Behavior of Proteins at Low Temperatures - ACS Publications

6 Behavior of Proteins at Low Temperatures

Downloaded by MICHIGAN STATE UNIV on February 19, 2015 | http://pubs.acs.org Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch006

OWEN FENNEMA University of Wisconsin—Madison, Department of Food Science, Madison, WI 53706

Protein behavior at below ambient temperatures has received l i t t l e attention compared to that of protein behavior at physio logical temperatures or above. This is, of course, understand able, but nonetheless unfortunate, since proteins are believed by many authorities to be involved in significant, if not substan t i a l , ways with the ability or inability of living matter and foods to tolerate a variety of low temperature situations. *The ability of some animals to hibernate and others to acclimate when exposed to a low, nonfreezing environment. *The ability of some plants to "winter harden" and thereby tolerate freezing conditions. *The ability of some fish to avoid freezing at water tempera tures normally low enough to cause freezing. *The ability of some microbial cultures and other small biological specimens to survive freezing, frozen storage and thawing. *The inability of humans to tolerate low nonfreezing body temperatures. *The inability of large biological specimens such as whole organs and small, whole animals to survive preservation by freezing. *The inability of plant and animal food tissue to withstand commercial freeze preservation procedures without under going detrimental changes, particularly with respect to water holding capacity and texture. In addition, it should be noted that a l l too many researchers pay insufficient attention to the effects of low temperature storage on proteinaceous samples, assuming simply, and often falsely, that the lower the temperature the better. As will be documented in this Chapter, the behavior of proteins at low temperatures often deviates from intuitive expectations. Functional properties, especially enzyme activity, can change nonlinearly as the temperature is lowered and this unexpected behavior becomes even more erratic when cellular systems are involved and/or freezing occurs. Changes in enzyme 0097-6156/82/0206-0109$07.75/0 © 1982 American Chemical Society In Food Protein Deterioration; Cherry, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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a c t i v i t y r e s u l t i n g f r o m a l o w e r i n g o f t e m p e r a t u r e c a n be r e v e r s i b l e , p a r t i a l l y r e v e r s i b l e o r i r r e v e r s i b l e d e p e n d i n g on f a c t o r s s u c h a s t h e k i n d o f enzyme, t h e n a t u r e o f t h e s a m p l e and the t i m e - t e m p e r a t u r e scheme e m p l o y e d . From what h a s a l r e a d y b e e n s a i d , a r e d u c t i o n i n t e m p e r a t u r e a l s o w o u l d be e x p e c t e d t o c a u s e s i g n i f i c a n t i n t r a - and i n t e r m o l e c u l a r c h a n g e s i n p r o t e i n s , and t h e s e a l t e r a t i o n s do i n f a c t o c c u r . The b e h a v i o r o f p r o t e i n s i n n o n c e l l u l a r and c e l l u l a r s y s t e m s w i l l be c o n s i d e r e d a t t e m p e r a t u r e s c o v e r i n g above and b e l o w f r e e z i n g c o n d i t i o n s . A t t e n t i o n w i l l be g i v e n t o b o t h c h a n g e s i n p r o t e i n f u n c t i o n a l i t y ( m a i n l y enzyme a c t i v i t y ) and c h a n g e s i n protein structure. Experimental Procedures One o b v i o u s r e a s o n f o r t h e l i m i t e d amount o f r e s e a r c h b e i n g g e n e r a t e d on p r o t e i n s a t l o w t e m p e r a t u r e s r e l a t e s t o t h e d i f f i c u l t i e s of conducting t h i s k i n d of r e s e a r c h , p a r t i c u l a r l y i n the p r e s e n c e o f i c e . O p t i c a l p r o c e d u r e s t h a t a r e r e l i e d on so h e a v i l y i n u n f r o z e n l i q u i d s a m p l e s a r e g e n e r a l l y u s e l e s s when an i c e phase i s p r e s e n t . F u r t h e r m o r e , g r o s s nonhomogeneity e x i s t s i n f r o z e n s a m p l e s and t h i s n o n h o m o g e n e i t y c h a n g e s w i t h f r o z e n storage. Procedures to a c c u r a t e l y assess the c h a r a c t e r i s t i c s of s a m p l e s o f t h i s n a t u r e s i m p l y do n o t e x i s t . T h u s , many r e s e a r c h e r s e v a l u a t e t h e e f f e c t s o f f r e e z i n g and f r o z e n s t o r a g e a f t e r t h a w i n g . F u r t h e r m o r e , a l l t o o many i n v e s t i g a t o r s f a i l t o d e s i g n t h e i r s t u d i e s so t h a t t h e e f f e c t s o f f r e e z i n g r a t e c a n be i s o l a t e d from the e f f e c t s of f r e e z i n g depth ( n a d i r ) . One a p p r o a c h t o s t u d y i n g e n z y m e - c a t a l y z e d r e a c t i o n s a t v e r y l o w t e m p e r a t u r e s (down t o a b o u t -100°C) i n v o l v e s t h e u s e o f aqueous-organic s o l v e n t s w i t h f r e e z i n g p o i n t s s u f f i c i e n t l y low t o a v o i d i c e f o r m a t i o n (_1_, 2). T h i s t e c h n i q u e was d e v e l o p e d p r i m a r i l y t o s t u d y r e a c t i o n i n t e r m e d i a t e s and h a s p r o v e n r e a s o n a b l y successful. Some c o n c e r n e x i s t s , h o w e v e r , t h a t h i g h c o n c e n t r a t i o n s of o r g a n i c s o l v e n t s might i n f l u e n c e r e a c t i o n pathways. F a c t o r s I n f l u e n c i n g Enzyme A c t i v i t y a t Low

Temperatures

I n t h i s s e c t i o n , enzyme a c t i v i t y i n s i m p l e , n o n c e l l u l a r s y s t e m s w i l l be c o n s i d e r e d f i r s t and t h i s w i l l be f o l l o w e d by d i s c u s s i o n o f enzyme a c t i v i t y i n c e l l u l a r s y s t e m s .

a

Enzymes i n n o n c e l l u l a r , s i m p l e s y s t e m s a t l o w t e m p e r a t u r e s . F a c t o r s c o n s i d e r e d t o be o f s i g n i f i c a n c e i n g o v e r n i n g t h e a c t i v i t y o f enzymes a t l o w t e m p e r a t u r e s i n c l u d e n a t u r e o f t h e enzyme, t e m p e r a t u r e d e p e n d e n c e , c o o l i n g - f r e e z i n g p r o c e d u r e s , and c o m p o s i t i o n o f t h e medium. 1) N a t u r e o f t h e enzyme. I t i s w e l l known t h a t enzymes d i f f e r g r e a t l y i n t h e i r r e s p o n s e t o a v a r i e t y o f c o n d i t i o n s so i t s h o u l d be no s u r p r i s e t h a t m a r k e d d i f f e r e n c e s i n t h e b e h a v i o r o f

In Food Protein Deterioration; Cherry, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.


