Overview on the Chemical Deteriorative Changes of Proteins and

1 Overview on the Chemical Deteriorative Changes of

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Proteins and Their Consequences ROBERT E . F E E N E Y Department of Food Science and Technology, University of California, Davis, CA 95616

The deteriorations and the deteriorative reactions of pro teins have been studied by scientists in many different fields for many centuries. In order to give proper tribute to the a l most ancient importance of proteins, it would be necessary to summarize the history of agriculture, medicine, food processing, and much of industry. Scientists and technologists have long recognized both the adverse and beneficial facets of deteriora tive changes in proteins. Putrefactive and coagulative processes might be considered two of the oldest and perhaps most investigated areas of protein chemistry. The disgustingly bad odors from the breakdown prod ucts of sulfur amino acids were, by perforce, of everlasting concern while the coagulative processes were probably part of ancient art, certainly of cooking, as well as of industrial and medical technologies. Most likely it is the ever obtrusive phenomenon of protein coagulation that even today may be re sponsible for the difficulty in differentiating between the initial, more delicate, steps of protein denaturation and the extensively devastating processes surrounding coagulations re sulting from extreme treatments such as boiling. The i s o l a t i o n , p r e s e r v a t i o n , and a n a l y s i s o f p r o t e i n s were among the primary areas o f p r o t e i n chemistry u n t i l the e a r l y 20th c e n t u r y . In n e a r l y every step o f i s o l a t i n g p r o t e i n s , workers encountered the problem o f preventing d e t e r i o r a t i v e r e a c t i o n s and, as a consequence, began t o study the d e t e r i o r a t i v e r e a c t i o n s themselves. Many o f these e a r l i e r s t u d i e s o f d e t e r i o r a t i v e r e a c t i o n s have now been d e s c r i b e d >n q u a n t i t a t i v e chemical terms, but many s t i l l elude the e f f o r t s o f c u r r e n t workers using modern techniques. The immensity o f t h i s subject a t f i r s t made i t seem t h a t an overview could o n l y be done one o f two ways, 1) e s s e n t i a l l y a many-page o u t l i n e o f the d e t e r i o r a t i o n s , o r 2) a s e l e c t i o n o f two or three d e t e r i o r a t i v e r e a c t i o n s and t h e i r coverage i n a compara t i v e and i l l u s t r a t i v e way. There were suggestions from several sources t h a t a more i l l u s t r a t i v e coverage could be based on the 0-8412-0543-4/80/47-123-001$11.75/0 © 1980 American Chemical Society Whitaker and Fujimaki; Chemical Deterioration of Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

2

CHEMICAL DETERIORATION OF PROTEINS

a u t h o r ' s long i n t e r e s t i n d e t e r i o r a t i v e r e a c t i o n s . A t h i r d approach has therefore been taken: a general coverage, w i t h more d e t a i l s f o r those s t u d i e s w i t h which the author has f i r s t h a n d knowledge. Omissions o f c i t a t i o n s to many areas are a conse quence o f these s e l e c t i o n s by the author as w e l l as o f the l a r g e amount o f m a t e r i a l . I t i s hoped t h a t the many f i n e a r t i c l e s i n t h i s volume w i l l compensate f o r these o m i s s i o n s . The Widespread Occurrence o f P r o t e i n D e t e r i o r a t i o n s D e t e r i o r a t i v e r e a c t i o n s o f p r o t e i n s are important i n almost every b i o l o g i c a l system, whether a l i v e or dead. U n t i l r e c e n t l y , most s t u d i e s d e a l t w i t h those d e t e r i o r a t i v e processes o c c u r r i n g on the death o f a system o r i n i t s storage or h a n d l i n g , such as i n food products. More r e c e n t l y , i n an e v e r - i n c r e a s i n g volume, p u b l i c a t i o n s have appeared c i t i n g s t u d i e s o f n a t u r a l l y o c c u r r i n g d e t e r i o r a t i v e changes, both b e n e f i c i a l and d e t r i m e n t a l . Two b i o l o g i c a l l y r e l a t e d processes which have r e c e i v e d a t t e n t i o n f o r many years are the c l o t t i n g s o f m i l k and blood. Blood c l o t t i n g , an exceedingly complex cascading system i n v o l v i n g numerous a c t i v a t i o n s o f zymogens, and subsequent a m p l i f i c a t i o n o f p r o d u c t s , i s a s e r i e s o f syntheses v i a degradations, i n each case i n v o l v i n g breakdown o f a p r e c u r s o r . Many other b i o l o g i c a l proc esses are today under s t r i n g e n t and extensive study. These processes i n c l u d e the a c t i v a t i o n s and i n a c t i v a t i o n s t h a t can occur by the a d d i t i o n s o r removals o f such substances as phos phate groups, carbohydrates, or fragments o f p e p t i d e s , as w e l l as by the l i m i t e d s c i s s i o n ( c l i p p i n g ) o f the peptide chains o f proteins. Denaturation The complex s t r u c t u r e o f p r o t e i n s and the many d i f f e r e n t kinds o f p r o t e i n s t r u c t u r e s are r e s p o n s i b l e f o r the d i f f e r e n t r e sponses o f p r o t e i n s to environmental s t r e s s e s . Denaturation i s a term which has been used w i t h many d i f f e r e n t meanings. In i t s broadest sense i t means "away from the n a t i v e s t a t e " . In i t s more s t r i c t thermodynamic sense, i t i s defined as "change from an ordered to a d i s o r d e r e d s t a t e - an increase i n entropy". A more p r a c t i c a l and everyday working d e f i n i t i o n i s "the change i n p r o t e i n s t r u c t u r e t h a t i s not accompanied by, or caused by, any mak ing o r breaking o f covalent bonds". Denaturation i s therefore a p h y s i c a l process r a t h e r than a chemical one, although i t i s eas i l y induced by chemical reagents, and consequently might be omitted from a d i s c u s s i o n o f chemical d e t e r i o r a t i o n o f p r o t e i n s . Any d i s c u s s i o n o f p r o t e i n d e t e r i o r a t i o n must, however, i n c l u d e at l e a s t a l i m i t e d d i s c u s s i o n o f denaturation because i t i s one o f the most important d e t e r i o r a t i v e r e a c t i o n s o f p r o t e i n s , and i t i s necessary to d i f f e r e n t i a t e between denaturation and chemical de t e r i o r a t i o n s . Denaturation should thus always be c o n s i d e r e d .

Whitaker and Fujimaki; Chemical Deterioration of Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1.

