Proteins at Low Temperatures - American Chemical Society

10 Denaturation of Fish Muscle Proteins During

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Frozen Storage JUICHIRO J. MATSUMOTO Department of Chemistry, Sophia University, Kioi-cho 7, Chiyoda-ku, Tokyo, Japan 102

Studies on the freeze denaturation of fish muscle proteins were reviewed with emphasis given to changes in their physico-chemical and biochemical properties during frozen storage. Denaturation of actomyosin commonly occurs during frozen storage and the side-to-side aggregation of myosin molecules appears to play a major role in this reaction. The author's group performed freezing studies with isolated preparations of proteins fromfishmuscle, i.e., actomyosin, myosin, H-meromyosin (HMM), L-meromyosin (LMM), and actin. Freeze denaturation occurred with individual proteins as well as with their subunits. Not only aggregation but also some conformational changes were observed. Denaturation was inhibited significantly in the presence of added monosodium glutamate (MSG). About 30 compounds were found to inhibit denaturation and their mechanisms of action are discussed.

Changes in the Sensory Attributes

of Fish

Muscle

s t u d i e s of p r o t e i n d e n a t u r a t i o n i n fish m u s c l e d u r i n g f r o z e n ^

storage

h a v e b e e n c a r r i e d o u t to g a i n scientific k n o w l e d g e a n d t o p r o v i d e a

basis f o r s u p p l y i n g foods of b e t t e r

quality.

E a r l y studies

d e m o n s t r a t e d t h a t f r o z e n storage is a n excellent means p u t r e f a c t i o n a n d autolysis of this p e r i s h a b l e c o m m o d i t y .

with

fish

of p r e v e n t i n g

However, it was

soon l e a r n e d that f r o z e n fish deteriorates m o r e r a p i d l y t h a n f r o z e n b o v i n e muscle.

F r o z e n fish c a n e x h i b i t s e v e r a l k i n d s of q u a l i t y d e t e r i o r a t i o n

d e p e n d i n g o n t h e state a t w h i c h i t i s e x a m i n e d . F o r e x a m p l e , a fish t h a t 0-8412-0484-5/79/33-180-205$5.00/0 © 1979 American Chemical Society In Proteins at Low Temperatures; Fennema, O.; Advances in Chemistry; American Chemical Society: Washington, DC, 1979.

206

PROTEINS

AT

LOW

TEMPERATURES

Table I. Proteins

Group


Proteins

S a r c o p l a s m i c proteins (albumins)

myogen

M y o f i b r i l l a r proteins (globulins)

(actomyosin) myosin H-meromyosin L-meromyosin actin tropomyosin t r o p o n i n , etc. collagen elastin

Stroma (scleroproteins) a

Values from many references were referred to and summarized.

has b e e n f r o z e n f o r a l o n g p e r i o d t h e n t h a w e d often exhibits a softened t e x t u r e , c o n s i d e r a b l e loss of fluid, a n d changes i n flavor a n d o d o r . W h e n this same fish is u s e d as a p r o c e s s i n g m a t e r i a l i t m a y differ f r o m f r e s h fish i n t e r m s o f t e x t u r e , w a t e r - h o l d i n g c a p a c i t y , b i n d i n g p r o p e r t i e s , a n d g e l l i n g strength. F i n a l l y , i t m a y differ f r o m f r e s h - c o o k e d fish w i t h respect to texture (toughness, coarseness, dryness, e t c . ) , fluid losses, flavor, a n d odor. M a n y of t h e changes i n f r o z e n fish m u s c l e are a t t r i b u t a b l e to d e n a t u r a t i o n of p r o t e i n s a n d studies of this o c c u r r e n c e w i l l be r e v i e w e d here. Several

earlier reviews

of

protein denaturation i n frozen

fish

have

a p p e a r e d i n t h e l i t e r a t u r e (1-8). Structure

and Constituents

of Fish

Muscle

A l t h o u g h fish m u s c l e is c h a r a c t e r i z e d b y t h e p r e s e n c e of d a r k m u s c l e and myocommata

( 9 ) , most w o r k s so f a r p u b l i s h e d concentrate o n the

n o r m a l o r w h i t e m u s c l e because i t c o m p r i s e s t h e m a j o r p a r t of t h e m u s c l e . T h e fibers of b o t h w h i t e a n d d a r k muscles consist of b u n d l e s

of

s t r i a t e d m y o f i b r i l s e a c h c o n t a i n i n g t h i n a n d t h i c k filaments a n d v a r i o u s s u b c e l l u l a r structures s u c h as the organelles

(9,10,11).

sarcoplasmic reticulum a n d

T h e size a n d shape of

fish

muscle

fibers

other and

m y o f i b r i l s differ s o m e w h a t f r o m the c o r r e s p o n d i n g c o m p o n e n t s of m a m m a l i a n muscles

(9,11).

T h e p r i n c i p a l constituents of fish m u s c l e a r e : w a t e r , 6 6 - 8 4 % ; p r o t e i n , 1 5 - 2 4 % ; l i p i d , 0 . 1 - 2 2 % ; a n d m i n e r a l substances, 0 . 8 - 2 %

(12).

Proteins,

t h e m a j o r c o n s t i t u e n t of t h e d r y m a t t e r , c a n b e classified i n t o t h r e e groups b a s e d o n s o l u b i l i t y . T h i s ch ssification is s h o w n i n T a b l e I ( 1 , 1 1 ) .

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

The

10.

MATSUMOTO

Denaturation

of Fish

207

Muscle

of Fish Muscle"


Localization

Amount per Total Proteins

Function

cell p l a s m a

g l y c o l y t i c enzymes

t h i c k filaments

contraction

t h i n filaments

contraction regulation regulation

myocommata and cell membranes

c o n n e c t i v e tissues

lS-30% 65-80%

3-5%

a m o u n t of s t r o m a p r o t e i n s is m u c h less i n fish m u s c l e ( 3 % ) t h a n i t is i n m a m m a l i a n s k e l e t a l m u s c l e s ( 1 5 % ) . M y o f i b r i l l a r p r o t e i n s are t h e m a j o r p r o t e i n s i n fish m u s c l e a n d t h e y are present i n l a r g e r a m o u n t s t h a n i n m a m m a l i a n skeletal muscle ( 5 7 - 6 8 % ). T h e size a n d shape o f m y o f i b r i l l a r p r o t e i n s of fish are s h o w n i n T a b l e I I . P r o p e r t i e s of fish p r o t e i n s i n c l u d i n g a m i n o a c i d c o m p o s i t i o n are g e n e r a l l y c o m p a r a b l e w i t h those of r a b b i t s k e l e t a l m u s c l e

(11,13,14,

H o w e v e r , t h e m y o f i b r i l l a r p r o t e i n s of fish differ f r o m those o f r a b b i t

15).

i n some respects: 1) fish actins go r e a d i l y i n t o s o l u t i o n i n a G u b a - S t r a u b medium μ =

0.5 a n d p H ~

6.5) to f o r m a viscous a c t o m y o s i n s o l u t i o n

t h a t h i n d e r s i s o l a t i o n of p u r e m y o s i n (11,13)

a n d 2 ) fish m y o s i n s are

m u c h m o r e l a b i l e t h a n m a m m a l i a n m y o s i n s w h e n stored at n o n f r e e z i n g t e m p e r a t u r e s (16),

o r w h e n exposed to proteases o r dénaturants s u c h as

u r e a a n d g u a n i d i n e - H C l (17).

I n the f o r m e r case ( n o n f r e e z e s t o r a g e ) ,

spontaneous side-to-side a g g r e g a t i o n occurs w i t h l i t t l e or no c h a n g e i n c o n f o r m a t i o n (16) Table II.

Protein Myosin Actin (G-form) (F-form) Tropomyosin Troponin a

w h e r e a s i n t h e l a t t e r case ( u r e a a n d g u a n i d i n e - H C l ) Size and Shape of Fish Myofibril Proteins"

Mol

Wt

500,000 43,000 68,000 80,000

Intrinsic Viscosity dL χ g'

Shape

S

1.8-2.3

fibrillar

3.3 S large 2.85 S

0.1-0.4

globular fibrillar rod globular

Sedimentation Coefficient 6.4

1

0.36

Values from many references were referred to and summarized.


208

PROTEINS

u n f o l d i n g of p e p t i d e c h a i n s occurs (17).

AT

LOW

TEMPERATURES

F u r t h e r m o r e , a g g r e g a t i o n is

f r e q u e n t l y e n c o u n t e r e d w i t h fish a c t o m y o s i n s .

