Properties of Protein-Water Systems at Subzero Temperatures

2 Properties of Protein-Water Systems at Subzero Temperatures

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I. D. KUNTZ Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143

The hydration of proteins at subzero temperatures is reviewed. The thermodynamics of the protein-water system and the water molecule dynamics are discussed. The hydration layer around a protein at low temperature is best thought of as being in a glass-like state with the water molecules selectively oriented near ionic and polar groups at the protein surface. Water motions in the nanosecond and microsecond range have been detected.

l

x

his p a p e r w i l l s u m m a r i z e t h e a v a i l a b l e i n f o r m a t i o n o n a n u m b e r o f p r o p e r t i e s o f p r o t e i n - w a t e r systems b e l o w 0 ° C . W e w i l l d i r e c t o u r

a t t e n t i o n t o t h e r m o d y n a m i c d a t a a n d t o e x p e r i m e n t s t h a t speak t o t h e d y n a m i c s o f t h e systems.

I n some cases, w e u s e results a t h i g h e r t e m -

peratures t o i n f e r l o w - t e m p e r a t u r e b e h a v i o r .

O u r p r i m a r y p u r p o s e is t o

o r g a n i z e t h e e x p e r i m e n t a l results. N o s i n g l e m o d e l i s l i k e l y t o c o v e r a l l the facts. Thermodynamic Phase component

Properties

Diagram.

W e will

treat p r o t e i n - w a t e r

systems

p o r t i n g electrolytes t h a t f o r m a l l y r e q u i r e t h r e e ( o r m o r e ) These

as t w o -

systems a l t h o u g h m a n y p r e p a r a t i o n s c o n t a i n buffer o r s u p -

additional components

c a n always b e expected

components.

t o influence a

n u m b e r o f measurements a n d w i l l alter t h e e n t i r e p h a s e d i a g r a m , i n c l u d ing the b u l k melting behavior, i n complex ways.

W e w i l l c a l l attention

to s u c h effects w h e r e a p p r o p r i a t e . 0-8412-0484-5/79/33-180-027$5.00/0 © 1979 American Chemical Society Fennema; Proteins at Low Temperatures Advances in Chemistry; American Chemical Society: Washington, DC, 1979.

28

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

T h e first i m p o r t a n t o b s e r v a t i o n is t h a t a n y p r o t e i n - w a t e r

system

w i l l c o n t a i n some w a t e r m o l e c u l e s t h a t d o not freeze i n t o a c r y s t a l l i n e state at t e m p e r a t u r e s f a r b e l o w z e r o

( J , 2, 3 ) .

A l t h o u g h the

lowest

t e m p e r a t u r e s r e a c h e d v a r y f r o m one t e c h n i q u e to a n o t h e r ( c a . — 7 0 ° C f o r N M R a n d — 1 9 6 ° C f o r i r ) n o e v i d e n c e for a first-order c r y s t a l l i z a t i o n f o r t h e " n o n f r e e z i n g " w a t e r has b e e n r e p o r t e d .

Further, annealing pro

cedures d o n o t i n d u c e c r y s t a l l i z a t i o n , s u g g e s t i n g s t r o n g l y t h a t a state w i t h some degree of s t a b i l i t y has b e e n a c h i e v e d . F o r m o s t p r o t e i n a c e o u s systems, the s t a r t i n g p r e p a r a t i o n contains w a t e r i n excess of t h e n o n f r e e z i n g w a t e r , a n d the excess w a t e r r e a d i l y freezes to a n o r m a l i c e p h a s e Downloaded by IOWA STATE UNIV on February 28, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch002

i f n o l o w m o l e c u l a r w e i g h t a d d i t i v e s are present. F o r p r e p a r a t i o n s w i t h b u l k w a t e r contents less t h a n 0.3-0.5 g w a t e r / g p r o t e i n , n o ice f o r m a t i o n is d e t e c t a b l e b y N M R at a n y l o w t e m p e r a t u r e . T h e d e v e l o p m e n t of v e r y small

s u b n u c l e a t i o n c r y s t a l l o i d s of

ice

has b e e n

suggested

in

some

cases ( 5 , β ) . W a t e r - p r o t e i n systems s h o w d i s t i n c t l y different f r e e z i n g b e h a v i o r t h a n aqueous solutions t h a t c o n t a i n m a n y l o w m o l e c u l a r w e i g h t

com

ponents. A d i l u t e N a C l solution, for example, w i l l have l i q u i d water i n stable e q u i l i b r i u m w i t h ice. T h e a m o u n t of u n f r o z e n w a t e r w i l l b e q u i t e temperature-dependent,

falling

attained, w h i c h for N a C l

smoothly

is — 2 1 ° C .

