Food Protein Deterioration - ACS Publications - American Chemical

14 Effect of Disulfide Bond Modification on the Structure and Activities of Enzyme Inhibitors Downloaded via TUFTS UNIV on July 10, 2018 at 18:13:55 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

MENDEL FRIEDMAN, OK-KOO K. GROSJEAN, and JAMES C. ZAHNLEY United States Department of Agriculture, Western Regional Research Center, Agricultural Research Service, Berkeley, CA 94710

Enzyme (trypsin, chymotrypsin, α-amylase, carboxypeptidase, etc.) inhibitors appear in many agricultural products (1-4) such as legumes (4), corn (5), wheat (6), and potatoes (7,8). Soybeans and other legumes containing active trypsin inhibi tors depress growth in rats compared to analogous feeding of inhibitor-free soybeans. Growth inhibition and its accompanying pancreatic hypertrophy are presumably partly due to the antitryptic activity of the inhibitor(s) (4, 9-13). Since naturally occurring protease inhibitors such as trypsin and chymotrypsin inhibitors are proteins that require their own structural integrity in order to inactivate proteolytic enzymes by complex formation (14-19), any alteration of the inhibitor's structure will inactivate it if interaction with the enzyme is thereby prevented. A reasonable strategy is to cleave the inhibitor's disulfide bonds, which often control protein structure (20). Reduced trypsin inhibitors are expected to be inactive. In addition to the cited primary benefit, i.e., inactivation of protease inhibitors, reductive cleavage of disulfide bonds of both inhibitors and structural proteins in legumes by, for example, cysteine or other thiols, should also offer an important secondary benefit, namely, improvement in the digestibility of the treated proteins, because disulfide cleavage is expected to decrease crosslinking thus permitting more rapid digestion. In this chapter we examine effects of 1) reductive S-pyridylethylation and S-quinolylethylation of disulfide bonds on analytical, structural, and inhibitory aspects of enzyme inhibitors; and, 2) cooperative and synergistic effects of heat and thiols in inactivating soybean and lima bean inhibitors.

This chapter not subject to U.S. copyright. Published 1982 American Chemical Society.

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

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FOOD P R O T E I N DETERIORATION

Experimental

Procedures

Enzymes, i n h i b i t o r s , and s u b s t r a t e s were o b t a i n e d f r o m W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n o r Sigma C h e m i c a l Co. Soy f l o u r was a g i f t o f A r c h e r D a n i e l s M i d l a n d C o r p o r a t i o n (Decatur, ID. L i m a bean f l o u r was p r e p a r e d f r o m raw beans p u r c h a s e d i n a l o c a l s t o r e . 2 - V i n y I p y r i d i n e was o b t a i ned f r o m A l d r i c h C h e m i c a l Co. and was r e d i s t i I Ied b e f o r e u s e . 2 - V i n y l q u i n o l i n e was f r o m Eastman Kodak and was v a c u u m - d i s t i l l e d b e f o r e u s e . T r i b u t y I phosph î ne was o b t a i ned f r o m A I d r i c h C h e m i c a l Co. and was s t o r e d under n i t r o g e n . S - 6 - ( 2 - P y r i d y l e t h y I ) - L - c y s t e i ne (2-PEC) was s y n t h e s i z e d a s d e s c r i b e d by F r i e d m a n and Noma (21 ) and S-Ê?( 2 - q u i n o l y l e t h y I ) - L - c y s t e i n e (2-QEC) a c c o r d i n g t o t h e p r o c e d u r e o f K r u I I e t a_[. ( 2 2 ) . O t h e r c h e m i c a l s were r e a g e n t g r a d e . Instrumental• A b s o r p t i o n s p e c t r a were r e c o r d e d on a C a r y 15 s p e c t r o p h o t o m e t e r ; and t r y p s i η was a s s a y e d by a b s o r b a n c e measured w i t h a Beckman DB s p e c t r o p h o t o m e t e r . A R a d i o m e t e r pHM 26 m e t e r and Beckman 39030 thiη-probe c o m b i n a t i o n e l e c t r o d e were used f o r most pH m e a s u r e m e n t s . Τitrimetric ( p H - s t a t ) e x p e r i ments were c a r r i e d o u t u s i n g a R a d i o m e t e r TTT1b T i t r a t o r , SBR 2c T i t r i g r a p h , S B U l a s y r i n g e b u r e t t e , and a TTA31 m i c r o t i t r a t i o n a s s e m b l y , e q u i p p e d w i t h a G2222c g l a s s e l e c t r o d e and a K4112 c a l o m e l e l e c t r o d e . C o n s t a n t t e m p e r a t u r e was ma i n t a i ned w i t h a Haake Model F E c i r c u l a t o r and a t h e r m o s t a t t i n g j a c k e t ( R a d i o m e t e r V 5 2 6 ) . Thermal d e n a t u r a t i o n measurements were made w i t h a DuPont Model 990 d i f f e r e n t i a l s c a n n i n g c a l o r i m e t e r ( D S C ) , a t a h e a t i n g r a t e o f 10 °/m i η and a n o m i n a l s e n s i t i v i t y o f 100 y e a I s " · C a l i b r a t i o n and o p e r a t i o n o f t h e DSC and t h e i n t e r p r e t a t i o n o f r e s u I t s have been d e s c r i bed ( 2 3 , 2 4 , 2 5 ) • F I u o r e s c e n c e s p e c t r a were r e c o r d e d on a T u r n e r Model 210 s p e c t r o f I u o r o m e t e r . S o l u t i o n s . B u f f e r s were p r e p a r e d a t room t e m p e r a t u r e by a d d i n g HCI o r NaOH t o a d j u s t t h e pH t o t h e d e s i r e d v a l u e . D i s t i I l e d , de i o n i z e d w a t e r was u s e d . Un I e s s o t h e r w i s e i n d i c a t e d , n a t i v e and S - p y r i d y I e t h y I a t e d t r y p s i n i n h i b i t o r s were d i s s o l v e d i n 0.05 M g l y c i n e a d j u s t e d w i t h 1 H_ HCI t o pH 3.0. T r y p s i n was d i s s o l v e d i n 2 mM HCI/20 mM C a C U , and k e p t a t 0-4°C. BenzoylD L - a r g i n i n e p - n i t r o a n i I i d e (ΒΑΡΝΑ) s t o c k s o l u t i o n s (0.1 M) were made w i t h d i m e t h y l s u I f o x i d e (DMSO), and N - a c e t y I - L - t y r o s î ne e t h y I e s t e r (ATEE) s t o c k s o l u t i o n s ( 0 . 2 M) were made w i t h 100$ ethanol· Mod i f i c a t i o n o f i n h i b i t o r s . 200 mg o f K u n i t z s o y b e a n t r y p s i n i n h i b i t o r ( S T I ) was d i s s o l v e d i n 10 ml o f pH 7.6 T r i s - 8 M u r e a b u f f e r . To t h i s s o l u t i o n were t h e n added 10 ml e t h a n o l , 0.2 ml t r i - n - b u t y I p h o s p h i ne and 0.2 ml o f 2 - v i n y I p y r i d i n e . N i t r o g e n was bubbIed i n f o r one m i n u t e . The r e a c t i o n m i x t u r e was s h a k e n and