T h e s e i n s t a b i l i t i e s of

fish

p r o t e i n s c l e a r l y are r e l a t e d to t h e ease w i t h w h i c h fish p r o t e i n denatures d u r i n g f r o z e n storage. Early

Investigations

D e n a t u r a t i o n of p r o t e i n s as r e l a t e d to q u a l i t y d e t e r i o r a t i o n of f r o z e n s t o r e d fish m u s c l e w a s first s t u d i e d b y F i n n (18).

R e a y (19)

revealed


that d u r i n g f r o z e n storage of h a d d o c k , g l o b u l i n s ( s a l t - s o l u b l e p r o t e i n s ) became insoluble whereas albumins (water-soluble proteins) affected.

D y e r (20)

were

not

d e m o n s t r a t e d the r e l a t i o n s h i p b e t w e e n t h e freeze

d e t e r i o r a t i o n of o r g a n o l e p t i c p r o p e r t i e s a n d d e n a t u r a t i o n of a c t o m y o s i n . H e f o u n d a significant c o r r e l a t i o n b e t w e e n f a v o r a b l e t a s t e - p a n e l scores a n d the a m o u n t of a c t o m y o s i n extractable f r o m f r o z e n - s t o r e d c o d , h a l i b u t ( 2 0 ) , p l a i c e (21),

a n d rosefish (22).

fillets

of

Sarcoplasmic proteins

(water-soluble proteins) underwent no appreciable change d u r i n g frozen storage. D y e r s o u t s t a n d i n g w o r k s t i m u l a t e d m a n y studies of a s i m i l a r n a t u r e l e a d i n g to the firm c o n c l u s i o n t h a t d e n a t u r a t i o n of a c t o m y o s i n occurs at a significant rate d u r i n g f r o z e n storage of fish m u s c l e a n d t h a t t h e r a t e of d e n a t u r a t i o n c a n b e u s e d to estimate t h e rate of q u a l i t y c h a n g e of t h e fish. M o s t of the e a r l y w o r k o n this subject w a s d o n e b y e x t r a c t i n g a c t o m y o s i n f r o m fish fillets f o l l o w i n g f r o z e n storage a n d t h a w i n g . o t h e r h a n d , m a n y studies d e s i g n e d

O n the

to d e t e r m i n e t h e m e c h a n i s m s

d e n a t u r a t i o n h a v e i n v o l v e d f r o z e n solutions or suspensions of

of

isolated

protein preparations. Factors Affecting

the Rate of

R a t e of Freezing.

Denaturation

I t is w i d e l y b e l i e v e d t h a t r a p i d f r e e z i n g

e r a l l y results i n less d e n a t u r a t i o n t h a n s l o w f r e e z i n g . studied cod

fillets

However,

a n d f o u n d t h a t s o m e i n t e r m e d i a t e rates of

freezing

r e s u l t e d i n m o r e d e t r i m e n t a l t e x t u r a l changes t h a n s l o w f r e e z i n g 25,26).

genLove (23,24,

T h e r a p i d l y f r o z e n fillets c o n t a i n e d s m a l l i n t r a c e l l u l a r i c e crystals

a n d e x h i b i t e d l i t t l e c h a n g e i n c e l l structure.

T h e slowly frozen

c o n t a i n e d l a r g e i n t e r c e l l u l a r i c e crystals w i t h m u s c l e shrunken and tightly grouped.

fibers

fillets

that were

F i l l e t s f r o z e n at i n t e r m e d i a t e rates c o n -

t a i n e d l a r g e i n t r a c e l l u l a r i c e crystals t h a t h a d g r o w n so l a r g e t h a t t h e y damaged the cell membranes. last instance (26).

A c t o m y o s i n w a s t h e least s o l u b l e i n t h e

S i m i l a r observations w e r e r e p o r t e d b y T a n a k a ( 2 7 ) .

Rates of d e n a t u r a t i o n of m u s c l e p r o t e i n s i n A l a s k a p o l l a c k , as i n f l u e n c e d b y f r e e z i n g r a t e a n d storage t e m p e r a t u r e , w e r e s t u d i e d b y M a t s u d a


10.

Denaturation

MATSUMOTO

a n d b y S u z u k i et a l . (29).

(28)

of Fish

209

Muscle

M u s c l e frozen i n l i q u i d nitrogen con-

t a i n e d s m a l l i n t r a c e l l u l a r ice crystals a n d less d e n a t u r a t i o n of a c t o m y o s i n t h a n m u s c l e f r o z e n i n a i r at — 2 0 ° C .

C o n t r a r y to t h i s

finding

Love

r e p o r t e d t h a t f r e e z i n g of c o d m u s c l e i n l i q u i d a i r c a u s e d t i g h t e n i n g of t e x t u r e , a c o a l e s c e n c e o f m y o f i b r i l s , a n d a decrease i n e x t r a c t a b l e p r o teins

(30,31). Temperature and D u r a t i o n of Storage.

D y e r (20,21,22)

a n d C o n n e l l (32)

A s s h o w n b y t h e w o r k s of

l o w e r i n g t h e storage

temperature

decreases the rate of p r o t e i n d e n a t u r a t i o n . L o v e ' s s t u d y , i n v o l v i n g f r e e z Downloaded by OHIO STATE UNIV LIBRARIES on June 2, 2012 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch010

i n g i n l i q u i d a i r , y i e l d e d results of a c o n t r a r y n a t u r e (30). S n o w (33)

Furthermore,

d i s s o l v e d c o d a c t o m y o s i n i n v a r i o u s i n o r g a n i c salt solutions

a n d f o u n d t h a t f r e e z i n g t o t e m p e r a t u r e s b e l o w the eutectic p o i n t s of the r e s p e c t i v e salts d e n a t u r e d the p r o t e i n , a p p a r e n t l y b e c a u s e of a d r a t i n g effect.

H o w e v e r , this r e s u l t is c o n t r a d i c t o r y w i t h t h e

dehygeneral

o b s e r v a t i o n t h a t p r o t e i n s of A l a s k a p o l l a c k a n d sea bass u n d e r g o

little

d e n a t u r a t i o n w h e n the fillets w e r e stored at — 2 0 ° C after f r e e z i n g i n liquid nitrogen

(29).

D u r i n g f r o z e n storage t w o events o c c u r s i m u l t a n e o u s l y — d e n a t u r a t i o n of a c t o m y o s i n a n d a n increase i n t h e a v e r a g e size of i c e crystals (34,35).

Moreover, a

fluctuating

storage t e m p e r a t u r e accelerates

the

d e c l i n e i n sensory score, the decrease i n t h e s o l u b i l i t y of a c t o m y o s i n , a n d t h e g r o w t h of i c e crystals (36,37).

T h u s , the l a t t e r o c c u r r e n c e m a y b e

r e l a t e d to t h e first t w o . I n g e n e r a l , r e f r e e z i n g has a d e t r i m e n t a l effect o n d e n a t u r a t i o n of p r o t e i n s i n fish m u s c l e S u p e r c o o l i n g (41)

(38,39,40).

a n d p a r t i a l f r e e z i n g (42)

w h i c h are a c c o m p a n i e d

b y n o o r l i t t l e i c e f o r m a t i o n at a t e m p e r a t u r e s l i g h t l y b e l o w t h e f r e e z i n g p o i n t of the s a r c o p l a s m a p p e a r to r e t a r d p r o t e i n d e n a t u r a t i o n as c o m p a r e d w i t h storage at the c o r r e s p o n d i n g t e m p e r a t u r e s after t h e greater p a r t of t h e w a t e r has b e e n f r o z e n b y exposure to t h e l o w e r t e m p e r a t u r e s . Fish Species

and Elapsed T i m e between Harvest

F r o m his d a t a o n f o u r k i n d s of fish, D y e r (21,22)

and Freezing.

suggested t h a t f a t t y

fishes are m o r e stable i n f r o z e n storage t h a n l e a n fishes. H o w e v e r , m o r e recent d a t a i n d i c a t e t h a t species

differences

i m p o r t a n t t h a n fat content (2,43,44).

m a y sometimes

be

more

F o r e x a m p l e , the p a t t e r n of

ice

c r y s t a l f o r m a t i o n differs b e t w e e n A l a s k a p o l l a c k a n d y e l l o w t a i l m u s c l e s (34)

a n d this m a y influence s t a b i l i t y . F r e s h n e s s o r p o s t - m o r t e m c o n d i t i o n of fish m u s c l e at t h e t i m e of

f r e e z i n g has a n i m p o r t a n t b e a r i n g o n t h e r a t e of freeze

denaturation

(1,2,38,45,46). T h e s t a b i l i t y of fish m u s c l e i n f r o z e n storage also v a r i e s w i t h t h e season a n d other b i o l o g i c a l c o n d i t i o n s s u c h as n u t r i t i o n a l status, d e g r e e of f a t i g u e , a n d s p a w n i n g status ( p r e , i n , or p o s t )

(45,47-50).