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3

Denaturation i s almost always a p p l i e d to changes i n g l o b u l a r p r o t e i n s . Fibrous p r o t e i n s , such as h a i r , can o b v i o u s l y be made to change t h e i r p h y s i c a l s t a t e by p h y s i c a l means, and such changes might therefore be c a l l e d d e n a t u r a t i o n , but these changes are u s u a l l y not described as d e n a t u r a t i o n . In t h i s d i s c u s s i o n we w i l l r e s t r i c t the coverage to g l o b u l a r p r o t e i n s . Denaturation was e a r l y observed to be a r e v e r s i b l e process. Indeed, Anson (1_) observed 35 years ago t h a t hemoglobin could be heat denatured i n a v a r i e t y o f ways and could be converted back to a s t a t e which had a l l the c h a r a c t e r i s t i c s o f i t s o r i g i n a l n a t i v e s t a t e , as determined by methods a v a i l a b l e at that t i m e . Almost a l l s t u d i e s o f p r o t e i n denaturation now r e v o l v e around not o n l y the denaturation i t s e l f , but a l s o i t s r e n a t u r a t i o n ; perhaps r e n a t u r a t i o n i s a more i n t e r e s t i n g and provocative f i e l d f o r modern r e s e a r c h . There have been many recent reviews o f denaturation and r e n a t u r a t i o n {2,3) and the many r e l a t e d t h e o r e t i c a l a r e a s , such as the e f f e c t s o f amino a c i d composition and microenvironment on p r o t e i n s t r u c t u r e ( 4 ) , the e m p i r i c a l p r e d i c t i o n o f p r o t e i n con formation ( 5 J , and the experimental and t h e o r e t i c a l aspects o f protein folding {§). The processes o f both denaturation and r e n a t u r a t i o n are i n t i m a t e l y r e l a t e d to the s t r u c t u r e s o f n a t i v e p r o t e i n s . Alpha h e l i c e s and 3-pleated sheets c o n s t i t u t e the main s t r u c t u r e s i n most a l l n a t i v e p r o t e i n s . How the h e l i c e s and sheets pack together de pends on the geometrical c h a r a c t e r i s t i c s o f t h e i r s u r f a c e s . Con t a c t s may e x i s t on a l l s i d e s and, although nonpolar (hydrophobic) s i d e chains are b u r i e d i n s i d e , water may be present i n c r e v i c e s as w e l l as i n pools on the s u r f a c e . I t i s through the d i s a r r a n g e ment and rearrangement o f a l l t h e s e , and more, s t r u c t u r e s t h a t the pathways o f denaturation and r e n a t u r a t i o n are d i r e c t e d . Modern t h e o r i e s o f p r o t e i n s t r u c t u r e s t a t e t h a t the amino a c i d sequence o f the p r o t e i n d i c t a t e s the f i n a l conformation o f the p r o t e i n . I f t h i s were s o , exposing the p r o t e i n to a de n a t u r i n g environment should not destroy the d i c t a t o r i a l powers o f the primary s t r u c t u r e ; consequently, p l a c i n g the p r o t e i n back i n to i t s former environment should a l l o w the p r o t e i n to resume i t s native structure. This simple concept i m p l i e s t h a t the n a t i v e form o f the p r o t e i n i s at i t s lowest free energy s t a t e . This i s i l l u s t r a t e d i n Figure 1. This simple thermodynamic p i c t u r e , how e v e r , i s not completely i n l i n e w i t h observed f a c t s . There ap pear to be " s t r u c t u r e s w i t h i n s t r u c t u r e s " i n the p r o t e i n which could introduce k i n e t i c pathways t h a t might put the p r o t e i n i n a d i f f e r e n t f i n a l s t a t e than t h a t represented by the minimal free energy. These " s t r u c t u r e s w i t h i n s t r u c t u r e s " have been termed LINCS ( l o c a l independently nucleated continuous segments) ( 7 ) . P r o t e i n f o l d i n g would then be l i k e t h a t shown i n Figure 2 , where the p r o t e i n does not r o l l up i n t o i t s o r i g i n a l g l o b u l a r b a l l shape i n one process, but r a t h e r assumes small areas o f n a t i v i t y , which then assume the f i n a l n a t i v e s t a t e .


4


-FOLDED

STATES-

-UNFOLDED

STATES -

UNFOLDING

θ

REFOLDING

CONFIGURATION SPACE Avi Publishing Company Figure 1. Highly schematic diagrams of the energy of a protein molecule as a function of chain conformation (4)

Polypeptide chain as synthesized

II. Local folding as dictated by local sequence - formation of LINCS

III.

Figure 2.

Protein folding in terms of the LINCS hypothesis (4)

Tertiary folding of chain at inter-LINC joints to minimize free energy of LINC structure Avi Publishing Company


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FEENEY

Deteriorative

Changes and Their

5

Consequences

I t must be emphasized t h a t denaturations are a l s o p o s s i b l e by many d i f f e r e n t r o u t e s , and the intermediate s t r u c t u r e s through which the p r o t e i n would pass i n assuming a completely denatured s t a t e would be d i f f e r e n t w i t h d i f f e r e n t denaturing c o n d i t i o n s . Denaturation i s a h i g h l y cooperative process. This i s eas i l y seen from the l a r g e values f o r the t r a n s i t i o n s t a t e denatura t i o n constants f o r p r o t e i n s (Table I ) . Very l a r g e ASt v a l u e s , the e n t r o p i e term, are seen - the e a r l y phase o f denaturation i s Table I .

T r a n s i t i o n S t a t e Denaturation Constants Various P r o t e i n s {2)

Protein

Trypsin Pepsin Hemoglobin Egg albumin Peroxidase, milk a

AFÎ(25°C)

aSt

(cal/mole)

(e.u.)

40,200 55,600 75,600 132,000 185,300

44.7 113.3 152.7 315.7 466.0

I n cal/mole/degree a t 25°C.

