Protein Alterations at Low Temperatures - American Chemical Society

1 Protein Alterations at Low Temperatures: A n Overview

Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

GEORGE TABORSKY Section of Biochemistry and Molecular Biology, Department of Biological Sciences, University of California, Santa Barbara, CA 93106

Physical and chemical alterations of protein structure and function, caused by the exposure of aqueous protein solutions to low temperatures, have been explored and explained by various investigators in terms of a broad range of hypotheses. This survey attempts to give a mostly retrospective overview of this range, emphasizing effects of low temperature and of freezing on hydrophobic interactions, hydrogen bonding, and interactions of the protein with the solvent, other solution components, the ice, and the ice-liquid interface. The effects of low temperatures per se are considered, followed by a discussion of those effects on protein structure and reactivity which are associated with ice formation and its consequences such as the freezing out of solutes. The effects of the admixture of organic cosolvents to aqueous protein solutions are noted with reference to cryoenzymological studies and the prevention of freezing injuries. Experimental and interpretive approaches and results of these approaches are illustrated by examples drawn selectively from the literature.

" V T T a t e r is, o f course, t h e p r i m a l m a t t e r . T h i s w a s as c l e a r t o t h e a n c i e n t * ^

p h i l o s o p h e r s o f G r e e c e a n d I n d i a as i t is c l e a r t o m o d e r n students

of t h e i r o w n i n t e r n a l a n d e x t e r n a l e n v i r o n m e n t s . T h e p h y s i c a l states of w a t e r , a n d t h e process a n d consequences of i t s p h a s e t r a n s i t i o n s , h a v e a l w a y s b e e n n e a r t h e center of t h e o v e r a l l interest i n w a t e r . T h e p h y s i c a l c h e m i s t is a t t r a c t e d b y t h e " a n o m a l i e s " associated w i t h these states a n d 0-8412-0484-5/79/33-180-001$6.50/0 © 1979 American Chemical Society

Fennema; Proteins at Low Temperatures Advances in Chemistry; American Chemical Society: Washington, DC, 1979.

2

PROTEINS A T L O W T E M P E R A T U R E S

phase t r a n s i t i o n s . T h e

geologist

is d r a w n b y

the abundance

of

the

aqueous c o v e r of t h e t e r r e s t r i a l surface a n d t h e d y n a m i c s of t h i s c o v e r i n its v a r i o u s states of a g g r e g a t i o n .

F o r t h e b i o l o g i s t , w a t e r is a substance

that is i n t i m a t e l y a n d p e r v a s i v e l y i n v o l v e d i n t h e m a i n t e n a n c e of t h e v i t a l i n t e g r i t y of b i o l o g i c a l structures a n d i n t h e expression of m a n y of t h e i r vital functions.

Indeed, the modern

biologist

c l e a r l y recognizes

the

essential r e c i p r o c i t y of t h i s i n v o l v e m e n t : t h e s t r u c t u r e of w a t e r is l i n k e d i n a m u t u a l r e l a t i o n s h i p w i t h the o r d e r a n d o r g a n i z a t i o n of b i o m a t t e r . T h e s e v a r i e d a n d f u n d a m e n t a l v a n t a g e p o i n t s w o u l d suffice b y t h e m selves to a c c o u n t f o r interest i n w a t e r a n d its p h y s i c a l states. B u t interest i n t h e p r o p e r t i e s of w a t e r a n d of aqueous systems, i n a l l states of a g g r e Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

g a t i o n , has b e e n s u s t a i n e d also b y d e m a n d s of p r a c t i c a l a p p l i c a t i o n s i n fields

as d i v e r s e as m e t e o r o l o g y , m e d i c i n e , a g r i c u l t u r e , t e c h n o l o g y ,

and

commerce—to name a few. D e s p i t e t h e l o n g h i s t o r y of p h y s i c a l a n d b i o l o g i c a l interest i n f r e e z i n g , a n effective j o i n i n g of p h y s i c a l - c h e m i c a l a n d b i o l o g i c a l insights is a r e c e n t achievement.

T h e e d i t o r of a l a n d m a r k r e v i e w of t h e m a n y facets

of

" c r y o b i o l o g y " has d u l y n o t e d t h a t a significant confluence of p h y s i c a l a n d b i o l o g i c a l t h o u g h t o n f r e e z i n g b a r e l y existed p r i o r to 1940, a n d e v e n i n t h e 1960s o n l y a h a n d f u l of investigators w e r e effectively e n g a g e d i n t h e s t u d y of c r y o b i o l o g y at the f u n d a m e n t a l l e v e l (1 ). The

last t w o

decades b r o u g h t

welcome

change

i n this regard.

A q u e o u s b i o l o g i c a l systems a r e b e i n g s t u d i e d at l o w t e m p e r a t u r e s o n a b r o a d f r o n t a n d i n a m a n n e r t h a t m a k e s i n t e g r a t i o n of d i v e r s e d i s c i p l i n a r y insights increasingly promising.

T h e t h e m e of this s y m p o s i u m

reaches

t h e c o r e of these efforts i n t h a t the b e h a v i o r of p r o t e i n s ( a n d , b y i n f e r ence, other m a c r o m o l e c u l e s ) at l o w t e m p e r a t u r e s m u s t b e v i e w e d as the k e y to a n y r e a l u n d e r s t a n d i n g of c r y o b i o l o g y .

Obviously, the book on

f r e e z i n g of b i o l o g i c a l systems is not a b o u t to b e c l o s e d .

B u t i t seems

e q u a l l y c e r t a i n t h a t o n - g o i n g e f f o r t s — m a n y of w h i c h are r e p o r t e d i n t h i s v o l u m e — a r e impressive a n d h i g h l y encouraging for the future. T h e r o l e a s s i g n e d to t h i s p a p e r is to p r o v i d e a n o v e r v i e w of p r o t e i n s at l o w t e m p e r a t u r e s . T h i s is a v e r y b r o a d assignment. W i t h i n a v a i l a b l e space, response to this a s s i g n m e n t m u s t b e u n a v o i d a b l y selective cursory.

B u t t h e t h e m e of t h i s c h a r g e is l a r g e l y coextensive

t h e m e of t h e s y m p o s i u m .

and

w i t h the

Its s p e c i a l aspects w i l l b e e x p l o r e d i n m o r e

c r i t i c a l l y s e a r c h i n g f a s h i o n t h r o u g h o u t the b u l k of this v o l u m e .

I shall

n o t e n c r o a c h o n those topics t h a t w i l l get expert c o v e r a g e i n t h e s p e c i a l i z e d chapters to f o l l o w .

Instead, I shall attempt to give a s u m m a r y

a c c o u n t t h a t w i l l b e l a r g e l y r e t r o s p e c t i v e . I t seems t h a t s u c h a n effort at t h e outset c o u l d b e of v a l u e to t h e extent t h a t i t p r o v i d e s a f a i r v i e w of t h e r a n g e over w h i c h p r o b l e m s of c r y o b i o l o g y h a v e b e e n a p p r o a c h e d a n d t h o u g h t about.


1.

TABORSKY

Protein

3

Alterations

A s s u b s e q u e n t chapters w i l l u n d o u b t e d l y s h o w , t h e i n t e l l e c t u a l a n d e x p e r i m e n t a l approaches t a k e n to c r y o b i o l o g y i n the r e l a t i v e l y r e c e n t p a s t h a v e c l e a r l y a d v a n c e d o u r u n d e r s t a n d i n g of f r e e z i n g p h e n o m e n a b u t , t h e advances of the last d e c a d e or t w o n o t w i t h s t a n d i n g , m a n y of t h e p r o b l e m s c o n t i n u e to c h a l l e n g e the c r y o b i o l o g i s t .

I t w o u l d b e p r e m a t u r e , i t seems,

to a t t e m p t to d r a w a n i n t e g r a t e d p i c t u r e of t h e state a n d d y n a m i c s of p r o t e i n s i n l o w t e m p e r a t u r e systems.

T o o m a n y features of t h e p i c t u r e

r e m a i n insufficiently clear f o r a p r e c i s e d e f i n i t i o n of t h e i r r e l a t i v e p l a c e w i t h i n the whole.

H o w e v e r , t h e p r o b l e m s c a n b e a d d r e s s e d one at a

time w h i l e keeping i n m i n d that fragmentary a n d oversimplified concepts m u s t e v e n t u a l l y m e r g e i n t o a n i n t e g r a t e d d e s c r i p t i o n i f t h e r e a l i t y of Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

f r e e z i n g p h e n o m e n a is not to b e m i s r e p r e s e n t e d .

Conceptual Approaches to the Study of Proteins at Low

Temperatures

I t is b y n o means c e r t a i n t h a t a g i v e n f r e e z i n g e x p e r i m e n t , d e s i g n e d o r i n t e r p r e t e d i n a p a r t i c u l a r c o n c e p t u a l setting, is o p t i m a l l y effective i n a d v a n c i n g o u r u n d e r s t a n d i n g of f r e e z i n g p h e n o m e n a .

T h e freezing litera-

t u r e is n o t free of a m b i g u i t i e s o r controversies t h a t stem f r o m hypotheses a d v a n c e d o n a too n a r r o w o r e v e n m i s t a k e n basis. T h e i d e n t i f i c a t i o n of the p r o p e r basis o n w h i c h to p l a n or evaluate experiments represents the k e y to progress. I t is l i k e l y t h a t a l l of t h e c o n c e p t u a l l y significant features of the f r e e z i n g process h a v e n o t y e t b e e n r e c o g n i z e d .

Thus, important

factors m a y e l u d e p r o p e r e x p e r i m e n t a l c o n t r o l . A l s o , w e m a y find o u r selves a t t r a c t e d b y reasonable notions t h a t t u r n out to b e not a m e n a b l e to d e f i n i t i v e e x p e r i m e n t a l tests. I t seems best to t r y to d e a l w i t h these reservations b y k e e p i n g t h e m i n focus. W i t h these reservations i n m i n d , i t seems safe a n d h e l p f u l to t a k e the p r a g m a t i c a p p r o a c h a n d p r o c e e d

w i t h o r g a n i z a t i o n of t h e

m a t t e r i n terms of t h e most f r e q u e n t l y e m p l o y e d w o r k i n g

subject

hypotheses.

T h e s e t e n d to b e r o o t e d i n p a r t i c u l a r features of the f r e e z i n g process o r t h e f r o z e n state.

I p r o p o s e to t a k e these u n d e r c o n s i d e r a t i o n i n t h e

a p p r o x i m a t e o r d e r of t h e i r e x p e r i m e n t a l or c o n c e p t u a l c o m p l e x i t y . T h e s i m p l e s t c o n c e p t u a l f r a m e w o r k f o r a l o w t e m p e r a t u r e or f r e e z i n g s t u d y is the a s s u m p t i o n t h a t a n y o b s e r v e d

difference

between

protein

s t r u c t u r e or b e h a v i o r at " n o r m a l " a n d at " l o w " t e m p e r a t u r e c a n

be

a c c o u n t e d f o r i n terms of t h e t e m p e r a t u r e difference alone. T h i s h y p o t h esis focuses a t t e n t i o n o n a c o l d p r o t e i n s o l u t i o n t h a t m a y b e h o m o g e n e o u s or i n contact w i t h f r o z e n solvent. I f t h e r e is i c e present, i t is c o n s i d e r e d of n o p a r t i c u l a r significance. W e c a n e x p a n d this v i e w next b y r e c o g n i z i n g t h a t f r e e z i n g has c e r t a i n consequences i n a d d i t i o n to a c h a n g e i n t e m p e r a t u r e . F u r t h e r m o r e , w e c a n take t h e t a c k t h a t t h e k e y to p r o t e i n alterations is the i n t e r a c t i o n of t h e p r o t e i n w i t h t h e f r o z e n f r a m e w o r k i n


4

PROTEINS A T L O W

w h i c h i t is e m b e d d e d .

TEMPERATURES

A n d l a s t l y , w e c a n d i r e c t o u r a t t e n t i o n at the

f r e e z i n g process i t s e l f r a t h e r t h a n t h e f r o z e n system i n its e q u i l i b r i u m o r q u a s i - e q u i U b r i u m state. I n this case, w e w o u l d t a k e c o g n i z a n c e of t h e p o s s i b i l i t y t h a t alterations of t h e p r o t e i n o c c u r w h e r e t h e d y n a m i c events of t h e p h a s e c h a n g e t a k e p l a c e , n a m e l y at o r n e a r t h e i c e - l i q u i d i n t e r f a c e . T h e s e events m a y b e l i n k e d d i r e c t l y t o the p r o d u c t i o n of s t r u c t u r a l changes

o r t h e y m a y p o t e n t i a t e s u c h changes,

to b e

expressed

later

through altered protein behavior. Proteins in Cold Solutions F o r o u r purposes, a p r o t e i n s o l u t i o n is " c o l d " w h e n e v e r its t e m p e r a Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

t u r e is n e a r or b e l o w t h e f r e e z i n g p o i n t of w a t e r . T h e f r e e z i n g p o i n t c a n b e r e a d i l y m a n i p u l a t e d b y a d d i n g l o w m o l e c u l a r w e i g h t solutes.

Thus,

the temperature range over w h i c h the solution can be studied i n the absence of i c e c a n b e a p p r e c i a b l y e x t e n d e d — b y 20 or m o r e degrees b e l o w zero.

T h e l o w e r H m i t of t h i s m a n i p u l a t i o n is d e f i n e d b y t h e

temperature(s)

of the p a r t i c u l a r system ( 2 ) .

eutectic

M o r e d r a s t i c d e p r e s s i o n of

t h e f r e e z i n g p o i n t ( t o —100° o r e v e n l o w e r ) c a n b e a t t a i n e d u p o n t h e a d m i x t u r e of c e r t a i n o r g a n i c solvents. T h e use of s u c h " c r y o s o l v e n t s " f o r the particular purpose

of p r o t e c t i n g cells against f r e e z i n g dates

back

a b o u t t h i r t y years, w h e n g l y c e r o l w a s first e m p l o y e d as a c r y o p r o t e c t a n t (3).

M o s t l y f o r the same p u r p o s e , m a n y o t h e r o r g a n i c solvents h a v e b e e n

u s e d a n d i n v e s t i g a t e d since t h e n (4).

