c o n
s i d e r a b l y , an d t h e r e f o r e , g r a p h s o f P /V v s . P w e re a l s o
m a d e . S u r f a c e a r e a s w e re c a l c u l a t e d fro m t h i s p l o t ,
a lo n g w i t h h e a t s o f a d s o r p t i o n , a s f o llo w s *
B r u n a u e r , E m m ett, a n d T e lle r-* -^>-^>-*-9 h a v e d e r i v e d
t h i s e q u a t i o n a s s u m in g e i t h e r u n i m o l e c u l a r a d s o r p t i o n
an d a l a r g e v a l u e o f c , o r m u l t i m o l e c u l a r a d s o r p t i o n
w i t h s m a ll v a l u e s o f P a n d l a r g e o n e s o f c .
P = PQ v P_
V V mc Vm
P Q - s a t u r a t i o n p r e s s u r e o f t h e g a s a t t h e g i v e n tem p
e r a t u r e
E i - F i r s t l a y e r h e a t o f r e a c t i o n
c - e ^ q = E i- E ^ Ej, - H e a t o f r e a c t i o n o n a l l
o t h e r l a y e r s
V m - v o lu m e o f a u n i m o l e c u l a r l a y e r o f g a s
a n d V m c a n b e fo u n d g r a p h i c a l l y , a n d t h e s u r f a c e
a r e a c a n b e fo u n d fro m t h i s v a l u e o f Ym m
I f i t b e a ssu m e d t h a t t h e m o le c u l e s o n t h e s u r f a c e
a r e p a e k e n i n t h e sam e m a n n e r a s m o le c u l e s i n t h e l i q u i d ,
a t t h e sam e t e m p e r a t u r e , t h e a r e a c o v e r e d p e r m o le c u le
1 6 B r u n a u e r , op?, c i t . , p . 6 *f.
1 7 B r u n a u e r , E n m e tt, an d T e l l e r , J . A m er. Chem.
S o c ., 6 0 , 309 (1 9 3 8 )
1 8 B r u n a u e r , D em ing, D em ing, a n d T e l l e r , I b i d . ,
6 2 , 1 7 2 3 ( 1 9 ^ )
w i l l b e :
A = / n _ \ 2 /3
\ m j
M — m o le c u l a r w e ig h t
N - A v o g a d ro 1s n u m b er
d - l i q u i d d e n s i t y
T h is i s t h e sam e a s s im p le c u b ic p a c k in g o f t h e m o le c u l e s .
S in c e t h e num ber o f m o le c u le s f o r m in g a u n i m o l e c u l a r l a y e r
c a n b e c a l c u l a t e d , t h e t o t a l s u r f a c e a r e a p e r gram o f p r o
t e i n c a n b e d e d u c e d .
The f o l l o w i n g v a l u e s o f d w e re t a k e n fro m t h e I . C . T .
t a b l e s : :
HC1: 0 .9 2 3 - 0 . 0 0 3 l 8 t g m s . / c . c . (-1 0 * f t o - 8 3 ° C .)
O.6 3 8 6 - 0 . 0 0 1 3 ^ g m s . / c . c . ( - 7 8 t o - 3 3 ° C .)
The n i t r o g e n r u n s w e re c a l c u l a t e d a s s u m in g t h e
2 1
B r u n a u e r , E m m ett, a n d T e l l e r e x p r e s s i o n f o r m u lt i m o l -
e c u l a r a d s o r p t i o n , a s w e re t h e h i g h p r e s s u r e HC1 r u n s .
20 I n t e r n a t i o n a l C r i t i c a l T a b l e s , o n . c i t
21 B r u n a u e r , o p . c i t . , p . 151,
3 1 ! -
G. TEST RUNS
E l l i s ^ m ade a s e r i e s o f t e s t r u n s om egg a lb u m in
w ith n i t r o g e n , an d cam e t o t h e f o l l o w i n g c o n c l u s i o n s :
1 . The v o lu m e s a d s o r b e d by t h e a p p a r a t u s w a l l s w e re
l e s s t h a n o n e p e r c e n t o f t h e t o t a l v o lu m e o f g a s a d d e d
t o a n e m p ty sa m p le t u b e .
2 . The t y p e o f p a c k in g a n d t h e am o u n t o f p r o t e i n i n
t h e s a m p le t u b e h a s l i t t l e e f f e c t u p o n t h e s p e c i f i c s u r f a c e
a r e a o f t h e p r o t e i n .
B la n k r u n s m ade w i t h HC1 an d NH3 g a s e s w ith em p ty
sa m p le t u b e s g a v e t h e sam e r e s u l t s . No t e s t s w e re m ade
w ith t h e s e g a s e s t o d e te r m i n e t h e e f f e c t o f p a c k in g .
22 E l l i s , o n . c i t .
CHAPTER IV
DATA 7
A* DESRIPTION OP ADSORPTION RUNS
The: a d s o r b e n t u s e d i n a l l c a s e s w as p o w d e re d eg g
a lb u m in p r e p a r e d i n M ay, 1 9 I +8 by E llis-* -. The m a t e r i a l
u s e d i n r u n s o n e , tw o , a n d f o u r w as s p r a y f r o z e n i n t o l i q
u i d n i t r o g e n fro m a tw o p e r c e n t s o l u t i o n .. R un t h r e e
u t i l i z e d p r o t e i n d i r e c t l y f r o z e n fro m a 5*6 p e r c e n t
s o l u t i o n , b y im m e rs io n i n a n a c e t o n e - c a r b o n d i o x i d e s l u s h .
I t i s o f i n t e r e s t t o n o t e t h a t E l l i s fo u n d t h a t t h e n i
t r o g e n a r e a s o f t h e p r o t e i n s p r e p a r e d fro m t h e sam e b a tc h
d e c r e a s e d w i t h a g e a t t h e r a t e o f a p p r o x i m a t e ly 0 . 3 m2/m o n th .
T h is i s f u r t h e r s t r e n g t h e n e d b y t h e r e i s u l t o o f r u n t h r e e .
The r u n w as m ade f i v e m o n th s a f t e r E l l i s fo u n d a s u r f a c e
a r e a o f 6 .0 m2/g m . p r o t e i n , a n d show s a d e c r e a s e o f t h e
sam e o r d e r a s E l l i s ' . R un o n e , w h ic h w as m ade a t t h e
sam e tim e a s tw o o f E l l i s ' , c h e c k s f a v o r a b l e w ith h i s
v a l u e s , t h e a v e r a g e o f w h ic h w as 1 2 .2 m ^/gm . The v a r
i a t i o n o f area„s i n r u n s o n e , tw o , a n d f o u r i s m o s t p r o b - .
a b l y d u e t o v a r i a t i o n s i n t h e s p r a y i n g p r o c e d u r e . The
a r e a a p p e a re d t o be v e r y s e n s i t i v e t o t h e a i r p r e s s u r e
u s e d i n s p r a y i n g . T hese, r u n s w e re m ade a t d i f f e r e n t t i m e s ,
1 E l l i s , I b i d .
36
o f c o u r s e , a s t h e p r o t e i n s w e re n e e d e d . R uns f i v e t o
e i g h t e e n w e re m ade o n o n e o f t h e f o u r s a m p le s , t h e n i t r o
g e n a r e a t h u s b e in g know n f o r e a c h r u n . . The B .E .T . ( n i
tr o g e n ) a r e a i s t h a t m e a s u re d b y m eans o f n i t r o g e n a d s o r p
t i o n , u s i n g t h e B .E .T . m u l t i m o l e c u l a r a d s o r p t i o n t h e o r y
t o t h e c a l c u l a t e t h e s u r f a c e a r e a s . T a b le V ,' w h ic h f o l l o w s ,
g i v e s a c o n c i s e p i c t u r e o f e a c h a d s o r p t i o n r u n . A ll a r e a s
a n d h e a t v a l u e s a r e c a l c u l a t e d a s su m in g t h e v a l i d i t y o f
t h e B .E .T . t h e o r y . The c o lu m n l a b e l e d R an g e g i v e s t h e
u p p e r l i m i t i n cm. Hg o f t h e l i n e a r p o r t i o n o f t h e g r a p h
p l o t t e d f o r t h a t r u n .