for

a

(cal/mole) 26,900 21,800 30,100 37,900 46,400 Avi Publishing Company

a h i g h l y c o o p e r a t i v e process; l a t e r phases could be considered cascading processes. Once the c r i t i c a l temperature range f o r de n a t u r a t i o n i s reached, s l i g h t l y increased s e v e r i t y o f the c o n d i t i o n s , such as a small increase i n temperature, g r e a t l y increases the speed o f d e n a t u r a t i o n . I t i s probably f o r t h i s reason t h a t p r o t e i n s are considered so s e n s i t i v e to denaturation i n commer c i a l processing procedures. There i s a d e l i c a t e temperature range, dependent on other environmental c o n d i t i o n s as w e l l , be yond which f u r t h e r treatment may r e s u l t i n undesirable denatured p r o d u c t s , f r e q u e n t l y ending i n coagulums. In common w i t h most l a b o r a t o r i e s engaged i n fundamental r e search on p r o t e i n s , our l a b o r a t o r y has s t u d i e d the denaturation and r e n a t u r a t i o n o f p r o t e i n s . Many o f these studies have been w i t h the two r e l a t e d homologous i r o n - b i n d i n g p r o t e i n s , human serum t r a n s f e r r i n and chicken o v o t r a n s f e r r i n . E a r l i e r s t u d i e s showed t h a t on the b i n d i n g o f i r o n these p r o t e i n s were g r e a t l y s t a b i l i z e d a g a i n s t denaturation by a v a r i e t y o f environmental s t r e s s e s as w e l l as to chemical s c i s s i o n o f t h e i r d i s u l f i d e bonds and to h y d r o l y s i s by p r o t e o l y t i c enzymes ( 8 , 9 ) . Such a seemingly simple question as to why these i r o n complexes, as w e l l as some other p r o t e i n s , are much more s t a b l e than others i s s t i l l impos s i b l e to answer w i t h p r e s e n t l y a v a i l a b l e i n f o r m a t i o n . Our l a b o r a t o r y has r e c e n t l y been concerned w i t h the de n a t u r a t i o n o f chicken egg-white o v o t r a n s f e r r i n by a c i d or urea


6


and the r e n a t u r a t i o n processes from each o f these treatments. When o v o t r a n s f e r r i n i s denatured by a c i d or u r e a , there i s an ex t e n s i v e change i n shape, r e s u l t i n g i n decreases i n both the s e d i mentation v e l o c i t y c o e f f i c i e n t and the d i f f u s i o n c o n s t a n t ; these are accompanied by a corresponding increase i n the v i s c o s i t y (10). O v o t r a n s f e r r i n , i n common w i t h i t s homologous p r o t e i n , serum t r a n s f e r r i n , has two separate i r o n - b i n d i n g s i t e s and i s reported to be the product o f gene d u p l i c a t i o n ( 1 1 ) , s u g g e s t i n g , i n present day terms, t h a t i t may c o n s i s t o f two domains. The p h y s i c a l changes observed on denaturation could be i n t e r p r e t e d as being due to an unfolding o f the molecule, a change which would perhaps be i n agreement w i t h a model o f two separate do mains unfolding a t , o r near, some p o s s i b l e connecting l i n k . An equal p o s s i b i l i t y , however, would be a simple s w e l l i n g o f the molecule. Our l a b o r a t o r y r e c e n t l y reported on a study o f the conforma t i o n a l p r o p e r t i e s o f o v o t r a n s f e r r i n , i t s denatured form (by treatment w i t h a c i d o r urea) and i t s renatured form. The samples were denatured i n 7.2 M urea o r i n a c i d i c (pH 3) c o n d i t i o n s f o r periods up to a few hours. Samples were renatured by d i l u t i o n and adjustment o f the pH to n e u t r a l i t y , o r by simple d i l u t i o n o f the u r e a . Combined data from q u a s i - e l a s t i c l i g h t s c a t t e r i n g and t r a n s i e n t e l e c t r i c b i r e f r i n g e n c e were used to estimate the mo l e c u l a r dimensions under the various c o n d i t i o n s . A n a l y t i c a l u l t r a c e n t r i f u g a t i o n was used to determine the changes i n s e d i mentation c o e f f i c i e n t , and changes i n h e l i c i t y were c a l c u l a t e d from c i r c u l a r d i c h r o i s m d a t a . The course o f r e n a t u r a t i o n as measured by the increase i n d i f f u s i o n during r e n a t u r a t i o n o f a c i d denatured o v o t r a n s f e r r i n i s seen i n F i g u r e 3. S t r u c t u r a l changes from c i r c u l a r d i c h r o i s m data o f the n a t i v e , urea-denatured, and renatured sample are seen i n F i g u r e 4. A summary o f the data and c a l c u l a t i o n s from the urea denaturation s t u d i e s i s i n Table I I . The c o n c l u s i o n from these data was t h a t , on d e n a t u r a t i o n , the p r o t e i n assumed a more expanded g l o b u l a r form than the n a t i v e sample; i n other words, i t s w e l l e d , r a t h e r than unfolded. Chemical Reactions o f Amino Acids o f Concern i n D e t e r i o r a t i o n s Approximately 150 d i f f e r e n t amino a c i d residues have been reported i n p r o t e i n s ( 1 5 ) . A t l e a s t h a l f o f these could undergo chemical d e t e r i o r a t i o n s under the c o n d i t i o n s o f s t r e s s u s u a l l y encountered. Many o f these d e t e r i o r a t i v e r e a c t i o n s i n v o l v e h y d r o l y t i c s c i s s i o n s , not o n l y o f peptide bonds but o f the many d i f f e r e n t nonprotein substances added c o v a l e n t l y to p r o t e i n s p o s t r i b o s o m a l l y . These s u s c e p t i b l e s i d e chain groups are i n d o l e , phenoxy, t h i o e t h e r , amino, i m i d a z o l e , s u l f h y d r y l , and d e r i v a t i v e s o f s e r i n e and threonine (such as 0 - g l y c o s y l o r 0 - p h o s p h o r y l ) , the d i s u l f i d e s o f c y s t i n e , and, o f c o u r s e , the amides (such as asparagine and g l u t a m i n e ) . With strong a c i d or a l k a l i , other r e s i d u e s , such as s e r i n e and t h r e o n i n e , a l s o are l e s s s t a b l e .


FEENEY

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Figure 3. Time development of the renaturation process of acid-denatured ovotransferrin. Concentration of ovotransferrin in the denatured state (pH 3) was approximately 10 mg/ml. The sample was diluted 10:1 in Tris buffer at pH 7.8. Note comparison values of D for steady-state native (A), and renatured (O) samples (12). t

210

220

230

WAVELENGTH (nm)

240

250

Figure 4. Circular dichroism spectra from 200 to 250 nm for ovotransferrin. Mean residue weight of 112 is used. Native, c = 1.04 mg/mL ( ); 7.2M urea-denatured sample, c = 1.04 mg/mL ( ); and renatured sample, c — 0.83 mg/mL.(- · -)(12).



z

2

2

b

w

1.91+0.13

z

+

6

0.72+0.08

"

-

0

2.17+0.32

¥w V

Sense of Biref.

(12)

« D / ( l - 3 o ) were

< [

Viscosity Corrected to w, 20°C

Urea Denaturation

D and D are the weight- and z-averaged d i f f u s i o n c o e f f i c i e n t s , r e s p e c t i v e l y . W L Weight averaging i s obtained d i r e c t l y by t r a n s i e n t e l e c t r i c b i r e f r i n g e n c e .