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361

5 ml s a m p l e s were removed a t v a r i o u s t i m e s . The s a m p l e s were d i a l y z e d a b o u t t w o days i n w a t e r - 2% m e t h a n o l , and l y o p h i l i z e d . The p r o c e d u r e w i t h o v o m u c o i d (OMI) was i d e n t i c a l t o t h a t d e s c r i b e d f o r S T I . R e d u c t i v e a l k y l a t i o n w i t h l i m a bean p r o t e a s e î n h i b i t o r (LB I) was c a r r i e d o u t w i t h 50 mg o f i nh i b i t o r , 5 ml pH 7.6-8M u r e a b u f f e r , 5 ml o f e t h a n o l , 0.05 ml o f n-Bu3P, and 0.05 ml 2-vînyI p y r i d i n e . C o n t r o l e x p e r i m e n t s were done i n pH 7.6 b u f f e r w i t h o u t u r e a and i n pH 7.6 b u f f e r w i t h o u t 11-BU3P and 2 - v i n y I p y r i d i n e . A s i m i l a r p r o c e d u r e was used t o t r a n s f o r m h a l f - c y s t i n e r e s i d u e s i n STI t o 2-QEC s i d e c h a i n s ( 2 6 ) . Reaction with t h i o l s . The f o l l o w i n g p r o c e d u r e w i t h s o y f l o u r i l l u s t r a t e s t h e g e n e r a l method used t o i n a c t i v a t e STI (18). Soy f l o u r (300 mg) and N - a c e t y I - L - c y s t e i ne ( Ν Α Ο (67.4 mg, 0.4 m i I I i mo I e s ) i n 30 ml o f 0.5 M T r i s b u f f e r were ρ laced i n a water bath a t a spec i f ied temperature f o r 1 h r . The r e a c t i o n m i x t u r e was t h e n c o o l e d i n i c e - w a t e r . Portions (0.05 m l ) o f t h e s u s p e n s i o n were d i I u t e d t o 0.5 ml w i t h pH 8.2, 0.05 M T r i s b u f f e r . C o n t r o l e x p e r i m e n t s w i t h o u t ΝAC were c a r r i e d out concurrent Iy. P u r e ST I was s t u d i e d s i m i I a r I y e x c e p t t h a t d i I u t i o n s were 1:300 w i t h t h e pH 8.2 T r i s b u f f e r . T h e r e m a i n i n g t r y p s î n i nh i b îtory a c t i v i t y was t h e n a s s a y e d a s d e s c r îbed b e l o w . A l I e x p e r i m e n t s were c a r r i e d o u t i n d u p I i c a t e . The r e p r o d u c i b i I i t y i s e s t i m a t e d t o be ± 3% o r b e t t e r . Ami no a c i d ana l y s i s . Ami no a c i d ana I y s e s o f a I i q u o t s o f h y d r o l y z e d w e i g h e d samp l e s d i s s o l v e d i n pH 2.2 b u f f e r were c a r r i e d o u t w i t h a s i n g l e - c o l u m n Durrum Amino A c i d A n a l y z e r , Model D500 under t h e f o I l o w i ng c o n d i t i o n s : s i n g I e - c o l u m n i o n e x c h a n g e c h r o m a t o g r a p h i c method ( Γ7 ); R e s i n , Durrum DC-4A, c i t r a t e b u f f e r pH, 3.25, 4.25, 7.90; P h o t o m e t e r , 590 and 440 nm; Column, 1.75 mm χ 48 cm; a n a l y s i s t i m e , 105 m i n u t e s .