T h e i r use w i t h p a r t i c u l a r e m p h a s i s

o n consequences at t h e m o l e c u l a r l e v e l , e s p e c i a l l y o n p r o t e i n s , is of r e l a tively recent vintage

(5,6).

Protein Conformation and L o w Temperature: General Considerations.

N a r r o w i n g t h e focus t o just t h e p r o t e i n solute a n d its i m m e d i a t e

l i q u i d e n v i r o n m e n t is a d m i t t e d l y a n o v e r s i m p l i f i c a t i o n . H o w e v e r , t a k i n g t h e n a r r o w v i e w , i f o n l y for t h e m o m e n t , has its a d v a n t a g e . I t w i l l e m p h a size t h a t w h a t e v e r else m a y o c c u r , t h e p r o t e i n w i l l a l w a y s adjust its conformation d u r i n g a temperature change i n accord w i t h the temperature d e p e n d e n c e of those forces t h a t m a i n t a i n the protein's A l t h o u g h a d e f i n i t i v e analysis is b e y o n d

confirmation.

the c a p a b i l i t i e s p r o v i d e d

by

theorists, a q u a l i t a t i v e l y reasonable a n d q u a n t i t a t i v e l y e m e r g i n g p i c t u r e is at h a n d a n d m a y b e u s e f u l to d e s c r i b e the effects of l o w temperatures on protein conformation.

I n o u r d i s c u s s i o n of this p i c t u r e , w e s h a l l r e l y

heavily on a recent pertinent review

(7).

I t is g e n e r a l l y a c c e p t e d t h a t t h e p r i n c i p a l c o n t r i b u t o r to the s t a b i l i t y of t h e " n a t i v e " c o n f o r m a t i o n of a p r o t e i n is t h e t e n d e n c y of its n o n p o l a r groups to a v o i d c o n t a c t w i t h w a t e r — t h a t is, to e n g a g e i n h y d r o p h o b i c i n t e r a c t i o n s . B a s e d o n m o d e l c o m p o u n d s a n d o n c u r r e n t theories of w a t e r s t r u c t u r e , t h e process is v i e w e d as l a r g e l y e n t r o p y - d r i v e n . T h e u n f a v o r a b l e l o w e n t r o p y of " o r d e r e d " w a t e r , w h i c h w o u l d s u r r o u n d n o n p o l a r


1.

TABORSKY

Protein

5

Alterations

g r o u p s w h e n these are exposed

to the aqueous

because the nonpolar groups f o l d i n w a r d .

m e d i u m , is

avoided

T h i s is a c c o m p a n i e d b y a n

e n t r o p y - i n c r e a s i n g release of o r d e r e d w a t e r to t h e r e l a t i v e l y d i s o r d e r e d state of the b u l k solvent. C o n s i d e r i n g t h a t b u l k w a t e r itself b e c o m e s m o r e extensively h y d r o g e n - b o n d e d a n d thus m o r e h i g h l y o r d e r e d as the t e m p e r a t u r e is l o w e r e d , t h e e n t r o p i e a d v a n t a g e of f o l d i n g n o n p o l a r groups i n w a r d is lessened at l o w temperatures.

I n this case, s t a b i l i z a t i o n b y

h y d r o p h o b i c i n t e r a c t i o n s w o u l d b e e x p e c t e d to decrease.

[It is g e n e r a l l y

u n q u e s t i o n e d t h a t " h y d r o p h o b i c i n t e r a c t i o n s " are of m a j o r i m p o r t a n c e w i t h r e g a r d to the c o n f o r m a t i o n a l i n t e g r i t y of p r o t e i n s a n d t h a t these i n t e r a c t i o n s suffer w h e n a p r o t e i n is " c o l d - d e n a t u r e d . " M a n y e x p e r i m e n t a l Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

d a t a are s u p p o r t i v e of this v i e w (see, f o r e x a m p l e , 8, 9 ) .

This support

n o t w i t h s t a n d i n g , c e r t a i n e x p e r i m e n t a l p a r a m e t e r s of p r o t e i n d e n a t u r a t i o n — p a r t i c u l a r l y t h e associated, u n e x p e c t e d l y s m a l l v o l u m e changes

(10,

c a u s e d questions to b e r a i s e d c o n c e r n i n g t h e strict a p p l i c a -

11)—have

b i l i t y of m o d e l s i n terms of w h i c h h y d r o p h o b i c i n t e r a c t i o n s , m a i n t a i n i n g p r o t e i n s i n a f o l d e d state, h a d f r e q u e n t l y b e e n a c c o u n t e d for. T h i s p r o b l e m is g i v e n c r i t i c a l a t t e n t i o n i n a n u m b e r of recent r e v i e w s (see, example,

for

7,12,13,14,15).]

A s t r o n g l y c o n t r a s t i n g b e h a v i o r m a y b e e x p e c t e d , at first sight, w i t h h y d r o g e n b o n d s t h a t c o n t r i b u t e to p r o t e i n s t a b i l i z a t i o n . L o w t e m p e r a t u r e s s h o u l d enhance h y d r o g e n b o n d f o r m a t i o n i n v i e w of t h e n e g a t i v e c h a n g e i n e n t h a l p y that characterizes this process.

W e w o u l d expect h y d r o g e n

b o n d s to b e c o m e stronger as t h e t e m p e r a t u r e drops. S u c h s t r e n g t h e n i n g s h o u l d a p p l y to i n t r a m o l e c u l a r b o n d s

(between donor

and

acceptor

g r o u p s of t h e p r o t e i n ) as w e l l as to i n t e r m o l e c u l a r b o n d s ( b e t w e e n p r o tein groups a n d water molecules).

T h e enhanced strength, particularly

of those i n t r a m o l e c u l a r b o n d s i n t h e protein's i n t e r i o r , m i g h t b e to e n h a n c e c o n f o r m a t i o n a l s t a b i l i t y — o r e v e n t o alter the

expected

conformation

b y u p s e t t i n g the o v e r a l l b a l a n c e of a l l forces t h a t m a i n t a i n c o n f o r m a t i o n . T h e r e appears, h o w e v e r , to b e l i t t l e l i k e l i h o o d that a d d i t i o n a l h y d r o g e n b o n d s w i l l f o r m i n the i n t e r i o r of the p r o t e i n d u r i n g c o o l i n g since m o s t of t h e p o s s i b l e b o n d s w o u l d b e e x p e c t e d to exist a l r e a d y at t h e h i g h e r , " n o r m a l " temperatures.

[ T h e v i e w t h a t most of the p o s s i b l e

hydrogen

b o n d s a l r e a d y exist i n the " n o r m a l " p r o t e i n m a y r e q u i r e q u a l i f i c a t i o n b y the postulate

(12)

t h a t the d e m o n s t r a t e d a c c e s s i b i l i t y of the p r o t e i n

i n t e r i o r to w a t e r ( a n d to H

+

or O H " ) is d u e to s t r u c t u r a l "defects," exist-

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

Accord-

i n g to this v i e w , some p o s s i b i l i t y r e m a i n s i n a " n o r m a l " p r o t e i n f o r t h e a c q u i s i t i o n of a d d i t i o n a l h y d r o g e n b o n d s as t h e t e m p e r a t u r e is l o w e r e d . ] T h e r e is l i t t l e t h a t w e c a n say a b o u t electrostatic forces i n c o n n e c t i o n w i t h l o w t e m p e r a t u r e effects.

U n d o u b t e d l y , the charged groups of

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


a

How-

6


ever, the m a g n i t u d e a n d d i r e c t i o n o f these c o n t r i b u t i o n s is b e y o n d o u r a b i l i t y to assess since i t w o u l d r e q u i r e i n f o r m a t i o n a b o u t t h e i r p r e c i s e l o c a t i o n o n the p r o t e i n surface a n d a b o u t the effective d i e l e c t r i c constant o n w h i c h t h e f o r c e of these i n t e r a c t i o n s d e p e n d s .

S u c h i n f o r m a t i o n is

usually lacking. T h u s , even though temperature w o u l d be expected

to

i n f l u e n c e c h a r g e d e n s i t y ( b e c a u s e a c i d d i s s o c i a t i o n e q u i l i b r i a are t e m perature dependent)

a n d t h e d i e l e c t r i c constant ( w h a t e v e r its v a l u e ) ,

o u r a b i l i t y t o p r e d i c t is severely c i r c u m s c r i b e d . Effects m a y b e s i g n i f i c a n t b u t t h e i r m a g n i t u d e c a n n o t b e assessed n o r is i t p o s s i b l e to d e t e r m i n e w h e t h e r s u c h effects w o u l d s t r e n g t h e n , w e a k e n , o r alter t h e p r o t e i n ' s conformation i n a temperature dependent manner. Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

I n s u m m a r y , w e c a n say t h a t l o w t e m p e r a t u r e s w o u l d b e

expected

to alter p r o t e i n c o n f o r m a t i o n s b y f a v o r i n g t h e f o r m a t i o n of a n d s t r e n g t h e n i n g h y d r o g e n b o n d s a n d d i m i n i s h i n g the i m p o r t a n c e of

hydrophobic

i n t e r a c t i o n s . T h e effect of l o w t e m p e r a t u r e s o n electrostatic i n t e r a c t i o n s i n p r o t e i n s is o u t s i d e o u r p a l e . W e s h o u l d also r e c a l l t h e e a r l i e r s t i p u l a t i o n that w h a t e v e r consequences the c o l d or f r o z e n state m i g h t h a v e o n p r o t e i n s p e r se, these c o n s e q u e n c e s m u s t b e i n t e g r a t e d , i n t h e

final

analysis, w i t h t h e c o n s e q u e n c e s o f c o o l i n g or f r e e z i n g o n n o n p r o t e i n c o m p o n e n t s of t h e system. T h e p r e s e n t " m i n i m a l " h y p o t h e s i s as w e l l as those w h i c h are y e t to b e c o n s i d e r e d , t a k e n i n d i v i d u a l l y , p r o v i d e o n l y a l i m i t e d v i e w of l o w t e m p e r a t u r e a n d f r e e z i n g p h e n o m e n a . Experimental Observations

of Putative Conformational

Ascribed or Ascribable to L o w Temperature Effects.

Changes

Some

of

the

p a t h - b r e a k i n g studies of the effects o f near-zero o r s u b z e r o t e m p e r a t u r e s o n several e n z y m e systems w e r e i n t e r p r e t e d o n the basis of t e m p e r a t u r e - d e p e n d e n t shifts i n e n z y m e c o n f o r m a t i o n .

assumed,

These conforma

t i o n a l changes w e r e p r e s u m e d to relate d i r e c t l y to t h e degree of i n t r a m o l e c u l a r h y d r o g e n b o n d i n g (16,

17, 18, 19, 20).

W h i l e the p h y s i c a l -

c h e m i c a l d e s c r i p t i o n of the observations is reasonable a n d s t r a i g h t f o r w a r d (cf.

20),

t h e h y p o t h e s i s m u s t b e v i e w e d w i t h reservations, n o t

only

b e c a u s e i t w a s p r o p o s e d at a t i m e w h e n l i t t l e w a s k n o w n a b o u t m e c h a n i s m s a n d forces b y w h i c h p r o t e i n c o n f o r m a t i o n is s t a b i l i z e d , b u t also b e c a u s e t h e e v i d e n c e i n s u p p o r t of the h y p o t h e s i s w a s i n f e r r e d r a t h e r t h a n d i r e c t . N e v e r t h e l e s s , these results s h o u l d b e n o t e d : t h e y w e r e the first results of a first a t t e m p t at q u a n t i t a t i v e i n s i g h t i n t o a h i g h l y r e f r a c t o r y p r o b l e m a n d they m a y still b e instructive. T a b l e I illustrates the nature of t h i s i n s i g h t . T h e k i n e t i c d a t a o b t a i n e d w i t h t h r e e e n z y m e s b i p h a s i c p l o t s of l o g k vs. 1 / Γ .

fitted

These plots y i e l d e d values for the apparent

A r r h e n i u s a c t i v a t i o n energies, w h i c h s h o w e d a m a j o r increase w h e n the t e m p e r a t u r e w a s l o w e r e d b e l o w t h e p o i n t at w h i c h t h e aqueous system began

to freeze.

Similar biphasic Arrhenius plots were

subsequently

r e p o r t e d f o r o t h e r e n z y m e s (e.g., a l k a l i n e p h o s p h a t a s e a n d p e r o x i d a s e )


1.

Protein

TABORSKY

Table I.

7

Alterations

Arrhenius Activation Energies of Some " F r o z e n " Enzyme Reactions 0

Arrhenius Enzyme

Above

Lipase Invertase Trypsin β

Energy

(kcal/mol) Below

0

C

(16).

T h e s e results w e r e i n t e r p r e t e d to m e a n t h a t l o w

f a v o r e d the f o r m a t i o n of e n z y m e bonds

(intramolecular)

and

molecules

caused,

0°

37.0 60.0 65.0

7.6 11.1 15.4

A l l data were taken from Sizer and Josephson

(18). Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

Activation

temperatures

with abundant

thereby,

hydrogen

a decrease i n

enzyme

a c t i v i t y . [ A l t h o u g h , as n o t e d , this i n t e r p r e t a t i o n predates t h e i n t r o d u c t i o n of t h e c o n c e p t of h y d r o p h o b i c

interactions a n d perforce

ignores

p u t a t i v e i m p o r t a n c e a t t r i b u t e d to these i n t e r a c t i o n s as t h e m a j o r f o r m a t i o n - m a i n t a i n i n g force, t h e n o t i o n of e n h a n c e d h y d r o g e n

the con

bonding

at l o w t e m p e r a t u r e s s h o u l d n o t b e s i m p l y d i s m i s s e d as o u t d a t e d .

The

r e c e n t l y p o s t u l a t e d " s t r u c t u r e defects" a t t r i b u t e d to i n c o m p l e t e l y h y d r o gen-bonded

structures i n t h e n a t i v e p r o t e i n i n t e r i o r (see R e f e r e n c e

are, i n p r i n c i p l e , f u l l y c a p a b l e of c o m p l e t i o n at l o w e r e d

12)

temperatures,

c a u s i n g c o n f o r m a t i o n a l r e a r r a n g e m e n t s o n a scale t h a t m a y b e significant.] It s h o u l d b e e m p h a s i z e d t h a t this h y p o t h e s i s w a s e v e n t h e n r e g a r d e d as o v e r l y s i m p l i s t i c i n t h a t i t p r o b a b l y o v e r l o o k e d a w h o l e r a n g e of o t h e r consequences of exposure to l o w t e m p e r a t u r e .