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v a r i o u s enzymes are observed during c o o l i n g and f r e e z i n g . Some enzymes, f o r example, e x h i b i t great t o l e r a n c e t o freeze-thaw treatments, whereas others do not (3, 4_» 5); and some e x h i b i t s i g n i f i c a n t a c t i v i t y i n p a r t i a l l y f r o z e n systems (e.g. oxidases and l i p a s e s ) whereas others do not (e.g. some p r o t e i n a s e s ) (6-10). Based l a r g e l y on a review of the Japanese and Russian l i t e r a t u r e , L o z i n a - L o z i n s k i i (11) concluded that f r e e z i n g causes greater changes i n f i b r i l l a r p r o t e i n s than i n g l o b u l a r p r o t e i n s . The r a m i f i c a t i o n s of t h i s c o n c l u s i o n , o f course, i n v o l v e more than j u s t enzyme a c t i v i t y . 2) Temperature dependence. Temperature i s , of course, a f a c t o r of primary importance i n determining enzyme a c t i v i t y and r e a c t i o n r a t e s . The r e l a t i o n s h i p between the r e a c t i o n r a t e constant and absolute temperature i s expressed by the w e l l known Arrhenius equation (k = se~^ ' ) , and i t i s normally expected that Arrhenius p l o t s ( l o g k v s . 1/T) w i l l be l i n e a r over reasonably small (20-30°C) temperature spans. T h i s e x p e c t a t i o n i s not normally met, however, f o r enzyme suspensions a t low or even mode r a t e l y low temperatures (12, 13, 14). For example, n o n l i n e a r Arrhenius p l o t s have been observed a t s l i g h t l y above-freezing temperatures f o r pyruvate carboxylase (15), glutamate carboxylase (16), a c e t y l CoA-carboxylase (17), pyruvate orthophosphate d i k i n a s e (18), l i p a s e (10) and f o r phosphatase and peroxidase (9). The behavior of l i p a s e i s shown i n Figure 1. I t has been suggested that these data conform e q u a l l y w e l l to a smooth curve and that more acceptable i n t e r p r e t a t i o n s are p o s s i b l e i n t h i s form (14). I t a l s o should be emphasized that the p l o t i n F i g u r e 1 i s obtained both i n the presence and absence o f i c e . Thus, the independent e f f e c t of decreasing temperature can cause enzymec a t a l y z e d r e a c t i o n s i n simple systems t o depart i n a s i g n i f i c a n t negative f a s h i o n from the Arrhenius equation. I t should not be assumed, however, that the e f f e c t s o f temperature lowering and the e f f e c t s of temperature lowering p l u s the formation of i c e always produce Arrhenius p l o t s that a r e superimposable. F r e e z i n g introduces many more c o m p l e x i t i e s (changes i n pH, i o n i c s t r e n g t h , s o l u t e c o n c e n t r a t i o n , e t c . ) than temperature lowering alone and the e f f e c t s of f r e e z i n g are t h e r e f o r e not always c o n s i s t e n t with the p a t t e r n i n F i g u r e 1. Although r a r e , f r e e z i n g can, f o r example, a c t u a l l y cause an i n c r e a s e i n the r a t e constant f o r some enzyme-catalyzed r e a c t i o n s (19, 20). The general p a t t e r n of a negative d e v i a t i o n from the Arrhenius r e l a t i o n s h i p as temperature i s lowered (with o r without f r e e z i n g ) probably i n v o l v e s conformational changes i n the enzyme and/or an a s s o c i a t i o n - d i s s o c i a t i o n type t r a n s i t i o n that leads to enzyme i n a c t i v i t y . In t h i s case, one of the assumptions underl y i n g the a p p l i c a b i l i t y t o the Arrhenius r e l a t i o n s h i p i s v i o l a t e d and l i n e a r i t y would not be expected. A second property of enzymes that i s sometimes temperature dependent i s t h e i r a f f i n i t y f o r s u b s t r a t e . Under a p p r o p r i a t e



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Figure 1. Log rate of hydrolysis (i.e., log reciprocal of time required for 1% hydrolysis) of tributyrin by lipase as influenced by temperature. (Reproduced, with permission, from Ref. 10. Copyright 1942, Institute of Food Technologists.)