TABLE V
G rap h G as
(R un)
Tem p. B .E .T .
a r e a
m2/g m .
HC1 o r Ex- E l
HH^a r e a c a l / m o l
m2/ffm .
R an g e R em arks
cm .Hg
i
n 2
n2H T 1 2
59 5
2 n2 w2 Ci)
21 618
3
n2 N2 (1 )
5 .1 6 3 5
h n2 t2 i
ro
H
V-■ '
11 52*f
5
bh. rm . 21 1 ^ 2 3 ,3 2 0
7
1 d a y e v a c .
( 2 1 °C . )
6 NH3 rm . 21 1 08 3 ,6 8 0 b 1 d a y e v a c .
7
nh3 -3 ¥ > C . 11 25*f 2 ,3 3 0
3
8 HC1 rm.. 12 2 76 5,180 3 d a y e v a c .
9
HC1 rm. 21 216 6,1**0 .6 3 d a y e v a c .
TABLE V ( e o n t . )
37
G rap h
(R un)
Gas Temp. B .E .T . HC1 o r
a r e a NH3 a r e a
m ^/gm . m2/g m .
Ei -E L R ange
c a l /m o l cm.Hg
R em ark s
10 HC1 rm . 21 1 6 2 T 7 5 2 5 .. . 2 1 d a y e v a c .
11 HC1 rm . 21
1 8 5
6 ,2 5 0 .6 3 h r s . e v a c .
1 2 HC1 -80O G . 21 1 ,1 6 0 1 ,9 0 0 h
1 3
HC1 -8 0 ° C .
5 .1
1 ,7 8 0
2 ,6 0 0
9 5 5
785
60 B .E .T . p l o t
L a n g m u ir
1 ^ HC1 - 8 0 ° C . 21 2 ,2 1 0
l , l f 60
1 ,^ 3 0 -
1 ,5 5 0
16 B .E .T . p l o t
L a n g m u ir
R un m ade a f t e r
e v a c . o f r u n
1 2 sa m p le
1 5
HC1 rm . 1 2 11 6 5 ,2 5 0 1 R un m ade a f t e r
i n i t i a l HC1
r u n 8
1 6 HC1 rm . 21 1 5 1
5 , 1 ^ 2. Made a f t e r
HC1 r u n
1 7
HC1 rm .
5 .1
221 it, 500 If R un m ade a f t e r
HC1 r u n o f 13
1 8 HC1 rm . 21 260 5 ,6 0 0
. 7
R un m ade a f t e r
1 d a y e v a c . o f
NH3 r u n 5
B . PROTEIN DENATURATI0 N
I n o r d e r t o d e te r m i n e i>f t h e p r o t e i n s w e re d e n a t
u r e d a f t e r t h e r u n s , s o l u b i l i t y t e s t s w e re m ade i n v a r i o u s
s o l u t i o n s . I t w as fo u n d t h a t a l l p r o t e i n s w h ic h h a d u n -
38
d e r g o n e NH3 r u n s w e re s t i l l s o l u b l e i n n e u t r a l s o l u t i o n ;
t h e HC1 r u n s y i e l d i n g i n s o l u b l e m a t e r i a l . A l l t e s t s o n
u n r e a c t e d p r o t e i n an d t h a t t a k e n fro m NH3 r u n s f i e l d t h e
f o l l o w i n g d a t a :
1 . S o lu b le i n w a t e r , .1M HC1, a n d 10M NaOH.
2 . I n s o l u b l e i n 10M HC1.
3 . The a d d i t i o n o f 10M HC1 t o a s o l u t i o n o f p r o t e i n
i n 10 M NaOH c a u s e s p r e c i p i t a t i o n a n d a l a r g e h e a t
r i s e ; h e a t i n g a lo n e d o e s n o t c a u s e p r e c i p i t a t i o n i n
a 10M NaOH s o l u t i o n o f t h e p r o t e i n .
F o r t h e p r o t e i n s r e a c t e d w ith HC1:
1 . S o lu b le i n . 1M a n d 10M NaOH.
2 . I n s o l u b l e i n w a t e r , . 1 , a n d 10M HC1.
T h ese d a t a i n d i c a t e t h a t t h e a d d i t i o n o f HC1 t o t h e
p r o t e i n i n t h e s o l i d s t a t e c a u s e s a r e a r r a n g e m e n t o f some
s o r t , o r t h e f o r m a t i o n o f some com pounds w h ic h lo w e r t h e
s o l u b i l i t y o f t h e p r o t e i n i n w a t e r a n d d i l u t e HC1. T h a t
t h i s lo w e re d s o l u b i l i t y c o u ld be c a u s e d b y t h e s l i g h t
a c i d i t y o f t h e s o l u t i o n i s r e n d e r e d u n t e n a b l e b y t h e s o l
u b i l i t y o f t h e m n r e a c te d p r o t e i n i n d i l u t e HC1.
I n o r d e r t o f i n d t h e a m o u n t o f HC1 p e r m a n e n tly
b o u n d t o t h e p r o t e i n , t h e .sa m p le o f g r a p h t h i r t e e n w as
e v a c u a te d f o r s i x d a y s u n t i l c o n s t a n t w e ig h t w as a t t a i n
e d . The am o u n t l e f t w as 3 5 .1 m g. H C l/g m . p r o t e i n , o r
O.9 6 m .m o le s /g m .
The sam e e x p e r im e n t d e a l i n g w i t h t h e NH3 a d s o r b
i n g p r o t e i n , g a v e t h e v a l u e 2 .6 0 mg. NH^/gm . p r o t e i n ,
o r 0 . 1 5 m .m o le s /g m .
W ith t h e s e r e s u l t s i n m in d , i t i s n o t a t a l l s u r
p r i s i n g t h a t t h e p r o t e i n a f t e r a n HC1 r u n w as d e n a t u r e d
a n d t h e p r o t e i n a f t e r a n NH^ r u n was n o t . The d e n a t u r -
a t i o n r e f e r r e d t o i s w i t h r e s p e c t t o s o l u b i l i t y .
INDIVIDUAL GRAPHS OF ALL RUNS
41
S am ple Egg A lb u m in
2 ' f o s o l u t i o n , s p r a y f r o z e n
K i t r o g e n
7 7 .1 + °A .
P re p
Gas
Temp
1,000
o
* — i
o
PU
500
70
a o 2
.3
p / p
42
G ra p h 1 a
Sam ple Egg A lb u m in
P r e p . 2 % s o l u t i o n , s p r a y f r o z e n
Gas N i t r o g e n
Temp. 77. b °A .
43
G raph 2
Sample Egg Albumin
Prep
Gas
Temp
2 % s o l u t i o n , s p r a y f r o z e n
N i t r o g e n 1
7 7 A °A .
08
06
02
44
G ra p h 2a
S am ple Egg A lb u m in
P r e p , 2 % s p r a y f r o z e n , fro m H2O s o l u t i o n
G as N i t r o g e n
Temp. 7 7 A °A .
10
P /P
P/(V)(P0-P )
45
G rap h 3
S am ple Egg A lb u m in
5,6% s o l u t i o n , c r u d e
N i t r o g e n
Temp. 7 7 A °A .
P re p
Gas
. 2
1
0
P /P
G rap h 3 a
S am ple Egg A lb u m in
5 . 6 $ s o l u t i o n , c r u d e
N i t r o g e n
7 7 . * * °A .