Recognized possibilities

i n c l u d e d : 1)

i n intermolecular inter

a v i s c o s i t y increase, 2 )

changes

actions i n v o l v i n g e n z y m e , substrate, solvent, a n d buffer i o n s , a n d 3 ) shifts i n i o n i z a t i o n e q u i l i b r i a of a n y of these c o m p o n e n t s .

T h u s , t h e stage w a s

set for f u t u r e i n v e s t i g a t i o n s of a m o r e s e a r c h i n g n a t u r e . O n c e the i m p o r t a n c e of h y d r o p h o b i c i n t e r a c t i o n s b e c a m e g e n e r a l l y e v i d e n t , i t b e c a m e c l e a r t h a t these i n t e r a c t i o n s m i g h t u n d e r g o i m p o r t a n t changes w h e n e v e r p r o t e i n s a r e exposed to l o w t e m p e r a t u r e s .

This view

d o m i n a t e s reports o n m u l t i m e r i c e n z y m e systems, m i c e l l e - l i k e aggregates, and

m e m b r a n o u s structures. I w i l l refer to s e v e r a l examples f r o m t h e

r e l a t i v e l y r e c e n t l i t e r a t u r e f o r purposes of i l l u s t r a t i o n . P r o m p t e d b y the s t r i k i n g i n i t i a l o b s e r v a t i o n t h a t m u l t i p l e e n z y m e forms are p r o d u c e d u p o n f r e e z i n g solutions of l a c t i c d e h y d r o g e n a s e

(21),

t h e p r o p e r t i e s of s e v e r a l other e n z y m e s w e r e e x p l o r e d u n d e r f r e e z i n g c o n d i t i o n s (22).

T h e s e studies m a y b e v i e w e d as t h e take-off p o i n t f o r

m a n y studies t h a t gave c a r e f u l a t t e n t i o n to t h e d i v e r s e p a r a m e t e r s t h a t c a n affect t h e o u t c o m e of a f r e e z i n g e x p e r i m e n t .

It produced the tenta

t i v e g e n e r a l i z a t i o n t h a t s u b u n i t d i s s o c i a t i o n m a y b e t h e p r i m a r y response of e n z y m e s to f r e e z i n g .


8


B y n o t i n g t h e b e h a v i o r of c a s e i n m i c e l l e s of g e n e t i c a l l y v a r i e d p o l y m o r p h i c c o m p o s i t i o n (23),

one c a n r e a d i l y sense t h a t f r e e z i n g m a y h a v e

o n l y s u b t l e effects o n t h e m o l e c u l a r structures of m a c r o m o l e c u l a r plexes.

com-

T h e p o l y m o r p h i s m o f casein stems f r o m m i n o r v a r i a t i o n s i n t h e

p r i m a r y s t r u c t u r e of casein s u b u n i t s . I t w a s f o u n d t h a t f r e e z i n g p r o d u c e d different changes i n m i c e l l e s t a b i l i t y d e p e n d i n g o n t h e genetic v a r i a n t b e i n g s t u d i e d . F o r e x a m p l e , i n the case of one p a r t i c u l a r v a r i a n t c o m p l e x , its s t a b i l i t y ( n o n f r o z e n ) e x c e e d e d t h a t of t h e average c a s e i n m i c e l l e , b u t u p o n f r e e z i n g t h i s same v a r i a n t c o m p l e x

b e c a m e less stable t h a n t h e

average m i c e l l e . A l l of this p r e s u m a b l y o c c u r r e d b e c a u s e of a f e w a m i n o a c i d substitutions. I t is a l m o s t necessary to assume t h a t the c h a n g e i n Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

s t a b i l i t y of the m i c e l l e reflects some r e a r r a n g e m e n t i n t h e m a n n e r i n which

subunits interact.

s u p p o r t this v i e w .

A n independent

observation

tends

(24)

to

L o n g f r o z e n storage w a s f o u n d to cause d i s t o r t i o n

a n d , u l t i m a t e l y , d e p o l y m e r i z a t i o n of t h e m i c e l l e . T h e e g g p r o v i d e s another system of interest b e c a u s e of the r o l e t h a t l i p o p r o t e i n complexes p l a y i n t h e y o l k structure. E g g y o l k gels o n freezing.

T h e r e is b r o a d u n a n i m i t y i n t h e l i t e r a t u r e , b a s e d o n a w e a l t h of

e x p e r i m e n t a l d a t a (25-30), protein aggregation.

t h a t t h i s response to f r e e z i n g i n v o l v e s l i p o -

R e p l a c e m e n t of n a t i v e h y d r o p h o b i c i n t e r a c t i o n s b y

a r t i f i c i a l ones m a y o c c u r because the n a t i v e a r r a n g e m e n t m a y b e d i s r u p t e d b y f r e e z i n g - i n d u c e d changes i n t h e l i p o p r o t e i n ' s aqueous

environment.

I m p o r t a n c e has also b e e n a s c r i b e d to t h e f a t t y a c i d s p e c t r u m of y o l k l i p i d s [ i n f l u e n c i n g the t e m p e r a t u r e of t h e r m a l transitions (26)]

a n d to

t h e salt c o n t e n t [affecting t h e i n t e g r i t y of the y o l k granules of w h i c h t h e y o l k l i p o p r o t e i n is a m a j o r c o m p o n e n t

(25, 27, 29)]

The formation

of a d d i t i o n a l h y d r o g e n b o n d s d u r i n g f r e e z i n g has also b e e n

suggested

(29). H y d r o p h o b i c i n t e r a c t i o n s are, of course,

c r u c i a l to

biomembrane

i n t e g r i t y . M e m b r a n e s , therefore, s h o u l d e x h i b i t v u l n e r a b i l i t y to f r e e z i n g , a n d t h e y do. I t seems, h o w e v e r , t h a t i n j u r i e s n e e d n o t p r i m a r i l y c o n c e r n the protein

chemist.

F o r example, the

fluorescence

of t h e p r o t e i n of

e r y t h r o c y t e membranjes undergoes a shift o n f r e e z i n g . T h i s is i n d i c a t i v e of a c h a n g e i n the p r o t e i n e n v i r o n m e n t , most l i k e l y i n v o l v i n g m e m b r a n e lipids

(31).

P r o t e i n s are a p p a r e n t l y not released

(32).

Nevertheless,

m e m b r a n e - b o u n d p r o t e i n s d o n o t escape t h e effects of l o w t e m p e r a t u r e altogether. I t has b e e n s h o w n , f o r e x a m p l e , t h a t the c i r c u l a r d i c h r o i s m e x h i b i t e d b y the e r y t h r o c y t e m e m b r a n e ( d u e , p r e s u m a b l y , to its p r o t e i n c o m p o n e n t s ) undergoes

significant changes

as the t e m p e r a t u r e of

the

m e m b r a n e suspension is v a r i e d over a w i d e r a n g e , d o w n to n e a r l y 0 ° C (33).

( S h a r p , c o o p e r a t i v e transitions r e f l e c t i n g d e n a t u r a t i o n w e r e s h o w n

to o c c u r o n l y at t e m p e r a t u r e s a b o v e 4 0 ° C u n d e r t h e c o n d i t i o n s of these experiments. )


1.

Protein

TABORSKY

The mitochondrial membrane content

during freezing.

dehydrogenase

9

Alterations

For

also sutlers alterations of

example,

freezing

alters

its l i p i d

ketoglutarate

a c t i v i t y a n d t h i s c h a n g e m a y result f r o m t h e i n h i b i t o r y

a c t i o n of free f a t t y acids t h a t h a v e b e e n f r e e z i n g - i n d u c e d a c t i v i t y of p h o s p h o l i p a s e s

generated, p r e s u m a b l y , (34).

I n another study, the

" a n t i r a d i c a l a c t i v i t y " of l i p i d s f r o m v a r i o u s f r o z e n organelles w a s s h o w n to d i m i n i s h ( 3 5 ) .

by

and thawed

liver

Studies w i t h f r o z e n b r a i n o r g a n -

elles l e n d a d d i t i o n a l s t r e n g t h to the v i e w t h a t p r o t e i n d a m a g e is n o t , p e r h a p s , the p r i m a r y , f u n c t i o n a l l y d e s t r u c t i v e event d u r i n g f r e e z i n g of membrane enzymes

systems.

among

I n these studies, some r e d i s t r i b u t i o n o f " m a r k e r "

s u b c e l l u l a r fractions w a s

found

b u t loss of

enzyme


a c t i v i t y w a s m i n i m a l , suggesting t h a t t h e m e m b r a n e b i n d i n g sites of these p r o t e i n s w e r e d a m a g e d r a t h e r t h a n the p r o t e i n s themselves

(36).

B e f o r e I t u r n to m o r e c o m p l e x matters, I w o u l d l i k e to e m p h a s i z e t h a t m a n y of t h e p u t a t i v e c o n f o r m a t i o n a l or other changes

mentioned

a b o v e o c c u r r e d i n a f r o z e n r a t h e r t h a n just a c o l d e n v i r o n m e n t .

Thus,

the changes o b s e r v e d p r o b a b l y i n v o l v e m o r e t h a n a s i m p l e exposure to l o w temperatures. H o w e v e r , i n some instances the effects of l o w t e m p e r a t u r e p e r se m a y p r e d o m i n a t e , a n d this s i m p l e h y p o t h e s i s a l w a y s m e r i t s consideration. Frozen Aqueous

Solutions:

As

Concentration Effects.

solute

is

r e j e c t e d b y the g r o w i n g i c e a n d as its c o n c e n t r a t i o n increases i n t h e s h r i n k i n g l i q u i d phase, the t e m p e r a t u r e d r o p s t o w a r d the eutectic p o i n t , w h e r e the entire system approaches

complete

solidification.

Of

major

i m p o r t a n c e is t h e f a c t that a h i g h l y c o n c e n t r a t e d l i q u i d phase c a n persist i n d e f i n i t e l y at a n y p o i n t a b o v e t h e eutectic t e m p e r a t u r e . T h i s t y p e of " f r e e z i n g - o u t "

has l o n g b e e n k n o w n a n d has

been

e x p l o i t e d i n i n d u s t r i a l settings. B u t , i n a r e s e a r c h s e t t i n g , a t t e n t i o n w a s focussed o n i t o n l y r e c e n t l y w h e n the c r y o c h e m i s t b e c a m e c o n c e r n e d w i t h " a n o m a l o u s " r e a c t i o n k i n e t i c s i n " f r o z e n " systems, a n d w h e n t h e c r y o b i o l o g i s t b e g a n l o o k i n g into t h e causes of f r e e z i n g i n j u r y . A n i l l u s t r a t i o n of s u c h studies, as t h e y p e r t a i n to p r o t e i n s , is n o w i n order. T h e t y p i c a l p r o t e i n s o l u t i o n contains a v a r i e t y of l o w w e i g h t solutes

(e.g.

substrates, cofactors,

buffer s a l t s ) .

molecular

Such

systems

s h o u l d e x h i b i t s e v e r a l " e u t e c t i c " p o i n t s . D u r i n g f r e e z i n g , the c o n c e n t r a tions of v a r i o u s m o l e c u l a r species, because t h e y h a v e different p o i n t s , w i l l c h a n g e r e l a t i v e to e a c h other. u n t i l the

final

eutectic

T h e s e changes w i l l c o n t i n u e

eutectic t e m p e r a t u r e has b e e n

attained.

For

example,

r e l a t i v e l y s i m p l e aqueous systems c o m p o s e d o n l y of s o d i u m a n d p o t a s s i u m salts of p h o s p h o r i c

acid were

shown

associated w i t h t h e m (see T a b l e I I ) .

to h a v e

eleven

eutectic

points

It is i m p o r t a n t f r o m a p r o t e i n -

o r i e n t e d p o i n t of v i e w t h a t t h e p H values a n d i o n i c strengths of these eutectic solutions r a n g e w i d e l y w i t h i n t h e i r f r e e z i n g r a n g e b e t w e e n


0°

10


Table II.

Eutectic Solutions of Phosphate Ionic Strength (M)

Solute(s) Na HP0 KH P0 Na HP0 + KH P0 NaH P0 NaH P0 + Na HP0 NaH P0 + KH P0 NaH P0 + Na HP0 + KH P0 K HP0 Na HP0 + K HP0 KH P0 + K HP0 Na HP0 + KH P0 + K HP0 2

4

2

4

2

4

2 2

4

2

4

2

2

4

2

4

2

4

4

2

4

2

4

4

2


2

4

4

2

2

4

2

4

2

4

4

2

4

2

4

Salts*

Freezing Point (°C)

0.33 0.92 2.50 3.42 3.60 3.84 4.10 8.55 9.00 9.40 9.72

8.9 4.1 5.5 3.3 3.6 3.4 3.6 9.3 9.3 7.5 7.5

-0.5 -2.7 -4.3 -9.7 -9.9 -11.2 -11.4 -13.7 -14.4 -16.7 -17.2

A l l data were taken from V a n den Berg and Rose (87) ; the ionic strength values were calculated from concentration data given in the reference. p H measurements were made at 25° after separation of the unfrozen liquid from the solid phase. a

&

a n d a b o u t — 17°. expected

T h u s , a d i s s o l v e d protein's s t r u c t u r a l i n t e g r i t y c a n b e

to be c h a l l e n g e d b y these v a r i a t i o n s .

M i l k , for e x a m p l e , i n

w h i c h the p r o t e i n s c a n experience f r e e z i n g - i n d u c e d changes, w i l l s h o w a d e c l i n e i n p H o n s l o w f r e e z i n g f r o m 6.6 to 5.8. F a s t f r e e z i n g causes n o i m m e d i a t e p H c h a n g e , b u t a l o w p H develops u p o n " f r o z e n "

storage—

p r e s u m a b l y b e c a u s e e q u i l i b r i u m is s l o w l y e s t a b l i s h e d b e t w e e n l i q u i d a n d s o l i d phases

(38).

F r e e z i n g - i n d u c e d changes

solutions a n d foods ( 3 9 )

i n p H occur

i n many

a n d i t is to s u c h p H v a r i a t i o n s t h a t

specific

e n z y m i c a c t i v i t y losses u p o n f r e e z i n g h a v e b e e n a t t r i b u t e d (40).