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circumstances, enzyme-substrate a f f i n i t y i s i n d i c a t e d by the m a g n i t u d e o f t h e M i c h a e l i s c o n s t a n t (Km), and r e p o r t s o f a s t r o n g t e m p e r a t u r e dependence o f t h i s v a l u e h a v e b e e n o b s e r v e d f o r s e v e r a l enzymes i n c l u d i n g l a c t a t e d e h y d r o g e n a s e s (21, 2 2 ) , p h o s p h o p h e n o l p y r u v a t e c a r b o x y l a s e ( 2 3 ) a n d p h e n y l a l a n i n e ammonia l y a s e ( 2 4 ) . The r e l a t i o n s h i p b e t w e e n Km and t e m p e r a t u r e f o r l a c t a t e d e h y d r o g e n a s e s f r o m v a r i o u s s o u r c e s i s shown i n F i g u r e 2. I n t h e s e e x a m p l e s , maximum a p p a r e n t e n z y m e - s u b s t r a t e a f f i n i t y (minimum Km) c o r r e s p o n d s v e r y c l o s e l y t o t h e h a b i t a t o r a c c l i m a t i o n t e m p e r a t u r e o f t h e h o s t f r o m w h i c h t h e enzyme i s d e r i v e d . A r e v e r s i b l e change i n c o n f o r m a t i o n n e a r t h e enzyme a c t i v e s i t e c o u l d be r e s p o n s i b l e f o r t h i s b e h a v i o r ( 2 3 ) . S i n c e s u b s t r a t e c o n c e n t r a t i o n s i n c e l l s a r e n o r m a l l y l o w ( u s u a l l y 1 mM o r l e s s ) and s e l d o m , i f e v e r , a t t a i n s a t u r a t i o n l e v e l s f o r most enzymes, t h e t e m p e r a t u r e dependence o f Km v a l u e s i n l i v i n g systems i s c o n s i d e r e d o f f a r g r e a t e r importance than t h e temperat u r e dependence o f V values (22). S t i l l a n o t h e r t e m p e r a t u r e - d e p e n d e n t p r o p e r t y o f enzymec a t a l y z e d r e a c t i o n s i s worthy of mention. T h i s concerns t h e u l t i m a t e extent t o which these r e a c t i o n s proceed. I t i s commonly observed t h a t t h e u l t i m a t e a c c u m u l a t i o n o f r e a c t i o n p r o d u c t s tends to decrease as the s u b f r e e z i n g temperature i s decreased. F o r example, t h i s r e s u l t has been observed f o r d e n a t u r a t i o n o f c h y m o t r y p s i n o g e n (25) and f o r r e a c t i o n s c a t a l y z e d b y l i p a s e (26), i n v e r t a s e ( 2 7 ) a n d l i p o x y g e n a s e (28), w i t h d a t a i l l u s t r a t i n g t h e l a t t e r example a p p e a r i n g i n F i g u r e 3. P o s s i b l e e x p l a n a t i o n s f o r t h i s behavior include; ^ R e v e r s i b l e c h a n g e s i n enzyme a c t i v i t y stemming f r o m r e v e r s i b l e c h a n g e s i n enzyme c o n f o r m a t i o n a n d / o r a s s o c i a tion. These changes c o u l d o c c u r because o f t h e d i r e c t e f f e c t s o f temperature o r the i n d i r e c t e f f e c t s o f f r e e z i n g (pH c h a n g e , i n c r e a s e i n i o n i c s t r e n g t h , e t c . ) . ^Restraints on d i f f u s i o n of substrate, reaction products a n d / o r enzyme. T h i s c o u l d o c c u r b e c a u s e o f e n t r a p m e n t i n i c e and/or slow d i f f u s i o n i n t h e c o n c e n t r a t e d , h i g h v i s c o s i t y , u n f r o z e n phase. I n l i q u i d systems, v i s c o s i t y i s known t o d e c r e a s e a s a f u n c t i o n o f 1/T, b u t t h e c o n s e q u e n c e s on enzyme a c t i v i t y , e v e n a t v e r y l o w t e m p e r a t u r e s , i s n o t c o n s i d e r e d i m p o r t a n t ( 2 9 ) . However, once i c e f o r m a t i o n occurs, v i s c o s i t y increases abruptly to a very high l e v e l and t h e e f f e c t o n enzyme a c t i v i t y , a l t h o u g h n o t w e l l known, i s l i k e l y t o be s u b s t a n t i a l . 3) C o o l i n g a n d f r e e z i n g p r o c e d u r e s . C o o l i n g and f r e e z i n g p r o c e d u r e s , e s p e c i a l l y t h e l a t t e r , c a n have pronounced e f f e c t s o n enzyme a c t i v i t y . F a c t o r s such as r a t e s o f c o o l i n g , f r e e z i n g and w a r m i n g , t e m p e r a t u r e n a d i r , and t h e t i m e and t e m p e r a t u r e o f f r o z e n s t o r a g e a l l a r e s i g n i f i c a n t (3, 8^, 30, 3 1 ) . I n e v a l u a t i n g t h e s e e f f e c t s , one s h o u l d be aware t h a t most i n v e s t i g a t o r s have m e a s u r e d enzyme a c t i v i t y f o l l o w i n g t h a w i n g r a t h e r t h a n d u r i n g f r o z e n s t o r a g e , and h a v e c o n f o u n d e d t h e e f f e c t s o f f r e e z i n g r a t e and t e m p e r a t u r e n a d i r . m a x


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20 LUNGFISH M - L D H ( 2 7 - 3 0 ° ) /

TUNA M-LDH C~I5°)

lEMATOMUS


M-LDH (-2°)

06h

10

20

40

30

TEMPERATURE (°C)

Figure 2. Temperature dependence of Michaelis constant (K ) of pyruvate in reactions catalyzed by lactate dehydrogenases. Temperatures indicate average tissue temperatures. (Reproduced, with permission, from Ref. 21. Copyright 1968, Pergamon Press.) m

ο

200

400 MINUTES

I

π ο—ό

600

Figure 3. Effect of sub free zing storage temperatures on the ultimate accumulation of oxidation products of linolenic acid. (All samples were rapidly frozen to —78.5 °C, then stored at temperatures indicated.) Each value is a mean of four deter minations. (Reproduced, with permission, from Ref. 28. Copyright 1980, Academic Press.)



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4) Composition of the sample. Enzyme p u r i t y and concent r a t i o n , pH o f the medium, the kind and q u a n t i t y o f e l e c t r o l y t e s present and the absence or presence o f p r o t e c t i v e agents (such as p r o t e i n s , g l y c e r o l , sugars, dimethyl s u l f o x i d e , etc.) can g r e a t l y i n f l u e n c e enzyme a c t i v i t y at low temperatures. For example, r e t e n t i o n of a c t i v i t y o f l a c t a t e dehydrogenase i n a simple s o l u t i o n f o l l o w i n g thawing i s favored when enzyme concentration i s high, s a l t concentration i s low, c h l o r i d e s r a t h e r than phosphates are present, other p r o t e i n s or g l y c e r o l are present, and pH i s a t the upper end o f the range 6.0-7.8 (31, 32). During f r e e z i n g , the concentration of s o l u t e s i n the unfrozen phase always increases and, as a r e s u l t , p r o p e r t i e s such as pH (31, 33, 34), i o n i c s t r e n g t h , surface t e n s i o n , concentration of d i s s o l v e d gases and v i s c o s i t y undergo marked changes. A l l o f these p r o p e r t i e s have important i n f l u e n c e s on enzyme a c t i v i t y . Enzymes i n c e l l u l a r systems a t low temperatures. Enzyme a c t i v i t y i n c e l l u l a r systems i s i n f l u e n c e d by a l l of the f a c t o r s p r e v i o u s l y discussed, with a d d i t i o n a l complications a r i s i n g because of the c e l l u l a r s t r u c t u r e . Two complications of p a r t i c u l a r importance i n v o l v e the behavior o f enzymes during c h i l l i n g i n j u r y of p l a n t s , and d e r e a l i z a t i o n of enzymes during f r e e z i n g of both p l a n t and animal t i s s u e s . It has long been recognized that p l a n t s of t r o p i c a l and s u b t r o p i c a l o r i g i n s undergo detrimental p h y s i o l o g i c a l changes, known as " c h i l l i n g i n j u r y , " when exposed to low nonfreezing temperatures. Two occurrences a s s o c i a t e d w i t h c h i l l i n g i n j u r y are 1) a l t e r a t i o n of membrane s t r u c t u r e i n o r g a n e l l e s (35, 36) and 2) d i s c o n t i n u i t i e s i n , o r a t l e a s t a sharp change i n the curvature o f , Arrhenius p l o t s f o r r e a c t i o n s c a t a l y z e d by membranebound enzymes. These d i s c o n t i n u i t i e s g e n e r a l l y c o i n c i d e w e l l with the maximum temperature f o r the onset o f membrane damage and c h i l l i n g i n j u r y (35, 3 7 ) . The l a t t e r occurrence e x e m p l i f i e s p r o t e i n behavior that i s apparently unique t o c e l l u l a r systems and i s worthy o f f u r t h e r c o n s i d e r a t i o n . Numerous enzymes, t y p i c a l l y nonsoluble (e.g. a s s o c i a t e d with membrane systems or s t a r c h grains) and from c h i l l i n g - s e n s i t i v e p l a n t s , e x h i b i t n o n l i n e a r Arrhenius p l o t s at above-freezing temperatures. An example i n v o l v i n g cytochrome oxidase a c t i v i t y i n i n t a c t mitochondria from tomato i s shown i n Figure 4. The most a t t r a c t i v e explanation f o r t h i s behavior i s that the enzyme i s a s s o c i a t e d with a l i p i d , and i t i s temperature-induced changes i n t h i s a s s o c i a t i o n , perhaps changes i n f l u i d i t y o f the l i p i d and/ or changes i n the geometry of the l i p i d molecules, that produce a conformational change i n the enzyme and r e v e r s i b l e i n a c t i v a t i o n . Although most r e p o r t s of the anamolous behavior mentioned above i n v o l v e enzymes that are attached t o membranes or other c e l l u l a r p a r t i c l e s , attachment i s not a p r e r e q u i s i t e f o r t h i s behavior. Enzymes experimentally detached from membranes o f