P re p
Gas
Temp
1.0
P /P
P/(V)(P0- P ) ix 10
47
G raph 4-
S am p le Egg A lb u m in
2% s o l u t i o n , s p r a y f r o z e n
N itr o g e n
77«*+ °A .
P r e p
G as
Temp
10
0
P /P
V (c.c./gm .)
48
S am ple Egg A lb u m in
2% s o l u t i o n , s p r a y f r o z e n
N i t r o g e n
77 A °A .
P re p
Gas
Temp
3
2
1
0
P/P,
49
G rap h 5
S am ple Egg A lb u m in , u n d e n a tu r e d
Ammonia
Temp • room ( 22°C •)
G as
o
•H
20
10
50
G ra p h 5 a
S am ple Egg A lb u m in , u n d e n a tu r e d
Ammonia Gas
20 Temp room
15
©
I
i
10
5
b P (cm . Hg)
6
2
8
51
G rap h 6
S am ple Egg A lb u m in , u n d e n a tu r e d
Aramoni a Gas
Temp room
20
1 5
rv
10
5
0 1 2 P (cm . Hg) 3 If
52
G rap h 6 a
S am ple Egg A lb u m in , u n d e n a tu r e d
Ammonia Gas
Temp room
16
12
2 P (cm. Hg) 3 0
1
53
G rap h 7
S am ple Egg A lb u m in , u n d e n a tu r e d
Ammonia
~3*+ °C .
Gas
Temp
12
r-i
10 P (cm . Hg)
54
G rap h 7a
S am ple Egg A lb u m in , u n d e n a tu r e d
Ammonia
Temp. -34- °C .
Gas
120
10 P (cm . Hg) 15 20
5
0
55
G rap h 8
S am ple . Egg A lb u m in , u n d e n a tu r e d
H y d ro g e n C h l o r id e Gas
Temp room
rH
h P (c m . Hg) 6 8 0 2
56
G raph 8a
Sam ple Egg Albumin.) u n d e n a tu re d
H ydrogen C h lo rid e Gas
Temp room
10
57
G rap h 8b
S am ple Egg A lb u m in , u n d e n a tu r e d
H y d ro g e n C h l o r i d e Gas
Temp room
100
60
P(cm . Hg)
58
G rap h 9
S am ple Egg A lb u m in , u n d e n a tu r e d
H y d ro g e n C h l o r id e Gas
Temp room
1C
oo
o o
59
G ra p h 9 a
S am ple Egg A lb u m in , u n d e n a tu r e d
Gas H y d ro g e n C h l o r id e
Temp room
20
. 2 P(cm . Hg) . 3 0 1
60
G ra p h 10
S am ple Egg A lb u m in , u n d e n a t u r e d
i
G as H y d ro g en ' C h lo r id e
room
•1 P(cm . Hg) . 2
61
G ra p h 1 0 a
S am ple Egg A lb u m in , Unden&pzfed
G as H y d ro g e n C h l o r id e
room
hO
30
>
20
1 0
0
1 0
62
G raph 11
S am ple Egg A lb u m in , u n d e n a tu r e d
H y d ro g e n C h l o r id e
Gas
Temp room
10
i
63
G ra p h 1 1 a
S am ple Egg A lb u m in
u n d e n a tu r e d
Gas H y d ro g e n C h l o r id e
Temp• room
10
* + P(em. Hg)
64
G rap h 1 2
S am ple Egg A lb u m in , u n d e n a tu r e d
H y d ro g e n C h l o r id e
Temp* - 8 0 ’ ° C .
Gas
a
%
2
2 P(cm . Hg)
0
65
G rap h 1 2 a
S am ple Egg A lb u m in , u n d e n a tu r e d
Gas H y d ro g e n C h l o r id e
- 8 0 °C . Temp
1+00
300
£
♦
b O
3
200
100
0 1 2 P(cm. Hg) 3
p / v X 10
66
G raph 13
Sample Egg A lbum in, u n d e n a tu re d
H ydrogen C h lo rid e
Temp. -8 0 °C .
Gas
6
C M
b
2
20 P(cm.Hg) 30 0
67
G raph 1 3 a
S am p le Egg A lb u m in , u n d e n a tu r e d
G as H y d ro g e n C h l o r id e
Temp. - 8 0 °C .
. 1 . 2 P /P 0 .3
68
G rap h 1 3 b
S am p le Egg A lb u m in , u n d e n a tu r e d
Gas H y d ro g e n C h l o r id e
Temp* - 8 0 °C .
800
e
W )
fi
600
U00
200
1
10 20 P(cm .H g) 30
* ♦ 0 50
69
G rap h l*f
S am p le Egg A lb u m in , d e n a t u r e d
H y d ro g e n C h lo r id e
-8 0 ° C .
Gas
Temp
20
1 0
8 P(cm .H g) 12
16 20
70
G rap h l**a
S am ple Egg A lb u m in , d e n a t u r e d
Gas H y d ro g e n C h l o r id e
Temp* - 8 0 ° C .
71
G rap h l*+b
S am ple Egg A lb u m in , d e n a t u r e d
Gas H y d ro g e n C h l o r id e
Temp. - 8 0 ° C .
600
200
20
8 P(cra. Hg) 12 16 0
72
60
G rap h 1 5
S am ple Egg A lb u m in , d e n a t u r e d
G as H y d ro g e n C h lo r id e
ffiemp room
o
H
20
P(cm . Hg) 0 1 2
3
73
G raph 1 5 a
S am ple Egg A lb u m in , d e n a t u r e d
G as
Temp room
30
20
1 0
0
P(cm . Hg) 1 3 2
74
G raph 16
S am ple Egg A lb u m in , d e n a t u r e d
H y d ro g e n C h l o r id e Gas
Temp room
1 0
8
6
if
2
2 P(em . Hg) if 0
6
75
Graph 16a
Sam ple Egg A lbum in, d e n a tu re d
Gas H ydrogen C h lo rid e
Temp room
60
20
0 2 P(cm. Hg) 1 + 6
76
Graph 17
Sam ple Egg A lbum in, d e n a tu re d
H ydrogen C h lo rid e Gas
Temp room
8
6
x
&
2
5 2 P(cm. Hg) 3 0 1
77
G rap h 1 7 a
S am p le Egg A lb u m in , d e n a t u r e d
G as H y d ro g e n C h l o r id e
Temp room
60
20
2 P(cm .H g) 3
5 0 1
78
G raph 18
Sam ple Egg A lbum in, a f t e r ru n
H ydrogen C h lo rid e Gas
Temp room
8
6
iH
2
. 2 P(cm. Hg) • 6 Q
79
G raph 18a
Sample Egg A lbum in, a f t e r N H -> ru n
Gas H ydrogen C h lo rid e 5
Temp. room /
H O
30
>
20
1 0
0
O H - P(cm. Hg) 108 12
80
TEMPERATURE RISE
G raph 19
Sam ple Egg A lbum in
Gas H y d ro g en C h lo r id e
A p p ro x . room B ase Temp
60
? o
%
£
ko
HC1 su fte r NH3 ( .0 9 5 8 gm. p r o t e i n )
HC1 ( .1 6 1 5 gm. p r o t e i n )
HC1 a f t e r HC1 ( .1 6 1 5 gm. p r o t e i n )
30
20
2 Time(min.) 3
81
TEMPERATURE RISE
G raph 20
Sam ple Egg A lbum in
Gas Ammonia
B ase Temp A p p ro x , room
KH3 a f t e r HC1 ( .1 6 1 5 gm. p r o t e i n )
NH3 a f t e r NH3 ( .0 9 5 8 gm. p r o t e i n )
NH3 ( .0 9 5 8 gm. p r o t e i n )
COMPOSITE GRAPHS OF ALL RUNS
83
Graph 21
Sample Egg Album in, u n d e n atu red
Ammonia Gas
Temp
P o in ts Runs 5 and 6
room
1 0
6 P(cm.Hg) 8 12 10
V (m g./gm ,)
60
protein sample denatured
by previous H Cl run
40
20
1 2
3
0 6 7
85
600
* ♦ 0 0
200
G raph 23
Sam ple Egg A lbum in
Gas H y d ro g en C h l o r id e
Temp. -8 0 ° C .