until

the

eutectic

point

A t a d e g r e e o r so b e l o w

is

this

temperature, no water signal can be detected b y conventional N M R t e c h n i q u e s , a n d i r w i l l i n d i c a t e a m i x t u r e of i c e a n d N a C l - H o O , a w e l l characterized crystalline monohydrate.

D i l u t e a q u e o u s p r o t e i n solutions

w i l l also s h o w i c e f o r m a t i o n b e g i n n i n g at a b o u t z e r o degrees, w i t h t h e a m o u n t of u n f r o z e n w a t e r d e c r e a s i n g r a p i d l y w i t h d e c r e a s i n g t e m p e r a t u r e u n t i l a v a l u e i n t h e 0.3-0.5 g H 0 / g p r o t e i n r a n g e is r e a c h e d ( u s u a l l y 2

at a b o u t — 1 0 ° C i n the absence of s a l t s ) . H o w e v e r , i n contrast to t h e e u t e c t i c b e h a v i o r d e s c r i b e d a b o v e f o r N a C l , the a m o u n t of f r o z e n w a t e r t h e n r e m a i n s essentially constant w i t h f u r t h e r decreases i n t e m p e r a t u r e . P r o t e i n c r y s t a l s — n o r m a l l y h y d r a t e d at r o u g h l y 1 g w a t e r / g p r o t e i n — s h o w s i m i l a r b e h a v i o r to a q u e o u s solutions c o n t a i n i n g t h e same p r o t e i n composition.

(However,

r e p o r t e d ( 7 , S, 9 ) .

s o m e exceptions

to t h i s p a t t e r n h a v e

been

These include tropomyosin (a muscle protein) and

a n u m b e r of p o l y p e p t i d e solutions t h a t s h o w a c o n t i n u a l decrease i n t h e f r a c t i o n of u n f r o z e n w a t e r , at least d o w n to the l o w t e m p e r a t u r e l i m i t of N M R . )

N o s i m p l e e x p l a n a t i o n has b e e n offered f o r these t w o t y p e s

of b e h a v i o r .

Perhaps the most straightforward qualitative explanation

is t h a t c o n v e n t i o n a l eutectic b e h a v i o r arises b e c a u s e of the i n t e r s e c t i o n of

two

phase

boundaries:

the

freezing

point

curve

that

represents

e q u i l i b r i u m conditions between the solution a n d ice a n d the m e l t i n g p o i n t c u r v e t h a t represents e q u i U b r i u m c o n d i t i o n s b e t w e e n t h e s o l u t i o n and (generally)

a w e l l defined crystalline hydrate.

P o l y m e r solutions

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

2.

Properties

KUNTZ

of

29

Protein-Water

have w e l l characterized freezing point curves, but they usually do

not

f o r m w e l l b e h a v e d c r y s t a l l i n e h y d r a t e s . T h u s t h e l a c k of a e u t e c t i c p o i n t is not too s u r p r i s i n g . T h e slope of t h e f r e e z i n g p o i n t c u r v e at t e m p e r a t u r e s f a r b e l o w z e r o is not easily d e t e r m i n e d b u t i t is t h o u g h t to d e p e n d o n a n u m b e r of p a r a m e t e r s s u c h as Δ Η

of f u s i o n a n d t h e F l o r y - H u g g i n s

i n t e r a c t i o n constant, a n d these c o u l d a c c o u n t f o r the r a n g e of b e h a v i o r d e s c r i b e d a b o v e . W e k n o w t h a t c e r t a i n solutions c o n t a i n i n g l o w m o l e c u l a r w e i g h t h y d r o x y l i c solutes (sugars, alcohols, etc.) c a n f o r m glasses at l o w temperatures.