210

PROTEINS A T L O W

Denaturation

of

Methods.

TEMPERATURES

Proteins

E a r l y i n v e s t i g a t i o n s r e l e v a n t to t e c h n o l o g i c a l analyses of

t h e f r e e z i n g operations

involved determining the amount

e x t r a c t a b l e i n salt solutions, s u c h as 5 %

of

protein

N a C l or 0 . 6 M K C 1 . Proteins

e x t r a c t e d i n this m a n n e r w e r e d e f i n e d as n a t i v e o r u n d e n a t u r e d . T h e n a q u e s t i o n arose c o n c e r n i n g t h e m e c h a n i s m b y w h i c h d e n a t u r a t i o n occurs. U n f o r t u n a t e l y , d e n a t u r e d p r o t e i n s are difficult to s t u d y b e c a u s e of t h e i r i n s o l u b i l i t y ; thus i n f o r m a t i o n a b o u t t h e state of p r o t e i n s i n fish h a d to b e


gained f r o m the soluble protein fraction. Actomyosin.

SOLUBILITY.

S t u d i e s h a v e d e a l t w i t h changes i n t h e

s o l u b i l i t y o f p r o t e i n s d u r i n g f r o z e n storage of fish m u s c l e o r solutions of isolated actomyosin

Analysis by gel

(33,51,52).

filtration

of t h e salt

extracts has s h o w n that t h e a c t o m y o s i n f r a c t i o n decreases i n s o l u b i l i t y d u r i n g f r o z e n storage w h e r e a s the s a r c o p l a s m i c p r o t e i n s r e m a i n essentially unchanged

(53). The reduced

VISCOSITY.

f r o z e n - s t o r e d fish m u s c l e

v i s c o s i t y of

the protein extracted

from

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

(44,54)

the

f r o z e n - s t o r e d solutions of i s o l a t e d a c t o m y o s i n decreases w i t h i n c r e a s i n g t i m e of storage (51,

52).

SEDIMENTATION.

E x a m i n a t i o n of extracts f r o m fish m u s c l e o r acto-

m y o s i n solutions b y u l t r a c e n t r i f u g i n g p r o c e d u r e s has i n d i c a t e d t h a t t h e actomyosin frozen appear.

components

storage

(20S-30S)

components

A f t e r p r o l o n g e d storage, n o c o m p o n e n t s

t h a n 20S are e v i d e n t (29,51), of

decrease w i t h i n c r e a s i n g t i m e

a n d several faster m o v i n g

actomyosin.

c o w o r k e r s (55,56,57)

w i t h movement

faster

suggesting a progressive polymerization

D e t a i l e d analyses

extracts f r o m f r o z e n - s t o r e d

cod

of

the u l t r a c e n t r i f u g a l patterns

muscle were

of

d o n e b y K i n g a n d his

a n d t h e y r e p o r t e d t h a t G - a c t o m y o s i n f o r m s as a n

i n t e r m e d i a t e p r o d u c t d u r i n g f r o z e n storage a n d t h a t s o m e a m o u n t G - a c t i n is e v e n t u a l l y d i s s o c i a t e d f r o m G - a c t o m y o s i n . that occur

of

T h e existence of

G - a c t o m y o s i n is q u e s t i o n a b l e a n d deserves f u r t h e r s t u d y T h e changes

of

simultaneously

(2).

i n viscosity a n d ultracentrifugal pattern

d u r i n g f r o z e n storage suggest d e f o r m a t i o n of a c t o m y o s i n

filaments

into

some denser, less a s y m m e t r i c f o r m . SALTTNG-OUT

ANALYSIS.

A n a l y s i s of s a l t i n g - o u t c u r v e s of

extracts

f r o m fish muscles b e f o r e a n d after f r o z e n storage has s h o w n t h a t f r o z e n storage shifts the a c t o m y o s i n p e a k to a h i g h e r s a t u r a t i o n l e v e l w i t h v a r i a b l e results f o r different fish species (44).

(44,58,59),

A similar experiment

o n a s o l u t i o n of i s o l a t e d c a r p a c t o m y o s i n has s h o w n a s i m i l a r shift of the p e a k f r o m 3 5 % to 3 8 % s a t u r a t i o n ( ( N H ) S 0 ) f o l l o w i n g f r o z e n storage, 4

2

4

suggesting the release o f some a c t i n f r o m the a c t o m y o s i n c o m p l e x


(59).

10.

Denaturation

MATSUMOTO

ATPASE

of Fish

ACTIVITY AND RELATED

211

Muscle D u r i n g frozen

PROPERTIES.

storage

of c o d fillets, a decrease is o b s e r v e d i n the A T P a s e a c t i v i t y of a c t o m y o s i n extracts, b u t the n u m b e r of free S H groups does not c h a n g e (32).

This

decrease i n A T P a s e a c t i v i t y also has b e e n r e p o r t e d f o r a c t o m y o s i n f r o m v a r i o u s o t h e r fishes i n s i t u d u r i n g f r o z e n storage

(60,61,62).

During

f r o z e n storage of solutions or suspensions of a c t o m y o s i n i s o l a t e d f r o m c a r p , A T P a s e a c t i v i t y i n i t i a l l y rises a n d t h e n declines to zero (42,51, 64).

63,

T h e i n i t i a l rise i n a c t i v i t y suggests a s l i g h t c o n f o r m a t i o n a l d e f o r m a -

t i o n a r o u n d the a c t i v e sites f o l l o w e d b y c o n v e r s i o n to a n i n a c t i v e r a n d o m Downloaded by OHIO STATE UNIV LIBRARIES on June 2, 2012 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch010

state. T h e decrease i n A T P a s e a c t i v i t y of a c t o m y o s i n is reflected i n t h e d e c l i n e i n its s u p e r p r e c i p i t a t i o n c a p a c i t y

(51,52)

a n d i n the rate of

v i s c o s i t y d r o p o n the a d d i t i o n of A T P (29, 5 9 ) . ANALYSIS

OF

SOLUBTXITY-STORAGE

CURVES.

The

solubility-storage

t i m e curves o f a c t o m y o s i n a n d m y o s i n e x t r a c t e d f r o m t h e same f r o z e n cod

fillets

f o r m a f a m i l y of

p a r a l l e l lines t h a t are not l i n e a r

w h e r e a s the s o l u b i l i t y of a c t i n does not c h a n g e w i t h storage t i m e

(65), (65,66).

T h u s C o n n e l l a t t r i b u t e d t h e freeze d e n a t u r a t i o n or loss of s o l u b i l i t y of a c t o m y o s i n to the m y o s i n c o m p o n e n t .

T h e authors group determined the

a m o u n t of s o l u b l e p r o t e i n that r e m a i n e d i n a f r o z e n s u s p e n s i o n of actom y o s i n i s o l a t e d f r o m c a r p a n d p l o t t e d this v a l u e o n l o g a r i t h m i c scale against storage t i m e . T h e p l o t consisted of t w o i n t e r s e c t i n g straight lines w i t h different slopes, suggesting t h a t the d e n a t u r a t i o n process i n v o l v e d m o r e t h a n one m e c h a n i s m (67).

T h e n a t u r e of the p l o t d i d not c h a n g e

w h e n the i o n i c s t r e n g t h of the storage s o l u t i o n w a s c h a n g e d b u t i t d i d c h a n g e w h e n the p H w a s a l t e r e d . R e p l o t t i n g t h e results of D y e r 22)

and C o n n e l l (65)

(20,21,

i n t h e m a n n e r d e s c r i b e d y i e l d e d plots f o r

fish

m u s c l e that w e r e s i m i l a r to t h e p l o t just d e s c r i b e d . A n a l y s i s of the s o l u b l e - p r o t e i n fractions r e m a i n i n g after f r o z e n storage of fish is b e i n g a c c o m p l i s h e d i n the author's l a b o r a t o r y b y s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l electrophoresis

(68).

Tropomyosin remains

i n s o l u t i o n after l o n g storage a n d i t seems to b e the m o s t stable p r o t e i n d u r i n g f r o z e n storage. A c t i n is the next most stable p r o t e i n . D u r i n g the e a r l y stages of freeze d e n a t u r a t i o n of p r o t e i n s i n fish b o t h m y o s i n a n d a c t i n a p p a r e n t l y f o r m a n i n s o l u b l e f r a c t i o n t h a t accounts for the o b s e r v e d decrease i n p r o t e i n s o l u b i l i t y . ELECTRON

MICROSCOPY.