T h e d i s p e r s i o n f a c t o r , 6 = 0.1 i n the f o l l o w i n g e q u a t i o n :

0.93+0.07

6.00+0.43

4.21+0.30

Renatured

a

0.81+0.11

3.86+0.21

2.70+0.15

Denatured 7.2 M urea

yS

0.82+0.14

1

7

Transient Electric Birefringence

6.14+0.43

cm s"

t

χ 1 0

9

+0.3

7

Conversion

4.31

- 1

χ 10

cm s

t

Quasi-elastic Light Scattering

Denaturation and Renaturation o f O v o t r a n s f e r r i n :

Native

Sample

Table I I .

0.31

21

42 22

68

84

67

Native

Denatured 7.2 M urea

Renatured


Reason ^Values values Values should

9

0.36

4.16

0.28

m

e

9 1 mg s o i n 32

19 29

η (H) 6 (0)

α%

Circular Dichroism f

59

75

57

R.C.%

results,

82

< 5 (0)

86

%

+3

Fe Binding

f o r l a r g e value i s not known at t h i s t i m e . i n p a r e n t h e s i s are c a l c u l a t e d from method o f Chen and Yang ( 1 3 ) , other by method o f G r e e n f i e l d and Fasman ( 1 4 ) . are c a l c u l a t e d from d i r e c t c o l o r determinations on s o l u t i o n s . True values be h i g h e r .

ν = 0 . 7 3 , m o l e c u l a r weight = 77,000 values used i n determining 6 j .

d—

e

6

d

Solvation Factor

Oblate e l l i p s o i d values were d i s c - l i k e , incompatible w i t h other reported

0.33

0.50

Ρ

b

Â

P e r r i n eq Prolate E l l i p s o i d

c

Denaturation and Renaturation o f O v o t r a n s f e r r i n : Urea Denaturation (12) (continued)

a

Sample

Table I I .

CNJ Ο

10


But even the r e l a t i v e l y r e s i s t a n t residues are attacked by free radicals. When p r o t e i n s are d e l i b e r a t e l y t r e a t e d w i t h chemicals i n order t o d e r i v a t i z e them, the r e a c t i o n c o n d i t i o n s may a l s o cause chemical d e t e r i o r a t i v e s i d e r e a c t i o n s . Some o f the more common ones are l i s t e d i n Table I I I . Inspection o f Table I I I shows t h a t many o f these e f f e c t s a r e those found i n d e t e r i o r a t i v e r e a c t i o n s Table I I I .

P o s s i b l e Chemical Side Reactions during Protein Modification

Treatment

Effects

Peptide bonds

A l k a l i n e pH A c i d i c pH A l k , heat

Hydrolysis ΓΜ) acyl s h i f t Racemization

Thiol

Oxidation

- S - S - , acids

D i s u l f i d e bonds

Reduction A l k a l i n e pH

-SH, mispairing Hydrolysis, 3 elimination

Methionyl

Oxidation

Oxy s u l f u r s

Amide groups

A l k a l i n e pH

Hydrolysis

O-Glycosyl

A l k a l i n e pH

3 Elimination

O-Phosphoryl

A l k a l i n e pH

3 Elimination

Groups

groups

groups

Biochemistry

r e s u l t i n g from other treatments; i n other words, they a r e , i n some c a s e s , environmentally produced r a t h e r than a d i r e c t r e s u l t o f the chemical procedure. An example o f a v a r i e t y o f r e a c t i o n s caused by a r e l a t i v e l y m i l d reagent are those w i t h hydrogen per oxide ( F i g u r e 5 ) . Hydrogen peroxide r e a d i l y r e a c t s w i t h three d i f f e r e n t s i d e chain groups under m i l d c o n d i t i o n s , and the extent o f the r e a c t i o n i s i n f l u e n c e d by the presence o f other sub s t a n c e s , such as organic a c i d s , t h a t can form more a c t i v e o x i d i z i n g agents. As i s the case w i t h some chemical changes o c c u r r i n g i n b i o l o g i c a l systems, such as the b l o o d - c l o t t i n g cascade system, de t e r i o r a t i v e r e a c t i o n s considered to have a b e n e f i c i a l e f f e c t are found i n foods. For example, the M a i l l a r d r e a c t i o n (17,18) i s used t o produce f l a v o r s and c o l o r s i n such foods as beverages and baked goods. Heat treatment ( i n v o l v i n g denaturation) has been found t o increase the n u t r i t i o n a l value o f raw soybean meal by


FEENEY

(i)

Deteriorative

Changes and Their

©-s-s-QJ^^-s-s-Gh

® _ s h M

®-SCH

3

[0] » ® - S 0 H 3

.(?)-SOH

(2)

Consequences

+ H 0 2

-M^^-SOpH-

> ®-S -CH

2

i

+H 0

3

2

0

(3)

(P)-SH + 3 H-cf° 0-OH

(4)

® - S C H - + 2 H-ct° 0-OH

(5)

® - / j

H

+ H 0 2

2

> (P)-S0,H + 3 H - c t ° OH

- ^ - >

J

> © - S - C H , + 2 H-cf Γ 3 OH 0

S e v e r a 1

products

Holden-Day Figure 5.

Oxidations of amino acids in proteins with peroxide (16)


12


i n a c t i v a t i n g the c o n s t i t u e n t i n h i b i t o r s (19,20) (Tables IV and V). Table I V .

Treatment

P r o t e i n N u t r i t i v e Value o f Raw and Cooked Red Gram (20)

Protein

Trypsin I n h i b i t o r

Efficiency Ratio

( u n i t s / 1 0 0 mg)

Raw

0.68

Cooked

1.43

10.8 Χ 1 0 "

3

Nil Avi Publishing Company

Table V,

Treatment

Comparison o f the E f f e c t s o f Heating Methods on P r o t e i n N u t r i t i v e Value o f Soy Meal (20)

Protein

Available

Efficiency Ratio

Lysine (%)