P o i n t s Runs 1 2 and 13
10 P (cm . Hg) 1 5 20
86
Graph 2b
Sample Egg Albumin
Gas Hydrogen C h lo rid e
6,000
u n d e n a tu re d p r o t e i n
(room tem p.)
o d e n a tu re d p r o t e i n
/ (room tem p.)
» H
2,000 low te m p e ra tu re
1 (rm . tem p.) 2
20(low tm.)3 0
Range (cm, Hg)
' . 3
0
8?
HYDROGEN CHLORIDE AREAS
Graph 25
Sample Egg Albmmin
Hydrogen C h lo rid e Gas
Temp room
u n d e n a tu re d p r o t e i n
200
d e n a tu re d p r o t e i n
o
100
Range (cm. Hg)
88
HYDROGEN CHLORIDE AREAS
G raph 26
Sam ple Egg A lbum in
H y d ro g e n C h l o r id e
Temp. -8 0 ° C .
Gas
2,000
o
1,000
10 20
R ange (cm . Hg)
CHAPTER; V
DISCUSSION OF RESULTS
T h is r e s e a r c h c o n c e r n s i t s e l f p r i m a r i l y w i t h t h e
§.M ition o f HC1 and NH3 g a s e s t o s o l i d egg a lb u m in and
t h e i r s u b s e q u e n t e f f e c t s u p o n t h i s com pound. T h is i n i t i a l
s t u d y i s i n t e n d e d a s a s t e p p i n g - s t o n e to w a rd s f u t u r e
u t i l i z a t i o n o f p o l a r g a s e s i n t h i s r e a lm .
C o n s i d e r a t i o n o f t h e s e m o le c u l e s a s e i t h e r e l e c t r o n
d o n o r s o r a c c e p t o r s ( b a s e s o r a c i d s ) w i l l b e h e l p f u l .
I n m o st r e a c t i o n s NH3 w i l l a c t a s a b a s e s i n c e i t h a s
a n e x t r a p a i r o f e l e c t r o n s a v a i l a b l e , and t h e HC1 w i l l
a c t a s a n a c i d , s i n c e t h e H c a n a c c e p t a p a i r o f e l e c t r o n s
t o fo rm a c o o r d i n a t e bond.
The d a t a o b t a i n e d fro m a d s o r p t i o n r u n s o f HC1 and
NH3 a t room an d lo w t e m p e r a t u r e s make t h e a s s u m p tio n o f
p h y s i c a l a d s o r p t i o n ( s m a ll h e a t e f f e c t s , e t c . ) u n t e n a b l e .
On t h e b a s i s o f t h e f o l l o w i n g s i x e f f e c t s , c h e m i s o r p t i o n ,
w i t h p r o b a b l e m u l t i m o l e c u l a r a d s o r p t i o n , i s i n d i c a t e d .
1 . L a rg e h e a t e v o l u t i o n .
2. . F l a t p o r t i o n s o n HC1 a d s o r p t i o n g r a p h s .
3 . I r r e v e r s i b l e b o n d in g o f h y d ro g e n c h l o r i d e and am m onia.
*+. L a rg e am o u n ts o f g a s a d s o r b e d .
5. In d e p e n d e n c e o f p o l a r g a s a d s o r p t i o n o n n i t r o g e n
a r e a s .
6 . C o r r e l a t i o n w i t h p r o t e i n s s t r u c t u r a l g r o u p i n g s .
1 . LARGE HEAT EVOLUTION
To e s t a b l i s h t h e p r e s e n c e o f a l a r g e t e m p e r a t u r e
r i s e u p o n r e a c t i o n , t h e a d d i t i o n o f HC1 a n d NH3 was c a r
r i e d o u t i n a sam ple t u b e e q u ip p e d w i t h a thermocouple"*"
I n a l l o f t h e f o l l o w i n g e x p e r i m e n t s , tw e n ty c . c . o f g a s
a t a p p r o x i m a t e ly f o r t y cm. Hg p r e s s u r e w ere a d m i t te d to
t h e sa m p le . The g a s b u r e t t e was f i l l e d to t h e " t w e n t y
c . c . m ark and t h e sam p le s to p c o c k o p e n e d . The te m p e r
a t u r e o f t h e sa m p le was t h e n d e te r m in e d a s a f u n c t i o n
o f tim e . S ix r u n s w ere m ade:
A. HC1 a d d ed t o t h e s a m p le .
B. HC1 a d d ed a f t e r t h e f i r s t r u n h a d b e e n e v a c u a te d
f o r a d a y .
C. HC1 a d d ed a f t e r a n NH3 r u n .
D. NH3 a d d ed t o t h e s a m p le .
E. NH3 a d d e d a f t e r t h e f i r s t r u n h a d b e e n e v a c u a te d
f o r a d a y .
F ., NH3 a d d ed a f t e r a n HC1 r u n .
T hese d a t a h a v e b e e n p l o t t e d i n G r a p h s ..n in e te e n
an d tw e n ty and y i e l d t h e f o l l o w i n g i n f o r m a t i o n :
1 See F i g u r e 5? P» 2*f.
91
a . Upon HC1 a d d i t i o n , t h e t e m p e r a t u r e r i s e i s g r e a t e s t
o n t h e new s a m p le s ; p r e v i o u s t r e a t m e n t by NH3 h a v in g l i t
t l e e f f e c t .
b . Upon NH3 a d d i t i o n , t h e g r e a t e s t r i s e i s o n t h e sam
p l e p r e v i o u s l y t r e a t e d w i t h HC1; t h e d i f f e r e n c e b e tw e e n
t h e new sam p le and t h e o n e a f t e r a d d i t i o n o f NH3 b e in g
s l i g h t .
As c a n be s e e n fro m t h e s e g r a p h s , t h e t e m p e r a t u r e
e f f e c t s v a r y fro m a r i s e o f 1 5 ° C ., i n t h e c a s e o f NH3
s
a d d ed t o new p r o t e i n , t o *+0°C., i n t h e c a s e o f HC1 a d d ed
t o p r o t e i n . An e s t i m a t e o f t h e h e a t e v o l u t i o n w h ic h to o k
p l a c e c a n be made a s f o l l o w s :
K now ledge o f t h e am ount o f g a s r e a c t i n g w ith a d e f i n i t e
w e ig h t o f p r o t e i n , a lo n g w ith t h e t e m p e r a t u r e r i s e
c a u s e d by t h i s r e a c t i o n , and t h e s p e c i f i c h e a t o f t h e
p r o t e i n , a llo w s a ro u g h c a l c u l a t i o n o f t h e e n t h a l p y
c h a n g e :
AH HC1 ad d ed
I n t h i s c a l c u l a t i o n , t h e am ount r e a c t i n g was e s t i m a t e d
a t t h e tim e o f maximum t e m p e r a t u r e r i s e . T h is v a l u e
t e n d s t o be s m a ll b e c a u s e o f two f a c t o r s :
A. T h e re i s a tim e l a g o f t h e th e r m o c o u p le u s e d t o
m e a s u re t h e t e m p e r a t u r e r i s e , t h u s y i e l d i n g a lo w e re d
maximum.
B. The h e a t c a p a c i t y o f t h e g a s p r e s e n t was i g n o r e d .
9 2
T h is l a r g e h e a t e f f e c t c a n n o t be e x p l a i n e d o n t h e b a s i s
o f p h y s i c a l a d s o r p t i o n , w h ic h w ould im p ly a v a l u e a b o u t
e q u a l t o t h e h e a t o f l i q u i f a c t i o n o f t h e HC1. T h is i s
o f t h e o r d e r o f two K c a l . / m o l e . I t m u s t, t h e r e f o r e , be
i n t e r p r e t e d o n t h e b a s i s o f a c h e m ic a l r e a c t i o n o f t h e
g a s w i t h t h e p r o t e i n .