Such

f r e e z i n g - i n d u c e d p H effects c a n b e c o u n t e r a c t e d b y t h e a d d i t i o n of " c r y o p r o t e c t a n t s " s u c h as g l y c e r o l or d i m e t h y l s u l f o x i d e , w h i c h are

effective

b e c a u s e t h e selective p r e c i p i t a t i o n of buffer salts w i l l o c c u r o n l y at l o w e r t e m p e r a t u r e s i n t h e i r presence

(41).

T h e r e are n u m e r o u s reports

p r o t e i n alterations a t t r i b u t e d to other c o n c e n t r a t i o n changes D e n a t u r a t i o n of c h y m o t r y p s i n (42),

a c t i v a t i o n of a

( 4 3 ) , a n d d i s r u p t i o n of y o l k g r a n u l e s (29)

as

of

well.

phosphodiesterase

h a v e b e e n a s c r i b e d , at least

i n p a r t , to increases i n salt c o n c e n t r a t i o n u p o n f r e e z i n g .

(Parenthetically,

w e m a y note some t e c h n i c a l l y i n t e r e s t i n g d e v e l o p m e n t s . has b e e n d e s c r i b e d t h a t p e r m i t s t h e freeze

A n apparatus

c o n c e n t r a t i o n of

proteins

themselves, o n a m u l t i l i t e r scale. T e n - f o l d increases i n c o n c e n t r a t i o n c a n b e o b t a i n e d b y t h i s means (44).

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

r e p o r t o n t h e use of p H i n d i c a t o r s i n f r o z e n solutions (45).

Finally, we

m a y t a k e note of a n e x p e r i m e n t t h a t is i n d i c a t i v e of t h e s e l e c t i v i t y of the freezing-out mechanism.

U p o n f r e e z i n g a m i x t u r e of l i g h t a n d

w a t e r ( d e u t e r a t e d ) , a n isotope e n r i c h m e n t a m o u n t i n g to 2 % achieved

heavy

could

(46).)


be

1.

Protein

TABORSKY

11

Alterations

I n g e n e r a l , i t is c l e a r t h a t f r e e z i n g c a n cause c o n c e n t r a t i o n of solutes to c h a n g e b y orders of m a g n i t u d e .

C h a n g e s i n c o n c e n t r a t i o n of t h i s

d e g r e e s h o u l d b e a b l e , p o t e n t i a l l y , to cause m a j o r alterations i n p r o t e i n s . Furthermore, changes.

s u c h alterations n e e d

not

be

confined

to

noncovalent

Substances c a p a b l e of r e a c t i n g c o v a l e n t l y w i t h a p r o t e i n m a y

u n d e r g o s u c h reactions w h e n p u s h e d b y t h e mass a c t i o n of t h e i r i n c r e a s e d concentrations. Reactions

in Frozen Aqueous

Systems.

Particularly

d u r i n g the

sixties, t h e r e w a s a w a v e of c o n c e r n a b o u t rates a n d m e c h a n i s m s

of

c h e m i c a l reactions that o c c u r i n t h e " f r o z e n state." E a r l i e r observations o f c h e m i c a l changes w e r e l a r g e l y a c c i d e n t a l a n d f r e q u e n t l y h e l d t o b e Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

o n l y of n u i s a n c e v a l u e .

T h e one m a j o r e x c e p t i o n c o n c e r n e d

cryobio-

l o g i c a l i n q u i r i e s i n t o t h e causes of f r e e z i n g i n j u r y . I n these cases, h o w ever, t h e c o m p l e x i t y of the m a t e r i a l effectively m a s k e d t h e c h e m i c a l roots of t h e m e c h a n i s m . reactions h a v e

E v e n t h o u g h most

involved low

studies of

molecular weight

"frozen"

chemical

reactants, t h e y

merit

c o n s i d e r a t i o n b e c a u s e t h e i r c o n c e p t u a l f r a m e w o r k is c e r t a i n l y a p p l i c a b l e to c o m p l e x b i o m o l e c u l e s or other b i o m a t t e r . M u c h of the interest i n these reactions s t e m m e d f r o m t h e a t t r a c t i v e ness of t h e p o s s i b i l i t y that s p e c i a l p r o p e r t i e s of i c e m a y b e r e s p o n s i b l e for novel reaction mechanisms.

F o r instance, p r o t o n m o b i l i t y is h i g h e r

i n ice t h a n i n l i q u i d w a t e r b y one o r t w o orders o f m a g n i t u d e a n d t h e effective d i e l e c t r i c constant i n ice is l o w e r t h a n t h a t of w a t e r b y a b o u t a n o r d e r of m a g n i t u d e (47).

U n d e r such conditions, mechanisms involv-

i n g p r o t o n t r a n s p o r t or c h a r g e i n t e r a c t i o n s m a y c h a n g e , e v e n q u a l i t a t i v e l y . C h a n g e s i n m e m b r a n e p e r m e a b i l i t y , for i n s t a n c e , m a y reflect changes i n dielectric properties of membranes resulting f r o m altered proton behavior at l o w t e m p e r a t u r e (48).

A n e n t i r e l y different s p e c u l a t i o n has

been

a d v a n c e d r e g a r d i n g the p o s s i b i l i t y of r e c i p r o c a l effects b e t w e e n i c e - l i k e structures a n d t h e s t a b i l i z a t i o n of e l e c t r o n i c a l l y e x c i t e d states of b i o m o l e cules, thus g i v i n g rise to mechanism

a hypothetical, novel energy

transduction

(49).

A m a j o r p o i n t w a s m a d e after a c r i t i c a l r e v i e w of n u m e r o u s reports o n r e a c t i o n rates i n f r o z e n systems (50).

Kinetic-mechanistic "surprises"

i n " f r o z e n " systems m a y n o t r e q u i r e e x c e p t i o n a l hypotheses. t i o n effects m a y a c c o u n t for t h e m .

Concentra-

E v e n i f a system appears to

be

c o m p l e t e l y s o l i d i f i e d a n d , therefore, n o t a m e n a b l e to analysis i n terms of unfrozen l i q u i d "puddles" (51), a l i q u i d phase should still be considered a p o s s i b i l i t y . A case i n p o i n t is p r o v i d e d b y the efficient e l e c t r o n transfer o b s e r v e d b e t w e e n ferrous a n d f e r r i c ions i n a n a q u e o u s system f r o z e n b e l o w its p u t a t i v e eutectic p o i n t (52).

T h i s s e e m e d to r e q u i r e a n i c e

s t r u c t u r e i n o r d e r to b r i d g e t h e d i s t a n c e b e t w e e n reactants t h a t w e r e c a l c u l a t e d to b e too f a r a p a r t f o r significant r e a c t i v i t y . H o w e v e r , i t w a s p o i n t e d out t h a t the a s s u m e d e u t e c t i c p o i n t w a s b a s e d o n l y o n t h e m a j o r


12

PROTEINS A T L O W

TEMPERATURES

solute c o m p o n e n t , w h e r e a s m i n o r c o m p o n e n t s — t h e reactants, i n t h i s case — c o u l d h a v e l o w e r e d t h e effective

eutectic p o i n t b e l o w

t e m p e r a t u r e , m a k i n g t h e a s s u m p t i o n of fluous

the r e a c t i o n

a special mechanism

super-

(50).

E l u c i d a t i o n of m e c h a n i s m s of f r o z e n reactions w i l l be h i n d e r e d i f t h e coexistence

of l i q u i d a n d s o l i d phases is v i e w e d necessarily as a

t h e r m o d y n a m i c a l l y defined concentrations.

phenomenon,

i m p l y i n g stable phases

and

F r o z e n reactions c o u l d o c c u r i n a l i q u i d p h a s e t h a t is

o n l y of t r a n s i e n t significance.

I n t h e t r i v i a l case, " p u d d l e s " m a y exist

s i m p l y b e c a u s e f r e e z i n g r e q u i r e s finite t i m e . I n o t h e r cases, m o r e sophist i c a t e d i n s i g h t i n t o t h e d y n a m i c s of t h e f r e e z i n g process m a y b e n e e d e d . Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

F o r e x a m p l e , transient p u d d l e s m a y exist i n a v a r i a b l e m a n n e r d e p e n d i n g o n t h e rate of c o o l i n g .

U n f o r t u n a t e l y , t h e r e q u i r e d i n s i g h t is elusive

because the process is too c o m p l e x . A h i g h l y r e l e v a n t , t h o u g h t f u l analysis of the d i s t i n c t i o n b e t w e e n c o o l i n g k i n e t i c s (i.e. t e m p e r a t u r e c h a n g e ) a n d f r e e z i n g k i n e t i c s (i.e.

solidification)

is a v a i l a b l e ( 5 3 ) .

A n important

feature of this d i s t i n c t i o n is t h a t f r e e z i n g v e l o c i t y d e p e n d s o n t h e a b i l i t y of t h e system to dissipate t h e heat of c r y s t a l l i z a t i o n . T h i s r e q u i r e s t i m e , d u r i n g w h i c h the s u p e r c o o l e d l i q u i d m u s t serve as a t e m p o r a r y heat sink w h i l e i t exists e n t r a p p e d w i t h i n a " s p o n g y " ice s t r u c t u r e ( 5 4 ) .

These

l i q u i d p o o l s c a n b e the m e d i u m i n w h i c h c h e m i c a l reactions t a k e p l a c e u n d e r c o n d i t i o n s t h a t are l a r g e l y i n d e t e r m i n a t e . F o r e x a m p l e , t h e u n i f o r m i t y of this s o l u t i o n i n terms of concentrations a n d t e m p e r a t u r e c a n n o t b e t a k e n for g r a n t e d , a n d a n y c o n c e n t r a t i o n a n d t h e r m a l g r a d i e n t s are, f o r a l l p r a c t i c a l purposes, b e y o n d p r e c i s e , q u a n t i t a t i v e d e s c r i p t i o n .

In

a d d i t i o n , r e l a t i v e l y l i t t l e c o n t r o l c a n b e exercised over the rate of heat w i t h d r a w a l a n d this d e t e r m i n e s the size of t h e l i q u i d p o o l s a n d

how

closely t h e ratios of the s o l i d a n d l i q u i d phases a p p r o a c h e q u i l i b r i u m . T h e r e also m a y b e i r r e g u l a r t h e r m a l c o n d u c t i v i t y gradients i n t h e system w h i c h , together w i t h features of gross g e o m e t r y

( surf a c e / v o l u m e

ratio

a n d s h a p e ) , w i l l h a v e a m a j o r i m p a c t o n the m a n n e r a n d s p e e d

with

w h i c h the heat of c r y s t a l l i z a t i o n is finally t a k e n u p b y t h e c o o l i n g source. T h i s is not a n e n c o u r a g i n g p i c t u r e b u t i t is a r e a l i s t i c o u t l i n e of

the

p r o b l e m s t h a t c a n n o t b e i g n o r e d — a t least not i n those cases i n w h i c h r a p i d , c o n c e n t r a t i o n - d r i v e n changes

m a y occur i n l i q u i d lacunae that

exist ( p e r m a n e n t l y or t r a n s i e n t l y ) w i t h i n the g r o w i n g ice. T h e r e are not m a n y reports of covalent, f r e e z i n g - i n d u c e d

changes

t h a t are r e l e v a n t to p r o t e i n c h e m i s t r y . A f e w examples c a n , h o w e v e r ,

be

c i t e d . A f r e e z i n g - d e p e n d e n t e n h a n c e m e n t of a d d i t i o n reactions i n v o l v i n g t h e f o r m y l groups of h e m e has b e e n n o t e d ( 5 5 ) .

Presumably, a concen-

t r a t i o n effect operates here, expressed t h r o u g h a p H shift t o w a r d the r e a c t i o n o p t i m u m . O f p e r h a p s greater significance is the h y p o t h e s i s t h a t f r e e z i n g i n j u r y of p r o t e i n s m a y c o m e a b o u t t h r o u g h t h e f o r m a t i o n of


1.

Protein

TABORSKY

13

Alterations

intermolecular disulfide bonds ( b y

a i r o x i d a t i o n or b y d i s u l f i d e i n t e r -

c h a n g e ) , p r o m o t e d b y the f r e e z i n g - i n d u c e d u n f o l d i n g a n d c o n c e n t r a t i o n of p r o t e i n s ( 5 6 ) .

F o r e x a m p l e , a set of e n z y m e s k n o w n to b e i n a c t i v a t e d

b y f r e e z i n g w a s r e c o g n i z e d some t i m e ago as b e i n g of the s u l f h y d r y l v a r i e t y . C r y o p r o t e c t i o n w a s afforded for some o f these e n z y m e s b y t h e a d d i t i o n of m e r c a p t o e t h a n o l ( 5 7 ) .

D e n a t u r a t i o n of m y o f i b r i l l a r p r o t e i n s

u p o n f r o z e n storage w a s a t t r i b u t e d t o the p r e s u m e d o x i d a t i o n of s u l f h y d r y l groups ( w h i c h w e r e s h o w n to u n d e r g o a loss as a c o n s e q u e n c e of freezing)

(58).

M o r e r e c e n t l y , o x i d a t i o n of a s u l f h y d r y l g r o u p i n a c t i n

w a s d e m o n s t r a t e d to o c c u r o n f r e e z i n g , r e s u l t i n g i n f o r m a t i o n of a d i m e r (59) . T h e freezing-induced inactivation could be reversed, or prevented, Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

i f a r e d u c i n g agent w a s a d d e d . H o w e v e r , i t seems t h a t u s i n g m e r c a p t a n s as c r y o p r o t e c t a n t s

w o u l d be counterproductive

whenever

the protein

d e p e n d s for its i n t e g r i t y o n the intactness of its d i s u l f i d e b r i d g e s . mercaptans enhanced

are d e s i r a b l e , t h e i r r e a c t i v e by

the

concentration

effect

effectiveness

associated

When

can usually

with

freezing.

be For

e x a m p l e , significant alterations o f p e a n u t p r o t e i n s c a n b e p r o d u c e d

by

r e d u c i n g agents, a n d t h e i r a d d i t i o n p r i o r to f r o z e n storage of s u c h p r o t e i n solutions c a n p r o m o t e alterations that are d e p e n d e n t o n r e d u c i n g agents ( 6 0 ) . W h e n e v e r p r o t e i n s are m o r e stable i n a r e d u c i n g e n v i r o n m e n t , this approach may be useful. A

final

comment should be made.