E x a m i n a t i o n of

fish

proteins b y

electron

m i c r o s c o p y c o n c l u s i v e l y shows t h a t a c t o m y o s i n aggregates d u r i n g f r o z e n storage fibrillar

(59,63,69).

T h e c h a n g e i n structures of t h e e x t r a c t e d

myo-

p r o t e i n s a n d of t h e m y o f i b r i l residues of f r o z e n - s t o r e d c o d m u s c l e

was studied by electron microscopy. actomyosin

filaments

T h e decrease i n the n u m b e r

of

a n d a n increase i n the n u m b e r a n d size of l a r g e

aggregate w e r e f o u n d ( 6 9 ) .

U n f r o z e n carp actomyosin, either dissolved

i n 0 . 6 M K C 1 or s u s p e n d e d i n 0 . 0 5 M K C 1 , exists i n a t y p i c a l a r r o w h e a d


212

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

s t r u c t u r e (59,63).

A f t e r f r e e z i n g f o l l o w e d b y i m m e d i a t e t h a w i n g the

a c t o m y o s i n filaments b e c o m e loosely j o i n e d together i n a crosswise f a s h i o n . A f t e r f r o z e n storage f o r s e v e r a l w e e k s , t h e n a t i v e s t r u c t u r e is lost a n d o n l y masses o r aggregates of r a n d o m l y e n t a n g l e d filaments a r e e v i dent.

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

actomyosin

filaments

a r e t h i n n e r a n d m o r e flexible t h a n those f o u n d i n

the u n f r o z e n p r e p a r a t i o n , i.e., t h e a r r o w h e a d s t r u c t u r e is n o apparent.

longer

A l s o e v i d e n t i n t h e b a c k g r o u n d of t h e p i c t u r e s are o t h e r

p a r t i c l e s t h a t m a y consist p a r t l y of a g g r e g a t e d masses of m y o s i n m o l e Downloaded by OHIO STATE UNIV LIBRARIES on June 2, 2012 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch010

cules.

T h e thinned entangled

filaments

filaments

i n t h e aggregates are p r o b a b l y

of F - a c t i n .

C h a n g e s i n t h e shape o f c a r p a c t o m y o s i n storage w a s also s t u d i e d at v a r i o u s p H ' s (67).

filaments

during frozen

P i c t u r e s of a c t o m y o s i n

f r o z e n a n d s t o r e d i n 0 . 9 M a n d 1 . 2 M K C 1 i l l u s t r a t e d t h e d i s s o c i a t i o n of actomyosin into actin a n d myosin Myosin.

AGGREGATION.

(1,2).

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

m y o s i n f r o m fish studies o n t h e b e h a v i o r of this p r o t e i n w e r e d e l a y e d . C o n n e l l s t u d i e d the b e h a v i o r o f c o d m y o s i n d u r i n g f r e e z i n g to t e m p e r a tures r a n g i n g f r o m a b o u t — 7 to — 7 8 ° C . B y u l t r a c e n t r i f u g a l analysis h e showed that the m y o s i n monomer polymerizes to dimers, trimers, a n d o t h e r l a r g e r m u l t i p l e s (70).

N e i t h e r t h e specific r o t a t i o n n o r t h e n u m b e r

of a c t i v e S H groups c h a n g e d a p p r e c i a b l y d u r i n g p o l y m e r i z a t i o n . C o n n e l l suggested t h a t m y o s i n m o l e c u l e s aggregate side-to-side w i t h o u t u n f o l d i n g or u n d e r g o i n g a n y c h a n g e i n i n t r a m o l e c u l a r c o n f o r m a t i o n . T h e a g g r e g a t i o n w a s a s c r i b e d n o t to S - S b o n d i n g b u t to l i n k a g e s of a n u n k n o w n nature.

C o n n e l l also suggested

that m y o s i n has a major role i n the

^ s o l u b i l i z a t i o n of a c t o m y o s i n d u r i n g f r o z e n storage

(2,65,66).

C h a n g e s i n t h e s o l u b i l i t y , u l t r a c e n t r i f u g a l b e h a v i o r , a n d n u m b e r of S H groups i n frozen m y o s i n f r o m trout were studied b y Buttkus ATPASE.

(71,72).

M y o s i n s i s o l a t e d f r o m v a r i o u s f r o z e n - s t o r e d fish m u s c l e s

e x h i b i t specific activities f o r A T P a s e t h a t are s l i g h t l y l o w e r t h a n t h e specific a c t i v i t i e s of m y o s i n f r o m f r e s h muscles (73,74).

T h e d e c l i n e of

A T P a s e a c t i v i t y also occurs w i t h m y o s i n i s o l a t e d f r o m c a r p , w h e n storage is c o n d u c t e d

at — 2 0 ° C

(64).

L i k e c a r p a c t o m y o s i n , t h e d e c l i n e is

p r e c e d e d b y a t e m p o r a r y rise i n a c t i v i t y . FILAMENT

FORMATION.

L i k e r a b b i t m y o s i n (74),

carp myosin i n

solutions of l o w i o n i c s t r e n g t h forms filaments t h a t are o b s e r v a b l e u n d e r the e l e c t r o n m i c r o s c o p e .

These

filaments

are either spindle shaped or

d u m b b e l l s h a p e d d e p e n d i n g u p o n t h e m e t h o d u s e d to p r e p a r e t h e n m . F i l a m e n t s f o r m e d f r o m e i t h e r f r o z e n - s t o r e d samples (filaments or m y o s i n s o l u t i o n ) w e r e n o t as p e r f e c t i n shape as those p r e p a r e d f r o m u n f r o z e n , i n t a c t m y o s i n . I n f r o z e n storage, t h e s p i n d l e - s h a p e d m y o s i n w a s m o r e stable t h a n t h e d u m b b e l l - s h a p e d m y o s i n , a n d m y o s i n i n t h e d i s s o l v e d state w a s least a b l e to f o r m

filaments

(64).


10.

MATSUMOTO

Denaturation

of Fish

Muscle

M a t s u m o t o et a l . (64)

Subunits of Myosin.

213 isolated H - m e r o m y o s i n

( H M M ) a n d L - m e r o m y o s i n ( L M M ) f r o m c a r p m u s c l e (15)

and studied

t h e i r stabilities at — 2 0 ° C . T h e A T P a s e a c t i v i t y of H M M d e c r e a s e d m u c h faster t h a n t h a t of m y o s i n a n d the c a p a c i t y of H M M to b i n d w i t h F - a c t i n as d e t e r m i n e d b y e l e c t r o n m i c r o s c o p y w a s lost. L M M also e x h i b i t e d a decreased c a p a c i t y to f o r m w e l l - o r d e r e d p a r a c r y s t a l s . T h e s e results t e n d to i n d i c a t e that f r o z e n storage

causes m y o s i n m o l e c u l e s

to

aggregate

side-by-side a n d myosin subunits to undergo conformational deformations. A c c o r d i n g to C o n n e l l ( 6 5 , 6 6 ) , a c t i n i n c o d m u s c l e r e m a i n s

Actin. Downloaded by OHIO STATE UNIV LIBRARIES on June 2, 2012 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch010

essentially u n m o d i f i e d d u r i n g storage for as l o n g as 30 w e e k s at — 1 4 ° C . T h i s statement is b a s e d o n a s t u d y of v a r i o u s p h y s i c o - c h e m i c a l p r o p e r t i e s s u c h as i n t r i n s i c v i s c o s i t y , m y o s i n c o m b i n i n g a b i l i t y , a n d u l t r a c e n t r i f u g a l behavior. O n the o t h e r h a n d , a c t i n extracts f r o z e n f o r either 96 o r 127 w e e k s at — 20 ° C u n d e r g o significant a l t e r a t i o n . T h i s w a s s h o w n i n t h e author's laboratory using actin isolated from carp (75).

F o r these studies a c t i n

w a s p r e p a r e d b y G u b a - S t r a u b ' s m e t h o d w i t h the e x c e p t i o n t h a t S p u d i c h W a t t ' s buffer A w a s u s e d . A c t i n i n either G - f o r m or F - f o r m w a s f r o z e n a n d stored at — 20° C , a n d its s o l u b i l i t y , r e d u c e d v i s c o s i t y , p o l y m e r i z i n g ability ( G - a c t i n ) , a n d appearance

under the electron microscope

(F-

a c t i n ) w e r e tested p e r i o d i c a l l y . B o t h G - a n d F - a c t i n s u n d e r w e n t d e n a turation d u r i n g frozen

storage.

j u d g e d b y its e l e c t r o p h o r e t i c

A s indicated earlier, actomyosin,

( S D S - d i s c electrophoresis)

comes i n c r e a s i n g l y i n s o l u b l e d u r i n g f r o z e n storage

behavior,

as be-

(68).