Norleucine was used as an internal standard (Figures 1-6). Extent of reaction was deduced from the amount of 2-PEC found. U l t r a v i o l e t s p e c t r o s c o p y o f 2-PEC and 2 - p y r i d y I e t h y I a t e d (2-PE) i n h i b i t o r s . E x t e n t o f r e a c t i o n o f 2 - v i n y I p y r i d ine w i t h t h e i nh i b i t o r s was a I s o d e t e r m i ned f r o m t h e d i f f e r e n c e between ^263 ^ equ i m o l a r c o n c e n t r a t i o n s o f t h e mod i f i e d and n a t i ve i n h i b i t o r s , u s i n g ε f o r 2-PEC o f 7070 a t pH 3.0. The v a l u e of ε a t pH 1, 7200, was s I i g h t I y h i g h e r t h a n p r e v i o u s I y f o u n d in 6 Ν HCI ( 2 j _ ) . To d e t e r m i n e ε f o r 2-PEC, a 2.7 mM s t o c k s o l u t i o n i n 2 mM HCI was made and d i I u t e d 1:20 i n a p p r o p r i a t e buffers. D u p I i c a t e d i l u t i o n s i n 0.05 M g l y c i n e / H C I (pH 3.0) gave ε va Iues c o n s î s t e n t w i t h i η 1$. S p e c t r a f o r 2 - p y r i d y I e t h y I a t e d i n h i b i t o r s were a I s o a n a l y z e d s i m u l t a n e o u s l y f o r c o n c e n t r a t i o n s o f 2-PE s i d e cha i ns and p r o t e i η by us i ng a two-wave I e n g t h two-component ana Iys i s (27)· One wave I e n g t h was 263 nm, t h e a b s o r p t i o n Ο Γ


362

food protein

deterioration


FRIEDMAN E T A L .

Disulfide

Bond

Modification


364

FOOD PROTEIN DETERIORATION

Asp

Ser


FRIEDMAN E T AL.

-J 10

ι 20

Disulfide

I 30

ι 40

Bond

Modification

ι 50

ι 60

ι 70

I 80

L 90

TIME (MINUTES) Figure 4.

A mino acid analysis of

2-PE-LBI.


366


Asp


FRIEDMAN ET AL.

Disulfide

Figure 6.

Bond

Modification

A mino acid analysis of

2-PE-OML


368

FOOD PROTEIN DETERIORATION e

e

maximum f o r 2-PEC a t pH 3, s i n c e P E C / p r o t e i η i s maximal a t t h i s wavelength. The s e c o n d w a v e l e n g t h , a t w h i c h e f i / pFÇ * maximal o r n e a r l y s o , was 285 nm f o r PE-OMI, 280 nm f o r P t - L B I , o r 289 nm f o r P E - S T I . V a l u e s o f e f j n and ερ^ς f o r w a v e l e n g t h s f o r w h i c h ε i s n o t l i s t e d were c a l c u Y a f e a f r o m s p e c t r a u s i n g t h e formuI a e

D

D

ε

r

o

x A

(

max

o

r

A

280

o

e

s

n

e

=

χ

r

e

m

a

Cor ε

x

2 8 0

)

)

Two s i m u l t a n e o u s e q u a t i o n s o f t h e f o r m : ε

A

£

C

= p r o t e i η χ V o t e in + P E C

were s o l v e d i n e a c h

χ PEC

case.

F o r e x a m p l e , e q u a t i o n s f o r PE-STI w e r e : A

A

263 289

=

=

6 S T I

e

263

STI

x

289

The ε v a l u e s λ ( nm) 263 280 285 289

x

C

C

STI STI

+

e

+

€

PEC

x

C

PEC

=

1 4

'

9 0 0

C

STI

+

7 0 7 0

C

PEC

=

1 2

'

6 0 0

C

STI

+

3

263

PEC

x

289

9

5

C

C

PEC

PEC

used a r e t a b u l a t e d below: £

ε

0Μ! 10,850 14,890 12,860

ε

PEC 7070 1460 700 395

STI 14,900 20,300 .

.

ε

LB 1 2480 2390

12,600

U l t r a v i o l e t s p e c t r o s c o p y o f 2-qu i n o I y I e t h y I - S T I

(2-QE-STI).