Unheated

0.63

58

Dry heat

1.00

53

Autoclave

1.75

46

Microwave

1.86

58 Avi Publishing Company

D e t e r i o r a t i v e Reactions I n v o l v i n g L y s i n e Amino groups are e x c e l l e n t n u c l e o p h i l e s , and there are many e p s i l o n amino groups o f l y s i n e s on p r o t e i n s . Three o f the most common types o f d e t e r i o r a t i o n s i n v o l v i n g l y s i n e s a r e : nonenzymatic browning ( M a i l l a r d ) w i t h reducing sugars, heat-induced damage i n v o l v i n g i s o p e p t i d e formation w i t h the carboxyl groups of a s p a r t i c and glutamic a c i d s o r t h e i r amides (Figure 6 ) , and formation o f c r o s s - l i n k e d products by i n t e r a c t i o n w i t h a l k a l i n e degradation products, such as dehydroalanine. The M a i l l a r d r e a c t i o n i n v o l v e s a t t a c k o f the n i t r o g e n o f the amino group on the carbon atom o f the c a r b o n y l , sometimes f o l l o w ed by removal o f water t o produce the S c h i f f base (17j (Figure 7 ) . D e t a i l e d coverage o f the M a i l l a r d r e a c t i o n i s given e l s e where i n t h i s volume by Hodges ( 1 8 ) , so o n l y a few examples, p a r t i c u l a r l y those w i t h which the author has had some r e l a t i o n -


FEENEY

Deteriorative

Changes

and Their

Consequences

A. By Amidation Prot-NH„

Prot-NH +

Prot-C

H0 2

Prot-C=0 V

B. By Transamidation Prot-NH

Prot-NH

0

+ NH

0

II Prot-C-NH

Figure 6.

Prot-C=0

0

Possible alternative reactions for formation of amide cross-linkages in proteins during heating

• R-NH 2

0

r ^

^

A-C-B I OH

r

-h o ?

^

^

A-C-B

Advances in Protein Chemistry

Figure 7.

Reaction mechanism of a strongly basic amine like an aliphatic amine or hydroxylamine with a carbonyl group (17)


14


s h i p , w i l l be given here. M a i l l a r d r e a c t i o n s are often considered to occur only under c o n d i t i o n s where heat i s a p p l i e d or i n d r i e d samples stored f o r c o n s i d e r a b l e periods o f time. In some m a t e r i a l s , however, such as l i q u i d chicken egg w h i t e , where there i s a high c o n c e n t r a t i o n o f glucose (0.5%) and an a l k a l i n e pH (greater than 9 ) , r e a c t i o n o f the amino groups o f l y s i n e w i t h glucose occurs w i t h i n a few days o f storage o f the i n t a c t s h e l l egg at room temperature (21). These r e a c t i o n s r e s u l t i n changes i n e l e c t r o p h o r e t i c patterns ( F i g u r e 8 ) , which caused confusion i n genetic s t u d i e s u n t i l the nature o f the u n c o n t r o l l e d d i s c r e p a n c i e s was understood. The products o f at l e a s t one M a i l l a r d r e a c t i o n caused the s u f f e r i n g o f m i l l i o n s o f n o n - s c i e n t i s t s before the problem was u n r a v e l l e d . This was the r e a c t i o n o c c u r r i n g i n d r i e d whole eggs, o f which m i l l i o n s o f pounds were eaten by American servicemen i n World War I I . When d r i e d whole eggs were transported i n the holds o f ships to the South P a c i f i c Islands and stored i n j u n g l e depots, the M a i l l a r d r e a c t i o n u s u a l l y r e s u l t e d i n products t h a t were so e x t e n s i v e l y p h y s i c a l l y a l t e r e d and had such v i l e and nauseating odors and f l a v o r s t h a t many shipments had to be d i s carded. Much o f t h i s , consumed by agonizing army men who r e ceived a p i l e o f such a disagreeable product as scrambled eggs i n t h e i r mess k i t s , was f r e q u e n t l y s u f f i c i e n t l y bad to cause the men to vomit. The author f e e l s a c l o s e k i n s h i p to the d r i e d egg develop ment because he was on the r e c e i v i n g end o f the devastating de t e r i o r a t i v e r e a c t i o n s when the products were dropped i n h i s mess k i t f o r many months i n New Guinea (now Papua and West I r i a n ) and the P h i l i p p i n e s i n 1944-45, and because half-a-dozen years l a t e r he was nominally i n charge o f the research group at the U . S . Department o f A g r i c u l t u r e ' s Western Regional Research Laboratory r e s p o n s i b l e f o r u n r a v e l l i n g the cause. The research was l e d by Dr. Leo K l i n e . Before Dr. K l i n e ' s work the foul products had been a t t r i b u t e d to a M a i l l a r d r e a c t i o n i n v o l v i n g the amino groups o f the p h o s p h o l i p i d s and carbonyls formed by o x i d a t i o n s and hydrolyses o f the l i p i d s (23). As a r e s u l t o f these f i n d i n g s , d r i e d eggs used by the m i l i t a r y f o r the Korean war were a c i d i f i e d before d r y i n g and were packed w i t h added sodium bicarbonate. The a c i d i f i c a t i o n slowed the M a i l l a r d r e a c t i o n , and the bicarbonate served to n e u t r a l i z e the a c i d on r e c o n s t i t u t i o n . The r e s u l t was a more s t a b l e product, but some d e t e r i o r a t i o n s t i l l occurred and the bicarbonate gave a soapy t a s t e . K l i n e ' s group showed there was a much s i m p l e r e x p l a n a t i o n f o r the source o f the carbonyls the glucose (24). Glucose had been overlooked because the de t e r i o r a t i v e r e a c t i o n occurred i n the l i p i d phase. Today the p o s s i b i l i t y o f a r e a c t i o n between the h y d r o p h i l i c head o f a p h o s p h o l i p i d and a water s o l u b l e component seems so obvious as to be t r i v i a l , but t h i r t y years p r e v i o u s l y i t was not. The g l u c o s e , accounting f o r n e a r l y 95% o f the reducing sugar, could be removed by fermentation (25) or by o x i d a t i o n w i t h glucose oxidase ( c a t a -


1.

FEENEY

A

Deteriorative Changes and Their Consequences

37°

Β Control C 37° D Control D 37° Ε Control F 37° Journal of Biological Chemistry Figure 8. Starch-gel electrophoretic patterns of incubated infertile eggs. Egg whites were all white Leghorn containing globulin A . Eggs were incubated at 37°C for 6 days or stored at 2°C for 6 days (controls). Letters refer to hen (22). t