2* FLAT PORTIONS OH H3TOR0GSN CHLORIDE ADSORPTION GRAPHS
To f u r t h e r s t r e n g t h e n t h e h y p o t h e s i s o f c h e m is o r p
t i o n , o n e may i n s p e c t G ra p h s e i g h t and n i n e , t h e a d s o r p
t i o n o f HC1 o n p r o t e i n a t room t e m p e r a t u r e , b o th o f w h ic h
show d e f i n i t e e v id e n c e o f f l a t p o r t i o n s a t p r e s s u r e s o f
0 .1 3 cm. Hg an d 0.3*+ cm. Hg, w i t h p o s s i b l e f l a t s a t o t h e r
p o i n t s . T h ese f a c t s i n d i c a t e t h e f o r m a t i o n o f a new
p h a s e a t t h e b e g in n in g o f t h e c o n s t a n t p r e s s u r e r e g i o n .
T h is i s m o s t e a s i l y e x p l a i n e d o n t h e b a s i s o f compound
f o r m a t i o n , a lt h o u g h t h e r e r e m a in s t h e d o u b t f u l p o s s i b i l
i t y o f t h e f o r m a t i o n o f a new s o l i d s o l u t i o n o f t h e HC1
and t h e p r o t e i n . I n o r d e r t o e x p l a i n t h e c o n s t a n t p r e s
s u r e r e g i o n , t h e new s o l i d s o l u t i o n w ould h a v e to b e o f
c o n s t a n t c o m p o s i t i o n ,o r a e u t e c t i c .
3 . IRREVERSIBLE BONDING OF HYDROGEN CHLORIDE AND AMMONIA
A f t e r a t y p i c a l .HC1 r u n a t room t e m p e r a t u r e , a
sam p le was e v a c u a te d a t 10 ' m.m. Hg p r e s s u r e f o r s i x
d a y s , u n t i l c o n s t a n t w e ig h t was a t t a i n e d . I t was fo u n d
93
t h a t t h e r e re m a in e d p e r m a n e n tly b o u n d , O.9 6 m .m o le s HC1/
gm. p r o t e i n . T h is w e ig h t c o r r e s p o n d s t o c h e m ic a l com pounds
o f h ig h s t a b i l i t y and v e r y lo w p a r t i a l p r e s s u r e s o f HC1.
An e x a m in a tio n o f G raph tw e n ty -tw o show s a d i f
f e r e n c e i n a d s o r p t i v e po w er b e tw e e n p r o t e i n s a f t e r p r e
v i o u s HC1 r u n s an d new p r o t e i n s . The d i f f e r e n c e i n am ount
a d s o r b e d o v e r a l a r g e p o r t i o n o f t h e r a n g e re m a in e d c o n
s t a n t a t a v a l u e o f 1.0*+ m .m o le s/g m . p r o t e i n . T h is i s
i n r e m a r k a b le a g re e m e n t w i t h t h e r e s u l t d e te r m in e d by
w e ig h in g - Thus i t a p p e a r s t h a t t h e r e a r e a num ber o f
e x tr e m e ly a c t i v e b a s i c s i t e s i n t h e egg a lb u m in m o le c u le ;
t h e s e b e in g d i s t r i b u t e d o v e r a v a p o r p r e s s u r e r a n g e o f
0 . 0 t o 0 - 3 cm. Hg, a s ; shown i n t h e a d s o r p t i o n c u r v e o f
HC1 o n p r o t e i n a t room t e m p e r a t u r e and a t a v a l u e o f Va(j s
o f 1 .0 0 m .m o le s/g m . S in c e t h i s : i r r e v e r s i b l y bound HC1
e x i s t s a t p r e s s u r e s o f up t o 0 *3 cm. Hg o n t h e a d s o r p t i o n
r u n , on e w ould n a t u r a l l y e x p e c t e v a c u a t i o n a t 1 0 ~^m.m.
Hg t o e l i m i n a t e su c h HC1. S in c e t h i s d o e s n o t o c c u r ,
i t i s p r o b a b l e t h a t a f t e r t h e i n i t i a l p s e u d o e q u i l i b r i u m
t a k e s p l a c e , a s lo w c h e m ic a l r e a c t i o n , o r r e a r r a n g e m e n t ,
t a k e s p l a c e w i t h i n t h e p r o t e i n m o le c u le l e a v i n g t h e a c t i v e
s i t e s d e a c t i v a t e d w ith r e s p e c t t o f u r t h e r a d s o r p t i o n o f
HC1 m o le c u le s and t h e HC1 p r e s e n t s t r o n g l y b o nded t o t h e
9 1 * -
p r o t e i n m o le c u l e .
The am ount o f NH3 p e r m a n e n tly hound w as fo u n d by
w e ig h in g t o b e 0 . 1 5 m .m o le s/g m . T h is s m a ll am ount b ond
ed f a l l s i n l i n e w ith t h e l a c k o f e f f e c t o n s o l u b i l i t y
o f t h e p r o t e i n by t h e NH3 w h ile t h e HC1 h a s t h e d e f i n i t e
2
e f f e c t a l r e a d y d i s e u s s e d . One c a n a l s o s e e t h a t t h e
a d d i t i o n o f NH3 h a s l i t t l e e f f e c t o n t h e t e m p e r a t u r e
r i s e o f t h e p r o t e i n w h e re a s t h e HC1 a d d i t i o n h a s a v e r y
p r o f o u n d e f f e c t ^ .
b , LARGE. AMOUNTS OF GAS ADSORBED
The am ount o f HC1 bound a t lo w t e m p e r a t u r e and
a t h i g h p r e s s u r e , a s show n i n G raph 1 3 b , r e a c h e s a v a l u e
a s h i g h a s 1 5 m .m o le s H C l/gm . p r o t e i n . T h is t a k e s p l a c e
a t a v a l u e o f B /P 0 o f o n l y 0 . 5 t h u s show ing t h a t t h e
s y ste m i s q u i t e f a r from c o n d e n sa tio n . One may c a k c u l a t e
t h a t a u n i m o l e e u l a r l a y e r o f n i t r o g e n o n t h e p r o t e i n s u r
f a c e c o n t a i n s a p p r o x i m a t e ly 0 . 2 m .m o le s H C l/gm . p r o t e i n .
T h u s, i f on e s t i p u l a t e s t h a t t h e HC1 u s e d t h e same s u r f a c e
a s t h e n i t r o g e n , and was a d s o r b e d by a m ech an ism o f m u l t i -
m o le c u l a r a d s o r p t i o n , s e v e n t y - f i v e l a y e r s w ould be n e e d e d .
T h is i s p o s s i b l e , b u t im p r o b a b le , a t a p r e s s u r e so f a r
rem oved fro m t h e s a t u r a t i o n v a l u e . One m ig h t e x p l a i n
t h e l a r g e a m o u n ts a d s o r b e d a s f o l l o w s :
2 S ee p a g e 3 8 .
3 S ee G raph 19
9 5
a . E ach o f t h e N ato m s i n t h e p x ro te in m o le c u le b in d s
o n e o r m ore HC1 m o l e c u l e s .
b* T h e re a r e s i t e s p r e s e n t , s u c h a s c a r b o n y l o x y g e n s ,
w h ere h y d ro g e n b r i d g i n g m ig h t be p r e s e n t .
c . Some m u l t i m o l e c u l a r a d s o r p t i o n t a k e s p l a c e .