S i n c e 2-QE-STI was I n s o l u b l e i n nondenaturÏng aqueous s o l v e n t s , i t was f i r s t d i s s o l v e d i n f o r m a m i d e a t 5-15 mg/ml a n d t h e n d i l u t e d i n aqueous s o l u t i o n s . A l i q u o f s o f s t o c k s o l u t i o n s o f 2-QEC o r 2-QE-STI were d i l u t e d t o A < 1.0. D i l u e n t s w e r e : 0.1 and 0.2 M H C I ; 0.1 N H S 0 : 0 . T and 0.2 M a c e t i c a c i d ; 0.2 M g l y c i n e — H C I , pH 3.0; 0.2 M a c e t a t e b u f f e r s , pH 3.6 and pH 5.0; 0.2 M 3 - ( N - m o r p h o l i n o ) p r o p a n e s u i f o n i c a c i d (MOPS)—NaOH b u f f e r , pH 7.0; 0.2 M g l y c i n e — N a O H b u f f e r , pH 9.0. S p e c t r a were r e c o r d e d a g a i n s t sol vent bIanks. 2

4

F I u o r e s c e n c e s p e c t r o s c o p y . 2-QE-STI s t o c k s o l u t i o n s were norma I I y d i l u t e d i n w a t e r o r 0.1 U H S 0 * t o A (1-cm I i g h t p a t h ) o f 0.04 o r l e s s , and t h e pH was a d j u s t e d a s needed w i t h 1 H H S 0 * and 1 Ν o r 4 Ν NaOH. F l u o r e s c e n c e was e x c i t e d a t 316 nm and 25°C. Y i e l d s were d e t e r m i n e d f r o m a r e a s o f t h e e m i s s i o n 2

2


14.

FRIEDMAN ET AL.

Disulfide

Bond

369

Modification

s p e c t r a , and i n t e n s i t i e s were measured a s n e t peak h e i g h t s a t maximum e m i s s i o n . T r y p s i η i nh i b i t o r assay. Many u n i t s a r e used t o d e f i n e t h e i n h i b i t o r y a c t i v i t y of protease i n h i b i t o r s (28). The f o l l o w i n g method, based on t h e d e f i n e d s t o i c h i o m e t r y o f enzyme, i n h i b i t o r , and s u b s t r a t e , was a d a p t e d from Mi k o I a and S u o l i n n a ( 2 9 ) . I n h i b i t i o n o f t r y p s i n a c t i v i t y was measured a t pH 8.2 and 37°C, w i t h ΒΑΡΝΑ a s s u b s t r a t e . I n h i b i t o r was i n c u b a t e d w i t h 25 yg o f t w i c e - c r y s t a l l i z e d b o v i n e t r y p s i n i n a t o t a l volume o f 0.75 ml f o r 5 min a t 37°C. ΒΑΡΝΑ (30 mg i n 1.00 ml o f DMSO) was d i l u t e d t o 100 ml w i t h 0.05 M T r i s - H C I , pH 8.2, c o n t a i n i n g 0.02 M CaCI . The d i l u t e d ΒΑΡΝΑ was k e p t a t 37°C and u s e d w i t h i n 1 h r . To s t a r t t h e r e a c t i o n , 3.0 ml o f s u b s t r a t e - b u f f e r m i x t u r e was added r a p i d I y t o t h e t r y p s i n - i n h i b i t o r m i x t u r e . The r e a c t i o n was s t o p p e d a f t e r 5 o r 10 min by a d d i n g 0.50 ml o f 30% acetic acid. 2

D e t e r m i n a t i o n o f a c t i v e t r y p s i η. The c o n c e n t r a t i o n o f a c t i ve t r y p s i η was d e t e r m i ned by a c t i ve s i t e t i t r a t i o n w i t h p_-n i t r o p h e n y I p_ -guan i d i n o b e n z o a t e (NPGB), e s s e n t i a I I y a c c o r d i ng t o C h a s e and Shaw (30). A b s o r b a n c e a t 410 nm was f o l lowed i n a C a r y 15 s p e c t r o p h o t o m e t e r , w i t h t h e 0.1 a b s o r b a n c e s I i dew i r e . NPGB (0.01 M) was d i s s o l v e d i n d i methyIformam i d e - a c e t o n i t r i I e (1:4). T r y p s i n was d i s s o l v e d i n 2 mM HCI/0.02 M C a C I a t 5-6 mg/ml, t h e n d i I u t e d 1:10 i n t h e same s o l v e n t . Each c u v e t t e c o n t a i n e d 0.05-0.09 M Na b a r b i t a l b u f f e r (pH 8.35 o r 8.39 a t 23°C), 0.02 M C a C I o , 0.1 M K C I . T r y p s i n (3-5 ηmo I o f a c t i v e enzyme) was added t o t h e samp l e c u v e t t e a s 0.20 ml o f t h e 1:10 d i I u t i o n o r 25 y g o f t h e s t o c k s o l u t i o n . A f t e r t h e b a s e I i ne was d e t e r m i ned, 5 yl_ o f NPGB was added t o each c u v e t t e . TotaI volume was 1.00 m l . I η o u r i n s t r u m e n t , 1 A ^ Q = 61.1 mol o f p_-n i t r o p h e n o l a t e (31 ). The t r y p s i n used was 61 % a ο enzyme, based on i t s p r o t e i η c o n t e n t a s d e t e r m i ned by ab?'* je at 280 nm and an actî ve s i t e t i t r a t i o n . !