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Figure 4. Arrhenius plot of cytochrome oxidase activity in mitochondria. (Reproduced, with permission, from Ref. 40. Copyright 1979, Academic Press.)



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c h i l l i n g - s e n s i t i v e p l a n t s w i l l e x h i b i t t h i s b e h a v i o r (39) and t h e r e i s a t l e a s t one r e p o r t o f a s o l u b l e enzyme, p h o s p h o p h e n o l p y r u v a t e c a r b o x y l a s e from tomato ( 2 3 ) , t h a t e x h i b i t s t h i s behavior. S i n c e t r e a t m e n t o f t h e enzyme w i t h a d e t e r g e n t , o r some o t h e r s u b s t a n c e t h a t w i l l remove l i p i d s , w i l l o f t e n ( b u t n o t a l w a y s ) c a u s e t h e enzyme t o y i e l d l i n e a r A r r h e n i u s p l o t s , i t i s t h e l i p i d - p r o t e i n i n t e r a c t i o n , and p e r h a p s t h e n a t u r e o f t h e l i p i d i t s e l f , that i s b e l i e v e d r e s p o n s i b l e f o r the n o n l i n e a r A r r h e n i u s p l o t s ( 3 8 , 39, 4 0 ) . A l t h o u g h most i n v e s t i g a t o r s b e l i e v e t h a t p r o t e i n s a r e n o t d i r e c t l y i n v o l v e d i n c h i l l i n g i n j u r y (41) t h i s v i e w i s n o t universally held (23). The b e h a v i o r o f enzymes d u r i n g f r e e z e - p r e s e r v a t i o n c a n v a r y g r e a t l y d e p e n d i n g o n w h e t h e r t h e enzyme i s l o c a t e d i n a c e l l u l a r o r n o n c e l l u l a r e n v i r o n m e n t . Many e n z y m e - c a t a l y z e d r e a c t i o n s i n c e l l u l a r s y s t e m s a c t u a l l y i n c r e a s e i n r a t e d u r i n g f r e e z i n g . Examp l e s i n c l u d e enzyme-catalyzed d e g r a d a t i o n of glycogen and/or a c c u m u l a t i o n of l a c t a t e i n f r o g , f i s h , beef or p o u l t r y muscle ( 4 2 - 4 6 ) ; enzyme-catalyzed degradation of h i g h energy phosphates i n f i s h , b e e f and p o u l t r y m u s c l e ( 4 2 , 4 7 - 5 2 ) ; e n z y m e - c a t a l y z e d h y d r o l y s i s o f p h o s p h o l i p i d s i n cod m u s c l e ( 5 3 ) ; e n z y m i c d e c o m p o s i t i o n o f p e r o x i d e s i n r a p i d l y f r o z e n p o t a t o e s and i n s l o w l y f r o z e n p e a s (54); and o x i d a t i o n o f L - a s c o r b i c a c i d i n r o s e h i p s (55) , s t r a w b e r r i e s ( 3 0 ) and B r u s s e l s s p r o u t s ( 5 6 ) . In n o n c e l l u l a r systems, t h i s b e h a v i o r i s v e r y uncommon, and has b e e n o b s e r v e d o n l y when the sample i s e x t r e m e l y d i l u t e p r i o r t o f r e e z i n g . The i n c r e a s e i n s o l u t e c o n c e n t r a t i o n t h a t o c c u r s i n t h e u n f r o z e n phase d u r i n g f r e e z i n g i s o f t e n suggested a s b e i n g responsible f o r instances o f freeze-induced increases i n reaction rates. For enzyme-catalyzed r e a c t i o n s i n c e l l u l a r systems, t h i s e x p l a n a t i o n i s i n a p p r o p r i a t e because: * E n h a n c e d r e a c t i o n r a t e s i n f r o z e n c e l l u l a r s y s t e m s o f t e n do n o t d e c r e a s e upon t h a w i n g ( 5 7 , 58, 5 9 ) . T h i s would not be so i f f r e e z e - i n d u c e d c o n c e n t r a t i o n s o f s o l u t e s w e r e t h e cause s i n c e thawing would r e v e r s e the c o n c e n t r a t i o n effect. *Enzyme-catalyzed r e a c t i o n s i n n o n c e l l u l a r systems r a r e l y a c c e l e r a t e d u r i n g f r e e z i n g , even though the freeze-concent r a t i o n phenomena i s f u l l y o p e r a t i v e . The most r e a s o n a b l e e x p l a n a t i o n f o r t h e f r e e z e - i n d u c e d r a t e enhancement o f e n z y m e - c a t a l y z e d r e a c t i o n s i n c e l l u l a r s y s t e m s i n v o l v e s membrane damage. I t i s w e l l known t h a t f r e e z i n g c a n d i s r u p t membranes and t h e r e b y c a u s e r e l e a s e o f enzymes. Documented i n s t a n c e s i n c l u d e r e l e a s e o f c y t o c h r o m e o x i d a s e f r o m m i t o c h o n d r i a i n b e e f and t r o u t m u s c l e and i n c h i c k e n l i v e r ( 6 0 ) ; r e l e a s e of a c i d l i p a s e s from lysosomes from rainbow t r o u t (61); and r e l e a s e o f m a l a t e and g l u t a m a t e d e h y d r o g e n a s e s , g l u t a m a t e pyruvate transaminase, glutamate or a l o a c e t a t e transaminase, a c o n i t a s e and f u m a r a s e f r o m m i t o c h o n d r i a o f b o v i n e and p o r c i n e muscle ( 6 2 ) . I t i s a l s o reasonable to expect that not o n l y