15

16


l a s e added to remove HoOo) to g i v e a s t a b l e product when packed i n the absence o f a i r (Figure 9 ) . There a r e , o f course, many carbonyl compounds formed by hyd r o l y t i c o r o x i d a t i v e d e t e r i o r a t i o n s o f l i p i d c o n s t i t u e n t s , and most o f these are p o t e n t i a l l y capable o f e n t e r i n g i n t o M a i l l a r d reactions with proteins. One such product i s reputedly malonaldehyde (26) (Figure 10). D e t e r i o r a t i o n s I n v o l v i n g D i s u l f i d e Linkages S u l f h y d r y l groups and d i s u l f i d e bonds, and t h e i r i n t e r r e l a t i o n s h i p s , are important groups a f f e c t i n g the p r o p e r t i e s o f the m a j o r i t y o f p r o t e i n s and are under continuous study by p r o t e i n chemists. Indeed, the r e d u c t i o n o f d i s u l f i d e s to form s u l f hydryl s , and the r e o x i d a t i o n o f these to re-form the c o r r e c t p a i r i n g s (Figure 11), are i n t i m a t e l y r e l a t e d to the e n t i r e sub j e c t o f p r o t e i n conformation and conformational changes (27). One o f the long-enduring problems i n v e s t i g a t e d i n the a u t h o r ' s l a b o r a t o r y has been t h a t o f the d e t e r i o r a t i v e breakdown o f t h i c k egg white and the egg white p r o t e i n s on the surface o f the y o l k membrane during the storage and/or i n c u b a t i o n o f s h e l l eggs (10). The breakdown can be simulated by the a d d i t i o n o f mercaptans or other d i s u l f i d e - b r e a k i n g agents (Figures 12 and 13). Reduction and r e o x i d a t i o n have a l s o been used to f o l l o w r e activations of biologically active proteins. I t was found t h a t an intermediate form o f turkey ovomucoid (Figure 1 4 ) , before com p l e t e o x i d a t i o n , was a c t u a l l y s l i g h t l y more a c t i v e as an i n h i b i t o r o f t r y p s i n than was e i t h e r the n a t i v e p r o t e i n or the com p l e t e l y r e o x i d i z e d product. H y d r o l y t i c s c i s s i o n s o f d i s u l f i d e s have been i n t e n s i v e l y s t u d i e d , p a r t i c u l a r l y by the l a b o r a t o r y o f Schoberl i n Germany (30). I t has been shown t h a t r e a c t i o n s such as these can occur on the a d d i t i o n o f small amounts o f metal i o n s , such as copper or mercury (31). Lysozyme, f o r example, i s r a p i d l y i n a c t i v a t e d by small amounts o f c u p r i c i o n (Figure 15). But i n many c a s e s , r e s u l t s o f t h i s nature have not been d e f i n i t e l y shown to be due to d i s u l f i d e bond s p l i t t i n g . Other p o s s i b l e causes, such as r a c e m i z a t i o n , must a l s o be c o n s i d e r e d . E f f e c t s o f A l k a l i on P r o t e i n s A l k a l i has long been used on p r o t e i n s f o r such processes as the r e t t i n g o f wool and c u r i n g o f c o l l a g e n , but more r e c e n t l y i t has r e c e i v e d i n t e r e s t from the food i n d u s t r y . A l k a l i can cause many changes such as the h y d r o l y s i s o f s u s c e p t i b l e amide and peptide bonds, racemization o f amino a c i d s , s p l i t t i n g o f d i s u l f i d e bonds, beta e l i m i n a t i o n , and formation o f c r o s s - l i n k e d products such as l y s i n o a l a n i n e and l a n t h i o n i n e . Our own l a b o r a t o r y has s t u d i e d these r e a c t i o n s and, i n par t i c u l a r , beta e l i m i n a t i o n s i n v o l v i n g d i s u l f i d e s (Figure 16) and


1.

FEENEY

Deteriorative

Changes and Their

17

Consequences

300

350

500

WAVE LENGTH IN MILLIMICRONS

Food Technology (Chicago)

Figure 9. Effect of glucose removal on storage-induced changes in absorption spectra of ether extracts of stored dried eggs. The two samples illustrated were spray-dried powders stored 5 weeks at 37.5°C (25). Left side, untreated right side, glucose-free. Control ( ); air pack ( );N pack(· -). ;

t

oxidation ->

Arachidonate or Linolenate

Malonaldehyde + Other products

NH

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0=CHCH=CHOH + Enzyme^ Malonaldehyde active\

ά

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NHCH ^ CH Intramolecular N=CH cross-linking inactive

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25

26

C H E M I C A L DETERIORATION OF PROTEINS

food, but there are no records o f t h i s , and there are no records o f any human ailment a s c r i b e d to t h i s agent. There i s always a p o s s i b i l i t y o f a t l e a s t minor changes, im portant o r unimportant, creeping i n t o processes when chemicals are used. In the enzymatic o x i d a t i o n o f glucose to g l u c o n i c a c i d to remove the carbonyl groups o f glucose i n c e r t a i n f o o d s t u f f s , such as i n the p r e p a r a t i o n o f d r i e d egg w h i t e , hydrogen peroxide i s a product o f the r e a c t i o n , r e q u i r i n g the a d d i t i o n o f c a t a l a s e for i t s decomposition. Hydrogen peroxide (see F i g u r e 5) i s o c c a s i o n a l l y used as a s t e r i l i z i n g agent and i s even added to food stuffs. A c t i v e S i t e S e l e c t i v e Reagents - N a t u r a l l y O c c u r r i n g Toxins and Laboratory Tools The term " a c t i v e s i t e s e l e c t i v e reagents" i s used to de s c r i b e s e v e r a l d i f f e r e n t kinds o f reagents t h a t r e a c t c o v a l e n t l y i n the a c t i v e center o f an enzyme. The term i s w i d e l y used to i n c l u d e agents t h a t r e a c t i n a p a r t i c u l a r p a r t o f a p r o t e i n doing a s p e c i f i c task i n some kind o f biochemical process. With an enzyme the reagent u s u a l l y resembles a s u b s t r a t e and by some r e a c t i o n remains i n the a c t i v e c e n t e r , thereby i n a c t i v a t i n g the enzyme o r l e a v i n g a piece o f the reagent i n the c e n t e r . This procedure i s used to l a b e l or f i n d the groups t h a t are i n the a c t i v e center as w e l l as t o i n a c t i v a t e the enzyme, although i t i s not necessary t h a t i n a c t i v a t i o n o c c u r . There are many d i f f e r e n t d e f i n i t i o n s d e s c r i b i n g the a c t i v e c e n t e r , a c t i v e s i t e , combining s i t e s , and a l l o s t e r i c s i t e s o f enzymes ( 1 6 ) . One o f the more commonly used d e f i n i t i o n s i s t h a t the a c t i v e center o f the enzyme i s t h a t area o r t h a t place i n the enzyme which contains the a c t i v e s i t e o f an enzyme and everything e l s e t h a t i s i n t h a t a r e a , u s u a l l y meaning at l e a s t p a r t o f the binding s i t e f o r the s u b s t r a t e and other groups t h a t are there i n order to maintain s t r u c t u r e , r e a c t w i t h water, o r provide a hydro phobic pocket, e t c . The a c t i v e s i t e i n t u r n i s u s u a l l y taken as t h a t p a r t o f the enzyme which does the work, i . e . , the c a t a l y t i c process. The term " a c t i v e s i t e s e l e c t i v e reagents" therefore r e a l l y should be " a c t i v e area s e l e c t i v e r e a g e n t s , " but the former term i s so e x t e n s i v e l y used t h a t we w i l l continue to employ i t here. A c t i v e s i t e s e l e c t i v e reagents can be c l a s s i f i e d i n various manners. One way i s to d i v i d e them according to how they r e a c t (Table I X ) . In such a c l a s s i f i c a t i o n one f i n d s substrates t h a t can be c o v a l e n t l y attached by chemical treatment o f the enzyme w h i l e i t i s c a t a l y z i n g some change i n the s u b s t r a t e . An example i s the r e a c t i o n o f f u n c t i o n a l amino groups by the enzyme muscle a l d o l a s e a c t i n g on glyceraldehyde and reduced by cyanoborohydride ( 1 6 ) . A second type o f a c t i v e s i t e s e l e c t i v e reagent i s when there i s a pseudosubstrate, such as d i i s o p r o p y l f l u o r o p h o s p h a t e . A c t i n g