P r o t e i n solutions at h i g h p o l y m e r concentrations

and/or

l o w t e m p e r a t u r e s h a v e m a n y features i n c o m m o n w i t h these glasses. Downloaded by IOWA STATE UNIV on February 28, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch002

F o r systems i n w h i c h t h e u n f r o z e n w a t e r content is i n d e p e n d e n t of t e m p e r a t u r e , t h e a m o u n t of u n f r o z e n w a t e r at a n y g i v e n t e m p e r a t u r e is r e l a t e d to the i o n i c c o m p o s i t i o n of t h e

the m o r e i o n i c , t h e m o r e u n f r o z e n w a t e r o b s e r v e d (4, 8 ) . are f o u n d

to b e

hydrated more

subfreezing

macromolecule— N e g a t i v e ions

extensively t h a n p o s i t i v e

Preparations containing oppositely charged macromolecules somes, v i r u s e s , a n d m e m b r a n e

proteins)

ions

(8).

(e.g., r i b o -

f r e q u e n t l y are less h y d r a t e d

t h a n w o u l d b e p r e d i c t e d b a s e d o n t h e b e h a v i o r of t h e f i x e d

components

T h i s suggests t h a t i o n i c g r o u p s b e c o m e b u r i e d i n a m i x e d system.

(4).

I a m n o t a w a r e of a g e n e r a l t r e a t m e n t of c o u n t e r - i o n effects o n m a c r o m o l e c u l a r h y d r a t i o n . T h e w i d e l y v a r y i n g eutectic p o i n t s i n s u c h systems m a k e a d e f i n i t i v e s t u d y q u i t e difficult. A p h a s e d i a g r a m f o r w a t e r - p r o t e i n systems has b e e n p r o p o s e d (4)

to s u m m a r i z e t h e a b o v e d i s c u s s i o n .

Enthalpy, Entropy, and Heat Capacity of Protein—Water Systems Below

0°C.

A number

of investigators h a v e r e p o r t e d t h e

apparent

e n t h a l p y of f u s i o n as a f u n c t i o n of t e m p e r a t u r e a n d c o m p o s i t i o n several hydrated proteins.

M a c K e n z i e a n d coworkers

a b s o r p t i o n isotherms at l o w

temperatures

and found

for

determined

(10)

t h a t : 1)

these

a b s o r p t i o n isotherms h a v e essentially t h e same s i g m o i d a l shapes as those o b s e r v e d a b o v e zero degrees; 2 ) the m a g n i t u d e s of the v a l u e s for p a r t i a l m o l a l e n t h a l p y a n d e n t r o p y increase as t h e content of u n f r o z e n w a t e r decreases;

3)

the h e a t of f u s i o n decreases as t h e c o n t e n t of

unfrozen

w a t e r decreases; a n d 4 ) t h e heat c a p a c i t y of the system increases as t h e content of u n f r o z e n w a t e r increases.

T a k i n g these

findings

a l l together,

the t h e r m o d y n a m i c p r o p e r t i e s of " u n f r o z e n ' w a t e r are n o t v e r y different f r o m those of s u p e r c o o l e d w a t e r at c o m p a r a b l e t e m p e r a t u r e s . O n e s h o u l d a l w a y s r e m e m b e r t h a t i t is difficult to p r o c e e d t h e r m o d y n a m i c measurements t o m o l e c u l a r p r o p e r t i e s . l a r l y t r u e i n m u l t i c o m p o n e n t systems.

from

T h i s is p a r t i c u

T h a t is, w a t e r - w a t e r a n d w a t e r -

p r o t e i n interactions a r e i n e x t r i c a b l y m i x e d w i t h p r o t e i n - p r o t e i n i n t e r actions w h e n one measures p a r t i a l m o l a l q u a n t i t i e s . A s s u m p t i o n s a n d e x p e r i m e n t s b e y o n d those y i e l d i n g t h e r m o d y n a m i c i n f o r m a t i o n are n e e d e d to d e t e r m i n e w h a t is h a p p e n i n g at t h e m o l e c u l a r l e v e l . T h e m a j o r cause


30

PROTEINS A T L O W

TEMPERATURES

f o r c o n c e r n , here, is the r e a l p o s s i b i l i t y t h a t significant changes i n p r o t e i n c o n f o r m a t i o n c a n o c c u r at l o w t e m p e r a t u r e s . T h e r e is some e v i d e n c e , f o r e x a m p l e , t h a t p r o t e i n d e n a t u r a t i o n is i m p o r t a n t i n some cases

(11)·

C l e a r l y , t h e concentrations of a n y solutes increase d r a m a t i c a l l y as b u l k w a t e r freezes.