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enzymes would be d e l o c a l i z e d by f r e e z i n g , but a l s o substrates and other c o n s t i t u e n t s that i n f l u e n c e r a t e s of enzyme r e a c t i o n s ( 8 ) . F i n a l l y , i t should be noted that c h i l l i n g , or f r e e z i n g and frozen storage, can a l s o a l t e r the b i n d i n g s t a t e s ( i o n i c a l l y bound v s . s o l u b l e ) , and the r e l a t i v e concentrations of isoenzymes i n c e l l u l a r systems ( 5 , 63, 64).


E f f e c t of Low Temperature on P r o t e i n

Structure

Although i t i s commonly b e l i e v e d that p r o t e i n s become more s t a b l e as the temperature i s lowered, i t has already been shown that many p r o t e i n s e x h i b i t i n s t a b i l i t y , as measured by p a r t i a l l o s s of f u n c t i o n a l i t y , at low, e s p e c i a l l y s u b f r e e z i n g , tempera tures. In t h i s s e c t i o n , a t t e n t i o n w i l l be d i r e c t e d to molecular changes that p r o t e i n s can undergo at low temperatures. These are important because they are no doubt r e s p o n s i b l e f o r observed a l t e r a t i o n s i n p r o t e i n f u n c t i o n a l i t y a t low temperatures. C l a s s i f i c a t i o n of molecular changes i n p r o t e i n s a t low temperatures. 1) D i s s o c i a t i o n of oligomers. According to Taborsky (65) and L o z i n a - L o z i n s k i i (11), d i s s o c i a t i o n of p r o t e i n oligomers i n t o subunits i s a primary response o f enzymes to f r e e z i n g . Examples of p r o t e i n s behaving i n t h i s manner i n c l u d e l a c t a t e dehydrogenase (66) , α-chymotrypsin (67) , glutamate dehydrogenase (68), an apoprotein of high-density serum l i p o p r o t e i n s (69), glucose-6-phosphate dehydrogenase (70) , pyruvate orthophosphate d i k i n a s e (18), albumins and g l o b u l i n s (71) and deoxyribonucleop r o t e i n s (72). % 2) Rearrangement of subunits w i t h i n oligomers. An example of t h i s kind i n v o l v e s l a c t a t e dehydrogenase ( 3 ) . 3) Aggregation. Low temperature a s s o c i a t i o n (a s p e c i f i c form o f aggregation u s u a l l y i n v o l v i n g subunits of oligomers and s p e c i f i c bonding s i t e s ) of p r o t e i n subunits i s comparatively r a r e . One example i n v o l v e s a t r a n s i t i o n of monomers to trimers i n a membrane proteinase from Streptococcus l a c t i s (73). Fink (2) a l s o has found that a s s o c i a t i o n of enzyme subunits can occur at low temperatures i n aqueous-organic s o l v e n t s . G e l a t i o n of egg yolk i s r o u t i n e l y observed during f r e e z i n g and thawing (74, 75). Low temperature aggregation has a l s o been reported f o r 17B-hydroxysteroid dehydrogenase (76) , urease (77) and myosin (78). Casein m i c e l l e s i n milk can a l s o aggregate during frozen storage. In the e a r l y stages, a f l o c c u l e n t p r e c i p i t a t e forms, and i n the more advanced stages, a g e l (79). 4) Changes i n conformation. Reports of conformâtional changes i n p r o t e i n s at low temperatures are reasonably abundant (11, 12, 80). Examples i n c l u d e p h o s v i t i n (81) , actomyosin (82), myosin Β (83), v i r i o n s of southern bean mosaic v i r u s (84), snake venom L-amino oxidase (85) and numerous instances of denaturation c i t e d by Brandts (12).



6.

FENNEMA

Behavior

of Proteins at Low

Temperatures

119

D i s c u s s i o n of changes i n p r o t e i n s t r u c t u r e a t low temperat u r e . The types o f changes c i t e d above can be best understood i f two premises, that appear to have general a p p l i c a b i l i t y to these s i t u a t i o n s , are accepted. F i r s t , i t i s g e n e r a l l y agreed that the t e r t i a r y n a t i v e s t r u c t u r e o f p r o t e i n i s maintained by s e v e r a l types of i n t e r a c t i o n s , namely hydrophobic, hydrogen-bonding, e l e c t r o s t a t i c and van der Waals f o r c e s (86). Among these, hydrophobic i n t e r a c t i o n s are regarded as being o f primary importance i n most p r o t e i n s (65, 80, 87) and the s t r e n g t h o f these i n t e r a c t i o n s i s weakened by lowering the temperature. Therefore, lowering the temperature w i l l tend to produce s t r u c t u r a l changes i n those p r o t e i n molecules that are dependent on hydrophobic i n t e r a c t i o n s f o r maintenance o f n a t i v e s t r u c t u r e (65, 87). I t should a l s o be recognized that t h i s statement a p p l i e s e q u a l l y w e l l t o both a s s o c i a t i o n - d i s s o c i a t i o n t r a n s i t i o n s and t o changes i n conformation (88) . Although t h i s c l e a r l y a p p l i e s to the s i t u a t i o n s being considered here, i t should nevert h e l e s s be recognized that the simple r e l a t i o n s h i p between hydrophobic i n t e r a c t i o n s and temperature i s not capable o f accounting f o r a l l p r o t e i n s t r u c t u r a l changes that are observed a t low temperatures. For example, f a c t o r s other than temperature have profound e f f e c t s on the s t r u c t u r e o f p r o t e i n s , and some o f these f a c t o r s (pH, i o n i c s t r e n g t h , s u r f a c e t e n s i o n , p r o t e i n concentrat i o n , c o n c e n t r a t i o n o f nonprotein s o l u t e s ) change s u b s t a n t i a l l y during f r e e z i n g . Second, a thermodynamic approach t o the molecular behavior of p r o t e i n s a t low temperatures provides i n f o r m a t i o n o f great importance and must t h e r e f o r e occupy a p o s i t i o n of c e n t r a l importance i n t h i s d i s c u s s i o n . A l l of the p r o t e i n a l t e r a t i o n s mentioned p r e v i o u s l y i n v o l v e the t r a n s f e r of some p o r t i o n o f a p r o t e i n molecule from an aqueous environment to an e s s e n t i a l l y organic environment (to the i n t e r i o r o f a f o l d e d p r o t e i n ) or the r e v e r s e . D i s s o c i a t i o n and u n f o l d i n g i n v o l v e i n c r e a s e d exposure of p r o t e i n s u r f a c e t o an aqueous environment (increased p r o t e i n water i n t e r a c t i o n ) . Whether d i s s o c i a t i o n and/or u n f o l d i n g w i l l a c t u a l l y occur depends s o l e l y on the change i n t o t a l f r e e energy. I f the change i n f r e e energy f o r i n c r e a s e d i n t e r a c t i o n with water i s n e g a t i v e , the d i s s o c i a t i o n - u n f o l d i n g type t r a n s i t i o n s w i l l occur spontaneously, but nothing w i l l be known about r a t e . I f the change i n f r e e energy o f i n c r e a s e d water i n t e r a c t i o n i s p o s i t i v e , the a s s o c i a t i o n - f o l d i n g - a g g r e g a t i o n type t r a n s i t i o n s w i l l occur spontaneously, but again, nothing w i l l be known about r a t e . These statements, of course, apply to the o v e r a l l behavior of the sample, not to i n d i v i d u a l molecules. Furthermore, changes i n temperature can a l t e r whether the t r a n s i t i o n s j u s t mentioned w i l l r e s u l t i n negative or p o s i t i v e changes i n f r e e energy, so at one temperature, i n c r e a s e d water i n t e r a c t i o n may be favored and at another temperature, the reverse may be t r u e . It i s now i n s t r u c t i v e t o examine some q u a n t i t a t i v e data f o r thermodynamic p r o p e r t i e s of g l o b u l a r p r o t e i n s exposed to low