T h e d i s a g r e e m e n t b e t w e e n C o n n e l l ' s results a n d those b y t h e author's g r o u p p r o b a b l y arises because of t h e different e x p e r i m e n t a l c o n d i t i o n s u s e d . I n C o n n e l l ' s s t u d y , a c t i n w a s i n s i t u , w h e r e a s i n the author's s t u d y i s o l a t e d a c t i n or a c t o m y o s i n w a s u s e d . Tropomyosin and Troponin.

T r o p o m y o s i n is a p p a r e n t l y t h e

stable o f the fish p r o t e i n s d u r i n g f r o z e n storage.

most

Tropomyosin can

e x t r a c t e d l o n g after a c t i n a n d m y o s i n b e c o m e i n e x t r a c t a b l e

T r o p o n i n s i s o l a t e d f r o m f r o z e n - s t o r e d b i g e y e t u n a , Tilapia

or

Beryx,

are less a c t i v e i n t h e i r r e g u l a t o r y f u n c t i o n t h a n those f r o m f r e s h muscles

be

(68). fish

(76).

Myofibrils and Tissues.

Microscopic

observations

m u s c l e w e r e first p u b l i s h e d b y L o v e (23,24,25,26) tions of other fish species w e r e r e p o r t e d b y T a n a k a To

determine

M a c K a y (77)

the d e g r e e of

developed

adherence

of

of

frozen

cod

a n d similar observa(27,34,48). myofibrils, L o v e

the " c e l l fragility method."

and

This method i n -

volves m e a s u r i n g t h e t u r b i d i t y of t h a w e d m u s c l e h o m o g e n a t e s p r e p a r e d i n 1.15%

formaldehyde.

I t has b e e n s h o w n t h a t t h e e x t i n c t i o n v a l u e

decreases w i t h i n c r e a s i n g l e n g t h of f r o z e n storage, i n d i c a t i n g r e d u c e d d i s p e r s i o n of t h e m y o f i b r i l s . T h i s m e t h o d has b e e n h e l p f u l i n s t u d y i n g s e v e r a l p r o b l e m s ( 30,78 ).


214

PROTEINS

AT

LOW

TEMPERATURES

E l e c t r o n m i c r o s c o p i c s t u d y of f r o z e n - s t o r e d c a r p m u s c l e h a v e s h o w n t h a t the u l t r a s t r u c t u r e of m y o f i b r i l s is, at least i n some specimens, w e l l p r e s e r v e d d u r i n g storage f o r 60 w e e k s at — 2 0 ° C ( 3 4 ) .

T h e ultrastruc-

t u r a l changes d u r i n g f r o z e n storage of c o d m u s c l e ( 7 9 ) , c o d m y o f i b r i l s , a n d solutions of m y o f i b r i l l a r proteins (80)

h a v e b e e n s t u d i e d u s i n g the

freeze e t c h i n g a n d the n e g a t i v e s t a i n i n g t e c h n i q u e s .

M u s c l e stored for a

l o n g p e r i o d at — 20 ° C s h o w e d d i s t u r b a n c e s i n t h e h e x a g o n a l p a t t e r n , d e f o r m a t i o n of the s a r c o p l a s m i c r e t i c u l u m , a n d a significant r e d u c t i o n i n the i n t e r f i l a m e n t s p a c i n g . A d d i t i o n a l studies of a s i m i l a r n a t u r e h a v e b e e n Downloaded by OHIO STATE UNIV LIBRARIES on June 2, 2012 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch010

published recently

(81,82).

Connective Tissue Proteins. of s k i n a n d m y o c o m m a t a .

C o l l a g e n comprises the major m a t e r i a l

L o v e a n d his c o - w o r k e r s s t u d i e d the p r o b l e m

of g a p i n g i n w h i c h slits or holes a p p e a r i n the m u s c l e a n d sometimes t h e fillet

falls a p a r t . T h i s d e f e c t is r e l a t e d to the b e h a v i o r of

p r o t e i n s (83-87).

myocommata

G a p i n g is m o r e severe i n fishes f r o z e n i n - r i g o r t h a n i n

those f r o z e n p r e - r i g o r , a n d the seriousness of this d e f e c t varies w i t h t h e b i o l o g i c a l c o n d i t i o n of t h e

fish

( i n f l u e n c e d b y season, size, age,

and

w h e t h e r t h e fish is h e a l t h y or s t a r v e d ) , a n d the fish species. O n f r e e z i n g , ice crystals sometimes f o r m i n t h e m y o c o m m a t a

and

this m a y influence t h e i n c i d e n c e of g a p i n g . D u r i n g studies w h e r e t h e p H a n d m o i s t u r e of t h e m u s c l e w e r e v a r i a b l e s , i t w a s l e a r n e d t h a t g a p i n g w a s c o r r e l a t e d w i t h t h e m e c h a n i c a l s t r e n g t h of m y o c o m m a t a

collagen

t h a t is less s t r o n g at l o w e r p H ' s . G a p i n g w a s f o u n d to v a r y w i t h

fish

species whereas the p h y s i c o - c h e m i c a l p r o p e r t i e s of c o l l a g e n f r o m v a r i o u s species w e r e f o u n d to b e i d e n t i c a l w i t h n o r m a l collagens. T a n a k a (34) r e p o r t e d t h a t m u s c l e - c e l l m e m b r a n e s of A l a s k a p o l l a c k b e c a m e t o u g h e r d u r i n g f r o z e n storage. Other Proteins.

Since Reay a n d D y e r discovered that denaturation

of m y o f i b r i l l a r p r o t e i n s is of s u c h p r o f o u n d i m p o r t a n c e , l i t t l e a t t e n t i o n has b e e n

g i v e n to the w a t e r - s o l u b l e p r o t e i n s i n c l u d i n g enzymes

and

other p r o t e i n s i n t h e s a r c o p l a s m , s u b c e l l u l a r organelles, a n d c e l l m e m branes.

R e c e n t l y reports h a v e a p p e a r e d o n t h e freeze d e n a t u r a t i o n of

enzymes.

T h e s e studies i n v o l v e d enzymes s u c h as catalase, A D H , G D H ,

L D H , a n d M D H f r o m sources other t h a n fish (88,89,90) w a s g i v e n to the effectiveness 90).

C o m p a r a b l e studies w i t h e n z y m e s

number (91).

a n d attention

of v a r i o u s c r y o p r o t e c t i v e substances f r o m fish m u s c l e are f e w

(89, in

Studies o n fish m u s c l e p r o t e i n s m u s t b e e x t e n d e d to this

area i f a c o m p l e t e p i c t u r e of the freeze d e n a t u r a t i o n of fish m u s c l e is to be obtained.

It s h o u l d b e n o t e d that freeze stable e n z y m e s m i g h t h a v e

i m p o r t a n t effects d u r i n g f r o z e n storage of

fish

(92,93).

M y o g l o b i n undergoes changes d u r i n g f r o z e n storage a n d this is t h e cause of d i s c o l o r a t i o n of r e d m u s c l e fishes s u c h as t u n a . D u r i n g f r o z e n storage m y o g l o b i n is d e n a t u r e d , p e r h a p s v i a m e t m y o g l o b i n .

Discoloration


10.

MATSUMOTO

Denaturation

of Fish

215

Muscle

of t u n a m y o g l o b i n c a n b e p r e v e n t e d b y r a p i d f r e e z i n g a n d storage at a b o u t — 6 0 ° C (94),

c o n d i t i o n s t h a t are e m p l o y e d c o m m e r c i a l l y i n J a p a n .

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

Cause

of

Denaturation

A c c o r d i n g to L o v e (41) proteins.

f r e e z i n g itself has a d e t r i m e n t a l effect o n

S o m e a g g r e g a t i o n of a c t o m y o s i n

filaments

has b e e n

i n specimens t h a w e d i m m e d i a t e l y after f r e e z i n g to — 2 0 ° C Downloaded by OHIO STATE UNIV LIBRARIES on June 2, 2012 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch010

is

(95).

observed

(59).

S e v e r a l proposals h a v e b e e n a d v a n c e d c o n c e r n i n g t h e causes a n d m e c h a n i s m s f o r freeze d e n a t u r a t i o n of fish m u s c l e p r o t e i n s b u t the v i e w s u s u a l l y h a v e some features i n c o m m o n .