T o the extent t h a t a p p r e c i a b l e d e n a t u r a t i o n o r s t r u c t u r a l

m o d i f i c a t i o n occurs, one c a n expect difficulties i n i n t e r p r e t i n g t h e r m o d y n a m i c measurements o n these systems. I n s u m m a r y , I suggest t h a t t h e p h e n o m e n o n of n o n f r e e z i n g w a t e r i n p r o t e i n - w a t e r systems at l o w t e m p e r a t u r e m i g h t arise f r o m t h e l a c k of e q u i l i b r i u m w i t h a w e l l d e f i n e d c r y s t a l l i n e p r o t e i n h y d r a t e phase.

For


p r o t e i n s i n w h i c h a c r y s t a l l i n e p h a s e is k n o w n to exist, M a c K e n z i e has s h o w n t h a t samples c a n b e t a k e n t h r o u g h t h e c r y s t a l l i z a t i o n r e g i o n w i t h n o c r y s t a l f o r m a t i o n (10).

T h i s l a c k of e q u i h b r i u m is q u i t e l i k e l y a k i n e t i c

p r o b l e m b e c a u s e of the v e r y h i g h v i s c o s i t y present. S u r f a c e effects m i g h t also c o n t r i b u t e s i g n i f i c a n t l y since t h e surface a r e a p e r u n i t v o l u m e is v e r y l a r g e for a n y m o l e c u l a r l y d i s p e r s e d p o l y m e r . O n t h e w h o l e , t h e t h e r m o d y n a m i c properties reported for nonfreezing water appear rather similar t o those of s u p e r c o o l e d

water.

Motions in Protein-Water

Systems

T h e most p o w e r f u l technique for studying molecular motions protein-water

systems

below

0°C

is m a g n e t i c

resonance.

in

Dielectric

r e l a x a t i o n measurements c a n b e u s e d , b u t these measurements are m o r e s u i t a b l e at h i g h e r t e m p e r a t u r e s i n h o m o g e n o u s solutions (13). the f r e q u e n c y d e p e n d e n c e of the m e h c a n i c a l p r o p e r t i e s of

Recently, biopolymers

has b e e n s h o w n to y i e l d c o n s i d e r a b l e k i n e t i c i n f o r m a t i o n (14).

I will

l i m i t d i s c u s s i o n to the salient results a t t a i n a b l e f r o m these t e c h n i q u e s . NMR.

M a g n e t i c resonance e x p e r i m e n t s at l o w t e m p e r a t u r e s h a v e

b e e n l i m i t e d l a r g e l y to p r o t o n a n d d e u t e r o n N M R of t h e w a t e r m o l e c u l e s i n w a t e r - p o l y m e r p r e p a r a t i o n s . T h i s is reasonable b e c a u s e of t h e sensi t i v i t y a t t a i n a b l e a n d b e c a u s e the most r a p i d l y m o v i n g m o l e c u l a r species ( w a t e r ) is t h e most easily detected. macroscopic

I w i l l discuss o n l y systems w i t h o u t

o r d e r (e.g., f r o z e n solutions of g l o b u l a r p r o t e i n s ) b u t t h e

i n t e r e s t e d r e a d e r w i l l find i n t r i g u i n g reports of n m r m e a s u r e m e n t s

on

p r o t e i n crystals a n d o n fibrous o r l a y e r e d m a t e r i a l s ( 1 5 , 1 6 , 1 7 ) . I m p o r t a n t results t h a t h a v e b e e n o b t a i n e d f r o m N M R analysis of p r o t e i n solutions at subzero t e m p e r a t u r e s a r e : (1)

T h e w a t e r p r o t o n l i n e w i d t h at — 20 ° C is 100 times s h a r p e r f o r

w a t e r t h a n f o r i c e , a n d e v e n at — 6 0 ° C t h e difference is v e r y m a r k e d . (2)

A l l systems w e h a v e s t u d i e d to date s h o w a m i n i m u m s p i n -

lattice relaxation time ( T i )

o r a m a x i m u m s p i n - l a t t i c e r e l a x a t i o n rate

( 1 / T i ) at c a . — 3 5 ° C f o r a l a r m o r f r e q u e n c y of 40 M H z . T h e m i n i m u m f o r Γ ι moves to l o w e r t e m p e r a t u r e s as t h e l a r m o r f r e q u e n c y

decreases.