2

Calculations. The f o l l o w i n g samp l e c a l c u l a t i o n was used t o e s t i m a t e t h e t r y p s i n i n h i b i t o r a c t i ν i t y p e r mg o f a c t i v e enzyme, where A^Q

= a b s o r b a n c e measured a t 410 nm = mol a r e x t î n c t i o n c o e f f i c i e n t V = r a t e o f ΒΑΡΝΑ h y d r o l y s i s by t r y p s i η i n t h e a b s e n c e o f inhibitor V. = r a t e o f ΒΑΡΝΑ h y d r o l y s i s by t r y p s i n î η t h e p r e s e n c e o f inhib itor TU = t r y p s i n un i t , def i ned a s t h e amount o f t r y p s i η t h a t c a t a I y z e s t h e h y d r o l y s i s o f 1 μ mol o f ΒΑΡΝΑ p e r min a t 37°C and pH 8.2 TIU = t r y p s i n î nh i b i t o r un i t , def i ned a s r e d u c t i o n i n a c t i v i t y o f t r y p s î η by o n e t r y p s i η u n i t ( T U ) . ε

Q


370


Usi ng ε^ f o r p_-n i t r o a n î I i ne = 8800 1/mol-cm ( 3 2 ) , and a t o t a l volume o f 4.25 ml , 1 TU c o r r e s p o n d s t o an i n c r e a s e i n A ^ Q of 2.07 m i n " . We f o u n d t r y p s i n a c t i v i t y o f 2.51 TU p e r mg o f a c t i v e enzyme ( s e e s a m p l e c a l c u l a t i o n ) . U s i n g m o l e c u l a r w e i g h t s o f 23,400 f o r t r y p s i n and 21,500 f o r S T I , we c a l c u l a t e d 2.92 T I U p e r mg o f ST I. F o r t r y p s i n o n l y , 0.358 A ^ i / 5 m i n ; R a t e , V = 0.0716/min; f o r t r y p s i n and i n h i b i t o r , 0.389 A ^ Q / 1 0 m i n . 1

Q

Q

ν V

ν i

v

AJIQ (no inh.)

A,

1

"

(inh.) T j

_ 0.358 5

0

= 0.0716 - 0.0389 = 0.0327 A

4

1

min"

0

1

f ^ I -

i

0.389 10

1

S i n c e 2.07 A min" i s equivalent t o 1 t r y p s i n amount o f i nhιbι t o r p r e s e n t i s : 4

the

"

n

unît ( T U ) ,

0.0158 TIU

F o r p u r e ST I t h e i n h i b i t o r c o n t e n t o f t h e samp l e u s e d i s : 0.0158 T I U _ , 2.92 TIU/mg STI ' A

5

1 Λ

4 1

X

S i n c e 1 mg o f a c t i v e

MrfuXg the

_ m

9

trypsin

=

trypsin

-3

1 0

6

'

2

9

x

1 0

S T I

=

5

*

4 1

^

S T I

= 2.51 TU, 0.0158 T I U r e p r e s e n t s "

3

m

9

a

c

t

i

v

e

t r

yP

s i n

inhibited in

p u r e ST I samp l e .

If i n h i b i t o r s o t h e r t h a n STI a r e a l s o p r e s e n t ( a s i s a p p a r e n t l y t h e c a s e i n most ρ I a n t f o o d s ) , a more p r e c i s e e s t i m a t e i s : TIU i n samp l e wt. o f s a m p l e

T

"

T

l

n

/

,

U

/

, M

9

(

O

R

» 9

K

The f o I l o w i ng a v e r a g e v a Iues d e m o n s t r a t e t h e r e p r o d u c i b i I i t y o f t h e a s s a y . P u r e c o m m e r c i a l ST I i nh î b i t e d 1.23 ± 0.03 mg o f a c t i v e t r y p s i n p e r mg STI ( e i g h t r e p I i c a t e s ) . (Th î s v a Iue i s h i gher t h a n un i t y b e c a u s e t h e r a t i o o f mol ecu I a r w e i g h t s o f t r y p s i n t o STI i s g r e a t e r t h a n o n e and b e c a u s e o f p o s s i b I e mi nor c o n t r i b u t i o n s o f l o w e r mo I e c u l a r w e i g h t i n h i b i t o r s t o t h e t o t a I enzyme i nh î b i t o r y p r o c e s s . ) F o r u n t r e a t e d soy f l o u r , t h e a v e r a g e f o r f o u r d e t e r m i n a t i o n s was f o u n d t o be 109.51 ± 1.52 T I U o r 43.63 ± 0.605 mg t r y p s i n i n h i b i t e d p e r gram of f l o u r . S i m i l a r l y , t h e t r y p s i n i n h i b i t o r a s s a y f o r LB I gave t h e f o l l o w i ng a v e r a g e va Iues f o r f o u r d e t e r m i n a t i o n s : 2.76 ± 0.041 TIU/mg c o m m e r c i a l LB I and 51.33 ± 0.994 T l U / g Iima bean f I o u r .