I n a l l instances, t h e f o r m a t i o n

of i c e crystals has effects, either d i r e c t l y or i n d i r e c t l y , t h a t are of m a j o r importance. Effect of Mineral Salts.

A s c e l l u l a r w a t e r is f r o z e n , m i n e r a l salts

a n d s o l u b l e - o r g a n i c substances b e c o m e c o n c e n t r a t e d i n t h e r e m a i n i n g u n f r o z e n phase. T h i s increase i n solute c o n c e n t r a t i o n , w i t h c o r r e s p o n d i n g changes i n i o n i c s t r e n g t h a n d p H , is b e l i e v e d to affect d i s s o c i a t i o n a n d / o r d e n a t u r a t i o n of p r o t e i n s (1,2,62,96-98).

E x p e r i m e n t s b y F u k u m i et a l .

( 9 9 ) s u p p o r t this theory. T h e y f o u n d t h a t freeze d e n a t u r a t i o n of w a s h e d a c t o m y o s i n f r o m A l a s k a p o l l a c k m u s c l e w a s a c c e l e r a t e d b y the presence of C a , M g 2 +

2 +

, K , a n d N a ions a n d r e d u c e d b y t h e i r r e m o v a l . +

+

Dehydration of Proteins.

Ice

crystal formation, especially w h e n

b r o u g h t a b o u t b y s l o w f r e e z i n g , results i n a r e d i s t r i b u t i o n of w a t e r . W h e n a c e l l is t h a w e d w a t e r has d i f f i c u l t y r e t u r n i n g to its o r i g i n a l sites (e.g., associated w i t h p r o t e i n s ) a n d some of this w a t e r leaves t h e tissue ( d r i p ) . D e n a t u r a t i o n or association of p r o t e i n m o l e c u l e s that m a y o c c u r d u r i n g f r e e z i n g a n d f r o z e n storage h i n d e r s the process of p r o t e i n r e h y d r a t i o n (1,2,4). A s i c e f o r m s a n d w a t e r m o l e c u l e s are r e m o v e d f r o m areas n e a r t h e p r o t e i n s , t h e p r o t e i n m o l e c u l e s m o v e closer together a n d the p o s s i b i l i t y of a g g r e g a t i o n t h r o u g h i n t e r m o l e c u l a r c r o s s - b o n d i n g increases.

If f r e e z -

i n g is c a r r i e d out to a v e r y l o w t e m p e r a t u r e , r e m o v a l of w a t e r f r o m p r o t e i n s m a y o c c u r t o s u c h a n extent t h a t t h e p r o t e i n s m i g h t b e affected. E v i d e n c e that a g g r e g a t i o n occurs d u r i n g f r o z e n storage is p r o v i d e d i n the f o r m of p h y s i c o - c h e m i c a l d a t a p r e s e n t e d earlier. E v i d e n c e f o r f r e e z e - i n d u c e d changes i n c o n f o r m a t i o n is p r o v i d e d f r o m t h e changes i n b i o c h e m i c a l p r o p e r t i e s of

m y o s i n a n d its s u b u n i t s

(e.g., changes i n

A T P a s e activity, superprecipitation capacity, actin-binding capacity, a n d a b i l i t y to f o r m p a r a c r y s t a l s ) as d e s c r i b e d earlier. T h e effects of

cryo-

p r o t e c t i v e substances, as m e n t i o n e d l a t e r , p r o v i d e f u r t h e r s u p p o r t f o r these v i e w s .


216

PROTEINS

AT

LOW

TEMPERATURES

B a s e d o n t h e decrease i n a c t i v e S H g r o u p s t h a t occurs i n t r o u t m y o s i n d u r i n g f r o z e n storage, B u t t k u s (71,72)

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

m o l e c u l a r S - S b o n d s as a m a j o r cause of a g g r e g a t i o n . M y o s i n aggregates w e r e s o l u b l e i n 6M g u a n i d i n e - H C l c o n t a i n i n g 0 . 5 M m e r c a p t o e t h a n o l b u t w e r e not s o l u b l e i n 1 M N a C l , 8 M u r e a , 6 M g u a n i d i n e - H C l , o r detergents. H y d r o p h o b i c a n d h y d r o g e n b o n d s w e r e suggested also as b e i n g i n v o l v e d i n t h e a g g r e g a t i o n process.

T h e proposed

m e c h a n i s m of

aggregation

c o n s i s t e d m a i n l y of S - S b o n d r e a r r a n g e m e n t a n d a n i n f l u e n c e of K C 1 at its eutectic p o i n t . Downloaded by OHIO STATE UNIV LIBRARIES on June 2, 2012 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch010

F i n d i n g s i n the a u t h o r s l a b o r a t o r y (100)

demonstrated that the

n u m b e r of cross b o n d s i n c a r p a c t o m y o s i n a n d m y o s i n increases d u r i n g f r o z e n storage a n d that s o l u b i l i t y of these p r o t e i n s decreases.

Based on

the types of c h e m i c a l s t h a t r e s o l u b i l i z e d these p r o t e i n s a t v a r i o u s rates, it w a s

concluded

that i o n i c b o n d s , h y d r o g e n

bonds,

covalent

bonds

( S - S ) , a n d h y d r o p h o b i c associations a l l are i n v o l v e d i n t h e a g g r e g a t i o n process. Effects o f L i p i d s .

D y e r w a s t h e first t o suggest t h a t l i p i d s a n d t h e i r

derivatives might be involved i n protein denaturation d u r i n g the frozen storage of fish (21,22).

S i n c e his studies m a n y w o r k e r s h a v e i n v e s t i g a t e d

t h i s p o s s i b i l i t y (101,102,103).

A series of elaborate w o r k s w e r e c a r r i e d

o u t b y K i n g , A n d e r s o n , a n d t h e i r collaborators to d e t e r m i n e w h e t h e r p r o t e i n - l i p i d i n t e r a c t i o n s are the cause o f p r o t e i n i n s o l u b i l i z a t i o n i n fish m u s c l e d u r i n g f r o z e n storage Effects of

(55,56,57,104,105).

linoleic acid and linoleic acid hydroperoxides

on

the

m y o f i b r i l s a n d t h e solutions of m y o f i b r i l l a r p r o t e i n s of c o d m u s c l e h a v e been proved using the electron microscopy

(80).

Linoleic acid hydro

p e r o x i d e s w e r e t e n times m o r e effective t h a n l i n o l e i c a c i d i n r e d u c i n g t h e a m o u n t of the p r o t e i n i n K C l - e x t r a c t s f r o m the m y o f i b r i l s i n c u b a t e d w i t h the a c i d or its h y d r o p e r o x i d e s .

L i n o l e i c a c i d s e e m e d to p r e v e n t

t h e d i s s o l u t i o n of the m y o f i b r i l f r a m e w o r k b u t a p p e a r e d not to i m p a i r t h e e x t r a c t i o n of

myosin w h i l e hydroperoxides

appeared

to

cause

a

r e t e n t i o n of Α - b a n d s ( m y o s i n ) i n the m y o f i b r i l s . N e v e r t h e l e s s after r e v i e w i n g t h e l i t e r a t u r e , C o n n e l l c o n c l u d e d t h a t p r o t e i n - l i p i d i n t e r a c t i o n s d o not a p p e a r t o b e a m a j o r cause of p r o t e i n d e n a t u r a t i o n d u r i n g f r o z e n storage of fish ( 2 ) . E f f e c t o f O t h e r Substances.

G A S . O x y g e n dissolved i n the b l o o d

a n d i n t e r n a l m e d i a of fish m a y e n c o u r a g e p r o t e i n s to f o r m S - S b o n d s d u r i n g f r o z e n storage.

F u r t h e r m o r e , b e c a u s e of its l o w s o l u b i l i t y i n i c e ,

nitrogen m a y be expelled f r o m the frozen m e d i u m , resulting i n b u b b l e f o r m a t i o n a n d surface d e n a t u r a t i o n of p r o t e i n s (27). p o s t u l a t e d b y T a n a k a (27)

fine

This was

because h e o b s e r v e d c o n s i d e r a b l e m o l e c u l a r

n i t r o g e n i n t h e holes of so c a l l e d " s p o n g e m e a t " of c o d t h a t h a d b e e n frozen for a long time.


10.

Denaturation

MATSUMOTO

METALS.

HEAVY

of Fish

217

Muscle

H e a v y metals are k n o w n to d e n a t u r e

A c c e l e r a t i o n of freeze d e n a t u r a t i o n of t r o u t m y o s i n b y C u observed

(72).