2.

Properties

KUNTZ (3)

of

31

Proteinr-Water

I n o u r w o r k , t h e p r o t o n l i n e w i d t h is a p p r o x i m a t e l y e q u a l to t h e

s p i n - s p i n r e l a x a t i o n rate ( 1 / Γ ) a n d this r e l a x a t i o n is n o t i c e a b l y faster 2

t h a n Ι / Τ Ι at a l l subzero t e m p e r a t u r e s . (4)

T h e s p i n - l a t t i c e r e l a x a t i o n rate ( 1 / Γ ι ) is r e p o r t e d to b e q u i t e

frequency-dependent

(see

(2)

above), and rotating frame

experiments

( l / T i p ) at l o w temperatures also i n d i c a t e f r e q u e n c y d e p e n d e n t r e l a x a tions

(18,19). (5)

N o r a p i d m o t i o n i n the m a c r o m o l e c u l a r c o m p o n e n t s

has b e e n

reported. Downloaded by IOWA STATE UNIV on February 28, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch002

C e r t a i n c o n c l u s i o n s c a n b e d r a w n f r o m these observations a l t h o u g h detailed interpretation w i l l depend on future quantitative developments. F i r s t , m o s t of the w a t e r m o l e c u l e s d e t e c t e d b y N M R are m o v i n g q u i t e r a p i d l y at s u b z e r o t e m p e r a t u r e s .

T h e sharp N M R s i g n a l f o r protons of

u n f r o z e n w a t e r is p e r h a p s the best e v i d e n c e that a n o n c r y s t a l l i n e state of w a t e r is present i n these samples.

Quantitative conclusions

about

p r o t o n m o b i l i t y c a n b e d e r i v e d f r o m the r e l a x a t i o n d a t a g i v e n a b o v e . W i t h o u t g o i n g into d e t a i l , a b o v e — 35 ° C the average r o t a t i o n a l c o r r e l a t i o n t i m e for w a t e r protons is c e r t a i n l y less t h a t 4 X 10~ seconds. I n o u r 9

e a r l y p a p e r s w e expressed t h e v i e w that this average c o r r e l a t i o n t i m e a p p l i e d to most of the w a t e r m o l e c u l e s t h a t c o n t r i b u t e to t h e n m r s i g n a l . It is n o w c l e a r t h a t there is n o h a r d e v i d e n c e i n s u p p o r t of this p o s i t i o n . T h e T i m i n i m u m is b r o a d a n d n o t as d e e p as s i m p l e t h e o r y (20)

would

p r e d i c t . C o n t r i b u t i o n s f r o m s p i n - s p i n d i f f u s i o n are r e a d i l y d e t e c t e d T h e r e is a r e a l c h a n c e t h a t a v e r y significant f r a c t i o n of

21, 22).

(17, the

unfrozen water molecules have rotational correlation times considerably shorter t h a n 10" seconds. T h e c o r r e l a t i o n t i m e f o r s u p e r c o o l e d w a t e r at 9

these t e m p e r a t u r e s is a b o u t 3 X 10~

10

seconds. T h e d e f i n i t i v e e x p e r i m e n t

w o u l d i n v o l v e m e a s u r e m e n t of h i g h f r e q u e n c y

dielectric dispersion i n

these m a t e r i a l s . S e c o n d , observations molecules

(3)

and (4)

e x h i b i t s l o w motions

suggest thas some of the w a t e r

(e.g., s l o w e r t h a n 4 Χ

T h e r e are t w o i m p o r t a n t restrictions here.

10"

seconds).

9

I t is n o t p o s s i b l e to b e v e r y

quantitative i n discussing slow motions because there is a substantial a m p l i f i c a t i o n f a c t o r f o r t h e w a y s l o w motions influence N M R l i n e w i d t h . A l s o , there is nô reason to assume t h a t a g i v e n w a t e r m o l e c u l e

experiences

a single r o t a t i o n a l m o t i o n . I t is m u c h m o r e l i k e l y t h a t a n y g i v e n m o l e c u l e samples a l l p o s s i b l e m o t i o n s f o r v a r y i n g p e r i o d s of t i m e . O b s e r v a t i o n of n o n e x p o n e n t i a l d e c a y of N M R signals is not, i n itself, e v i d e n c e of s l o w l y e x c h a n g i n g p o p u l a t i o n s of w a t e r m o l e c u l e s (17,

21, 22).