14.

FRIEDMAN ET AL.

Disulfide

Bond

371

Modification

Chymotrypsin I n h i b i t i o n assay* I n h i b i t i o n of chymotrypsin was d e t e r m i n e d t i t r i m e t r i c a l I y , w i t h 0.01 M ATEE a s s u b s t r a t e ; t h e p r o c e d u r e was a d a p t e d f r o m t h a t o f W i l c o x ( 3 3 ) . Inhibitor was i n c u b a t e d w i t h c h y m o t r y p s i n (25 μ I o f 0.44 mg/ml s o l u t i o n ) a t room t e m p e r a t u r e , t h e n e q u i l i b r a t e d a t 25°C, i n 1.9 ml o f 5 mM T r i s / H C I , 0.1 M i n C a C U , pH 8.0. A t l e a s t 10 min was a l l o w e d f o r enzyme and i n h i b i t o r t o r e a c t . After the baseline u p t a k e o f t i t r a n t (0.1 Ν NaOH) was r e c o r d e d , 100 μΙ o f 0.2 M ATEE was i n j e c t e d i n t o t h e s o l u t i o n . I n i t i a l r a t e s were d e t e r mined f r o m t h e l i n e a r p o r t i o n o f t h e p l o t o f base u p t a k e a g a i n s t t i m e . C h a r t speed was 2 cm/min. M e a s u r e m e n t s o f t h e n o n e n z y m a t i c breakdown o f ATEE and t h e e f f e c t o f o r d e r o f a d d i n g ATEE and c h y m o t r y p s i n ( n o i n h i b i t o r ) were a l s o made and used t o c o r r e c t assays of i n h i b i t i o n . P r o t e o l y t i c d i g e s t i o n assay* T r e a t e d s o y f l o u r was d i a l y z e d f o r t h r e e d a y s a g a i n s t d i s t i l l e d w a t e r and l y o p h i l i z e d . Samples were d i s s o l v e d o r d i s p e r s e d (5 mg/ml) i n 0.05 M KCI w i t h s t i r r i n g and a d j u s t e d t o pH 8.5. T o d e t e r m i n e d i g e s t i b i l i t y by t r y p s i n , a I i q u o t s (5 mg o f p r o t e i n ) were p i p e t t e d i n t o p l a s t i c t i t r a t i o n v e s s e l s ( R a d i o m e t e r V 524) and d i l u t e d t o 4.0 ml w i t h 0.05 M K C I . S a m p l e s were i n c u b a t e d under N t o m i n i m i z e C 0 absorption. In a t y p i c a l r u n , t h e s u b s t r a t e was b r o u g h t t o pH 8.5 and 37°C, and t h e b a s a l i ne r a t e o f u p t a k e o f t i t r a n t ( 0 . 0 2 NaOH) was d e t e r m i n e d . T h e n , 20 μΙ_ o f b o v i n e t r y p s i n (5 mg/ml in 2 mM HCI-0.02 M C a C I ) was a d d e d . F u r t h e r a d d i t i o n s o f enzyme were made when t h e u p t a k e o f a l k a l i became v e r y s l o w . When t h e r e a c t i o n was e s s e n t i a l l y c o m p l e t e , t h e n e t u p t a k e was d e t e r m i n e d . 2

2

2

S i n c e l i b e r a t i o n o f h y d r o g e n i o n s by p r o t e o l y s i s i s incom p l e t e a t pH v a l u e s where some new α-amîno g r o u p s a r e i o n i z e d ( 3 4 ) , c a l c u l a t i o n o f t h e number o f p e p t i d e bonds c l e a v e d r e q u i r e s a v a l u e f o r t h e a v e r a g e pK o f t h e n e w l y formed p e p t i d e α - a m i n o groups. I f we assume a pK o f 7.5 ( 3 5 ) , f r e e h y d r o g e n i o n s w i l l be p r o d u c e d i n 90% o f t h e p e p t i d e bond c l e a v a g e s a t an a s s a y pH 8.5. To c a l c u l a t e t h e a c t u a l a v e r a g e number o f p e p t i d e bonds c l e a v e d p e r c h a i n c o r r e c t e d f o r i n c o m p l e t e l i b e r a t i o n o f hydrogen i o n s , we a l s o assumed an a v e r a g e m o l e c u l a r w e i g h t o f 23,000 f o r the soy p r o t e i n ( 3 6 ) . Q

ResuIts The

2 - P E - d e r i v a t i v e s o f a l I t h r e e i n h i b i t o r s showed

absorp

tion maxima at 263-264 nm i n acidic (pH 3.0) solution (Figures 7-10), coinciding with that f o r free 2-PEC. Reaction was rapid in buffers containing 8 M urea, especially i n t h e cases of STI and chicken 0ΜΙ. A l l 4 half-cystine groups of STI and 12 of 18 half"cystine groups o f 0ΜΙ reacted within 30 min under these conditions, based on an increase i n A (Figure 8 and Table I ) . As expected, LBI reacted more slowly (figure 10 and Table I ) . Only 5 to 6 of the 14 half-cystines, on the average, reacted



PROTEIN

ALKYLATED

REDUCED

PROTEIN

REDUCED

PROTEIN

9

3

TRIBUT Y L PHOSPHINE

4

9

2

HCI

S

2

oc

it

ϊ

Figure 7.