P r o t e i n structure is m a i n t a i n e d

b y m a n y " w e a k " interactions. D u r i n g freezing, w e must be prepared for the p o s s i b i l i t y t h a t c o n f o r m a t i o n - m a i n t a i n i n g forces m a y b e d r i v e n to seek a n e w b a l a n c e , v i a a c o n f o r m a t i o n a l adjustment.

This could occur

i n response to i n t e r a c t i o n of the p r o t e i n w i t h " l i g a n d s " present i n t h e s o l u t i o n , e s p e c i a l l y i f these l i g a n d s m i m i c the r o l e of p r o t e i n

groups

i n v o l v e d i n w e a k interactions i n the n a t i v e structure. T h e s e l i g a n d s m a y c o m p e t e w i t h i n t r a m o l e c u l a r l y i n t e r a c t i n g groups. concentration

of

such ligands m a y

The freezing-induced

result i n significant

conformation

changes e v e n w i t h l i g a n d s t h a t w o u l d , u n d e r " n o r m a l , " m o r e d i l u t e c o n d i t i o n s b i n d too w e a k l y to h a v e a n effect.

( F o r a relevant discussion,

see R e f . 6 1 ) . Cryoenzymology (62).

R e c e n t l o w t e m p e r a t u r e studies of e n z y m e s

m e r i t separate c o n s i d e r a t i o n .

T h e y w i l l be g i v e n s p e c i a l emphasis i n a

later c h a p t e r of this v o l u m e .

These investigations have opened a n e w

w i n d o w t h r o u g h w h i c h w e c a n g a i n insights i n t o the d y n a m i c s of e n z y m e a c t i o n w i t h a r e s o l v i n g p o w e r u n a t t a i n a b l e , or a t t a i n a b l e o n l y w i t h diffic u l t y , at " n o r m a l " temperatures. cryosolvents

T h i s c a p a b i l i t y hinges o n t h e use

t h a t p e r m i t a t t a i n m e n t of

very

low

temperatures

of

while

a v o i d i n g the c o m p l i c a t i o n s of the f o r m a t i o n a n d p r e s e n c e of a s o l i d phase. T h e t o p i c w a s r e c e n t l y r e v i e w e d a n d t h e results of

cryoenzymological

studies w e r e i n t e g r a t e d w i t h results g a i n e d f r o m i n v e s t i g a t i o n s of e n z y m e


14


d y n a m i c s i n t h e c r y s t a l l i n e state (63,

64).

T h e m a j o r a t t r a c t i o n of t h i s

a p p r o a c h lies i n its p o t e n t i a l f o r y i e l d i n g i n f o r m a t i o n a b o u t

enzymic

processes u n d e r c o n d i t i o n s w h e r e o t h e r w i s e i n a c c e s s i b l e i n t e r m e d i a t e s a c c u m u l a t e a n d b e c o m e a m e n a b l e to c h a r a c t e r i z a t i o n . O b v i o u s l y , a r t i facts of d o u b t f u l significance to t h e t r u e b i o l o g i c a l m e c h a n i s m c o u l d b e e x p e c t e d to f o r m i n t h e p r e s e n c e of o r g a n i c solvents. H o w e v e r , c a r e f u l assessment of s u c h u n d e s i r e d effects is p o s s i b l e a n d t h e r e is a l r e a d y a n a p p r e c i a b l e n u m b e r of e n z y m i c systems f o r w h i c h m e c h a n i s t i c a l l y i m p o r t a n t features of t h e c a t a l y t i c process h a v e b e e n i d e n t i f i e d

(63).

T h e p o s s i b i l i t y of p r e p a r i n g p r o t e i n crystals i n e q u i l i b r i u m w i t h cryosolvents Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

X-ray

m u t u a l l y enhances the v a l u e of c r y s t a l analysis b y

(64)

d i f f r a c t i o n at l o w

approach.

i n t e r m e d i a t e forms respectively

temperatures

a n d of

the

cryoenzymological

B y this means i t has b e c o m e p o s s i b l e to c o m p a r e r e a c t i v e trapped i n the

c r y s t a l l i n e state a n d i n s o l u t i o n ,

(63).

Cryosolvents

and Protection Against Freezing Injury,

Cryosol-

vents c a n assist t h e b i o l o g i s t w h o s e g o a l is to p r o t e c t b i o l o g i c a l systems f r o m f r e e z i n g i n j u r y a n d t h e r e b y e n a b l e l o n g - t e r m p r e s e r v a t i o n of f u n c t i o n a l l y i n t a c t tissues a n d c e l l s .

P r o t e c t i v e cryosolvents m a y n o t a l t e r

the p r o t e i n s i n a p a r t i c u l a r l y significant m a n n e r , r a t h e r t h e y h e l p assure t h a t the p r o t e i n s i n t e g r i t y r e m a i n s essentially unaffected.

T h e effects of

cryoprotectants w i l l not be completely understood u n t i l i t becomes more f u l l y c l e a r w h a t t h e i r effects m a y b e o n t h e s t r u c t u r e o f t h e a q u e o u s m e d i u m o n w h i c h , i n t u r n , the c o n f o r m a t i o n a l features of t h e p r o t e i n c o m p o n e n t so i n t i m a t e l y d e p e n d .

I n addition, the eventual explanation

of c r y o p r o t e c t i o n i n m o l e c u l a r terms w i l l r e q u i r e m o r e i n s i g h t i n t o t h e n a t u r e of the d i r e c t i n t e r a c t i o n s b e t w e e n t h e p r o t e i n a n d t h e p r o t e c t i v e solvent c o m p o n e n t .

( R e l e v a n t discussions are p r o v i d e d , f o r e x a m p l e , i n

References 7, 65, 66, 67).

A n o t h e r aspect of the effectiveness of c r y o p r o -

tectants is t h e fact t h a t t h e y p e n e t r a t e m e m b r a n e s w i t h ease a n d at least p a r t of t h e i r f u n c t i o n m a y b e to e x t e n d t h e r a n g e of t e m p e r a t u r e s o v e r w h i c h the c e l l contents c a n exist i n a l i q u i d state a n d to a v o i d d e v e l o p m e n t of d e l e t e r i o u s l y h i g h c o n c e n t r a t i o n s of c e l l u l a r constituents d u r i n g f r e e z i n g . S e l e c t i o n of these p r o t e c t i v e cryosolvents is l i k e l y t o b e b a s e d i n c r e a s i n g l y o n i n f o r m a t i o n o b t a i n e d f r o m c r y o e n z y m o l o g i c a l studies t h a t r e v e a l f u n d a m e n t a l characteristics of c r y o s o l v e n t - p r o t e i n i n t e r a c t i o n s as a "by-product." T h e m a t t e r of c r y o p r o t e c t i o n , w i t h c l i n i c a l a p p l i c a t i o n s i n m i n d , w i l l u n d o u b t e d l y b e c o m e b e t t e r u n d e r s t o o d w i t h t i m e . O b v i o u s l y , measures of c e l l o r tissue v i a b i l i t y m u s t b e t h o u g h t of w i t h m o r e s u b t l e a n d s t r i n g e n t c r i t e r i a i n m i n d t h a n those n o r m a l l y u s e d i n m o s t studies of tissue p r e s e r v a t i o n . A s s e s s i n g v i a b i l i t y i n terms of one o r a f e w


bio-

1.

Protein

TABORSKY

15

Alterations

c h e m i c a l p a r a m e t e r s r a t h e r t h a n i n terms of a w h o l e s w e e p of p h y s i o l o g i c a l characteristics o f a g i v e n tissue spells t h e difference

between

a d e q u a c y f o r a l i m i t e d p u r p o s e , s u c h as i n v i t r o e x p e r i m e n t s , a n d s u i t a b i l i t y for, say, tissue t r a n s p l a n t a t i o n . T h e c o m p l e x i t y of t h i s p r o b l e m w i l l increase f u r t h e r w h e n c o n s i d e r a t i o n is g i v e n to p o t e n t i a l i n t e r a c t i o n s b e t w e e n cryoprotectants a n d other p h a r m a c o l o g i c a l l y a c t i v e agents a n d t o t h e m e t a b o l i c state of t h e tissue. A l t h o u g h t h e art of c r y o p r o t e c t i o n is n o t y e t at a p o i n t w h e r e these considerations c a n b e effectively d e a l t w i t h , progress is b e i n g m a d e

(68).


Proteins in the Presence of

Ice

A c r i t i c a l r e l a t i o n s h i p exists b e t w e e n w a t e r a n d p r o t e i n ( 6 9 ) .

I have

f o u n d i t c o n v e n i e n t to d e a l e a r l i e r w i t h one m a n i f e s t a t i o n o f t h i s r e l a t i o n s h i p , n a m e l y h y d r o p h o b i c i n t e r a c t i o n s . I t seems o b v i o u s , h o w e v e r , t h a t this r e l a t i o n s h i p s h o u l d be affected b y a n y significant alterations i n t h e state of t h e a q u e o u s c o m p o n e n t , regardless

of

w h e t h e r t h e w a t e r is

a t t e m p t i n g to a v o i d contact w i t h n o n p o l a r groups o r is a t t r a c t e d to p o l a r groups. A l t h o u g h this assertion is o b v i o u s l y t r u e , difficulties arise w h e n a r i g o r o u s analysis is a t t e m p t e d .

The problem

begins w i t h our l i m i t e d

u n d e r s t a n d i n g of w a t e r i t s e l f — m o r e t h a n one m o d e l of w a t e r s t r u c t u r e can be

fitted

to a v a i l a b l e d a t a (70).

F u r t h e r m o r e , theories of

p r o t e i n interactions are i n c o m p l e t e a n d t e n t a t i v e (14).

water-

T h u s , an accurate

d e s c r i p t i o n of w a t e r - p r o t e i n i n t e r a c t i o n s i n c o m p l e x b i o l o g i c a l systems is n o t yet p o s s i b l e

(71).

[It is n o t e w o r t h y t h a t the authors of a

recent

r e v i e w of t h e role of solvent i n t e r a c t i o n s i n p r o t e i n c o n f o r m a t i o n compelled

to n o t e t h a t t h e " v e r i t a b l e j u n g l e of d a t a a n d

produced

b y m a n y investigators " w o r k i n g i n closely a l l i e d

felt

hypotheses" fields"

w i t h a " g e n e r a l l a c k o f c o o r d i n a t i o n " m i g h t i n v i t e the c o n s i d e r a t i o n

but of

" a m o r e p u r p o s e f u l , joint attack o n some of t h e p r o b l e m s " w h i c h " m a y w e l l be timely a n d y i e l d valuable dividends"

(13).]

A g a i n s t this s o m e w h a t f o r b i d d i n g b a c k g r o u n d , I w i l l engage i n a c u r s o r y r e v i e w of some of t h e p r o b l e m s associated w i t h p r o t e i n - w a t e r i c e systems w h e r e i n ice is suggested, o r a c k n o w l e d g e d , to h a v e a r o l e i n p r o t e i n alterations. A t this p o i n t , a t t e n t i o n w i l l b e g i v e n to t h e effects of f r e e z i n g o n h y d r a t i o n of p r o t e i n s .

S u c h effects m u s t l e a d to alterations

of p r o t e i n s t r u c t u r e i f t h e h y d r a t i o n s h e l l p l a y s a n i n t e g r a l r o l e i n m a i n t e n a n c e of p r o t e i n structure. ( H y d r o p h o b i c

interactions have been dealt

w i t h a l r e a d y i n the context of p r o t e i n b e h a v i o r i n c o l d solutions. I w i l l s i m p l y a d d here t h a t i t is c l e a r l y u n a v o i d a b l e t h a t d i m i n i s h i n g t h e l i q u i d e n v i r o n m e n t of t h e p r o t e i n b y f r e e z i n g w i l l r e m o v e t h e r a i s o n d'etre of these i n t e r a c t i o n s a n d w i l l therefore w e a k e n this f o r m of s t r u c t u r e s t a b i l i -


16

PROTEINS A T L O W

zation.)

TEMPERATURES

O u r d i s c u s s i o n of f r e e z i n g effects o n p r o t e i n h y d r a t i o n c a n b e

b r i e f since m u c h o f t h e p e r t i n e n t i n f o r m a t i o n a n d m a n y of t h e r e l e v a n t ideas h a v e b e e n t h o u g h t f u l l y r e v i e w e d q u i t e r e c e n t l y ( 1 5 , 67)

and will

be dealt w i t h later i n this volume. Protein Dehydration U p o n Freezing.

M o s t of t h e c u r r e n t m o d e l s

of p r o t e i n h y d r a t i o n p o s t u l a t e t h e existence of s e v e r a l classes of w a t e r molecules

t h a t are d i s t i n g u i s h e d b y

different

degrees of

"structure,"

m o b i l i t y , a n d s t r e n g t h of i n t e r a c t i o n w i t h the p r o t e i n . T a b l e I I I p r o v i d e s an "orders-of-magnitude"-type

s u m m a r y of these classes.

"Site-bound"

w a t e r consists of w a t e r m o l e c u l e s b o u n d i n d i v i d u a l l y a n d s t o i c h i o m e t r i c a l l y to specific, t y p i c a l l y c h a r g e d or p o l a r sites. T h i s w a t e r is b o u n d w i t h Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

r e l a t i v e l y great s t r e n g t h a n d e x h i b i t s g r e a t l y r e d u c e d m o b i l i t y c o m p a r e d to b u l k w a t e r . " S u r f a c e " w a t e r is w a t e r adjacent to t h e p r o t e i n s t r u c t u r e , held w i t h moderate

strength, intermolecularly hydrogen-bonded,

and

e x h i b i t i n g h i n d e r e d m o b i l i t y . W h i l e " w a t e r of h y d r a t i o n " m u s t s t i l l b e c o n s i d e r e d as a n o p e r a t i o n a l l y d e f i n e d t e r m (different "classes" of w a t e r b e i n g identified differently, d e p e n d i n g on the particular experimental m e t h o d chosen to reveal t h e m ) , most experimental determinations

of

p r o t e i n h y d r a t i o n are l i k e l y to d e a l , m o r e o r less, w i t h these t w o categories

Table III. Classes of Water Molecules in Protein " H y d r a t i o n Shells""

Class Protein-bound site-bound surface bulk-like

Binding strong medium loose

Rota tional Relaxation Time (sec) 10~ 10" 10'

6 9

1 1

Monolayer

—

—

Protein-free liquid ice

— —

10" 10"

Molecules of Water per Residue

Degree of Hydra tion (g/g)

Freezability

0.1-0.5 1-3

0.02-0.1 0.2 - 0 . 6

—

—

— —

4-7 11

5

— —

0.7

-1.2

— —

+

—

+

—

Information collected in this table is based on data from Kuntz and Kauzmann (67) and from Richards (16). Numerical values given here represent approximations based on numerous proteins and diverse experimental techniques. The class desig nated as Monolayer is included for purposes of comparison with "Surface" water in particular. The latter is, presumably, an actual layer of uneven thickness that is variable from protein to protein. The former is an idealized concept of a uniform layer, one molecule in thickness, covering the protein surface. "Freezability" denotes whether or not a particular class of water is capable of transition into normal ice. β


1.