One c a n s e e t h a t t h i s a d s o r p t i o n p r o c e s s d e a l s w i t h a n
a d s o r b e n t w hose po w er i s n o t l i m i t e d t o t h e s u r f a c e o f
l a r g e p a r t i c l e s - T h is a p p e a r s t o be a p r o c e s s i n t i m a t e
l y r e l a t e d t o t h e c h e m ic a l g r o u p i n g s w i t h i n t h e p r o t e i n
m o le c u le i t s e l f an d i s p r o b a b l y a phenom enon f a r rem oved
fro m p h y s i c a l a d s o r p t i o n o n a p a r t i c l e s u r f a c e , b r o u g h t
a b o u t by Van d e r W aa l’ s a t t r a c t i v e f o r c e s .
5 . INDEPENDENCE OP POLAR GAS ADSORPTION ON NITROGEN AREAS
F o r e a c h p r o t e i n sa m p le p r e p a r e d , t h e s u r f a c e a r e a
was d e te r m in e d by. m eans o f a B .E .d). p l o t o f a n i t r o g e n
r u n . I t was fo u n d t h a t v a r i a t i o n s i n t h i s a r e a , w h ic h
r a n g e d fro m 5«1 t o 21 m ^/gm . p r o t e i n , h a d no- e f f e c t u p o n
t h e a d s o r p t i o n c h a r a c t e r i s t i c s o f t h e HC1 and NH^ g a s e s .
I f t h e same s u r f a c e s w e re u s e d f o r t h e a d s o r p t i o n o f t h e s e
g a s e s , on e w ould e x p e c t a f a r g r e a t e r d e p e n d e n c e o n t h e
n i t r o g e n a r e a s . T h e r e f o r e , we a g a i n s e e t h a t t h i s i s a
p r o c e s s d e p e n d in g n o t u p o n t h e g r o s s s u r f a c e a r e a o f a
p r o t e i n p a r t i c l e , b u t u p o n t h e a c t u a l m akeup o f t h e p a r t
i c l e i t s e l f .
A good d e g r e e o f c o r r e l a t i o n was o b t a i n e d f o r a l l r u n s
by m eans o f a- L angm uir p l o t ( B .E .T . 'a t lo w P /P Q) and
t h i s w i l l be d i s c u s s e d i n P a r t 7»
6 . CORRELATION WITH PROTEIN'S STRUCTURAL GROUPINGS
On t h e b a s i s o f t h e ab o v e d a t a , i t was d e c id e d to
a t t e m p t t o c o r r e l a t e t h e am ount o f g a s p e r m a n e n tly bound
w i t h t h e c h e m ic a l .g r o u p in g s w i t h i n t h e p r o t e i n m o le c u le
w h ich w ere a v a i l a b l e f o r r e a c t i o n * I t was assum ed i n
t h i s c a l c u l a t i o n t h a t a l l o f t h e a lp h a am ino and c o r r e s
p o n d in g c a r b o x y l g ro u p s w e re t i e d up i n p e p t i d e l i n k a g e .
T a b le VI shows t h a t v a r i o u s am ino a c i d s p r e s e n t i n egg
a lb u m in h a v e p o s s i b l e r e a c t i v e a c i d and b a s i c g r o u p i n g s .
TABLE VI
Amino A cid
m .m o le s p e r
gm. egg a l
bum in
S t r u c t u r e
C O O H C Q O H
C y s t i n e
0 . 0 7 ^
C H N H p CHNHp
H p C - S — S - C H p
T ry p to p h a n 0*0 58 Q j-Jj-C H ^ G H m ^ C O O H
H
T y r o s in e 0 .2 1 9 ^ “CH^CHNH^COOH
TABLE. V I ( c o n t . )
9 7
Amino A c id
m .m o les p e r
gm. egg a l
bum in
S t r u c t u r e
H i s t i d i n e 0 .0 9 6
„ r ^NH-CH
^ N— c - ch2- chnh2cooh
A r g i n i n e 0 . 3 2 ?
rm ''NH-(CH2) 3-CHNH2COOH
L y s in e o.,3*fO
CH2NH2- CCH2) 3-CHNH2CO0H
H y d ro x y g lu ta rn i c
A cid
O.O83 H00C- CH2- CHOH- CHNH2C00H
Ammonia* 0 .7 2 3 nh3
A s p a r t i c A c id 0 .6 0 8 H00C- ch2- chnh2 cooh
G lu ta m ic A c id
1 .0 9 ?
H00C— ( CH2) 2-CHNH2C00H
M e th io n in e
0 .3 5 1 H0QC- CHNH2- ( CH2) 2-S-C H 3
$ jc
The, ammonia p r e s e n t i s bound up i n am ide l i n k a g e su c h
a s i n g lu ta m in e (HOQC-CHMHg-(CH2) 2 “ C0NH2)• The d a t a w ere
t a k e n fro m Cohn and E d s a l l ^ .
I t i s o f g r e a t i n t e r e s t t o c o m p a re , w i t h t h e s e d a t a
i n m in d , t h e am ount o f HC1 i r r e v e r s i b l y bound t o t h e p r o
t e i n w i t h t h e num ber o f r e a c t i v e b a s i c g ro u p s p r e s e n t .
The two g ro u p s w h ic h w ould h a v e t h e g r e a t e s t p r o b a b i l i t y
o f r e a c t i o n w i t h t h e HC1 a r e t h e e p s i l o n am ino g ro u p o f
4 Cohn a n d E d s a i l , P r o t e i n s . Amino A c id s , and P e p
t i d e s . (New Y orks R e in h o ld P u b l i s h i n g Company, 19^+3^ •
9 8
l y s i n e and t h e am in o g ro u p o f a r g i n i n e , w h ic h ad d up t o
0 .6 6 5 m .m o le s/g m . p r o t e i n - I f we a l s o i n c l u d e a s a\..-pos
s i b i l i t y , t h e e x p o se d end sHH g ro u p o f a r g i n i n e , th e . sum
i s 0 .9 9 0 m .m o le s /g m ., a v a l u e w h ic h c o m p a re s m o s t f a v o r
a b l y w i t h 1 .0 0 m .m o les/g m a s a n a v e r a g e f o r t h e i r r e v
e r s i b l y b o u n f HC1. O th e r p o s s i b l e r e a c t i v e g ro u p s i n
c y s t i n e , t r y p t o p h a n , arid h i s t i d i n e w ould n o t c h a n g e t h e
o r d e r o f m a g n itu d e o f t h i s sum s i n c e t h e y a r e p r e s e n t
i n su c h s m a ll q u a n t i t y i n egg a lb u m in . I t seems; q u i t e
c e r t a i n , t h e n , t h a t t h e o r d e r o f m a g n itu d e o f t h e sum
o f t h e p r o b a b l e b a s i c g r o u p in g s i s su c h a s t o w a r r a n t
f u r t h e r s t u d y .
The m o st p r o b a b l e a c i d g ro u p s f o r W H3 a d d i t i o n
a r e t h e s u l f u r s o f c y s t i n e a n d m e t h i o n i n e , a n d t h e f r e e
c a r b o x y l g ro u p s o f a s p a r t i c , g l u t a m i c , and h y d ro x y g lu ta m -
i c a c i d s - S in c e t h e num ber o f t h e s e g ro u p s i s much g r e a t
e r t h a n t h e am ount o f NH3 bound i r r e v e r s i b l y , o n e i s l e d
t o t h e c o n c l u s i o n t h a t t h e s t r e n g t h o f b o n d in g i s much
w e a k e r t h a n t h a t w ith HC1, a ll o w i n g t h e com pounds fo rm ed
t o be e v a c u a t e d - T h ere a l s o r e m a in s t h e p o s s i b i l i t y t h a t
t h e r e a r r a n g e m e n t i s much s l o w e r , i f t h e r e b e a r e a r r a n g e
m e n t. T h is i s b o r n o u t b y t h e lo w e r t e m p e r a t u r e r i s e
fo u n d i n t h e NH3 a d d i t i o n to egg a lb u m in . I t i s i n a l l
99
p r o b a b i l i t y a f o r t u i t o u s c ir c u m s ta n c e t h a t t h e NH^ p e r
m a n e n tly bound i s e q u a l a lm o s t e x a c t l y t o t w i e e t h e c y s t
i n e p r e s e n t . Work p la n n e d b n a d s o r p t i o n by am ino a c i d s
w i l l c l e a r up many p o i n t s w ith r e g a r d t o r e a c t i v i t y o f
s p e c i f i c g r o u p i n g s .