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temperatures. At the o u t s e t , i t should be emphasized that data i s not abundant s i n c e the thermodynamic p r e r e q u i s i t e of r e a c t i o n r e v e r s i b i l i t y s e v e r e l y l i m i t s the c o n d i t i o n s that can be used experimentally. Furthermore, the data presented here were developed on the assumption that the t r a n s i t i o n s under c o n s i d e r a t i o n (associâtionjdissociation; n a t i v e ^ e n a t u r e d ) i n v o l v e only two s t a t e s . Reaction i n t e r m e d i a t e s , i f they occur, must not be s t a b l e under the c o n d i t i o n s used. T h i s assumption i s considered reasonable f o r many p r o t e i n s , i n c l u d i n g those considered here, but not n e c e s s a r i l y f o r a l l p r o t e i n s (12, 8 9 , 9 0 , 91). Shown i n Figure 5 i s the temperature dependence of the standard f r e e energy (AG° or AF°) of denaturation f o r chymotrypsinogen at three d i f f e r e n t pH v a l u e s . Conditions were such that a s s o c i a t i o n d i d not occur and observations t h e r e f o r e p e r t a i n to n a t i v e j u n f o l d e d t r a n s i t i o n s of monomers. Since accurate values of AG° could be d i r e c t l y determined only w i t h i n the range of ±2,000 c a l o r i e s , the remainder of the data were c a l c u l a t e d from a four-term power s e r i e s i n v o l v i n g absolute temperature (92). The data i n Figure 5 c l e a r l y i n d i c a t e that AG° i s not a l i n e a r f u n c t i o n of temperature as had been f r e q u e n t l y assumed p r i o r to p u b l i c a t i o n of these data. Instead, the r e l a t i o n s h i p i s p a r a b o l i c i n nature, with a temperature of maximum s t a b i l i t y ( T ) occurr i n g at about 10-12°C, i . e . r a i s i n g or lowering the temperature from T can r e s u l t i n denaturation ( u n f o l d i n g ) . Thus, low temperature denaturation i s c l e a r l y p o s s i b l e . Lowering the pH of the sample causes the e n t i r e curve to s h i f t to a more negative value of AG° and thereby reduce the temperature at which a value of AG° = 0 ( f o r the high-temperature l e g of the graph) i s a t t a i n e d . According to Brandts (12), the e s s e n t i a l f e a t u r e s of these data are undoubtedly t y p i c a l of denaturation r e a c t i o n s f o r most p r o t e i n s . The data i n F i g u r e s 6 and 7 provide c l e a r evidence that the c o n c l u s i o n s drawn from Figure 5 are v a l i d . The data i n F i g u r e 6 r e l a t e to chymotrypsin at pH 1.47 and i t i s evident that t h i s p r o t e i n does, i n f a c t , e x h i b i t maximum s t a b i l i t y at about 12°C, with increased denaturation o c c u r r i n g at higher or lower temperatures. T f o r chymotrypsin i s t h e r e f o r e almost i d e n t i c a l to max chymotrypsinogen i n Figure 5. Figure 7 i s a van't Hoff p l o t ( l o g e q u i l i b r i u m constant, Κ = unfolded/native v s . 1/T) f o r β-lactoglobulin A showing T at about 35°C (90). A s u f f i c i e n t number of other i n v e s t i g a t o r s have reported i n c i d e n c e s of low temperature denaturation to v e r i f y that t h i s behavior i s a reasonably common occurrence (12, 25). From the slopes of the curves i n Figure 7 i t i s p o s s i b l e to c a l c u l a t e the enthalpy of t r a n s i t i o n from the n a t i v e to the denatured s t a t e , assuming a two-state process at a l l temperatures. From such c a l c u l a t i o n s , i t i s found that the u n f o l d i n g r e a c t i o n f o r β-lactoglobulin A i s exothermic below 35°C and endothermic above t h i s temperature (90). C a l c u l a t e d thermodynamic parameters f o r the a-chymotrypsinom a x

m a x

m a x

T

f o r

m a x


FENNEMA

Behavior


6.

of Proteins at Low

121

Temperatures

pH 3.0, 0.01 M Cl

J 0

1 10

I 20

I 30

TEMPERATURE

I 40

I 50

X60

Figure 5. Temperature dependence of the free energy of denaturation of chymotrypsinogen at different pH values and ionic strengths. (Reproduced from Ref. 92. Copyright 1964, American Chemical Society.)



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j

Ο

ι

10

ι

20

i

30

T E M P E R A T U R E (°)

PROTEIN

I

40

DETERIORATION

Ι

50

Figure 6. Temperature dependence of the extinction coefficient at 293 nm for chymotrypsin at pH 1.47. The dashed lines at the top and bottom of the plot indicate the extinction coefficients for the denatured and native states, respectively, (Reproduced, with permission, from Ref. 12. Copyright 1967, Academic Press.)