TABORSKY

of w a t e r .

Protein

17

Alterations

I t is n o t e w o r t h y f o r o u r purposes t h a t " s i t e - b o u n d w a t e r " or

" s u r f a c e w a t e r " is n o t c a p a b l e of f o r m i n g n o r m a l i c e — i t is m o r e

mobile

t h a n w a t e r m o l e c u l e s i n ice, e v e n a t l o w t e m p e r a t u r e s , b u t i t is less m o b i l e t h a n w a t e r i n t h e b u l k solvent at t h e same t e m p e r a t u r e

(67).

A l t h o u g h w a t e r of h y d r a t i o n appears to b e " s t r u c t u r e d , " i t is not " i c e - l i k e " (72).

T h e t h i r d class, d e s i g n a t e d as " b u l k - l i k e , " is i n most w a y s i n d i s -

tinguishable

from

b u l k solvent.

Its existence

is a c k n o w l e d g e d

only

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

of

t h e p r o t e i n . P r e s u m a b l y , t h e d e m a r c a t i o n b e t w e e n " f r e e " solvent w a t e r a n d " h y d r a t i o n " w a t e r is not a sharp one.

T h e " b u l k - l i k e " w a t e r is

e n v i s a g e d as r e p r e s e n t i n g t h e t r a n s i t i o n f r o m one to the other. Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

It s h o u l d b e stressed t h a t this classification is a n o v e r s i m p l i f i c a t i o n . T h e p r o p e r t i e s of w a t e r of h y d r a t i o n most l i k e l y c h a n g e m o r e g r a d u a l l y t h a t is i m p l i e d b y "classes" (67,

73).

N e v e r t h e l e s s , t h e y are u s e f u l i n

that t h e y f a c i l i t a t e a reasonable c o n c e p t u a l i z a t i o n of m a n y of the e x p e r i m e n t a l d a t a t h a t b e a r o n p r o t e i n h y d r a t i o n . B u t i t is p r u d e n t to k e e p i n m i n d that these classes p r o b a b l y represent a v e r a g e d segments of a c o n t i n u u m of w a t e r structures. I n a n y case, w h a t e v e r m a y b e its p r e c i s e d e f i n i t i o n , the r e a l i t y of the h y d r a t i o n s h e l l is p r o f u s e l y d o c u m e n t e d

(67).

I t m u s t b e v i e w e d as a n i n t e g r a l p a r t of the p r o t e i n , t h e f u n c t i o n a l l y significant e n t i t y . I n d e e d , p r o t e i n h y d r a t i o n is of c r i t i c a l i m p o r t a n c e to t h e protein's f u n c t i o n a l i n t e g r i t y i n s o f a r as t h i s f u n c t i o n is a n i n h e r e n t m o l e c u l a r p r o p e r t y — a specific m a n i f e s t a t i o n of p r o t e i n structure.

The

r e a l i t y o f t h e h y d r a t i o n s h e l l appears also f r o m the v a n t a g e p o i n t of a r e a c t i v e l i g a n d to w h i c h the h y d r a t i o n s h e l l seems to represent a p h y s i c a l b a r r i e r to b e s u r m o u n t e d before t h e f u n c t i o n a l l y significant, d i r e c t i n t e r action between protein and l i g a n d can be accomplished.

T h i s has b e e n

s h o w n , f o r e x a m p l e , w i t h respect to t h e i n t e r a c t i o n b e t w e e n

myoglobin

a n d o x y g e n , or c a r b o n d i o x i d e , w h e r e t h e i n i t i a l association

between

l i g a n d a n d p r o t e i n is seen as a process t h a t is slower t h a n d i f f u s i o n l i m i t e d a n d is a t t r i b u t e d to l i g a n d e n t r y f r o m b u l k solvent t h r o u g h t h e h y d r a t i o n s h e l l , a process n o t i c e a b l e

d o w n to t e m p e r a t u r e s as l o w as

about —60° ( i n glycerol-water mixtures)

(74).

I n t e r p r e t i n g d a t a r e l a t i n g to the effects of f r e e z i n g o n p r o t e i n i n t e g r i t y a n d p r o t e i n h y d r a t i o n is difficult since most studies p r o v i d e

inferences

rather t h a n proof. T h e closest to " p r o o f are those studies i n v o l v i n g t h e attributes of d r i e d p r o t e i n s a n d t h e i r r e l a t i o n to p r o t e i n s i n f r o z e n systems. W h a t seems necessary is to s h o w t h a t d i s t u r b a n c e of the h y d r a t i o n s h e l l , p e r h a p s b y ice crystals or b y m o r e subtle changes i n t h e structure of w a t e r of h y d r a t i o n , has n o t a b l e consequences o n p r o t e i n structure. T h a t the d r y i n g of p r o t e i n s has major c o n s e q u e n c e s of this sort has b e e n s h o w n . T h i s is, of course, as w e w o u l d expect.

I f the m o r e i n t i m a t e l y b o u n d


18


l a y e r s of t h e h y d r a t i o n s h e l l are r e m o v e d , the o r i g i n a l p r o t e i n c o n f o r m a t i o n w o u l d h a v e to a c c o m m o d a t e l a r g e stresses p r o d u c e d b y t h e s t r u c t u r a l v o i d s . T r a n s c o n f o r m a t i o n or d e n a t u r a t i o n w o u l d b e t h e a p p r o p r i a t e a n d accessible response of t h e p r o t e i n to s u c h stresses

(67).

S o m e examples m a y serve to s u p p o r t this a r g u m e n t . T h e h e m e i r o n of c y t o c h r o m e c experiences a l i g a n d exchange

(replacing a methionine

b y , p e r h a p s , a l y s i n e s i d e c h a i n ) u p o n r e m o v a l of w a t e r b y l y o p h i l i z a t i o n (75).

B o t h , d e s i c c a t i o n a n d f r e e z i n g of catalase h a v e s i m i l a r consequences

o n t h e a c t i v i t y of t h e e n z y m e (76).

F r e e z i n g a n d t h a w i n g a p p e a r to alter

c e r t a i n p h y s i c a l - c h e m i c a l p r o p e r t i e s of some p r o t e i n s a n d s i m i l a r

but

greater changes are o b s e r v e d d u r i n g f r e e z e - d r y i n g , w h i c h is p r e s u m a b l y


a m o r e d r a s t i c m e t h o d f o r affecting t h e h y d r a t i o n s h e l l (77).

Thermal

transitions a n d i r r e v e r s i b l e s t r u c t u r a l changes o c c u r i n t h e l i p o p r o t e i n of e g g y o l k d u r i n g f r e e z i n g a n d these changes are s t r o n g l y d e p e n d e n t w a t e r c o n t e n t w h e r e w a t e r is r e d u c e d b e l o w a c r i t i c a l v a l u e of

on

20%

( w h i c h corresponds a p p r o x i m a t e l y to t h e t w o " n o n f r e e z a b l e " classes of w a t e r i n T a b l e I I I ) (78).

T h e r e are, of course, n u m e r o u s — m o s t l y c a s u a l

— r e f e r e n c e s i n t h e l i t e r a t u r e to p r o t e i n d e n a t u r a t i o n , or e n z y m e i n a c t i v a t i o n , d u r i n g f r o z e n storage.

T h e s e observations are f r e q u e n t l y

coupled

w i t h the suggestion that the extensive ( o r c o m p l e t e ? ) t r a n s i t i o n of w a t e r to i c e affected o r d e s t r o y e d t h e h y d r a t i o n s h e l l of t h e p r o t e i n thus c a u s i n g a structural change.

T h e s e suggestions are f o r t h e most p a r t b a s e d o n

inadequate experimental evidence. " U n e x p l a i n e d " Effects.

S o m e observations are d i f f i c u l t t o a t t r i b u t e

t o a n y of t h e f r e e z i n g m e c h a n i s m s c o n s i d e r e d so f a r . M o s t a m e n a b l e to conceptual accommodation

are those cases w h e r e a p a r t i c u l a r r e a c t i v e

i n t e r m e d i a t e , or a p a r t i c u l a r c o n f o r m e r , appears to h a v e b e e n " t r a p p e d . " T h i s is b e l i e v e d to o c c u r at v e r y l o w t e m p e r a t u r e s w h e r e m o b i l i t y at t h e a t o m i c l e v e l is severely l i m i t e d p a r t l y b e c a u s e of l i t t l e t h e r m a l m o t i o n a n d p a r t l y b e c a u s e of t h e p h y s i c a l c o n s t r a i n t p r o v i d e d b y the glassy or i c y structures of t h e m e d i u m . N o r m a l l y u n s t a b l e f r e e r a d i c a l d e r i v a t i v e s of p r o t e i n s c a n b e s t u d i e d b y t r a p p i n g t h e m at l o w t e m p e r a t u r e s 79).

T h i s t e c h n i q u e also c a n b e u s e d to s t a b i l i z e w h a t a p p e a r to

(e.g. be

specific c o n f o r m a t i o n a l isomers of n o r m a l structures. F o r e x a m p l e , this a p p r o a c h has e n a b l e d t h e d e t e c t i o n of a n a m m o n i a - c a t a l a s e c o m p l e x (SO), conformers

of t h e i r o n p r o t e i n s c o n a l b u m i n a n d t r a n s f e r r i n ( 8 1 ) , a n d a

c o n f o r m e r of t h e

flavoprotein

L - a m i n o a c i d o x i d a s e (82,

83).

A n i n t e r e s t i n g s p e c i a l case is p r o v i d e d b y aldolase. I f f r o z e n i n the presence

of a l k y l a t i n g agents, i t u n d e r g o e s

alkylation

(84).

inactivation b y sulfhydryl

H o w e v e r , this i n a c t i v a t i o n a p p e a r s

to o c c u r

during

t h a w i n g , c o n d i t i o n e d s u p p o s e d l y b y c o n f o r m a t i o n a l stresses t h a t d e v e l o p d u r i n g f r o z e n storage.


1.

Protein

TABORSKY

Protein Alterations with Dynamic

19

Alterations

and Behavior

Aspects of

Associated

Freezing

I t seems a p p r o p r i a t e to d e a l s o m e w h a t

more

explicitly w i t h

the

d y n a m i c aspects of the f r e e z i n g process i n contrast to the effects of a g i v e n state of a c o l d o r f r o z e n system.

T h i s is p r o b a b l y also t h e best

p l a c e to a c k n o w l e d g e a m a j o r o m i s s i o n t h r o u g h o u t t h i s o v e r v i e w .

None

of t h e d i s c u s s i o n has d e a l t s p e c i f i c a l l y w i t h the process of t h a w i n g .

It

m u s t b e t a k e n f o r g r a n t e d t h a t s o m e " f r e e z i n g " effects are i n r e a l i t y " t h a w i n g " effects a l t h o u g h i n v e r y f e w cases is t h e r e c o n c l u s i v e on this point.

evidence

I t c a n b e a s s u m e d that w h a t e v e r a r g u m e n t s m a y

be


r e a s o n a b l y a d v a n c e d r e g a r d i n g the d y n a m i c s of f r e e z i n g a n d its conseq u e n c e s also m a y b e a d v a n c e d , b y s u i t a b l e extension, i n v e r s i o n , o r i n f e r ence, to t h e d y n a m i c s of the m e l t i n g process.

Undoubtedly, "melting

effects" are r e a l , h o w e v e r n e g l e c t e d t h e i r separate c o n s i d e r a t i o n m a y be. Protein Antifreezes.

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

n o t i c e n o t because f r e e z i n g affects

t h e m b u t b e c a u s e t h e y affect

the

f r e e z i n g process. F i s h e s i n h a b i t i n g p o l a r w a t e r s h a v e r e c e n t l y b e e n s h o w n to possess u n u s u a l l y s t r u c t u r e d , a l a n i n e - r i c h s e r u m g l y c o p r o t e i n s depress t h e f r e e z i n g p o i n t of aqueous process ( 8 5 , 86). m u s s e l (87).

that

systems b y s o m e n o n c o l l i g a t i v e

S i m i l a r antifreeze proteins also h a v e b e e n f o u n d i n t h e

T h e s e p r o t e i n s h a v e a m a r k e d effect o n the m o d e of

ice

g r o w t h , i n d i c a t i n g t h a t t h e y m a y exert t h e i r f r e e z i n g i n h i b i t i o n b y

be-

c o m i n g a d s o r b e d onto p a r t i c u l a r facets o f t h e g r o w i n g i c e c r y s t a l . T h e presence according

of antifreeze p r o t e i n s i n fish b l o o d has b e e n s h o w n to v a r y to a n a n n u a l c y c l e

Circular dichroic

(88).

measurements

i n d i c a t e t h a t some antifreeze p r o t e i n s are d i s o r d e r e d i n s o l u t i o n w h i l e others h a v e a great h e l i x c o n t e n t — s u g g e s t i n g t h a t t h e r e is n o p a r t i c u l a r c o n f o r m a t i o n to w h i c h the antifreeze p r o p e r t y c a n be a t t r i b u t e d

(89).

S t u d i e s i n v o l v i n g R a m a n s p e c t r a t e n d to c o n f i r m t h i s suggestion

and

i n d i c a t e also that these p r o t e i n s d o not affect the b u l k p r o p e r t i e s of w a t e r or i c e t h a t exist i n t h e i r presence ( 9 0 ) . copolymer—modeling

the natural

A synthetic alanine-aspartic a c i d

antifreeze

proteins—was

shown

to

depress the f r e e z i n g p o i n t of w a t e r t o a b o u t o n e - t h i r d t h e extent of t h e antifreeze p r o t e i n s of fish (91).