Thus, the con-

servative c o n c l u s i o n is t h a t a s m a l l f r a c t i o n of t h e w a t e r m o t i o n s at, say, — 35 ° C

are c o n s i d e r a b l y

s l o w e r t h a n 10"

9

and might approach

seconds.


10"

6

32

PROTEINS A T L O W

TEMPERATURES

T h i r d , t h e r e is n o N M R e v i d e n c e f o r r a p i d t u m b l i n g of t h e m a c r o m o l e c u l e s o r f o r r a p i d m o t i o n of t h e i r s i d e c h a i n s . H o w e v e r , these m o t i o n s w o u l d b e difficult to detect.

C a r e f u l e x p e r i m e n t s of p r o t e i n r e l a x a t i o n i n

c o n c e n t r a t e d solutions a n d gels are n e e d e d b e f o r e a c c u r a t e c a n b e d r a w n w i t h r e g a r d to m o t i o n s of

conclusions

macromolecules.

T h u s w e i n t e r p r e t t h e N M R results t o date as p r o v i d i n g r e a s o n a b l e e v i d e n c e f o r t w o classes of m o t i o n i n f r o z e n p r o t e i n solutions i n t h e subzero range.

T h e first a n d most p r o m i n e n t m o t i o n is the q u i t e r a p i d

t u m b l i n g of n o n f r o z e n w a t e r m o l e c u l e s . T h e s e c o n d , m o r e p o o r l y d e f i n e d m o t i o n i n v o l v e s m a n y f e w e r w a t e r p r o t o n s at a n y i n s t a n t of t i m e a n d these h a v e c o r r e l a t i o n t i m e s of a p p r o x i m a t e l y 10" to 10" seconds i n t h e Downloaded by IOWA STATE UNIV on February 28, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch002

5

7

t e m p e r a t u r e r a n g e of — 20 to — 5 0 ° C . Dielectric Measurements.

R e c e n t d i e l e c t r i c e x p e r i m e n t s at a b o v e -

z e r o t e m p e r a t u r e s h a v e d e t e c t e d a n u m b e r of m o l e c u l a r m o t i o n s m i g h t o c c u r at l o w t e m p e r a t u r e s ( 2 3 , 24, 2 5 ) .

that

I n particular, sidechain

m o t i o n s a n d the m o t i o n s of counter-ions c o u l d c o n t r i b u t e t o t h e w a t e r r e l a x a t i o n processes. F r e q u e n c i e s i n the r a n g e of 1 0 suggested

4

to 1 0

8

have

been

f o r s u c h effects, b u t , at present, there is n o d i r e c t l i n e of

e v i d e n c e c o n n e c t i n g these m o t i o n s to t h e l o w f r e q u e n c y n m r results. Mechanical Properties.

H i l t n e r a n d coworkers have measured the

d y n a m i c p r o p e r t i e s of h y d r a t e d p r o t e i n s a n d p o l y p e p t i d e s at l o w t e m p e r a t u r e s (6,14).

T h i s technique involves direct mechanical deformation

a n d has a v e r y l o w f r e q u e n c y " w i n d o w " f o r m o t i o n s at 1 H z . C o l l a g e n , f o r e x a m p l e , shows t w o processes, t h e faster one m o v i n g t h r o u g h t h e 1 H z w i n d o w at a p p r o x i m a t e l y — 1 2 5 ° C w h i l e the s l o w e r process is seen at — 7 5 ° C .

H i l t n e r suggests t h a t the first m o t i o n is r e l a t e d to p o l y m e r

s i d e c h a i n effects, w h i l e the s e c o n d is a s s i g n e d to a specific w a t e r - p r o t e i n interaction.