(2-PEC)

3

2

HYDROLYSIS

S-PYRIDYL-ETHYLATION

1 REDUCTION

Reductive S-pyridylethylation of a protein.

?

CH -S„

S-/Q —(2-PYRIDYLETHYL)—L —CYSTEINE

2 Ν

α » ,

HOOC-CH-CH -S-CH -CH - \ 2

Ν

(? - V I N Y L P Y R I D I N E )

9

CH =CH

4

F?-SH + HS-R-SH + ( n - C H ) - P = 0

r

P _S-S-f?-SH + ( n - C H ) - P + H 0

ο

S ο

Η W

w

Η m

SO Ο

to

14.

FRIEDMAN ET AL.

240

Disulfide

260

280

Bond

373

Modification

300

320

340

360

W A V E L E N G T H (nm) Figure 8. UV spectra of trypsin inhibitors and their 2-PEC derivatives at pH 3.0. Lyophilized proteins were dissolved in 0.1 M KCl by adjusting the pH to 3.2 at 7.8 mg/mL (native) or 3.1-3.7 mg/mL (modified). Aliquots were diluted to 0.5 mg/mL (2.3 χ 10 M) in 0.05 M glycine/HCl (pH 3.0). Key: a, native STI; and b, modi fied STI. 5



374

ob

ι

240

ι

ι

260

ι

1

280

ι

Γ ι

300

I 320

I

l340

WAVELENGTH (nm) Figure 9. UV absorption spectra of chicken ovomucoid inhibitors (OMl). Protein samples were dissolved and diluted to 0.25 mg/mL. Key: a, untreated OMl: 8.2 X 1CT M; and b, treated OMl: 7.6 X 10 M. 6

e


14.

FRIEDMAN ET AL.

Disulfide

Bond

375

Modification

d

240

260

280

300

320

340

W A V E L E N G T H (nm) Figure 10. The UV absorption spectra of lima bean trypsin inhibitors (LBI) is shown. Control samples in the presence or absence of urea gave almost identical spectra; therefore, only one is shown. Key to protein concentrations (w/w): a, untreated, 2.28 X 10 M; controls (urea), 2.36 χ 10 M; b, treated 30 min without urea, 2.17 X 10 M ; c, treated 30 min with urea, 2.10 X 10 M; and d, treated 240 min, 2.48 χ 10~ M . s

5

s

s

5



376

Table

I.

Protein

2-PE-STI

2-PEC Cha I f - c y s t i n e ) c o n t e n t s o f r e d u c e d a l k y l a t e d S T I , LB I , and OMl d e t e r m i n e d by amino a c i d a n a l y s i s and u l t r a v i o l e t s p e c t r o s c o p y .

Time o f Reduction (min )

Mean ± 2-PE-OMl

2-PE-LB 1

Amino a c i d

analysis

4.0 4.6 4.4

30 60 120 240

1

M o l e s 2-PEC/mole p r o t e i η UV

spectra

4.372

4.38^ 4.47*

4.24 S.D.

30 60 240 30 no u r e a 30 8M u r e a 240

4.3±0.3

z

4.361.10

11.4 12.3 15.3

13.27* 13.63^ 14.89

7.4

2.0 , 5.6^ 7.1

z

3

3

M o l e c u l a r w e i g h t s = STI = 21,500; OMl = 29,600; LB I = 9 0 0 0 . D e t e r m i n e d by s i m u l t a n e o u s e q u a t i o n s ( s e e t e x t ) . D e t e r m i n e d by d i r e c t s u b t r a c t i o n o f 2-PE LB I and LB I a b s o r b a n c e s a t 263 nm ( p e a k max imum f o r 2 - P E C ) .


14.

FRIEDMAN ET AL.