FENNEMA

Behavior

of Proteins at Low

Temperatures

σ* ο

3.5 1/Tx Figure

7.

unfolded^ ^

Van't Hoff

3.6

10 ( ° K ~ )

plots (logarithm of

3

1

the equilibrium constant, Κ

reciprocal of absolute temperature) for β-lactoglobulin

=

A in urea at

various concentrations. (Reproduced from Ref. 90. Copyright 1968, American Chemical Society.)


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gen data (native^unfolded) i n F i g u r e 5 (pH 3.0) are shown i n Figure 8 ( 9 2 ) . Values f o r ΔΗ° and AS° are h i g h l y temperature dependent, both e x h i b i t i n g l a r g e p o s i t i v e values at 65°C, decreasing to values o f zero at about 10°C and becoming negative below 10°C. The change i n heat c a p a c i t y (ACp) between the n a t i v e and unfolded forms a t a given temperature i s l a r g e and p o s i t i v e f o r a l l temperatures i n F i g u r e 8. The l a r g e p o s i t i v e v a l u e s of ACp are r e s p o n s i b l e f o r the temperature dependency of both d(AH) ΔΗ° (dT

= ACp) and AS° (AS = IC 2 S&dT) 1 and f o r the occurrence of a temperature of maximum p r o t e i n s t a b i l i t y (Figures 5 and 7) (88, 90, 92, 93). Although ACp i s temperature dependent i n Figure 8, t h i s i s not e s p e c i a l l y important nor i s t h i s c h a r a c t e r i s t i c true f o r a l l d e n a t u r a t i o n reactions ( 8 8 ) . The l a r g e heat c a p a c i t y values apparently a r i s e from exposure of a p o l a r groups to water d u r i n g the d e n a t u r a t i o n p r o c e s s . In water, a p o l a r groups e x h i b i t a p a r t i a l molal heat c a p a c i t y that i s about three times greater than t h e i r heat c a p a c i t y i n an organic environment, such as i n the i n t e r i o r of the n a t i v e p r o t e i n . Consequently, c o n s i d e r a b l e energy must be expended during temperature i n c r e a s e s to p a r t i a l l y d i s r u p t the s t r u c t u r a l water e x i s t i n g around apolar groups at low temperature ( 2 5 ) . The general importance of the heat c a p a c i t y e f f e c t at atmospheric pressure was emphasized by Edelhock and Osborne (80) i n t h e i r statement that ". . .most, i f not a l l , p r o t e i n r e a c t i o n s i n which nonpolar groups are exposed to water, i . e . , d e n a t u r a t i o n , l i g a n d d i s s o c i a t i o n , the d i s s o c i a t i o n of macromolecules or microscopic s t r u c t u r e s composed of a l a r g e number of s u b u n i t s , are c o n t r o l l e d , i n l a r g e p a r t , by a p o s i t i v e heat c a p a c i t y change which appears to increase w i t h temperature." D i s s o c i a t i o n of o l i g o m e r i c p r o t e i n s i n t o subunits at low temperatures probably occurs more e a s i l y than simple u n f o l d i n g s i n c e the b i n d i n g f o r c e s i n v o l v e d i n d i s s o c i a t i o n are not as strong as those i n v o l v e d i n u n f o l d i n g . In F i g u r e 9 the tempera ture dependence o f heats o f d i s s o c i a t i o n ( e n t h a l p i e s , ΔΗ°) are shown f o r glutamate dehydrogenase at pH 7 (68), α-chymotrypsin at pH 4.12 (67) and apo A - I I p r o t e i n a t pH 7.4 from h i g h - d e n s i t y serum l i p o p r o t e i n (69) . The e n t h a l p i e s f o r glutamate dehydro genase and α-chymotrypsin are negative (exothermic) a t low temperatures and p o s i t i v e (endothermic) a t high temperatures. The enthalpy of d i s s o c i a t i o n f o r carboxymethylated (Cm) apo A - I I i s negative below, and p o s i t i v e above, 23°C, and c o n t r a r y to glutamate dehydrogenase and α-chymotrypsin, which y i e l d g l o b u l a r molecules upon d i s s o c i a t i o n , Cm apo A - I I d i s s o c i a t e s i n t o molecules that are randomly c o i l e d . Thus, d i s s o c i a t i o n i n t h i s instance i s accompanied by major changes i n secondary and tertiary structure. T



6.

FENNEMA

Behavior

of Proteins at Low

Temperatures

TEMPERATURE ( ° ) Figure 8. Temperature dependence of enthalpy (àH°), entropy (AS°), and heat capacity (àC ) pertaining to denaturation of chymotrypsinogen. (Reproduced from Ref, 92, Copyright 1964, American Chemical Society.) p


125

126

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DETERIORATION


FOOD

20 TEMP. (°C)

40

60

Figure 9. Temperature dependence of heats of dissociation for a-chymotrypsin (O) (61), glutamate dehydrogenase (A) (68), and reduced and carboxymethylated apo A-II protein (•) (69). (Reproduced, with permission, from Ref. 80. Copyright 1976, Academic Press.)



6.