I t is n o t e w o r t h y t h a t t h i s p o l y p e p t i d e

c o n t a i n e d no c a r b o h y d r a t e sidechains as d o the n a t u r a l a n t i f r e e z e p r o t e i n s . I t is n o t k n o w n w h e t h e r s u c h n a t u r a l c r y o p r o t e c t a n t s o c c u r w i d e l y d i s t r i b u t e d a m o n g a n i m a l species.

H o w e v e r , a relevant experiment was

c o n d u c t e d w i t h b r a i n slices, f r o m w a r m - a d a p t e d a n d h i b e r n a t i n g h a m sters, b e f o r e a n d after f r e e z i n g (92).

T i s s u e slices f r o m t h e h i b e r n a t i n g

h a m s t e r e x h i b i t e d h i g h e r t h a n n o r m a l o x y g e n c o n s u m p t i o n rates after freezing.

T h i s r e s u l t d i d n o t o c c u r w i t h slices f r o m t h e w a r m - a d a p t e d


20

PROTEINS A T L O W

TEMPERATURES

a n i m a l . T h e s e e x p e r i m e n t s p r o m p t e d t h e suggestion t h a t , i n t h e h i b e r n a t i n g a n i m a l , m e m b r a n e p r o t e i n s of t h e b r a i n m a y b e m o d i f i e d i n a w a y t h a t enhances t h e i r antifreeze p o t e n t i a l . T w o e a r l y observations m a y h a v e s o m e r e l e v a n c e to t h e m e c h a n i s m b y w h i c h a n t i f r e e z e p r o t e i n s act. P o l y m e r gels of h i g h l y r a m i f i e d , n e t - l i k e structures h a v e b e e n s h o w n to depress t h e f r e e z i n g p o i n t of w a t e r a n d cause t h e ice crystals to assume a d o m i n a n t g r o w t h o r i e n t a t i o n ( 9 3 ) . is also n o t e w o r t h y ,

perhaps, that certain amino

n u c l e a t o r s w h e r e a s others are n o t

acids

are

good

It ice

(94).

Events O c c u r r i n g at or N e a r the Water—Ice Interface.

It would


t a k e us too f a r afield to a t t e m p t to d e a l i n d e t a i l w i t h theories a n d s p e c u lations c o n c e r n i n g the s m a l l z o n e e x i s t i n g b e t w e e n s u p e r c o o l e d

liquid

a n d the g r o w i n g i c e mass. B u t , i n v i e w of the p o t e n t i a l i m p o r t a n c e this z o n e m a y h a v e w i t h r e g a r d to f r e e z i n g effects, some d i s c u s s i o n of i t is appropriate.

I already acknowledged

t h e significance of this i n t e r f a c e

w h e n c o n s i d e r a t i o n w a s g i v e n to the increase i n solute c o n c e n t r a t i o n t h a t o c c u r s w h e n solutes are rejected b y the g r o w i n g i c e crystals. A l s o , the e a r l i e r d i s c u s s i o n of factors t h a t i n f l u e n c e t h e f o r m a t i o n of t r a n s i e n t l y existing, concentrated

" p u d d l e s " h a d clear implications regarding this

interface. I s h a l l n o w c o n s i d e r this i n t e r f a c e i n m o r e d e t a i l . O f h i s t o r i c a l interest is a r e v i e w of a century's w o r t h of e x p e r i m e n t a n d t h o u g h t d i r e c t e d at the n a t u r e of the ice surface

(95).

A

major

c o n c l u s i o n i n this r e v i e w is t h a t a t r a n s i t i o n l a y e r of w a t e r m o l e c u l e s does i n f a c t exist at t h e ice surface. T h i s r e v i e w is c o n c e r n e d , h o w e v e r , w i t h interfaces b e t w e e n i c e a n d t h e v a p o r p h a s e or a n o t h e r s o l i d phase.

More

d i r e c t l y p e r t i n e n t to o u r purposes is another f a s c i n a t i n g r e v i e w t h a t takes a d e t a i l e d l o o k at the i n t e r f a c e "as seen f r o m the l i q u i d s i d e " ( 9 6 ) .

This

assessment of r e l e v a n t e v i d e n c e also results i n the firm c o n c l u s i o n t h a t a n i n t e r f a c e l a y e r w i t h s p e c i a l p r o p e r t i e s exists. O f p a r t i c u l a r i m p o r t a n c e to t h e present d i s c u s s i o n is t h e n o t i o n t h a t t h e t r a n s i t i o n l a y e r consists of water molecules i n an oriented, polar arrangement.

T h e p o l a r i z a t i o n of

this l a y e r is a s c r i b e d to t h e n e e d to r e l i e v e strains i n t h e i c e l a t t i c e . T h e s e strains arise f r o m the e n l a r g i n g of t h e n o r m a l h y d r o g e n - o x y g e n angles i n o r d e r to fit the r e q u i r e m e n t s of the t e t r a h e d r a l s t r u c t u r e i n ice. T h i s s t r a i n r e l i e f process w o u l d result, a m o n g other t h i n g s , i n t h e m a i n t e n a n c e of a n e l e c t r i c p o t e n t i a l b e t w e e n t h e l i q u i d a n d s o l i d phases a n d w o u l d a c c o u n t for the experimentally observed

selective i n c o r p o r a t i o n of ions i n t h e

ice structure. T h e n o t i o n of a p o l a r , o r i e n t e d i n t e r f a c e l a y e r of w a t e r m o l e c u l e s — w h i l e b a s e d o n a n a d h o c a r g u m e n t — i s consistent w i t h e x p e r i m e n t a l d a t a . I t is i n a c c o r d , f o r e x a m p l e , w i t h the l a r g e e l e c t r i c p o t e n t i a l s t h a t d e v e l o p


1.

Protein

TABORSKY

21

Alterations

across t h e w a t e r - i c e interface d u r i n g f r e e z i n g of d i l u t e salt solutions ( 9 7 ) . T h i s o c c u r r e n c e p r o b a b l y results f r o m t h e often d e m o n s t r a t e d i n c o r p o r a t i o n of ions ( i n most cases, a n i o n s ) i n t o t h e i c e (e.g.

selective

98-106).

Some time ago, I observed a freezing-induced conformational change i n a p r o t e i n t h a t a p p e a r e d to b e associated w i t h selective r e j e c t i o n of ions at t h e l i q u i d - i c e i n t e r f a c e (107).

T h e results o f t h i s s t u d y a p p e a r e d t o

m a k e a g o o d case f o r t h e c l a i m t h a t s u c h solute r e d i s t r i b u t i o n s m a y h a v e i m p o r t a n t effects o n proteins d u r i n g f r e e z i n g , p a r t i c u l a r l y since a v e r y high

concentration

of protons

m a y develop

i n the interface

region.

A l t h o u g h this c o n c e n t r a t i o n of protons is t r a n s i e n t , i t p r e s u m a b l y persists for t h e d u r a t i o n of c r y s t a l g r o w t h . Downloaded by 80.82.77.83 on May 17, 2018 | https://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch001

T h e r e a l i t y o f a n interface r e g i o n i n w h i c h solute concentrations m a y b e h i g h d i n i n g t h e f r e e z i n g process w a s s h o w n b y d i r e c t measurements. I t w a s f o u n d that t h e i n t e r f a c e l a y e r m a y h a v e a thickness o n t h e o r d e r of 100-1000 n m , t h e exact v a l u e v a r y i n g i n v e r s e l y w i t h t h e rate of i c e propagation

(108).

I t has also b e e n suggested that as t h e i c e surface grows, t h e solutee n r i c h e d l a y e r just a h e a d of i t w i l l assume a l o w e r f r e e z i n g p o i n t t h a n t h e s o l u t i o n of l o w e r c o n c e n t r a t i o n t h a t exists f u r t h e r a w a y f r o m t h e i c e surface. T h i s s i t u a t i o n w o u l d b e most l i k e l y to o c c u r d u r i n g r a p i d f r e e z i n g w h e n t h e diffusive r e d i s t r i b u t i o n o f solute ( r e j e c t e d b y t h e i c e ) is s l o w c o m p a r e d w i t h t h e rate of its c o n c e n t r a t i o n b u i l d - u p . T h i s c o u l d l e a d to nucleation i n advance

of t h e f r e e z i n g b o u n d a r y ,

thereby

causing

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

(109).

I t s h o u l d b e n o t e d t h a t o n a gross scale this w o u l d encourage

more

u n i f o r m solute d i s t r i b u t i o n i n t h e i c e b u t o n a m i c r o s c a l e ( w h i c h w o u l d b e r e l e v a n t f o r m o l e c u l a r i n t e r a c t i o n s ) t h e system w o u l d b e d e c i d e d l y heterogeneous a n d c o n c e n t r a t i o n effects s h o u l d b e great. It seems u s e f u l to c o n c l u d e this section w i t h d a t a i l l u s t r a t i n g t h e effects of v a r i o u s solutes o n t h e p r o p a g a t i o n rates of i c e ( T a b l e I V ) . D i s c u s s i o n of t h e effects that solutes h a v e o n t h e r e l a t i o n s h i p b e t w e e n i c e g r o w t h rate a n d t h e degree of s u p e r c o o l i n g is a v a i l a b l e (III).

A more

recent

elsewhere

d i s c u s s i o n of e q u i l i b r i u m a n d n o n e q u i l i b r i u m

aspects of f r e e z i n g p r o v i d e s a m o s t h e l p f u l a i d i n t h e i n t e g r a t e d c o n s i d e r a t i o n of aqueous

solutions subjected

reference to b i o l o g i c a l systems

to f r e e z i n g , w i t h p a r t i c u l a r

(112).

Possible "Interface Effects" on Proteins.

I n t e r f a c e effects t h a t arise

d u r i n g g r o w t h of i c e crystals c a n influence proteins b u t t h i s issue is rarely addressed i n a direct fashion. D u r i n g freezing, the egg yolk protein p h o s v i t i n has b e e n s h o w n to u n d e r g o t o w a r d a n o r d e r e d structure.

a major conformational

T h i s is caused

change

b y the transiently h i g h

c o n c e n t r a t i o n of protons s h o w n to exist i n t h e i c e - l i q u i d i n t e r f a c e r e g i o n


22


Table I V .

Retardation of Ice Propagation by Diverse Rate

of Ice Growth Solute

Solute Acetates lithium sodium potassium ammonium Chlorides lithium


sodium

Solutes"

(cm/sec)

at 1

0%

10%

1.41

0.13 0.33 0.62

2.76 2.69 1.74 1.32

Given

Concentration "

0.35 0.10 0.48

potassium

3.09 2.82

ammonium

2.09

0.54

Thiocyanates sodium potassium

3.02

0.85

2.82

—

ammonium

2.09

0.96

acetic glycolic

2.14 2.14

0.76 0.83

malic citric

1.66

0.78 0.60

1.08

Acids

aminoacetic

1.29 2.19

—

Alcohols methanol

1.20

0.25

ethanol

0.43

propanol glycerol

0.54 1.38

0.09 0.12

Sugars glucose fructose sucrose

1.02 1.20 0.85

0.41 0.38 0.46 0.36

Proteins lysozyme albumin N o n e (pure water)

0.81 0.76

0.56

ca. 7

—

—

"Original data from Lusena (110). In the original article, values were i n logarithmic form. Above data were recalculated to give dimensions of velocity. A l l experiments carried out with solutions supercooled 10° below their respective freezing points. The data have relative significance. A11 experiments yielded a biphasic relationship between the logarithm of the rate of ice growth and the concentration of the solute. A t low concentrations of all solutes, the rate of ice growth fell drastically within a narrow concentration range. Further rate diminution occurred over a range of higher concentrations where the log rate vs. concentration relationship was linear. Extrapolation of the linear portion of this linear relationship to zero concentration produces the values listed under the heading " 0 % . " T h e data i n the " 1 0 % " concentration column reflect growth rates which tend to be associated with the lower end of the concentration range i n which the linear relationship holds. &


1.

TABORSKY

Protein

23

Alterations

during crystal formation

(113).

I t is also n o t e w o r t h y

that a highly

ordered p o l y m e r forms d u r i n g freezing of a solution of polyadenylic a c i d . T h i s t r a n s c o n f o r m a t i o n m a y also b e a n " i n t e r f a c e effect"

(114).

W h e n p r o t e i n alterations o c c u r as a r e s u l t of e x p e r i m e n t a l c o n d i t i o n s t h a t a r e e x p e c t e d t o i n f l u e n c e t h e d y n a m i c s o f t h e f r e e z i n g process, i t seems reasonable to p o s t u l a t e t h a t i n t e r f a c e effects m a y b e o p e r a t i v e . H o w e v e r , s u c h experiments a r e r a r e l y d e s i g n e d f o r t h e p u r p o s e o f e l u c i d a t i n g t h e m e c h a n i s m b y w h i c h f r e e z i n g causes p r o t e i n a l t e r a t i o n . T h e r e fore, t h e m e c h a n i s m r e m a i n s l a r g e l y a m a t t e r o f conjecture.

F r o m among

t h e m a n y c a n d i d a t e s f o r a n i n t e r f a c e effect, a f e w w i l l b e c i t e d — e s s e n t i a l l y r a n d o m l y — s i m p l y to u n d e r p i n t h e suggestion t h a t these

effects


m a y h a v e a b r o a d e r significance i n t h e context of p r o t e i n c r y o c h e m i s t r y t h a n is g e n e r a l l y a c k n o w l e d g e d . T h e p o s s i b i l i t y o f i n t e r f a c e effects h a s b e e n s p e c i f i c a l l y n o t e d i n a study i n v o l v i n g freezing of lipoamide dehydrogenase

(115).

This possi

b i l i t y w a s also m e n t i o n e d i n a s t u d y o f c o n a l b u m i n a n d t r a n s f e r r i n , r e f e r r e d to e a r l i e r (81).

A n i n t e r f a c e m e c h a n i s m also m a y b e o p e r a t i v e

w i t h r e g a r d to f r e e z i n g - i n d u c e d alterations of p h y c o e r y t h r i n (116), dehydrogenase

(117),

catalase (118),

w e r e r e f e r r e d to e a r l i e r (22).

lactic

a n d o t h e r p r o t e i n s , some of w h i c h

F i n a l l y , i t m a y be noted that interface

effects, e s p e c i a l l y t h e t r a n s i e n t d i s t r i b u t i o n of solutes, m a y u n d e r l i e s o m e of t h e a n o m a l o u s k i n e t i c s t h a t h a v e b e e n d e s c r i b e d f o r c e r t a i n reactions i n t h e " f r o z e n state"

(119).