I n stored G a d o i d m u s c l e , C u

f o r m a t i o n of t r i m e t h y l a m i n e o x i d e to H C H O

accelerates

2 +

proteins. has b e e n

2 +

the trans-

via trimethylamine and

d i m e t h y l a m i n e , i n t u r n , a n d the H C H O m i g h t b i n d to p r o t e i n s , r e s u l t i n g i n their denaturation (106). WATER

SOLUBLE

PROTEINS.

I n studies w i t h f r o z e n m i n c e d A l a s k a

p o l l a c k , the p o s s i b i l i t y has b e e n r a i s e d t h a t r e m o v a l of w a t e r

soluble

p r o t e i n s m a y r e n d e r the r e s i d u a l a c t o m y o s i n m o r e stable to f r o z e n storage Downloaded by OHIO STATE UNIV LIBRARIES on June 2, 2012 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch010

(107).

W h e t h e r the effect o f the w a s h i n g is b e c a u s e of t h e r e m o v a l of

the w a t e r - s o l u b l e proteins or of t h e o r g a n i c substances of l o w e r m o l e c u l a r w e i g h t is left to f u r t h e r s t u d y . Control

of

Denaturation

Because

p r o t e i n d e n a t u r a t i o n u s u a l l y does not

involve

a

single

specific r e a c t i o n , c o n t r o l of this o c c u r r e n c e at first w a s t h o u g h t to b e v e r y difficult. E a r l y attempts to r e d u c e d e n a t u r a t i o n d e p e n d e d a l m o s t e n t i r e l y o n c o n t r o l of t e m p e r a t u r e d u r i n g f r o z e n storage a n d o n c o n t r o l of p r o p e r t i e s (freshness, b i o l o g i c a l c o n d i t i o n , etc.) p r i o r t o f r e e z i n g .

fish

Now,

c o n t r o l of freeze d e n a t u r a t i o n is b e i n g a p p r o a c h e d t h r o u g h the a d d i t i o n of c r y o p r o t e c t i v e

substance a n d t h i s a p p r o a c h is l i k e l y to increase i n

importance. F r o z e n M i n c e of A l a s k a P o l l a c k M u s c l e .

I n 1959 researchers

at

H o k k a i d o Fisheries Research Station, Japan, headed b y Nishiya, develo p e d a t e c h n i q u e b y w h i c h f r e e z e d e n a t u r a t i o n of p r o t e i n s i n A l a s k a p o l l a c k m u s c l e c o u l d b e p r e v e n t e d (108,109).

T h e t e c h n i q u e consists of:

1 ) r e m o v i n g the m i n e r a l salts a n d w a t e r - s o l u b l e o r g a n i c substances f r o m m i n c e d m u s c l e b y w a s h i n g w i t h w a t e r a n d 2 ) a d d i t i o n of 1 0 %

sucrose

a n d 0 . 2 - 0 . 5 % p o l y p h o s p h a t e s o r a m i x t u r e of 5 %

sorbitol

and polyphosphate

p r i o r to f r e e z i n g .

sucrose, 5 %

B y this m e a n s ,

actomyosin

of

A l a s k a p o l l a c k , w h i c h is r e m a r k a b l y u n s t a b l e i n f r o z e n storage, is p r o tected

from

denaturation.

Within

a few

years, this n e w

technique

s t i m u l a t e d g r o w t h of a n i n d u s t r y t h a t n o w has a n a n n u a l p r o d u c t i o n of 400,000 tons. S c i e n t i f i c analyses f o l l o w e d t h e i n i t i a l i n v e n t i o n a n d these p r o v e d the benefits of r e m o v i n g the m i n e r a l s a n d w a t e r - s o l u b l e o r g a n i c substances

( i n c l u d i n g water-soluble proteins)

protective chemicals

a n d of a d d i n g t h e c r y o -

(99).

Cryoprotective Compounds.

C r y o p r o t e c t i v e effects of sugars, p o l y -

alcohols, a n d c o m p o u n d s of o t h e r f a m i l i e s h a v e b e e n k n o w n since t h e 1940s.

M a n y w o r k s o n those effects h a v e b e e n r e p o r t e d i n t h e fields of

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


218

PROTEINS A T

POLYPHOSPHATES.

LOW

TEMPERATURES

T r e a t i n g r o u n d fish o r fillets w i t h p h o s p h a t e s o l u -

tions p r i o r to f r e e z i n g has p r o v e n to h a v e a p r o t e c t i v e effect o n p r o t e i n s T r i p h o s p h a t e a n d hexametaphosphates are m o r e

(97,110,111,112,113).

effective t h a n p y r o p h o s p h a t e a n d o r t h o p h o s p h a t e is of little benefit. A d d i t i o n a l studies h a v e b e e n c o n d u c t e d o n f r o z e n samples of actom y o s i n i s o l a t e d f r o m c a r p . A c t o m y o s i n w a s either s u s p e n d e d i n 0 . 0 5 M K C 1 o r d i s s o l v e d i n 0 . 6 M K C 1 . T h e s e studies s h o w e d t h a t t r i p h o s p h a t e is m o r e c r y o p r o t e c t i v e t h a n p y r o p h o s p h a t e w h e n a c t o m y o s i n is s u s p e n d e d i n 0 . 0 5 M K C 1 , w h e r e a s b o t h c h e m i c a l s are ineffective w h e n a c t o m y o s i n Downloaded by OHIO STATE UNIV LIBRARIES on June 2, 2012 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch010

is d i s s o l v e d

in 0.6M KC1.

Orthophosphate

was

ineffective

in

both

situations ( 5 2 ) . I n the f r o z e n m i n c e of A l a s k a p o l l a c k t h e r e w a s a m a r k e d s y n e r g i s t i c effect b e t w e e n p y r o p h o s p h a t e s o r p o l y p h o s p h a t e s a n d sucrose o r s o r b i t o l , b u t this effect w a s less significant i f 2 . 5 % N a C l w a s present i n the m i n c e A l s o for carp actomyosin solution i n 0 . 6 M K C 1 , no appre-

(97,110,114).

c i a b l e s y n e r g i s t i c effect w a s f o u n d a m o n g these a d d i t i v e s , w h i l e some s y n e r g i s t i c effect w a s f o u n d i n 0 . 0 5 M K C 1 . W h e n a suspension of c a r p m y o f i b r i l s w a s u s e d i n s t e a d of the a c t o m y o s i n p r e p a r a t i o n , the s y n e r g i s t i c effect w a s greater t h a n w i t h t h e i s o l a t e d p r o t e i n (115,116,117).

This

suggests that t h e s y n e r g i s t i c c r y o p r o t e c t i v e effect of these substances is d e p e n d e n t o n the m o d e of association of a c t i n a n d m y o s i n a n d t h e i o n i c state of these p r o t e i n s . ORGANIC

COMPOUNDS.

Sucrose, glucose, o t h e r sugars, a n d s o r b i t o l

h a v e c r y o p r o t e c t i v e effects o n f r o z e n m i n c e of A l a s k a p o l l a c k and

carp

actomyosin

g l y c e r o l (121,122),

(117,119,120).

a n d c i t r a t e (123)

(60,118)

F u r t h e r m o r e , ethylene

glycol,

h a v e c r y o p r o t e c t i v e effects o n the

p r o t e i n s i n muscles of A l a s k a p o l l a c k a n d c o d . I n t h e a u t h o r s l a b o r a t o r y , a n i n v i t r o test w a s d e v i s e d to e v a l u a t e the c r y o p r o t e c t i v e effect of a n y c o m p o u n d o n fish p r o t e i n . T h e test system c o n s i s t e d of c a r p a c t o m y o s i n e i t h e r i n s o l u t i o n ( i n 0 . 6 M K C 1 ) o r i n s u s p e n s i o n ( i n 0 . 0 5 M K C 1 ) . B y means of this system a b o u t 150 p o u n d s w e r e screened, o u t of w h i c h a b o u t 30 c o m p o u n d s

were

comfound

to b e m a r k e d l y effective a n d a n o t h e r 20 c o m p o u n d s w e r e f o u n d to m o d e r a t e l y effective. (MSG)

be

A m o n g the former group, monosodium glutamate

was particularly outstanding.

A d d i t i v e s w e r e u s e d at a

con-

c e n t r a t i o n of 0 . 1 - 0 . 2 M i n t h e final m i x t u r e a n d t h e p H w a s a d j u s t e d to 7

before freezing

(51,52,116,117,119,124,125,126).

have been done b y other workers

Similar

studies

(60,127).

T h e effective c o m p o u n d s f o u n d t h u s f a r are d i s t r i b u t e d o v e r s e v e r a l classes of c h e m i c a l s , n a m e l y a m i n o acids, d i c a r b o x y l i c acids, h y d r o c a r boxylic acids, polyalcohols, carbohydrates, a n d polyphosphates.