A l a r g e a c t i v a t i o n b a r r i e r w o u l d m o v e these m o t i o n s i n t o

t h e N M R f r e q u e n c y r a n g e at h i g h e r temperatures. I n s u m m a r y , a s u b s t a n t i a l n u m b e r of p o s s i b l e m o t i o n s exist f o r w a t e r m o l e c u l e s associated w i t h p r o t e i n a c e o u s m a t e r i a l s at l o w t e m p e r a t u r e s . A v e r y w i d e r a n g e of f r e q u e n c i e s exists: f r o m a f e w H z to a f e w G H z . A c o m b i n a t i o n of studies, i n v o l v i n g N M R measurements of t h e f r e q u e n c y d e p e n d e n c e of r e l a x a t i o n rates a n d t h e d i e l e c t r i c a n d m e c h a n i c a l t e c h n i q u e s d e s c r i b e d a b o v e , w i l l b e r e q u i r e d to c h a r a c t e r i z e a n d assign a l l these m o t i o n s . O u r present i n t e r p r e t a t i o n is t h a t t h e w a t e r m o t i o n s a p p e a r to reflect the t o t a l s p e c t r u m of k i n e t i c events i n t h e system.

Acknowledgment S u p p o r t f r o m t h e N a t i o n a l Institutes of H e a l t h a n d t h e D i v i s i o n of R e s e a r c h Resources is g r a t e f u l l y a c k n o w l e d g e d .


2.

KUNTZ

Properties

of

Protein-Water

33


Literature Cited 1. Migchelsen, C.; Brendsen, H . J. C.; Rupprecht, A. J. Mol. Biol. 1968, 37, 235. 2. Kuntz, I. D.; Brassfield, T. S.; Law, G. D.; Purcell, G. V. Science 1969, 163, 1329. 3. Falk, M.; Poole, A. G.; Goymour, C. G. Can. J. Chem. 1970, 48, 1536. 4. Kuntz, I. D.; Kauzmann, W. Adv. Protein Chem. 1974, 28, 239. 5. Luyet, B. J. Ann. Ν. Y. Acad. Sci. 1965, 125, 502. 6. Nomura, S.; Hiltner, Α.; Lando, J. B.; Baer, E . Biopolymers 1977, 16, 231. 7. Blanshard, J. M. V.; Derbyshire, W. In "Water Relations of Foods"; Duck worth, R. B., Ed.; Academic: New York, 1975; p 559. 8. Kuntz, I. D. J. Amer. Chem. Soc. 1971, 93, 514. 9. Ramirez, J. E.; Cavanaugh, J. R.; Purcell, J. M. J. Phys. Chem. 1974, 78, 80. 10. MacKenzie, A. P. In "Water Relations in Foods"; Duckworth, R. B., Ed.; Academic: New York, 1975; p 477. 11. Kuntz, I. D.; Brassfield, T. S. Arch. Biochem. Biophys. 1971, 142, 660. 12. Resing, Η. Α.; Wage, C. G. ACS Symp. Ser. 1976, 34. 13. Takashima, S.; Fishman, Η. H., Eds., "Electric Properties of Biological Polymers, Water, and Membranes", Ann. Ν. Y. Acad. Sci. 1977, Vol. 303. 14. Shiraisi, H.; Hiltner, Α.; Baer, E . Biopolymers 1977, 16, 2801. 15. Resing, Η. Α.; Garroway, A. N.; Foster, K. R. ACS Symp. Ser. 1976, 34. 516. 16. Woessner, D. E.; Snowden, B. S. J. Colloid Interface Sci. 1970, 34, 290. 17. Bryant, R. G. Adv. Biophys. Bioengr. 1978, Vol. 8. 18. Kuntz, I. D.; Zipp, Α.; James, T. L. ACS Symp. Ser. 1976, 34. 499. 19. Zipp, A; Kuntz, I. D.; James, T. L. Arch. Biochem. Biophys. 1977, 178, 435 20. Bloembergen, N.; Purcell, Ε. M.; Pound, R. V.; Phys. Rev. 1948, 73, 679. 21. Edzes, H. T.; Samulski, Ε. T. Nature 1977, 265, 521. 22. Kimmich, R.; Noack, F. Ber. Bunsenges Phys. Chemie. 1971, 75, 269. 23. Cole, R. H. Ann. Ν. Y. Acad. Sci. 1977, 303, 59. 24. Mandel, M. Ann. Ν. Y. Acad. Sci. 1977, 303, 74. 25. Minakata, A. Ann. Ν. Y. Acad. Sci. 1977, 303, 107. RECEIVED June 16,

1978.


Properties of Protein-Water Systems at Subzero Temperatures

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