Disulfide

Bond

Modification

377

w i t h i n 30 min i n t h e p r e s e n c e o f u r e a . Even a f t e r 2 4 0 m i n , o n l y a b o u t 7 o f 14 had r e a c t e d . O m i t t i n g u r e a from t h e r e a c t i o n medium s l o w e d t h e a l k y l a t i o n o f LB I , s u g g e s t i n g t h a t t h e r a t e of r e a c t i o n and p o s s i b l y t h e s e l e c t i v i t y f o r d i f f e r e n t d i s u l f i d e b r i d g e s c o u l d be m o d i f i e d by a p p r o p r i a t e c h o i c e o f r e a c t i o n conditions. T a b l e II shows t h e e f f e c t o f e x t e n t o f r e d u c t i o n and a l k y l a t i o n on t r y p s i n i n h i b i t i o n . E s s e n t i a l l y complete loss o f a c t i v i t y o f LB I was o b s e r v e d when 7 ha I f - c y s t i nes p e r m o l e c u l e , on t h e a v e r a g e , were m o d i f i e d . T h i s r e s u l t i s c o n s i s t e n t w i t h t h e f i n d i n g s o f F e r d i n a n d e t a l . ( 3 7 ) . L o s s o f a c t i v i t y was d i s p r o p o r t i o n a t e t o t h e e x t e n t o f m o d i f i c a t i o n a t lower l e v e l s . When an a v e r a g e o f 1 d i s u l f i d e b r i d g e p e r LB I m o l e c u l e was l o s t , a p p r o x i m a t e l y 2/3 o f t h e i n h i b i t o r y a c t i v i t y was l o s t . When m o d i f i c a t i o n was a l m o s t 40% com ρ I e t e ( a v e r a g e o f 3 d i s u l f i d e b r i d g e p e r LBI m o l e c u l e ) a l m o s t 90% o f t h e i n h i b i t o r y a c t i v i t y was l o s t . The c o n t r o l s show t h a t l i t t l e o r no i n a c t i v a t i o n o f i n h i b i t o r was due t o t h e p r o c e d u r e i t s e l f and t h a t , under t h e s e c o n d i t i o n s , the e f f e c t o f urea i s r e v e r s i b l e . E s s e n t i a l l y c o m p l e t e r e d u c t i o n and a l k y l a t i o n o f STI and OMl a b o l i s h e d t h e i r t r y p s i n i n h i b i t o r y a c t i v i t i e s , a s shown in T a b l e I I . T h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h o s e o b t a i n e d with other reducing agents. S i n c e some v a r i a n t s o f LBI a l s o i n h i b i t c h y m o t r y p s i n ( 3 8 ) , t h e e f f e c t o f m o d i f i c a t i o n on i n h i b i t o r y a c t i v i t y a g a i n s t c h y m o t r y p s i n was a l s o d e t e r m i n e d t o s e e w h e t h e r t h e s e n s i t i v i t y o f a n t i c h y m o t r y p s i n a c t i v i t y t o r e d u c t i o n and a l k y l a t i o n d i f f e r e d from t h a t o f t r y p s i n i n h i b i t i o n . A s shown i n T a b l e I I , t h e l o s s e s o f i n h i b i t o r y a c t i v i t y a g a i n s t b o t h enzymes were similar. L e s s t h a n 30% o f t h e LBI c o n s i s t e d o f v a r i a n t s t h a t i n h i b i t chymotryps i n . F r e e 2-PEC (0.01 M) i n h i b i t e d c h y m o t r y p s i n w e a k l y ( 1 3 ? ) a t a mol r a t i o o f - 5 χ 1 0 . ΡΕ-OMl (0.8 nmol) d i d n o t i n h i b i t 0.4 nmol o f c h y m o t r y p s i n (Mol r a t i o o f 2-PEC m o i e t y t o enzyme 30-40). T h u s , t h e 2-PEC d o e s n o t a p p e a r t o b i n d e f f e c t i v e l y t o chymotrypsiη· Where I i t t I e o r no i n h i b i t o r y a c t i v i t y i s found , f r e e t r y p s in o r c h y m o t r y p s in c a n a t t a c k t h e i n h i b i t o r , i f s i t e s o f potentiaI cleavage are accessi ble. T h u s , t h e mod i f i e d i n h i b i t o r s may we I I show i n c r e a s e d s u s c e p t i b i I i t y t o p r o t e o l y s i s . T a b l e I I I shows t h a t t h e 2 - P E - i n h i b i t o r s a r e a t t a c k e d more r e a d iI y by t r y p s i n o r c h y m o t r y p s in t h a n a r e t h e n a t i v e i n h i b i t o r s , even t h o s e t h a t d o n o t i n h i b i t c h y m o t r y p s i η. Digestion was i n c o m p l e t e , h o w e v e r , under t h e cond i t i o n s u s e d . (Both pr imary and s e c o n d a r y s p e c i f i c i t i e s o f c h y m o t r y p s i η were c o u n t e d in deducing t h e p o t e n t i al s i tes.) I η t h e c a s e o f LB I , s u s c e p t i b i I i t y t o d i g e s t i o n by t r y p s i η o r c h y m o t r y p s i η was n o t i n c r e a s e d u n t i I t h r e e o r more d i su If i d e br i d g e s were b r o k e n . These r e s u I t s and t h o s e i n T a b l e I I show t h a t i n h i b i t o r y a c t i ν i t i e s o f LB I a r e more e a s iI y d e s t r o y e d by t h i s mod i f i c a t i o n p r o c e d u r e t h a n a r e the s t r u c t u r a l elements r e s p o n s i b l e f o r r e s i stance t o p r o t e o l y s i s .



378

Table

II.

E f f e c t o f m o d i f i c a t i o n on i n h i b i t o r y a g a i n s t t r y p s i n and c h y m o t r y p s i n .

Reductive treatment ;

Time (min ) Inhibitor

STI 2-PE-STI

LBI LBI-controls 2-PE-LBI

1

% Initial specific inhibitory a c t i v i t y agai nst :

Tryps î η

100

100 0

30^ 30 240

14 40 51

100 92 32 10

Food Protein Deterioration - ACS Publications - American Chemical

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