Conclusions I set o u t t o g i v e a n o v e r v i e w o f f r e e z i n g - i n d u c e d alterations o f p r o teins. I a p p r o a c h e d t h e task w i t h a bias t h a t , I h o p e , b e c a m e c l e a r as the d i s c u s s i o n p r o g r e s s e d .

I t seems to m e t h a t i n q u i r i e s i n t o p r o t e i n

b e h a v i o r at l o w t e m p e r a t u r e s a n d i n f r e e z i n g systems a r e r i p e f o r m o r e s h a r p l y focussed studies o f t h e m e c h a n i s m s b y w h i c h " f r e e z i n g operate.

of l o w t e m p e r a t u r e exposure. provide

effects"

A massive a m o u n t o f d a t a exists c o n c e r n i n g t h e consequences A common

i n s i g h t i n t o u n d e r l y i n g causes.

deficiency

is t h e f a i l u r e t o

Intellectually satisfying a n d

p r a c t i c a l l y u s e f u l g e n e r a l i z a t i o n s a r e n e e d e d a n d these s h o u l d e m e r g e i n the f u t u r e . I n d e e d , t h e b r o a d r a n g e o f topics i n c l u d e d i n this s y m p o s i u m m a k e s i t e v i d e n t t h a t o n - g o i n g investigations a r e effectively m o v i n g t o w a r d this goal.

Literature Cited 1. Meryman, H . T., Ed. "Cryobiology"; Academic: London and New York, 1966; pp vii-x. 2. Rey, L. R. Ann. Ν. Y. Acad. Sci. 1960, 85, 510. 3. Polge, C.; Smith, A. U.; Parkes, A. S. Nature (London) 1949, 164, 666.



24

PROTEINS AT LOW TEMPERATURES

4. Nash, T. In "Cryobiology"; Meryman, H . T., Ed.; Academic: London and New York, 1966; p 179. 5. Doucou, P. Biochimie 1971, 53, 1135. 6. Fink, A. L. Acc. Chem. Res. 1977,10, 233. 7. Edelhoch,H.;Osborne, J. C., Jr. Adv. Protein Chem. 1976, 30, 183. 8. Brandts, J. F. J. Am. Chem. Soc. 1964, 86, 4302. 9. Brandts, J. F.; Hunt, L. J. Am. Chem. Soc. 1967, 89, 4826. 10. Brandts, J. F.; Oliveira, R. J.; Westort, C. Biochemistry 1970, 9, 1038. 11. Zipp, Α.; Kauzmann, W. Biochemistry 1973, 12, 4217. 12. Franks, F. Philos. Trans. R. Soc. London, Ser. Β 1977, 278, 89. 13. Franks, F.; Eagland, D. CRC Crit. Rev. Biochem. 1975, 3, 165. 14. Scheraga, H. A. Ann. Ν. Y. Acad.Sci.1977, 303, 2. 15. Richards, F. M. Annu. Rev. Biophys. Bioeng. 1977, 6, 151. 16. Sizer, I. W.; Josephson, E. S. Food Res. 1942, 7, 201. 17. Kavanau, J. L . J. Gen. Physiol. 1950, 34, 193. 18. Maier, V. P.; Tappel, A. L.; Volman, D. H. J. Am. Chem. Soc. 1955, 77, 1278. 19. Hultin, E. Acta Chem. Scand. 1955, 9, 1700. 20. Tappel, A. L . In "Cryobiology"; Meryman, H. T., Ed.; Academic: Lon don and New York, 1966; p 163. 21. Markert, C. L . Science 1963, 140, 1329. 22. Chilson, O. P.; Costello, L . Α.; Kaplan, N. O. FedProc.,Fed. Am. Soc. Exp. Biol. 1965, 24, S-55. 23. El-Negoumy, A. M. J. DairySci.1974, 57, 1170. 24. Hood, L. F.; Seifried, A. S. J. FoodSci.1974, 39, 121. 25. Hasiak, R. J. Vadehra, D. V.; Baker, R.C.;Hood, L. J. FoodSci.1972, 37, 913. 26. Smith, M. B.; Back, J. F. Biochim. Biophys. Acta 1975, 388, 203. 27. Sato, Y.; Aoki, T. Agric. Biol. Chem. 1975, 39, 29. 28. Sato, Y.; Takagaki, Y. Agric.Biol.Chem. 1976, 40, 49. 29. Chang, C. H.; Powrie, W. D.; Fennema, O. J. Food Sci. 1977, 42, 1658. 30. Parkinson, T. L. J. Sci. Food Agric. 1977, 28, 806. 31. Grek, A. M.; Lugovoi, V. I.; Bondarenko, T. P.; Morozov, I. A. Aktual. Vopr. Kriobiol. Kriomed., Mater. Simp. 1974, 43; Chem. Abstr. 1976, 84, 146529. 32. Skrabut, Ε. M.; Crowley, J. P.; Catsimpoolas, N.; Valeri, C. R. Cryo biology 1976, 13, 395, 33. Jackson, W. M.; Kostyla, J.; Nordin, J. H.; Brandts, J. F. Biochemistry 1973, 12, 3662. 34. Araki, T. Biochim. Biophys. Acta 1977, 496, 532. 35. Dzhafarov, A. I.; Kol's, O. R. Biofizika 1976, 21, 653; Chem. Abstr. 1976, 85, 107064. 36. Stahl, W. L.; Swanson, P. D. Neurobiology 1975, 5, 393. 37. Van den Berg, L.; Rose, D. Arch. Biochem. Biophys. 1959, 81, 319. 38. Van den Berg, L. J. Dairy Sci. 1961, 44, 26. 39. Van den Berg, L. Cryobiology 1966, 3, 236. 40. Soliman, F. S.; Van den Berg, L. Cryobiology 1971, 8, 73. 41. Van den Berg, L.; Soliman, F. S. Cryobiology 1969, 6, 93. 42. Pincock, R. E.; Lin, W.-S. J. Agric. Food Chem. 1973, 21, 2. 43. Sheppard, H.; Tsien, W. H. Biochim. Biophys. Acta 1974, 341, 489. 44. Janson, J.C.;Ersson, B.; Porath, J. Biotechnol. Bioeng. 1974, 16, 21. 45. Orii, Y.; Morita, M. J. Biochem. (Tokyo) 1977, 81, 163. 46. Posey, J.C.;Smith, H. A. J. Am. Chem. Soc. 1957, 79, 555. 47. Eigen, M.; DeMaeyer, L. Proc. R. Soc. London, Ser. A 1958, 247, 505. 48. VonHippel, A. R.; Runck, A. H.; Westphal, W. B. Gov. Rep. Announce. (U.S.) 1974, 74, 59. 49. Szent-Gyorgyi, A. Science 1956, 124, 873. ;


1.

TABORSKY

Protein Alterations


50. 51. 52. 53. 54. 55. 56.

25

Pincock, R. E . Ace. Chem. Res. 1969, 2, 97. Wang, S. Y. Nature (London) 1961, 190, 690. Horne, R. A. J. Inorg. Nucl. Chem. 1963, 25, 1139. Luyet, B. J. Biodynamica 1957, 7, 293. Macklin, W. C.; Ryan, B. F. J. R. Meteorol. Soc. 1962, 88, 548. Orii, Y.; Iizuka, T. J. Biochem. (Tokyo) 1975, 77, 1123. Levitt, J. In "Cryobiology"; Meryman, H . T., Ed.; Academic: London and New York, 1966; p 495. 57. Levitt, J. Cryobiology 1966, 3, 243. 58. Khan, A. W.; Davidkova, E.; Van den Berg, L . Cryobiology 1968, 4, 184. 59. Ishiwata, S. J. Biochem. (Tokyo) 1976, 80, 595. 60. Cherry, J. P.; Ory, R. L . J. Agric. Food Chem. 1973, 21, 656. 61. Weber, G. Adv. Protein Chem. 1975, 29, 1. 62. Fink, A. L. Biochemistry 1976, 15, 1580. 63. Makinen, M. W.; Fink, A. L . Annu. Rev. Biophys. Bioeng. 1977, 6, 301. 64. Petsko, G. A. J. Mol. Biol. 1975, 96, 381. 65. Franks, F. Philos. Trans. R. Soc. London, Ser. Β 1977, 278, 33. 66. Timasheff, S. N. Acc. Chem. Res. 1970, 3, 62. 67. Kuntz, I. D., Jr.; Kauzmann, W. Adv. Protein Chem. 1974, 28, 239. 68. Shlafer, M. Fed. Proc., Fed. Am. Soc. Exp. Biol. 1977, 36, 2590. 69. Hagler, A. T.; Scheraga, Η. Α.; Nemethy, G. Ann. Ν. Y. Acad. Sci. 1973, 204, 51. 70. Eisenberg, D.; Kauzmann, W. "The Structure and Properties of Water"; Oxford University Press: New York and Oxford, 1969; pp 254 ff. 71. Drost-Hansen, W. Ann. N. Y. Acad. Sci. 1973, 204, 100. 72. Sussman, M. V.; Chin, L. Science 1966, 151, 324. 73. Parungo, F. P.; Wood, J. J. Atmos. Sci. 1968, 25, 154. 74. Austin, R. H.; Beeson, K. W.; Eisenstein, L.; Frauenfelder, H.; Gunsalus, I. C. Biochemistry 1975, 14, 5355. 75. Aviram, I.; Schejter, A. Biopolymers 1972, 11, 2141. 76. Darbyshire, B. Cryobiology 1974, 11, 148. 77. Hanafusa, N. Bull. Inst. Int. Froid, Annexe 1973, 5, 9; Chem. Abstr. 1974, 81, 165554. 78. Smith, M. B.; Back, J. F. Biochim. Biophys. Acta 1975, 388, 203. 79. Bray, R. C.; Lowe, D. J.; Capeillere-Blandin, C.; Fielden, Ε. M. Biochem. Soc. Trans. 1973, 1, 1067. 80. Ehrenberg, Α.; Estabrook, R. W. Acta Chem. Scand. 1966, 20, 1667. 81. Price, Ε. M.; Gibson, J. F. J.Biol.Chem. 1972, 247, 8031. 82. Curti, B.; Massey, V.; Zmudka, M. J.Biol.Chem. 1968, 243, 2306. 83. Coles, C. J.; Edmondson, D. E.; Singer, T. P. J. Biol. Chem. 1977, 252, 8035. 84. Friedrich, P.; Foldi, J.; Varadi, K. Acta Biochim. Biophys. 1974, 9, 1. 85. Raymond, J. Α.; Lin, Y.; DeVries, A. L. J. Exp. Zool. 1975, 193, 125. 86. Raymond, J. Α.; DeVries, A. L . Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 2589. 87. Theede, H.; Schneppenhelm, R.; Beress, L. Mar. Biol. 1976, 36, 183. 88. Fletcher, G. L. Can. J. Zool. 1977, 55, 789. 89. Raymond, J. Α.; Radding, W.; DeVries, A. L . Biopolymers 1977, 16, 2575. 90. Tomimatsu, Y.; Scherer, J. R.; Yeh, Y.; Feeney, R. E . J. Biol. Chem. 1976, 251, 229. 91. Ananthanarayanan, V. S.; Hew, C. L. Nature (London) 1977, 268, 560. 92. Tower, D. B.; Goldman, S. S.; Young, Ο. M. J. Neurochem. 1976, 27, 285. 93. Kuhn, W.; Bloch, R.; Laeuger, P. Kolloid-Z. 1963, 193, 1. 94. Barthakur, N.; Maybank, J. Nature (London) 1963, 200, 866. 95. Jellinek, H. H. G. J. Colloid Interface Sci. 1967, 25, 192. 96. Drost-Hansen, W. J. Colloid Interface Sci. 1967, 25, 131.


26

97. 98. 99. 100. 101. 102. 103. 104. 105. 106.


107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119.


Workman, E . J.; Reynolds, S. E . Phys. Rev. 1950, 78, 254. Lodge, J. P.; Baker, M. L.; Pierrard, J. M. J. Chem. Phys. 1956, 24, 716. Jaccard, C.; Levi, L . Z. Angew. Math. Phys. 1961, 7, 70. Heinmets, F. Trans. Faraday Soc. 1962, 58, 788. DeMicheli, S. M.; Iribarne, J. V. J. Chim. Phys., Phys.-Chim. Biol. 1963, 60, 767. Gross, G. W. J. Geophys. Res. 1965, 70, 2291. Parreira, H. C.; Eydt, A. J. Nature (London) 1965, 208, 33. Anantha, N. G.; Chalmers, B. J. Appl. Phys. 1967, 38, 4416. LeFebre, V. J. Colloid Interface Sci. 1967, 25, 263. Pruppacher, H . R.; Steinberger, E . H.; Wang, T. L . J. Geophys. Res. 1968, 73, 571. Taborsky, G. J.Biol.Chem. 1970, 245, 1063. Terwilliger, J. P.; Dizio, S. F. Chem. Eng. Sci. 1970, 1331. Himes, R. C.; Miller, S. E.; Mink, W. H.; Goering, H . L. Ind. Eng. Chem. 1959, 51, 1345. Lusena, C. V. Arch. Biochem. Biophys. 1955, 51, 277. Pruppacher, H. R. J. Colloid Interface Sci. 1967, 25, 285. MacKenzie, A. P. Philos. Trans. R. Soc. London, Ser. Β 1977, 278, 167. Taborsky, G. J.Biol.Chem. 1970, 245, 1054. Zimmerman, S. B.; Coleman, N. F. Biopolymers 1972, 11, 1943. Visser, J. Meded. Landbouwhogesch. Wageningen 1970, 70-7, 90. Leibo, S. P.; Jones, R. F. Arch. Biochem. Biophys. 1964, 106, 78. Greiff, D.; Kelly, R. T. Cryobiology 1966, 2, 335. Fishbein, W. N.; Winkert, J. W. Cryobiology 1977, 14, 389. Oakenfull, D. G. Aust. J. Chem. 1972, 25, 769.

RECEIVED June 16,

1978.


Protein Alterations at Low Temperatures - American Chemical Society

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