1.

FEENEY

Deteriorative

Changes and Their

Table I X .

A c t i v e S i t e S e l e c t i v e Reagents

Type

Consequences

27

Mechanism

Substrate

Normal intermediate product can be c o v a l e n t l y a t t a c h e d , e . g . , by r e duction

Pseudosubstrate

Product i s poor l e a v i n g group, e . g . , DFP

A f f i n i t y Reagent: General

"Double-headed" - one l i k e s u b s t r a t e , other c h e m i c a l l y r e a c t i v e

Photoaffinity

"Double-headed" - one l i k e s u b s t r a t e , other converted to c h e m i c a l l y reac t i v e group by p h o t o a c t i v a t i o n

Product o f Enzyme Reaction-"Suicide" Reagent

Is a s u b s t r a t e , part o f which i s con verted to c h e m i c a l l y r e a c t i v e group by enzyme c a t a l y s i s


28

C H E M I C A L DETERIORATION O F PROTEINS

on the enzyme t r y p s i n , such a pseudosubstrate may c o n t a i n a poor l e a v i n g group and thereby remain on the a c t i v e s i t e as a d i i s o propylphosphoryl e s t e r o f the s e r i n e i n t r y p s i n . A t h i r d type i s the one t h a t i n c l u d e s a number o f s u b c l a s s i f i c a t i o n s , a l l o f which come under the general term " a f f i n i t y s u b s t r a t e s ( r e a g e n t s ) " . The two types described above a l s o have a f f i n i t y c h a r a c t e r i s t i c s , but these l a t t e r ones are d i f f e r e n t . A sketch o f an a f f i n i t y reagent i s shown i n Figure 20. In t h i s , the b i n d i n g group i s what the enzyme recognizes and b i n d s , w h i l e the group marked X , the c o v a l e n t l y r e a c t i v e group, now i s able to form a c o v a l e n t bond somewhere i n the v i c i n i t y o f the a c t i v e c e n t e r o r a t i t s p e r i p h e r y , p r o v i d i n g , o f course, t h a t there i s a s u i t a b l y s u s c e p t i b l e amino a c i d s i d e chain i n these l o c a t i o n s . The a f f i n i t y reagent i s t h e r e f o r e always a double-headed one, one head resembling the s u b s t r a t e and the other head the working head to form a c o v a l e n t bond. There are a t l e a s t three kinds o f a f f i n i t y reagents: those i n which the c o v a l e n t l y r e a c t i n g group i s a l r e a d y present i n the reagent, those i n which the c o v a l e n t r e a c t i v e group must be generated by an external a c t i o n such as p h o t o a c t i v a t i o n , and those i n which the enzyme i t s e l f generates the r e a c t i v e group. These l a t t e r have been termed " k t r e agents" because t h e i r i n t e r a c t i o n occurs as a r e s u l t o f the enzymatic c a t a l y s i s to form the r e a c t i v e group, o r " s u i c i d e r e agent" because the enzyme k i l l s i t s e l f by a c a t a l y t i c a c t i o n (41-44). These have a very much higher s p e c i f i c i t y than other a f f i n i t y reagents because they not o n l y have the s p e c i f i c i t y o f b i n d i n g i n common w i t h the o t h e r s , but they a l s o have the spec i f i c i t y o f c a t a l y s i s which the others do not have. In t h i s respect they should thus be a "perfect drug". These " s u i c i d e reagents" w i l l be described i n d e t a i l e l s e where i n t h i s volume ( 4 4 ) , so o n l y one phase w i l l be b r i e f l y mentioned here. Of p a r t i c u l a r i n t e r e s t to food and n u t r i t i o n r e searchers are the n a t u r a l l y o c c u r r i n g t o x i n s which i n v o l v e a " s u i c i d e " mechanism (42_). Some o f these can be consumed i n foods or feeds and commonly occur i n a number o f d i f f e r e n t p l a n t sources. A very common t o x i n i s the b e t a - a m i n o p r o p i o n i t r i l e present i n l a t h y r i t i c legumes, and another i s the w i l d f i r e t o x i n (42) ( F i g u r e 2 1 ) . c a

Chemical D e t e r i o r a t i o n s to Purposely D e r i v a t i z e P r o t e i n s Under t h i s heading might be placed many o f the r e a c t i o n s a l ready d i s c u s s e d , but there are several t h a t f i t more a p p r o p r i a t e l y i n such a c l a s s i f i c a t i o n . One o f t h e s e , w i t h which the author has been a s s o c i a t e d , i s the formation o f i n a c t i v e d e r i v a t i v e s o f p r o t e o l y t i c enzymes by a l k a l i n e beta e l i m i n a t i o n o f a d e r i v a t i v e o f the a c t i v e s i t e s e r i n e o f t r y p s i n (45) (Figure 2 2 ) . This m o d i f i c a t i o n uses an a f f i n i t y reagent followed by a second chemi cal m o d i f i c a t i o n , the a l k a l i n e beta e l i m i n a t i o n , to form the product. The products o f the r e a c t i o n w i t h t r y p s i n and chymo-


FEENEY

Deteriorative

Changes and Their

Consequences

Binding Group ovalently Reactive Group

Figure 20. Diagram of principle of affinity labelling of a reactive site. In affinity labelling there is (a) a binding group that resembles the type of substance (sub strate, antigen, etc.) with which the protein normally interacts specifically, and (b) an additional group, a covalently reactive group, capable of forming a covalent bond in the reactive site. Affinity reagents are usually classified into three different types: general affinity, photoaffinity, and "suicide" affinity (40).