FENNEMA

Behavior

of Proteins at Low

Temperatures

127

As mentioned a t the beginning o f t h i s s e c t i o n , examples do e x i s t f o r both a s s o c i a t i o n and d i s s o c i a t i o n type r e a c t i o n s i n p r o t e i n s exposed t o low temperatures, and the type of t r a n s i t i o n favored w i l l be that which e x h i b i t s a decrease i n f r e e energy. From the expression AG = ΔΗ - TAS (constant Τ and P) i t i s evident that AG w i l l become more negative as AH becomes more negative (exothermic) or AS becomes more p o s i t i v e (greater d i s o r d e r or g r e a t e r p r o b a b i l i t y of e x i s t e n c e ) . S i t u a t i o n s do e x i s t where AH i s negative and AS i s p o s i t i v e , but i t i s more common f o r both terms t o have the same s i g n (94). Based on the data presented, the thermodynamic p r o p e r t i e s of u n f o l d i n g and d i s s o c i a t i o n r e a c t i o n s f o r p r o t e i n s can be summarized as shown i n Table I . The two assumptions mentioned e a r l i e r apply to these data. Since these assumptions are con s i d e r e d v a l i d f o r many p r o t e i n s (12), these data, and the a s s o c i a t e d behavior d e p i c t e d i n F i g u r e s 5-7, can be considered reasonably t y p i c a l . Omitted from Table I are the a s s o c i a t i o n type t r a n s i t i o n s that some p r o t e i n s undergo a t low temperatures. In these i n s t a n c e s , i n t e r a c t i o n s ( e l e c t r o s t a t i c , hydrogen bonding, d i s u l f i d e interchange) other than hydrophobic may p l a y d e c i s i v e r o l e s , or hydrophobic i n t e r a c t i o n s may be i n f l u e n c e d s i g n i f i c a n t l y by f a c t o r s other than temperature (pH, i o n i c s t r e n g t h , e t c . ) . Thus, the data i n Table I are*an o v e r s i m p l i f i c a t i o n , but they n e v e r t h e l e s s are b e l i e v e d to r e p r e s e n t , reasonably w e l l , the behavior of many p r o t e i n s a t low temperatures. Beginning w i t h a d i s c u s s i o n o f the data at intermediate t o high temperatures (row 1 o f Table I ) , i t i s evident that the e n t h a l p i e s and e n t r o p i e s of t r a n s i t i o n to the u n d i s s o c i a t e d or unfolded s t a t e are both p o s i t i v e . E i t h e r o f these t r a n s i t i o n s are favored (-AG) when the temperature i s high enough and AS i s l a r g e enough so that TAS > AH. The entropy term, i n a l l i n s t a n c e s i n Table I i s composed o f two components, one i n v o l v i n g p r o t e i n s t r u c t u r e and the other water s t r u c t u r e . U n f o l d i n g o r d i s s o c i a t i o n would i n v o l v e a p o s i t i v e change i n entropy f o r the p r o t e i n and a negative change i n entropy f o r water, the l a t t e r o c c u r r i n g because groups exposed t o water during u n f o l d i n g or d i s s o c i a t i o n are predominantly hydrophobic, thus promoting i n c r e a s e d water s t r u c t u r e (88). As the temperature i s r a i s e d i n the r e g i o n above the temperature o f maximum s t a b i l i t y ( T ) f o r the p r o t e i n , water e x h i b i t s a reduced tendency t o a s s o c i a t e i n an ordered manner around hydrophobic groups, and more hydrophobic groups become exposed. Thus, the change i n p r o t e i n s t r u c t u r e becomes an i n c r e a s i n g l y dominant component of AS, and AS assumes a l a r g e r p o s i t i v e v a l u e , and AG a l a r g e r negative v a l u e , as the tempera ture i s r a i s e d above T . At T , the values of AH and AS both decrease to near zero (row 2, Table I and F i g u r e 8 ) . Since AG assumes the l a r g e s t p o s s i b l e p o s i t i v e value a t t h i s temperature, AH and AS cannot both have values o f zero. Small changes i n e i t h e r AH o r AS near m a x

m a x

m a x



128

FOOD

PROTEIN

DETERIORATION

T a b l e I . Thermodynamic s o f p r o t e i n u n f o l d i n g o r d i s s o c i a t i o n when h y d r o p h o b i c i n t e r a c t i o n s a r e o f m a j o r i m p o r t a n c e

Thermodynamic d a t a Temperature AG Sufficiently to cause disordering

ACp

2

AH

AS

|TAS/AH|

+

+

>1

@55°C

-0

small -

>1

~+2 @10°C

-

>1

H-1.5 @-10 C

-

|ΔΗ|, w i t h t h e AS t e r m b e i n g d o m i n a t e d b y t h e change i n w a t e r s t r u c t u r e . A t s t i l l l o w e r t e m p e r a t u r e s (row 4» T a b l e I ) AG p r e s u m a b l y becomes n e g a t i v e ( l o g Κ > 0» where AG = -RT I n K ; F i g u r e 7), i n w h i c h c a s e t h e a b s o l u t e v a l u e o f AH(-) must e x c e e d t h e a b s o l u t e v a l u e o f TAS ( - ) . m a x


m a x

Conclusions P o s s i b l e adverse e f f e c t s o f low temperatures on p r o t e i n s i n c l u d e d e r e a l i z a t i o n o f p r o t e i n s i n c e l l u l a r s y s t e m s and a l o s s of p r o t e i n s o l u b i l i t y and f u n c t i o n a l i t y . Factors influencing the d e g r e e o f damage d u r i n g f r e e z i n g i n c l u d e t h e t y p e o f p r o t e i n , the c o m p l e x i t y o f t h e s y s t e m ( c e l l u l a r v s . n o n c e l l u l a r ) , s t o r a g e t e m p e r a t u r e ( r e l a t e d t o w a t e r a c t i v i t y ) , s t o r a g e t i m e , and t h e c o n d i t i o n s o f f r e e z i n g and t h a w i n g . P r o t e i n s a t l o w t e m p e r a t u r e may u n d e r g o c h a n g e s i n c o n f o r m a t i o n a n d t h e d e g r e e o f a s s o c i a t i o n , w i t h t h e degree o f r e v e r s i b i l i t y depending on t h e c o n d i t i o n s . C a u s a t i v e f a c t o r s i n f l u e n c i n g p r o t e i n damage d u r i n g f r e e z i n g i n c l u d e c h a n g e s i n s a l t c o n c e n t r a t i o n , c h a n g e s i n pH, m e c h a n i c a l e f f e c t s o f i c e o n membranes and d e h y d r a t i o n e f f e c t s . D i f f e r e n t p r o t e i n s d i f f e r g r e a t l y i n t h e i r responses t o low t e m p e r a t u r e s , and t h e b e h a v i o r o f a s i n g l e p r o t e i n a t a g i v e n l o w t e m p e r a t u r e c a n be u n u s u a l and d i f f i c u l t t o p r e d i c t . The l a t t e r i s t r u e because l o w t e m p e r a t u r e s c a n have a d i r e c t i n f l u e n c e o n p r o t e i n s t r u c t u r e and f u n c t i o n a l i t y , a n d b e c a u s e low t e m p e r a t u r e s , e s p e c i a l l y s u b f r e e z i n g t e m p e r a t u r e s , c a n i n d u c e d r a m a t i c changes i n t h e p r o t e i n ' s environment. A proper perspec t i v e i s perhaps best achieved by r e c o g n i z i n g that a great d e a l r e m a i n s t o b e l e a r n e d a b o u t t h e muçh-studied b e h a v i o r o f p r o t e i n s a t a m b i e n t t o h i g h t e m p e r a t u r e s , a n d t h a t f a r more r e m a i n s t o b e l e a r n e d about t h e l i t t l e - s t u d i e d b e h a v i o r o f p r o t e i n s a t l o w temperatures. Acknowledgments R e s e a r c h s u p p o r t e d by t h e C o l l e g e o f A g r i c u l t u r a l and L i f e Sciences, U n i v e r s i t y of Wisconsin-Madison.


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