7 . CORRELATION BY: B .E .T . THEORY
As; p r e v i o u s l y m e n tio n e d , a good d e g r e e o f c o r r e l
a t i o n was o b t a i n e d f o r t h e r u n s o n b o th g a s e s , a t room
and a t lo w t e m p e r a t u r e s , by m eans o f a L an g m u ir o r
T 3.E.T . p l o t . , The B*E„T. p l o t becom es e q u i v a l e n t t o a
L an g m u ir p l o t a t lo w v a l u e s o f P /P 0 w h ic h i s u s u a l l y
t h e c a s e i n t h e g r a p h s p l o t t e d . T h e re a p p e a r e d , h o w e v e r,
a s m a l l , b u t w e l l - d e f i n e d c u r v a t u r e i n t h e g r a p h s , w i t h
i n c r e a s i n g p r e s s u r e .
T h e o r e t i c a l l y , t h e a s s u m p tio n s t h a t go i n t o t h e s e
t h e o r i e s a p p e a r h i g h l y im p r o b a b le i n l i g h t o f t h e e v i d
e n ce a l r e a d y p r e s e n t e d .
A. L an g m u ir a s s u m p tio n s
a . A d so rb e d l a y e r i s u n im o le c u la r .
b . No a t t r a c t i o n f o r g a s m o le c u l e s by t h o s e a l r e a d y
a d s o r b e d . (HC1 i s h i g h l y p o l a r ) .
c . The h e a t o f r e a c t i o n i s c o n s t a n t th r o u g h o u t t h e
a d s o r p t i o n r u n .
d . No i n t e r a c t i o n among a d s o r b e d m o le c u l e s .
1 0 0
B. B .E .T . a s s u m p tio n s
a* C o n s ta n t h e a t o f r e a c t i o n o v e r f i r s t l a y e r .
b . H e a t o f r e a c t i o n e q u a l t o h e a t o f l i q u i f a c t i o n
o v e r a l l o t h e r l a y e r s .
I t c a n be s e e n t h a t t h e B .E .T , t h e o r y m ig h t a t l e a s t
g i v e a n i d e a o f t h e h e a t s and a r e a s t o be d e a l t > d th i n
t h e a d s o r p t i o n . I f one a ssu m e s t h e c u r v a t u r e o f t h e g r a p h s
due to, c h a n g e s i n t h e h e a t o f a d s o r p t i o n o n s u b s e q u e n t
l a y e r s , e a c h p o r t i o n o f t h e c u r v e w ould g i v e an i n d ic a 4 —
t i o n , a a t l l e a s t , o f t h e m a g n itu d e o f t h e h e a t s and a r e a s
i n v o l v e d . I t m u st be e m p h a siz e d a g a i n t h a t t h e v a l u e s
o b t a i n e d p r o b a b l y h a v e l i t t l e p h y s i c a l v a l u e , and u n t i l
m ore c o n c l u s i v e d a t a c a n be o b t a i n e d , s h o u ld b e t h o u g h t
o f a s c o n s t a n t s t o b e s u b s t i t u t e d i n t o a n e m p i r i c a l a d
s o r p t i o n i s o t h e r m e q u a t i o n .
G raph t w e n t y - f o u r i s a p l o t o f E i-E j, v e r s u s t h e
u p p e r l i m i t o f t h e p r e s s u r e r a n g e f o r w h ic h a l i n e a r
L an g m u ir p l o t was o b ta in e d .. One c a n s e e t h a t t h e h e a t s
o f a d s o r p t i o n d e c r e a s e w i t h i n c r e a s i n g r a n g e , a r e s u l t
w h ic h i s t o be e x p e c te d i f t h e m ore a c t i v e s i t e s i n t h e
m o le c u le a r e a t t a c k e d f i r s t . I f o n e be i n c l i n e d t o
a t t a c h a n y s i g n i f i c a n c e t o t h e v a l u e s o f E i , i t i s e a s i l y
c a l c u l a t e d t o be a p p r o x i m a t e ly 9 ,0 0 0 c a l . / m o l e f o r HC1
a t room t e m p e r a t u r e , and 8 ,0 0 0 c a l . / m o l e f o r NH3 g a s .
1 0 1
(T h e se v a l u e s do n o t f a l l I n t o l i n e w ith t h e h e a t m e a s u re
m e n ts m a d e .) F o r the" p r o t e i n d e n a t u r e d by HClj t h e v a l u e
i s 8 ,0 0 0 c a l . / m o l e , a b o u t 1 ,0 0 0 c a l . / m o l e l e s s t h a n f o r t h e
new p r o t e i n . S in c e t h e m ore a c t i v e s i t e s h a v e p r o b a b l y
b e e n d e a c t i v a t e d i n t h e d e n a t u r e d p r o t e i n , t h i s i s to
be e x p e c te d . Such l a r g e r e s u l t s i n f e r t h a t we a r e d e a l i n g
w i t h a c h e m ic a l ( h i g h b i n d in g e n e r g i e s ) ty p e o f a d s o r p t i o n
i n s t e a d o f a p h y s i c a l t y p e . The lo w t e m p e r a t u r e r u n w i t h '
HC1 y i e l d e d a v a l u e o f 5»000 c a l . / m o l e . I f c h e m ic a l com
p o u n d s o f d e f i n i t e v a p o r p r e s s u r e s a r e fo rm e d , lo w e r in g
o f t e m p e r a t u r e w ould c a u s e t h e com pounds t o be fo rm ed
a t much lo w e r e q u i l i b r i u m p r e s s u r e s . T hu s, t h e h i g h en
e r g y com pounds w ould be fo rm e d a t lo w p r e s s u r e s , an d t h e
e n e r g i e s f o r t h e lo w t e m p e r a t u r e r u n s , c a l c u l a t e d a t e q u a l
p r e s s u r e s a s t h e h i g h t e m p e r a t u r e r u n s w ould be c o n s i d
e r a b l y l e s s .
From G raph t w e n t y - f i v e o n e may s e e t h a t t h e a r e a ,
a s m e a su re d b y a B^JE.T.- p l o t , i n c r e a s e s a lm o s t l i n e a r l y
w i t h t h e u p p e r l i m i t o f t h e p r e s s u r e r a n g e f o r t h e s t r a i g h t
l i n e p o r t i o n o f t h e c u r v e . The v a l u e s o b t a i n e d by e x t r a p
o l a t i o n o f t h e s e p l o t s t o z e r o p r e s s u r e a r e a s f o l l o w s :
University of S outhern California Liters^
TABLE V II
1 0 2
Gas Tefanperature
°C*
Area.
HC1 20 100;
HC1. -8 0 1 ,0 0 0
NH3 20 60
m 3
— 3 ^
200
T hese v a l u e s a r e so d e p e n d e n t u p o n t e m p e r a t u r e t h a t l i t t l e
p h y s i c a l s i g n i f i c a n c e c a n be a t t a c h e d t o them . A t t h e
m o s t, t h e y i n d i c a t e t h a t egg a lb u m in i s a b e t t e r a d s o r b e n t
f o r HC1 t h a n f o r NH3 and i s m ore e f f i c i e n t a t lo w tem p
e r a t u r e s .