However,

n o t a l l m e m b e r s of e a c h class a r e effective. A m o n g t h e a m i n o a c i d s n o n e


10.

MATSUMOTO

Denaturation

of Fish

219

Muscle

of those w i t h l a r g e n o n p o l a r g r o u p s , s u c h as v a l i n e a n d l e u c i n e effective.

Furthermore, monocarboxylic

acids

are

ineffective.

are With

c a r b o h y d r a t e s , effectiveness is present o n l y i n those that are s m a l l e r t h a n inulin.

T h u s s t a r c h is ineffective unless h y d r o l y z e d i n t o s m a l l e r f r a g -

ments. D i f f e r e n c e s are also f o u n d i n the effectiveness of v a r i o u s isomers of m o n o s a c c h a r i d e s

(52,117,119).

S i m i l a r studies h a v e b e e n c o n d u c t e d u s i n g i s o l a t e d c a r p m y o s i n as a test system above

a n d the results w e r e

essentially the

Mechanism of the Cryoprotective Effect. Downloaded by OHIO STATE UNIV LIBRARIES on June 2, 2012 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch010

same

as

described

(128).

compounds

T h e results o n a l l of t h e

tested w e r e s y s t e m a t i c a l l y e v a l u a t e d a n d a n a t t e m p t w a s

m a d e to c o r r e l a t e m o l e c u l a r s t r u c t u r e of the test c o m p o u n d c r y o p r o t e c t i v e effectiveness.

w i t h its

T h i s e v a l u a t i o n l e d to a p r o p o s e d

list of

c h e m i c a l attributes t h a t seem to b e c h a r a c t e r i s t i c of c r y o p r o t e c t i v e s u b stances: 1) m o l e c u l e has to possess one essential g r o u p , e i t h e r - C O O H , - O H , or - O P O 3 H 2 , a n d m o r e t h a n one s u p p l e m e n t a r y g r o u p , - C O O H , - O H , - N H , - S H , - S 0 H , a n d / o r - O P 0 H ; 2) 2

3

3

2

the f u n c t i o n a l groups

m u s t be s u i t a b l y s p a c e d a n d p r o p e r l y o r i e n t e d w i t h e a c h other a n d 3 ) the m o l e c u l e m u s t b e c o m p a r a t i v e l y s m a l l

(52,117).

B y t a k i n g these r e q u i r e m e n t s into a c c o u n t , a m e c h a n i s m f o r cryoprotective

effect w a s

proposed.

Each

cryoprotective

the

compound

appears to f u n c t i o n as a c o a t i n g m a t e r i a l b y associating w i t h the p r o t e i n b y i o n i c b o n d i n g o r h y d r o g e n b o n d i n g , the means d e p e n d i n g o n n a t u r e of the c o m p o u n d .

T h e i o n i c c o a t i n g p r e s u m a b l y occurs

the with

a c i d i c or b a s i c a m i n o acids a n d w i t h d i c a r b o x y l i c acids. I n these instances t h e c o m p o u n d associates t h r o u g h t h e i r i o n i c groups w i t h t h e o p p o s i t e l y c h a r g e d sites of p r o t e i n s , t h e r e b y i n c r e a s i n g t h e net c h a r g e of p r o t e i n , i n c r e a s i n g its electrostatic r e p u l s i o n , a n d h i n d e r i n g a g g r e g a t i o n of p r o t e i n m o l e c u l e s d u r i n g f r o z e n storage.

T h e i n c r e a s e d n e t c h a r g e also

may

augment protein hydration. O n t h e other h a n d , the h y d r o g e n - b o n d c o a t i n g appears to b e o p e r a t i v e w h e n c a r b o h y d r a t e s a n d p o l y a l c o h o l s are u s e d .

I n these instances,

t h e a d d e d c o m p o u n d p r e s u m a b l y covers t h e p r o t e i n m o l e c u l e s b y h y d r o g e n b o n d i n g w i t h the O H g r o u p s of the p r o t e i n . T h e " e x t r a " O H g r o u p s of t h e a d d i t i v e m o l e c u l e s w o u l d h y d r o g e n b o n d w i t h w a t e r ,

thereby

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

(52,117). alternate m e c h a n i s m has r e c e n t l y b e e n

hydrates a n d polyalcohols

(129,130).

c o n s i d e r e d for

carbo-

T h e s e a d d i t i v e s m i g h t alter the

state of l i q u i d w a t e r so as to i m p e d e i c e - c r y s t a l f o r m a t i o n . T h i s v i e w finds

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

at l o w t e m p e r a t u r e s

(130).


220

PROTEINS

Cysteine apparently functions

b y coating the protein

t h r o u g h S - S b o n d i n g b e c a u s e this c o m p o u n d of M S G

A T L O W TEMPERATURES

molecules

s u p p l e m e n t s t h e effect

(116,117).

Sodium Glutamate and Its Effect on Muscle Proteins.

M S G is a

h i g h l y effective c o m p o u n d f o r p r o t e c t i n g c a r p a c t o m y o s i n against freeze d e n a t u r a t i o n a n d i t f u n c t i o n s at a c o n c e n t r a t i o n as l o w as 0 . 0 2 5 M . W h e n c a r p a c t o m y o s i n i s stored f r o z e n i n t h e presence o f 0 . 1 - 0 . 2 M g l u t a m a t e , l i t t l e o r n o d e n a t u r a t i o n occurs d u r i n g 10 w e e k s storage a t — 2 0 t o — 3 0 ° C (denaturation w a s monitored b y measuring various physico-chemical Downloaded by OHIO STATE UNIV LIBRARIES on June 2, 2012 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch010

and

biochemical properties of the protein samples).

I t is p a r t i c u l a r l y

r e m a r k a b l e t h a t samples o f a c t o m y o s i n , w h e n s t o r e d i n t h e presence of M S G , exhibit little change i n appearance (electron microscope) d u r i n g f r o z e n storage (51,63,131 MSG LMM,

).

i s also a n effective c r y o p r o t e c t i v e a g e n t f o r m y o s i n , H M M ,

a n d actin that have been isolated from carp muscle

(63,64).

C o l l e c t i v e l y , these results suggest t h a t t h e c r y o p r o t e c t i v e effect o f M S G extends to e a c h c o n s t i t u e n t p r o t e i n a n d e a c h s u b u n i t o f t h e m y o f i b r i l s . E l e c t r o n m i c r o s c o p y studies o f a c t o m y o s i n t h a t h a v e b e e n

frozen

a n d stored i n t h e presence o f 1 M glucose i n d i c a t e t h a t glucose i s essent i a l l y as effective as M S G

(132).

Summary N o w i t c a n b e said that t h e nature a n d mechanisms of protein d e n a t u r a t i o n i n f r o z e n fish m u s c l e a r e b e c o m i n g clearer. D e n a t u r a t i o n is e v i d e n t n o t o n l y at t h e l e v e l o f h i g h l y o r g a n i z e d w h o l e m u s c l e b u t also a t t h e l e v e l of less o r g a n i z e d i n t r a c e l l u l a r constituents i n v o l v i n g associated p r o t e i n systems, i n d i v i d u a l p r o t e i n m o l e c u l e s , a n d s u b u n i t s o f p r o t e i n s . D e t a i l e d i n f o r m a t i o n o n changes i n i n t r a m o l e c u l a r c o n f o r m a t i o n d u r i n g f r e e z i n g is s t i l l l a c k i n g a n d is n e e d e d .

A l s o needed is further

i n f o r m a t i o n o n t h e effect of f r e e z i n g o n i n t r a c e l l u l a r organelles. C o n t r o l l i n g freeze d e n a t u r a t i o n , w h i c h a p p e a r e d v e r y difficult o n l y a short t i m e a g o , i s n o w w i t h i n r e a c h as f a r as m i n c e d m u s c l e is c o n cerned. agent.

M S G h a s p r o v e d t o b e a n e s p e c i a l l y effective

cryoprotective

S t i l l t o b e a c c o m p l i s h e d is t h e c o n t r o l o f p r o t e i n d e n a t u r a t i o n i n

i n t a c t fish m u s c l e .

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120. Arai,K.; Takashi,R.; Saito,T. Bull. Jpn. Soc. Sci. Fish. 1970,36,232236.

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121. 122. 123. 124.


335.

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127. Nakamura,M. Mon. Rep. Hokkaido Municpl. Fish. Exp. Stn. 1970,27, 127-132.

128. 129. 130. 131.

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132. Tsuchiya,Y.; Tsuchiya,T.; Matsumoto,J. J.; unpublished data. RECEIVED June 16, 1978.


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