Ο

I NH —ÇH—C—NH—CHCOr 3

CHOH

I

Enz—B:

CH

a

Ο R

- a Enz—B-

-OH

NH

V *NH, Ο

I

II

' R - —CH—CH —CH—C—NH—CHCO, f

CHOH

I CH, Accounts of Chemical Research

Figure 21.

Inhibition of glutamine synthetase by wildfire toxin (42)


30

C H E M I C A L DETERIORATION OF PROTEINS

>

X:~^C-^OS0 Ar 2

X

—

< \

+ ATS0 "

(1 )

3

OH~ H

„

Κ /

H

\

C—C £

H

• OS0 Ar 2

/ C = C

+ ArS0 ~ + H 0 3

(2)

2

Η Holden-Day

Figure 22.

Displacement of an aromatic sulfonate (weakly basic) by (1) nucleophilic attack or (2) β elimination with alkali (16)


1.

FEENEY

Deteriorative

Changes and Their

Consequences

31

t r y p s i n are c a l l e d anhydrotrypsin and anhydrochymotrypsin, r e s p e c t i v e l y . The r e a c t i o n i s s i m i l a r i n mechanism to the a l k a l i n e beta e l i m i n a t i o n o f O-phosphoryl o r 0 - g l y c o s y l groups described above. The products, anhydrotrypsin or anhydrochymotrypsin, are very useful i n enzyme chemistry because the o v e r a l l s t r u c t u r e and conformation o f an enzyme i s very l i t t l e a f f e c t e d . They have been used i n several d i f f e r e n t s t u d i e s , one o f which i s the i n t e r a c t i o n o f p r o t e o l y t i c enzymes w i t h s p e c i f i c p r o t e i n i n h i b i t o r s . The anhydro d e r i v a t i v e s w i l l form h i g h l y a s s o c i a t e d com plexes w i t h the i n h i b i t o r s i n a manner very s i m i l a r to t h a t o f the n a t i v e c a t a l y t i c a l l y a c t i v e enzymes (Table X ) . In f a c t , i n some cases they may be as e f f e c t i v e , or even b e t t e r , i n combining w i t h the i n h i b i t o r s than the n a t i v e enzyme. These data have been used as evidence t h a t c a t a l y t i c a c t i o n , i n c l u d i n g formation o f a t e t r a h e d r a l adduct o r an enzyme a c y l bond, i s not necessary f o r the formation o f the i n h i b i t o r y complex ( 4 7 ) . Another s i m i l a r type o f r e a c t i o n has been the use o f an a f f i n i t y reagent ( 2 , 3 * - e p o x y p r o p y l 3 - g l y c o s i d e o f d i - ( N - a c e t y l D-glucosamine) to r e a c t w i t h a carboxyl group o f an a s p a r t i c a c i d i n the a c t i v e c e n t e r o f the enzyme lysozyme (48) (Figure 2 3 ) . Then t h i s reagent can be removed from the enzyme by r e d u c t i o n . Since the bond between the a f f i n i t y reagent and the carboxyl group o f lysozyme i s an e s t e r bond, the carboxyl group o f as p a r t i c a c i d o f the enzyme i s now changed by r e d u c t i o n to an a l c o h o l . The new residue i s therefore an a s p a r t i c a c i d w i t h a carboxyl group changed to a hydroxyl to g i v e 2-amino-4-hydroxyb u t y r i c a c i d (homoserine). The homoserine lysozyme has proper t i e s so s i m i l a r t o those o f the o r i g i n a l enzyme t h a t i t forms t i g h t complexes w i t h s u b s t r a t e s (Table X I ) . A m o d i f i c a t i o n embodying several o f the d i f f e r e n t d e t e r i o r a t i v e r e a c t i o n s discussed i n t h i s a r t i c l e was r e c e n t l y s t u d i e d i n our l a b o r a t o r y (50). Two d i f f e r e n t avian ovomucoids w i t h d i f f e r ent i n h i b i t o r y p r o p e r t i e s a g a i n s t p r o t e o l y t i c enzymes were modi f i e d by the a l k a l i n e b e t a - e l i m i n a t i o n r e a c t i o n so as to form new c o v a l e n t c r o s s - l i n k s c o n s i s t i n g o f l a n t h i o n i n e and l y s i n o a l a n i n e . One ovomucoid was t u r k e y , which has a double-headed c h a r a c t e r w i t h independent s i t e s f o r forming an i n h i b i t o r y complex w i t h bovine t r y p s i n a t one s i t e and bovine alpha-chymotrypsin at the other s i t e . Both o f these s i t e s are r e l a t i v e l y strong b i n d i n g s i t e s as compared to the s t r e n g t h s o f b i n d i n g o f other i n h i b i tors. In a d d i t i o n , the alpha-chymotrypsin b i n d i n g s i t e w i l l a l s o accept the b a c t e r i a l enzyme s u b t i l i s i n , which has an a f f i n i t y f o r the i n h i b i t o r o f about the same order o f magnitude as does a l p h a chymotrypsin. Consequently, the two enzymes compete about e q u a l l y f o r the same s i t e . In c o n t r a s t , penguin ovomucoid has the same two s i t e s as turkey ovomucoid, one f o r t r y p s i n and one f o r chymotrypsin, but the r e l a t i v e a f f i n i t y f o r the d i f f e r e n t enzymes i s q u i t e d i f f e r e n t . The t r y p s i n s i t e i s r e l a t i v e l y weak, and the chymotrypsin s i t e i s q u i t e strong f o r s u b t i l i s i n but much weaker f o r alpha-chymotrypsin. When penguin and turkey ovomu1


32

C H E M I C A L DETERIORATION O F PROTEINS

Table X .

Comparison o f A s s o c i a t i o n E q u i l i b r i u m Constants f o r I n a c t i v e and A c t i v e Enzymes (46)

I n a c t i v e Enzyme

Inhibitor

Κ inactive/ a Inactive a

Anhydro trypsin

Bovine p a n c r e a t i c ( K u n i t z , BPTI) Reduced BPTI Bovine p a n c r e a t i c (Kazal) Chicken ovomucoid Soybean i n h i b i t o r Lima bean i n h i b i t o r (unfractionated)

Anhydro chymotrypsin

Potato i n h i b i t o r Lima bean ( I I I ) Bovine p a n c r e a t i c (Kunitz)

Methylchymotrypsin

Turkey ovomucoid Duck ovomucoid Golden pheasant ovomucoid

A 6

inactive active (kcal)

>0.2

200. 1.0

0.010 0.014 0.010

0.0

Overview on the Chemical Deteriorative Changes of Proteins and

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