8 . KINETICS AND OTHER GENERAL OBSERVATIONS
As f a r a s t h e k i n e t i c s o f t h e a d s o r p t i o n r u n s f o r
b o th HC1 and NH3 a r e c o n c e r n e d , t h r e e q u a l i t a t i v e r e s u l t s
w ere o b t a i n e d :
A. The l o n g e r t h e tim e o f e v a c u a t i o n o f t h e sam p le
p>rior t o a r u n , t h e s h o r t e r th e tim e f o r e a c h p o i n t to
a t t a i n e q u i l i b r i u m .
B. The tim e f o r e a c h p o i n t to a t t a i n e q u i l i b r i u m i n
c r e a s e d a s t h e r u n p r o g r e s s e d ; a s t h e p r e s s u r e i n c r e a s e d .
T h is tim e r a n g e d fro m f i v e m in u te s a t t h e s t a r t o f a r u n
t o o v e r a d a y a t t h e end o f some r u n s .
C* A t e q u a l p r e s s u r e s , lo w t e m p e r a t u r e r u n s to o k l o n g
e r t o a p p r o a c h e q u i l i b r i u m t h i n d i d t h o s e a t r&om tem p
e r a t u r e .
B o th t h e h e liu m d e a d s p a c e m e a su re m e n ts and t h e
n i t r o g e n lo w t e m p e r a t u r e a d s o r p t i o n r u n s r e a c h e d e q u i l
ib r iu m w i t h i n t h r e e m in u t e s a t a l l p o i n t s *
The tim e f o r t h e p o l a r g a s e s t o a t t a i n e q u i l i b
riu m may be a t t r i b u t e d t o two f a c t o r s : ;
a . The d i f f u s i o n i n t o , o r i n t e r p e n e t r a t i o n o o f , t h e
p r o t e i n m o le c u l e , and s u b s e q u e n t r e a r r a n g e m e n t o f th e
m o le c u le .
b . The t e m p e r a t u r e r i s e - c a u s e d by t h e ' l a r g e h e a t e f f e c t s
d u e t o c h e m ic a l r e a c t i o n , and t h e s u b s e q u e n t tim e l a g
b e f o r e a t t a i n m e n t o f t e m p e r a t u r e e q u i l i b r i u m .
By lo o k in g a t G rap h s n i n e t e e n an d tw e n ty , o n e c a n se e
t h a t t h e t e m p e r a t u r e e f f e c t i s i n c o n s e q u e n t i a l , a s i t
l a s t s a p p r o x i m a t e ly f i v e m in u t e s ,w h e r e a s t h e tim e f o r
e q u i l i b r i u m t o be a t t a i n e d i s u s u a l l y much g r e a t e r t h a n
t h i s . Thus one may s a f e l y assum e t h a t t h e c o n t r o l l i n g
f a c t o r i n t h e r a t e o f a d s o r p t i o n o f t h e s e g a s e s i s t h e
p e n e t r a t i o n o f t h e p r o t e i n m o le c u le b y t h e p o l a r g a s e s .
The e f f e c t o f i n c o m p le te e v a c u a t i o n p r i o r t o a
r u n i s v e r y p ro n o u n c e d w i t h r e s p e c t t o t h e d i s a p p e a r a n c e
lo b
o f f l a t s o n t h e a d s o r p t i o n i s o t h e r m s . An e v a c u a t i o n o f
o n ly o n e d a y , I n s t e a d o f t h r e e , b e f o r e a r u n , e l i m i n a t e s
t h e a p p e a ra n c e o f t h e f l a t s a t (t h e p r e s s u r e s o f 0 .1 3 and
0.3*f cm. Hg o n t h e HC1 a d s o r p t i o n i s o t h e r m s a t room tem p
e r a t u r e . H ow ever, t h e t o t a l am ount a d s o r b e d o v e r t h e
w h o le r a n g e was n o t n o t i c e a b l y a f f e c t e d . T h is d i s a p p e a r
a n ce o f f l a t s i s p r o b a b l y c a u s e d by w a te r a d s o rb e d from
t h e a i r . I n a l l p r o b a b i l i t y , i t s l a c k o f e f f e c t on t h e
o v e r a l l r u n i s due to t h e r e p la c e m e n t o f t h e w a te r by
HC1 m o le c u l e s . T hus, w a t e r a p p e a r s t o be r e v e r s i b l y bound
t o t h e p r o t e i n w h e re a s t h e HC1 i s n o t .
I t s h o u ld be n o te d t h a t G raphs tw e n ty - o n e , tw e n ty -
tw o , and t w e n t y - t h r e e i n d i c a t e good r e p r o d u c i b i l i t y o f
r e s u l t s o v e r many r u n s .
The w ork w h ic h h a s b e e n a c c o m p lis h e d i s by no m eans
c o m p le te w i t h i n i t s e l f . I t m e r e ly p o i n t s t h e way to w a rd s
f u t u r e and p o s s i b l y m ore p r o f i t a b l e r e s e a r c h . An e n o r
mous am ount o f w ork d e a l i n g w ith t h e p in n in g -d o w n o f t h e
r e a c t i v e g ro u p s i s p o s s i b l e . T h is m ig h t be com posed o f
t h e f o l l o w i n g s e c t i o n s :
A. V a r i a t i o n o f t h e a c i d i t y and b a s i c i t y o f t h e p o l a r
g a s e s b e in g u s e d , s u c h a s a s e r i e s o f g a s e o u s a m in e s ,.
BF3 , H2 S, HpO, and many o t h e r s .
1 0 5
B.. Use o f t h e s e g a s e s i n a d s o r p t i o n w ork d e a l i n g w ith
t h e c o n s t i t u e n t s o f p r o t e i n s , t h e am ino a c i d s ,
C. C h e m ic a lly b lo c k in g some o f t h e s u s p e c t e d r e a c t i v e
g r o u p s and t h e n m aking a d s o r p t i o n r u n s .
K i n e t i c s t u d i e s , a lo n g w ith d a t a o n t h e h e a t e f f e c t s
o f p o l a r g a s a d s o r p t i o n , w ould do much to w a rd s e x p o s in g
t h e -m echanism o f t h e r e a c t i o n , and t h i s i n t u r n w ould
g i v e a c l e a r e r i n s i g h t i n t o t h e o v e r a l l s p a t i a l c o n f i g
u r a t i o n o f t h e p r o t e i n m o le c u le .
An a n a l y s i s o f t h e i n f r a - r e d a d s o r p t i o n s p e c t r a
o f t h e p r o t e i n s and amino a c i d s b e f o r e and a f t e r a d s o r p t i o n
o f t h e g a s e s w ould c e r t a i n l y be o f g r e a t a s s i s t a n c e .
I n c l o s i n g , i t m ig h t be w e l l to s t a t e t h a t t h e
f u l l pow er o f t h e s o l i d p h a s e - g a s p h a s e s a d s o r p t i o n m e th
od w i t h p o l a r g a s e s h a s o n ly b e e n u t i l i z e d t o an i n s i g
n i f i c a n t d e g r e e . I t s a p p l i c a t i o n to o t h e r b io c h e m ic a l
and m e d i c i n a l p ro b le m s h a s n o t a s y e t b e e n c o n s i d e r e d
t o a n y g r e a t d e g r e e . The s t r e n g t h o f t h e m eth o d seem s
w e l l e s t a b l i s h e d b y t h e w ork a c c a m p li s h e d . I t s f u t u r e
s h o u ld be one o f g r e a t s e r v i c e t o h u m a n ity
BIBLIOGRAPHY
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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Seehof, Jerrold Morton (author)

Core Title The adsorption of gaseous hydrogen chloride and ammonia on egg albumin surfaces

Contributor Digitized by ProQuest (provenance)

Degree Master of Science

Degree Program Chemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-794352

Unique identifier UC11348864

Identifier EP41580.pdf (filename),usctheses-c17-794352 (legacy record id)

Legacy Identifier EP41580.pdf

Dmrecord 794352

Document Type Thesis

Rights Seehof, Jerrold Morton

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA