Xenobiotics in Foods and Feeds - American Chemical Society

2

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 6, 2015 | http://pubs.acs.org Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

Protease and Amylase Inhibitors in Biological Materials JOHN R. WHITAKER Department of Food Science and Technology, University of California, Davis, CA 95616

Proteins which specifically inhibit enzymes by forming tight inactive complexes with the enzyme are widely distributed in biological materials. With the exception of a few of the protease inhibitors and the α-amylase inhibitors, very l i t t l e work has been done on the mechanism of action of the inhibitors or of their nutritional and physiological importance. The trypsin and chymotrypsin inhibitors appear to form specific complexes with the proteases because trypsin and chymotrypsin recognize the protein inhibitors as substrates. However, the normal sequence of cataly tic steps cannot be completed, perhaps because of a conformational change accompanying complexation. On the other hand, the α-amylase inhibitors may not bind at the active site of α-amylase. Rather, the initial complex may undergo a conformational change which destroys the catalytic ability of the α-amylase but leaves the substrate binding ability intact. Some of the animal protease inhibitors are known to serve a protective function against proteases. There is speculation that the plant protease and amylase in hibitors may serve as a protection against insects and microorganisms, but this has not been proven. There is a great deal of work yet to be done on the naturally-occurring protein and peptide inhibi tors of enzymes. Better knowledge of their proper ties and their physiological, nutritional and medical roles is essential. Any compound which decreases the a c t i v i t y of an enzyme i s an i n h i b i t o r . Inhibitors of enzymes include: (a) small molecules which combine with an essential group of the active s i t e (examples include heavy metal ions, acylating and alkylating reagents) or remove an essential part of the active site (an example i s removal of essential cations by chelating agents) and compounds which sim0097-6156/83/0234-0015$09.00/0 © 1983 American Chemical Society

In Xenobiotics in Foods and Feeds; Finley, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

X E N O B I O T I C S IN F O O D S


16

AND

FEEDS

u l a t e the substrates (competitive i n h i b i t o r s , i n c l u d i n g products of the r e a c t i o n ) and (b) l a r g e polymeric molecules which i n h i b i t enzymes ( p r o t e i n and peptide i n h i b i t o r s of proteases; p r o t e i n , peptide and carbohydrate i n h i b i t o r s of α-amylases). This chapter w i l l deal only with s e l e c t e d examples from the second group. pH, temperature, denaturing agents and p r o t e o l y s i s , which decrease enzyme a c t i v i t y , are excluded from the d e f i n i t i o n of an i n h i b i t o r given above. Study of n a t u r a l l y - o c c u r r i n g enzyme i n h i b i t o r s i s of impor tance f o r s e v e r a l reasons. These i n c l u d e : (a) the p h y s i o l o g i c a l importance of an i n h i b i t o r i n b i o l o g i c a l m a t e r i a l , (b) the n u t r i t i o n a l importance of an i n h i b i t o r when the m a t e r i a l i s consumed as a food or feed, ( c ) the use of i n h i b i t o r s to c o n t r o l enzymatic a c t i o n , such as that of polyphenol oxidase, (d) the use of i n h i b i t o r s f o r a n a l y s i s and f o r p u r i f i c a t i o n purposes and (e) a b e t t e r understanding of s p e c i f i c i n t e r a c t i o n s among complex molecules such as p r o t e i n s (examples i n c l u d e antigen-antibody r e a c t i o n s , subunit i n t e r a c t i o n s i n p r o t e i n s , enzymatic a c t i o n s ) . Occurrence of Enzyme I n h i b i t o r s Compounds i n b i o l o g i c a l m a t e r i a l s which i n h i b i t proteases and amylases were noted as early as the 1930's. Kunitz and Northrop, during the p u r i f i c a t i o n of trypsinogen and t r y p s i n from beef pan creas, found and i s o l a t e d a t r y p s i n i n h i b i t o r from the same source ( p a n c r e a t i c secretory t r y p s i n i n h b i t o r ; 1). About the same time, Chrzaszcz and J a n i c k i (2j3) reported that there was something i n c e r t a i n plant e x t r a c t s which i n h i b i t e d α-amylase. Since that time, many enzyme i n h i b i t o r s have been discovered, p u r i f i e d and p a r t i a l l y c h a r a c t e r i z e d . Whitaker (4) has l i s t e d 54 protease i n h i b i t o r s i n animal t i s s u e s , 44 protease i n h i b i t o r s i n plant t i s s u e s , s i x protease i n h i b i t o r s i n microorganisms and some 25 i n h i b i t o r s of n o n - p r o t e o l y t i c enzymes. While these data are important i n showing the great numbers of i n h i b i t o r s present i n b i o l o g i c a l m a t e r i a l s , the numbers are rather meaningless i n most part because many i n h i b i t o r s have yet to be discovered, the numbers r e f l e c t ( i n part) i s o i n h i b i t o r s which have been reported and the i n t e r r e l a t i o n s h i p s and homology among the i n h i b i t o r s are r e l a t i v e l y unknown (see below). Discovery of Enzyme I n h i b i t o r s . Discovery of enzyme i n h i b i t o r s i n b i o l o g i c a l m a t e r i a l s occurs p r i m a r i l y i n four ways. One of the most frequent i s the observation that the percentage recovery of an enzyme a c t i v i t y during p u r i f i c a t i o n suddenly increases at one step i n the p u r i f i c a t i o n (_5). An example i s shown i n Table I. The v a l i d i t y of such an observation must be c o l l a b o r a t e d by observing a decrease i n a c t i v i t y when some of the removed f r a c t i o n i s added back to the enzyme-containing f r a c t i o n . This method only works, of course, when the a c t i v i t y of an enzyme of the b i o l o g i c a l system i s reduced by an i n h i b i t o r i n the same p r e p a r a t i o n . In the


2.

WHITAKER

Table I.

Protease

and Amylase

Inhibitors

17

E f f e c t of Presence of I n h i b i t o r on A c t i v i t y of T r y p s i n During P u r i f i c a t i o n 3

Sp.


Preparation

a

Activity

IX c r y s t a l l i z e d

100

3Χ

crystallized

120

5Χ c r y s t a l l i z e d

120

8Χ c r y s t a l l i z e d

140

3Χ c r y s t a l l i z e d ·+ ppt'd with TCA

160

K u n i t z and Northrop (1) ; c r y s t a l l i z e d from the TCA

b

b

A polypeptide i n h i b i t o r supernatant l i q u i d .

was

case of the α-amylase i n h i b i t o r s described below, they do not i n h i b i t α-amylase(s) of the same source. A second i n d i c a t i o n of the presence of an i n h i b i t o r i s when experimental animals f a i l to grow as w e l l on raw as heat-treated b i o l o g i c a l m a t e r i a l s . This was e s p e c i a l l y valuable i n research on raw soybean f l o u r . A t h i r d method of d e t e c t i n g i n h i b i t o r s , of rather l i m i t e d u t i l i t y , i s the observation of m u l t i p l e p H - a c t i v i t y optima f o r what i s otherwise a pure enzyme. For example, Schwimmer (_6) ob served that potato i n v e r t a s e showed a double pH optima. It was discovered that the explanation of the double pH optima was due to an i n v e r t a s e i n h i b i t o r i n the potato (7). This observation i s shown schematically i n Figure 1. The f o u r t h method of d e t e c t i n g the presence of n a t u r a l l y o c c u r r i n g enzyme i n h i b i t o r s i n b i o l o g i c a l m a t e r i a l s i s to combine e x t r a c t s with a s o l u t i o n of the enzyme being t e s t e d . This i s the most systematic way. The procedure may be no more than a s e r i e s of t e s t tubes, c o n t a i n i n g the enzyme, to which b i o l o g i c a l t i s s u e e x t r a c t s are added. There are some hazards associated with t h i s method and a d d i t i o n a l experiments are r e q u i r e d . A u s e f u l technique has been the cross-wise a p p l i c a t i o n of enzyme and i n h i b i t o r to an agar p l a t e c o n t a i n i n g substrate of the enzyme. This method was o r i g i n a t e d to detect the presence of pro tease i n h i b i t o r s i n microorganisms ( 8 ) ; i t has since been a p p l i e d to the search f o r amylase i n h i b i t o r s ( 9 ) . The p r i n c i p l e of the technique i s shown i n Figure 2 f o r protease i n h i b i t o r s . The buf fered agar gel contains abut 1% c a s e i n . C e l l u l o s e paper s t r i p s saturated with the b i o l o g i c a l extract to be tested are l a i d on the gel surface i n the v e r t i c a l d i r e c t i o n . A f t e r 15-20 minutes, these s t r i p s are removed and replaced i n the h o r i z o n t a l d i r e c t i o n with c e l l u l o s e paper s t r i p s saturated with the enzyme s o l u t i o n to be t e s t e d . A f t e r 15-20 minutes, these l a t t e r s t r i p s are removed and the gel p l a t e i s incubated overnight at 25-35°C. Protease



X E N O B I O T I C S IN F O O D S A N D F E E D S

P

Figure

1. Schematic

Enzyme

Biological Extract

Figure

H

representation of effect of enzyme inhibitor curve of an enzyme.

1 ι

1 •

1

1 ι

1 ι

2

2. Schematic representation presence of protease

1 ι

1 ι

3

1 ι

1 ι

4

1 ι

1 ι

5

on

ι

pH-activity

1—r ι

6

of casein-agar plate technique for inhibitors in biological extracts.

detecting


2.

WHITAKER

Protease

and Amylase

Inhibitors

19

a c t i v i t y i s i n d i c a t e d by a white band due to h y d r o l y s i s and prec i p i t a t i o n of the c a s e i n . If the b i o l o g i c a l extract contains an i n h i b i t o r , the white band w i l l be narrow at the cross point of the two s t r i p s ( F i g u r e 2). The system can be made more s p e c i f i c by e l e c t r o p h o r e s i s of the b i o l o g i c a l extract on the c e l l u l o s e paper s t r i p p r i o r to a p p l i c a t i o n to the agar-casein g e l . Many samples can be run simultaneously by t h i s method.


Types of Protease and Amylase I n h i b i t o r s The n a t u r a l l y - o c c u r r i n g i n h i b i t o r s of proteases and amylases can be p r o t e i n s , peptides of various s i z e s and non-proteins. Protease I n h i b i t o r s . The protease i n h i b i t o r s can be enzyme s p e c i f i c and/or group s p e c i f i c . Based on t h e i r mechanism of a c t i o n , the proteases are d i v i d e d into four groups: (a) the s e r i n e proteases, (b) the s u l f h y d r y l proteases, ( c ) the carboxyl ( a c i d ) proteases, and (d) the métallo proteases. Laskowski and Kato (10) have concluded that there are no examples of i n d i v i d u a l i n h i b i t o r s which are a c t i v e against proteases from two or more of the groups. For example, human plasma ot^-trypsin i n h i b i t o r i s a c t i v e against t r y p s i n , chymotrypsin, e l a s t a s e and plasmin ( a l l s e r i n e proteases) but i t i s not a c t i v e against proteases from any of the other groups ( 1 1 ) . The t h i o l protease i n h i b i t o r of chicken egg white (papain i n h i b i t o r ; 12,13) i s a c t i v e against f i c i n , papain, bromel a i n , cathepsin B, and s t r e p t o c o c c a l protease ( a l l s u l f h y d r y l proteases), but i t i s not a c t i v e against proteases from any of the other three groups. There are examples of i n h i b i t o r s which are s p e c i f i c f o r only one protease w i t h i n a group. The best known examples i n c l u d e the Kunitz soybean ( G l y c i n e max) i n h i b i t o r (14) and i s o i n h i b i t o r s I and II of the Great Northern bean (Phaseolus v u l g a r i s ) ( 1 5 ) . Even these two examples are not c l e a r cut as there i s some small nons t o i c h i o m e t r i c combination and i n h i b i t i o n of a-chymotrypsin. Chicken (16) and Japanese q u a i l (17) egg white ovomucoids only inhibit trypsin. One p o s s i b l e exception to the above dictum that a protease i n h i b i t o r cannot i n h i b i t proteases from two or more groups i s o^macroglobulin. T h i s very l a r g e g l y c o p r o t e i n (725,000 daltons) has a very broad s p e c i f i c i t y i n that i t binds to proteases from a l l four groups (18-23). The p o s s i b i l i t y that t h i s i s due to nons p e c i f i c adsorption i s i n d i c a t e d by the observation that the enzyme-inhibitor complex r e t a i n s a c t i v i t y on small s u b s t r a t e s , although a c t i v i t y on p r o t e i n s i s l o s t (due to s t e r i c hindrance?). Other apparent exceptions are the small peptide protease i n h i b i t o r s produced by s e v e r a l species of Streptomyces. For example, the leupeptins are a c t i v e against plasmin and t r y p s i n ( s e r i n e proteases) and papain and cathepsin Β ( s u l f h y d r y l proteases)(_24). A n t i p a i n (25) and the chymostatins are a l s o a c t i v e against enzymes from both the s e r i n e and s u l f h y d r y l groups of proteases ( 2 6 ) .


XENOBIOTICS IN FOODS A N D F E E D S


20

Amylase I n h i b i t o r s . There are four types of amylases. These a r e : (a) α-amylases, (b) β-amylases, ( c ) glucoamylases and the (d) p u l lulanase-type (debranching) amylases. The only w e l l - d e s c r i b e d type of n a t u r a l l y - o c c u r r i n g p r o t e i n amylase i n h i b i t o r s are those against the α-amylases from higher animals and i n s e c t s . These i n h i b i t o r s , with the p o s s i b l e exception of the one from corn ( 2 7 ) , are not e f f e c t i v e against higher plant and m i c r o b i a l α-amylases or against the other three types of amylases. Quite r e c e n t l y , there have been d i s c u s s i o n s of the p o s s i b i l i t y of a glycoamylase-type inhibitor (28). There are a l s o small peptide i n h i b i t o r s of α-amylase found i n c e r t a i n Streptomyces ( 2 9 ) . Two carbohydrate α-amylase i n h i b i t o r s , Acarbose and Amylostatin, have been described (30-32). Their s t r u c t u r e s are shown i n Figure 3. The i n h i b i t o r s are very s i m i l a r in structure. Heterogeneity

of Protease and Amylase

Inhibitors

There are a number of well-documented examples of hetero geneity among the protease and amylase i n h i b i t o r s . This hetero geneity i s a r e s u l t of (a) i n h i b i t o r s against m u l t i p l e enzymes from a s i n g l e b i o l o g i c a l f l u i d , (b) i s o l a t i o n from d i f f e r e n t s t r a i n s ( v a r i e t i e s ) of a s p e c i e s , ( c ) i s o i n h i b i t o r s , (d) proteo l y s i s , ( e ) chemical m o d i f i c a t i o n d u r i n g i s o l a t i o n , and ( f ) molecu l a r heterogeneity. I n h i b i t o r s of D i f f e r e n t Enzymes i n a S i n g l e B i o l o g i c a l F l u i d . A few examples w i l l i l l u s t r a t e the m u l t i p l i c i t y of i n h i b i t o r s found i n b i o l o g i c a l f l u i d s (Table I I ) . Human plasma contains at l e a s t eight d i f f e r e n t types of i n h i b i t o r s , chicken egg white contains three d i f f e r e n t types, the soybean contains four d i f f e r e n t types, and the white potato s i x d i f f e r e n t types. These are c l e a r l y d i f f e r e n t compounds as they i n h i b i t q u i t e d i f f e r e n t enzymes and they can be separated from each other. I s o l a t i o n from D i f f e r e n t S t r a i n s ( V a r i e t i e s ) of a Species. Very s i m i l a r protease i n h i b i t o r s have been i s o l a t e d from s e v e r a l d i f f e r e n t s t r a i n s ( v a r i e t i e s ) of the same species or from s i m i l a r s p e c i e s . In some cases, the i n h i b i t o r s are very s i m i l a r , i n others they are q u i t e d i f f e r e n t . Three examples w i l l i l l u s t r a t e this. Ovomucoids are probably found i n a l l species of b i r d s . They are p r o t e i n s of 28,000 daltons and c o n t a i n ~20% carbohy d r a t e . They are q u i t e s i m i l a r i n other p r o p e r t i e s as w e l l . How ever, they can be q u i t e d i f f e r e n t i n t h e i r i n h i b i t o r y p r o p e r t i e s against proteases. Chicken (16) and Japanese q u a i l (17) ovomu coids i n h i b i t only t r y p s i n . Tinamou ovomucoid i n h i b i t s chymotryp s i n and s u b t i l i s i n (66) and turkey ( 67) and penguin (68) ovomu c o i d s i n h i b i t t r y p s i n , chymotrypsin and s u b t l l i s i n . The reason f o r t h i s d i f f e r e n c e among the ovomucoids i s due to more than one b i n d i n g s i t e f o r proteases i n the ovomucoid molecule. As shown by



WHITAKER

Figure

Protease

3. Structures and amylostatin

and Amylase

Inhibitors

of the carbohydrate α-amylase inhibitors acarbose (30,31) (32) (m = 0 to 8, η = 1 to 8, and m + η = 1 to 8).



Soybean

Chicken egg white

Human plasma

Source

12,700

Thiol proteinase (papain) i n h i b i t o r

Bowman-Birk

8,000

21,700

4 6,500

Ovoinhibitor

Kunitz

28,000

Ovomucoid

Cathepsins Β and Η inhibitors

cathepsin Β

T r y p s i n , chymotrypsin

Trypsin

Papain, f i c i n ,

T r y p s i n , chymotrypsin, subt i l i s i n , A. oryzae protease

Trypsin

Cathepsins Β and H

F i c i n , papain, c a t h e p s i n Β and bromelain

90,000

Thiol proteinase inhibitor

T r y p s i n , chymotrypsin, l e s s e r extent plasmin Chymotrypsin

160,000

Int er-α-1ryρ s i n inhibitor

C^ protease, plasmin, k a l l i k r e i n , others

Thrombin, other s e r i n e proteases of blood c l o t t i n g sequence

Very broad

T r y p s i n , chymotrypsin, e l a s t a s e , plasmin

Specificity

α γ-Anti chymotrypsin

104,000

62,00067,000

725,000

54,000

Molecular Weight

of Protease I n h i b i t o r s i n B i o l o g i c a l F l u i d s

C^ i n a c t i v a t o r

Antithrombinheparin c o f a c t o r

2

a -Macroglobulin

(Xj-Trypsin inhibitor

Type

Table I I . Examples of M u l t i p l i c i t y


55, 56

U_, 52 - 54

12, 13

50, 51

J^6, _48, _49

k]_

45, 46

j£

41_ - 44.

37^- 40

35, 36

18 - 23

11, 33, 34

Reference

m w σ 00

Ζ U *τΐ

>

CO

α

Ο Ο

τ\

ο

DO

χ m z ο


Potato

Soybean (continued)

Source

Papain

inhibitor

Carboxypeptidases A and Β i n h i b i t o r

pKI-56, pKI-64

Proteinase inhibitor l i b

Proteinase inhibitor H a

Chymotrypsin inhibitor I

Components I-IV

Elastase

Type

Table I I . (continued)

80,000

3,100

39,000

7,000-8,000

Molecular Weight

_59 60 _61, J52 63 64 65

Trypsin Chymotrypsin Chymotrypsin, nagarse, trypsin Chymotrypsin, nagarse Kallikrein Carboxypeptidases A and Β Papain,

chymotrypsin

58

E l a s t a s e , a l s o t r y p s i n and chymotrypsin

Reference 57

Specificity


OS

a ft.

Co

m

Η >

X

3


24

XENOBIOTICS

IN F O O D S A N D

FEEDS

Laskowski et a l . (69), the ovomucoids contain at l e a s t three domains each with a p o t e n t i a l binding s i t e f o r a protease. Two of these s i t e s are s p e c i f i c f o r t r y p s i n and one f o r chymotrypsin ( s u b t i l i s i n binds c o m p e t i t i v e l y with chymotrypsin). In chicken, Japanese q u a i l and tinamou ovomucoids, only one of the s i t e s i s capable of binding a protease while i n turkey and penguin ovomu coids two binding s i t e s are expressed and i n duck ovomucoid (67) a l l three s i t e s are expressed. This i s shown d i a g r a m a t i c a l l y i n Figure 4. The t r y p s i n i n h i b i t o r s from s e v e r a l d i f f e r e n t v a r i e t i e s of Phaseolus v u l g a r i s (common bean) are q u i t e v a r i a b l e i n amino a c i d composition (Figure 5). ASN, SER and 1/2CYS are found i n the l a r g e s t amounts i n most of the i n h i b i t o r s . MET and TRP are q u i t e low i n a l l the i n h i b i t o r s and absent i n a s u b s t a n t i a l number of them. PRO i s present i n l a r g e r amounts than i n most p r o t e i n s . PHE and TYR are i n low amounts. I s o i n h i b i t o r s (Genetic Heterogeneity). The t h i r d type of h e t e r o geneity i s that of i s o i n h i b i t o r s . These i s o i n h i b i t o r s , obtained from one organ or organism, have the same s p e c i f i c i t i e s f o r b i n d ing proteases. However, they d i f f e r i n one or more p r o p e r t i e s such as chromatographic and e l e c t r o p h o r e t i c behavior, heat s t a b i l i t y , molecular weight, or amino a c i d composition, as w e l l as q u a n t i t a t i v e l y i n binding with proteases. S n a i l epidermis contains at l e a s t s i x t r y p s i n - k a l l i k r e i n i n h i b i t o r s with molecular weights ranging from 6431 to 6591 (70-72). The soybean contains two b a s i c types of protease i n h i b i t o r s , the Kunitz i n h i b i t o r of 21,500 daltons (73) and the Bowman-Birk i n h i b i t o r of 7975 daltons (74). The two are quite d i f f e r e n t pro t e i n s as shown i n Figure 6. The Great Northern bean (Phaseolus v u l g a r i s ) has at l e a s t three t r y p s i n i s o i n h i b i t o r s ranging i n molecular weight from 8086 to 8884 (15). There are four and p o s s i b l y s i x i s o i n h i b i t o r s of t r y p s i n i n lima bean (Phaseolus lunatus)(75). Recently, Whitaker and S g a r b i e r i (76) and S g a r b i e r i and Whitaker (77) reported there are at l e a s t four i s o i n h i b i t o r s of t r y p s i n i n B r a z i l i a n pink beans (Phaseolus v u l g a r i s L. v a r . Rosinha G2). These are separable on a DEAE-cellulose column (Figure 7) but not by a f f i n i t y chromatography (76). P r o p e r t i e s of three of the i s o i n h i b i t o r s were i n v e s t i g a t e d . They are d i f f e r e n t by d i s c g e l e l e c t r o p h o r e s i s , have s l i g h t l y d i f f e r e n t amino a c i d compositions but have i d e n t i c a l molecular weights (within e x p e r i mental e r r o r ) and a l l contain one residue of mannose per mol. The molecular weights are 20,000, about twice the s i z e of most BowmanB i r k type i n h i b i t o r s i n beans· They each bind two mol of t r y p s i n and one mol of chymotrypsin per mol i n h i b i t o r . One of the l a r g e s t d i f f e r e n c e s among the three i s o i n h i b i t o r s i s i n the a f f i n i t y f o r t r y p s i n and chymotrypsin (77). As shown i n Table I I I , the d i s s o c i a t i o n constants f o r t r y p s i n range from 1.8 χ 10 M^ to 8.5 χ 10~ M^while those f o r chymotrypsin range from 2.8 χ 10 M_ to 10


2.

Protease

WHITAKER

and Amylase

Inhibitors

25

Figure 4. Schematic representation of multiple binding sites for proteases, and E , on an inhibitor, I. In the case of duck ovomucoid, E is trypsin and is chymotrypsin.


B

E E

A

A

5

15

5

15

5

15

5

15

5

15

5

B

15

CO

8r

5

15

5

15

5

15

5

15

5

15

5

15

INHIBITOR N U M B E R Figure 5. Amino acid composition of several protease inhibitors from soybean ( G l y c i n e m a x ) and several species and varieties of P h a s e o l u s . The results are expressed as amino acid residues per 10,000 grams. The inhibitors are 1, Kunitz soybean trypsin inhibitor; 2, Bowman-Birk soybean trypsin inhibitor; 3-6, lima bean (Phaseolus lunatus) isoinhibitors I, II, III, and IV; 7-9, French bean (Phase o l u s c o c c i n e u s ) isoinhibitors 2, 3, and 4; 10, mung bean ( P h a s e o l u s a u r e u s Roxb.) inhibitor; 11-13, Brazilian pink bean (Phaseolus vulgaris) isoinhibitors A, B, and C; 14, kidney bean (Phaseolus vulgaris) inhibitor; 15-17, Great Northern bean (Phaseolus lunatus) isoinhibitors I, II, III, and IV; 7-9, French bean (Phasvulgaris) isoinhibitor; and 19 and 20, pinto bean (Phaseolus vulgaris) isoinhibitors I and II (97).



26

XENOBIOTICS

IN F O O D S A N D

FEEDS

Figure 6. Primary structures of the Kunitz soybean trypsin inhibitor (A) and the Bowman-Birk soybean trypsin inhibitor (B). The solid black circles on the primary structure of the Bowman-Birk soybean trypsin inhibitor represent LYSSER and LEU-SER and are the binding sites for trypsin and chymotrypsin, respectively. (Reproduced with permission from (A) Ref. 73, copyright 1973, E . J . o f B i o c h . and (B) Ref. 74, copyright 1973, J . F o o d B i o c h e m J


Protease


WHITAKER

and Amylase

Inhibitors

1.2

18

~

A

g

β ο

H»

GO c\i 0.8

>^

13.5

_l

UJ

E 9.0 I

ο ζ

< CD

oc Ο·

0.5

X

0.4 ζ

liJ

0.3 Ior ω 0.2 ·ο

4

4.5

Ο

(/)

ί

m

Ho..

< 64

96

FRACTION NO.

128

160

(4.7 ml)

Figure 7. DEAE-cellulose chromatography of the trypsin-chymotrypsin isoin hibitors from Brazilian pink bean (Phaseolus vulgaris var. Rosinha G2. (Repro duced with permission from Ref 76. Copyright 1981, J . F o o d B i o c h e m . )


28

X E N O B I O T I C S IN

Table I I I .

FEEDS

1^ Values for the Binding of α-Chymotrypsin and T r y p s i n with B r a z i l i a n Pink Bean I n h i b i t o r s A, Β and C (77) *i

Inhibitors A Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 6, 2015 | http://pubs.acs.org Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

FOODS AND

α-Chymotrypsin (CT) 4.4

Β

2.8

C

3.0

χ

8.5

10"

1.8

1

χ

10""

χ

1

W

T r y p s i n (T)

6.8

10"

K (CT)/K (T) 1

1

χ 10~

iU

520

χ 10"

10

160

χ 10"

10

44

7

4.4 χ 10" M. The r a t i o s of K ( C T ) / K ( T ) are: A, 520; B, 160; C, 44. These binding p r o p e r t i e s c l e a r l y i n d i c a t e that the three i s o i n h i b i t o r s are d i f f e r e n t and most l i k e l y are not the r e s u l t of a r t i f a c t s ( p r o t e o l y s i s or binding with phenols) produced by the p u r i f i c a t i o n procedure. Another w e l l studied example of genetic heterogeneity i s that of the wheat α-amylase i n h i b i t o r s . There appear to be at l e a s t three d i f f e r e n t molecular weight species of i n h i b i t o r s (60,000, 24,000 and 12,000) as w e l l as d i s t i n c t species w i t h i n each of these molecular weight groups. Granum and Whitaker (78) have p u r i f i e d and c h a r a c t e r i z e d three of the α-amylase i n h i b i t o r s from Anza wheat ( T r i t i c u m aestivum var. Anza). Their chemical, p h y s i c a l and i n h i b i t o r y p r o p e r t i e s were q u i t e d i f f e r e n t (Table IV). They a l s o d i f f e r e d i n l y s i n e , a r g i n i n e , h i s t i d i n e , alanine ( 1 ^ L

1

Table IV. P r o p e r t i e s of Three α-Amylase I n h i b i t o r s of Wheat ( T r i t i c u m aestivum var. Anza)(78) Inhibitors Property Molecular weight Hedrick-Smith method (79) Sedimentation e q u i l i b r i u m pi S p e c i f i c i t y on α-amylases Human s a l i v a r y Hog pancreatic Bacillus subtilis A s p e r g i l l u s oryzae a

R

m

3

0.19

1^ 0.28

1^

0.55

24,000 29,000

18,500 14,500

30,000

5.9

5.2

4.2

+ + -

± -

+ -

= E l e c t r o p h o r e t i c m o b i l i t y r e l a t i v e to bromophenol blue.


2.

WHITAKER

Protease

and Amylase

Inhibitors

29

0 · 2 8 o n l y ) , v a l i n e ( R 0 . 2 8 only) and phenylalanine ( R 0 . 2 8 only) contents. They a l l had r e l a t i v e l y high contents of p r o l i n e and half-cystine. De Ponte et a l . ( 8 0 ) have r e c e n t l y proposed a model that might e x p l a i n the r e l a t i o n s h i p among a l l the known α-amylase i n h i b i t o r s i n wheat (see Figure 8 ) . How d i f f e r e n t the 1 2 , 0 0 0 d a l t o n subunits of the three i n h i b i t o r s are from the proposed a n c e s t r a l 1 2 , 0 0 0 d a l t o n molecule i s not known. U n f o r t u n a t e l y , the RJJJ 0 . 5 5 amylase i n h i b i t o r of MW 3 0 , 0 0 0 reported by Granum and Whitaker ( 7 8 ) does not f i t t h i s model.


m

m

Proteolytic Artifacts. The f o u r t h type of heterogeneity reported i s that produced by p r o t e o l y s i s . This must be of p a r t i c u l a r con cern i n the i s o l a t i o n of any p r o t e i n s i n c e the h y d r o l y s i s of one or two peptide bonds w i l l give r i s e to a number of products. Re c e n t l y , i t has been reported that some of the protease i n h i b i t o r s p r e v i o u s l y i s o l a t e d from winged bean are the r e s u l t of p r o t e o l y s i s (81). The evidence f o r t h i s i s q u i t e convincing, l e a d i n g to the p o s s i b i l i t y that some of the i s o i n h i b i t o r s reported i n the l i t e r a ture are the r e s u l t of p r o t e o l y s i s . Whitaker and S g a r b i e r i ( 7 6 ) addressed t h i s i s s u e i n d e t a i l , p r o v i d i n g s e v e r a l data to i n d i c a t e that t h i s i s probably not the case f o r the i s o i n h i b i t o r s of B r a z i l i a n pink beans. Peptide mapping of i s o i n h i b i t o r s would be a v a l u a b l e t o o l i n t h i s respect, but i t has not been p r e v i o u s l y a p p l i e d to t h i s problem. Chemical M o d i f i c a t i o n . The f i f t h type of heterogeneity i s due to chemical m o d i f i c a t i o n , other than p r o t e o l y s i s , d u r i n g i s o l a t i o n of the i n h i b i t o r s . Beans, f o r example, c o n t a i n phenolic compounds and various amounts of polyphenol oxidase. I f polyphenol oxidase i s a c t i v e d u r i n g the i s o l a t i o n procedure there i s the r e a l p o s s i b i l i t y that some of the products (benzoquinones) formed w i l l r e a c t with the e-amino group of l y s i n e residues of the i n h i b i t o r ( s ) , thereby producing e l e c t r o p h o r e t i c a l l y and chromatographically d i s t i n c t components. P u s z t a i ( 8 2 ) p u r i f i e d an i n h i b i t o r from kidney beans which contained a f i r m l y bound p i n k i s h - b l u e pigment that was not removed by ammonium s u l f a t e p r e c i p i t a t i o n , gel f i l t r a t i o n or by chromatography on a DEAE-Sephadex column. For i n h i b i t o r s which are g l y c o p r o t e i n s (ovomucoids, some of the Phaseolus v u l g a r i s i n h i b i t o r s , the red kidney bean α-amylase i n h i b i t o r s ) , v a r i a b l e amounts of carbohydrate attached to the p r o t e i n w i l l produce i s o inhibitors. Molecular Heterogeneity. The l a s t type of heterogeneity we s h a l l d i s c u s s i s that of molecular heterogeneity. This type of hetero geneity was mentioned under the s e c t i o n on i n h i b i t o r s from d i f f e r ent v a r i e t i e s of a s p e c i e s . I t i s w e l l known that many of the i n h i b i t o r s have more than one b i n d i n g s i t e f o r proteases, e i t h e r f o r the same protease or f o r d i f f e r e n t proteases. Examples of t h i s molecular heterogeneity are shown i n Table V. In the case of the b i r d egg white ovomucoids, Laskowski et a l . ( 6 9 ) have shown that



XENOBIOTICS

IN F O O D S A N D

Figure 8. Possible interrelationships among the protein α-amylase families from hexaploid wheats (80). All three inhibitors may have from a common ancestral protein of 12,000 daltons.

FEEDS

isoinhibitor originated



a

n.d. n.d.

+

-K

τι ο ο

ο

Η

δ

χ m z ο


LB

GB

ΒΒ

LB

GB

H

eu

™

10 Cys •Asp-Gln-Cy s-Ala- Cys-Thr

Continued on next page

20 t •Pr o-Pro-Gln-Cy s-•Arg Cys- -Ser-Asp-Lys-rSer-j-Asn30 ( V a l , Cys)Thr - A l a + S e r f l l e •Pro-Pro-Gln(Cys i l e , Cys ,Thr, Asx, 30 1 ! Cys- -As η-Hi s -Cy s -tA l a Cys-Thr -Lys-j-Ser-j-Ile- -Pr o-Pro-Gln-Cy s--Arg- Cys •Thr" •AspSer L^ 40 30 -Ile- Cys -Ala-Leu- SerMet+Arg-Leuf-Asn- Ser-Cy s-His-Ser-Ala-Cys50 ! I 40 Val)Arg-LeufAsx-Ser-Cys-His-Ser-Ala-Cys- Lys-Ser-Cys -Met- Cys -Thr-Arg- •Ser50 j ; 40 ILeutfArg-Leuf-Asp- Ser-Cy s-His-Ser-Ala-Cy s- Lys-Ser-Cys -Ile- •Cys--Thr-Leu- •Ser -

10 Ser-Gly-His-His-Glu-His-Ser ·-Thr-Asp-Glx- -Pro-Ser-Glx- Ser-Ser- Lys-Pro-Cys-

LB

BB

Asp-Asp-Glu- Ser-Ser- •Lys-Pro-Cys-

Amino Acid Sequence Homology Among the Bowman-Birk Soybean T r y p s i n I n h i b i t o r (BB: 89), Lima Bean T r y p s i n I n h i b i t o r IV (LB; 75) and Great Northern Bean T r y p s i n I n h i b i t o r I I (GB; 90)

BB

Table V I I I .


u>

ο

•?

ε-

ν;

δ

H > m 50

3


LB

GB

BB

LB

GB

BB

Table V I I I .

7

0

Asn Asp-Asp-Ly s-Glir J 80 LysfSer-Asx-Ser-Gly-Glx-Asx-Asx 80 tAsnfAsn Lys-Ser-Ser-His-Ser-Asp-Asp-Asp-Asn

!

60 50 Tyr4Pro|-Ala-Gln CysfPhe+Cys •Val-Asp-Ile-Thr-Asp-Phe+Cys-Tyr-fGlu-Pro-[Cysi 70 60 Met -fPr of-Gly-Ly s-j-Cy s fAr g-fCy s j-Leu-Asx-Thr-Thr-Asx-Ty r+Cy s-TyrfLys-Ser4Cys 70 60 ί CysIle-fPrc^Ma-Gln+C^

— ι Ly s f P r o-Ser-Glu

Continued


α τ\ m m α

Ζ

•Α ο ο σ >

η 05

Η

δ

x m ζ ο β

ON


2.

WHITAKER

Protease

and Amylase

37

Inhibitors

There appears to be sequence homology between the pineapple stem bromelain i n h i b i t o r s and some of the small molecular weight i n h i b i t o r s from the leguminosae (91). Human i n t e r - a - t r y p s i n i n h i b i t o r contains two domains with great s i m i l a r i t y to the domains of the Kunitz-type i n h i b i t o r s (44, 92-94). The o v o i n h i b i t o r s from Japanese q u a i l and chicken egg whites contain s i x tandem domains which are homologous to the Kazal p a n c r e a t i c s e c r e t o r y i n h i b i t o r and to the ovomucoids (69)· Considerable homology e x i s t s w i t h i n the binding s i t e s of sev e r a l of the i n h i b i t o r s as shown i n Table VII. It has been sug gested that t r y p s i n i n h i b i t o r s require a peptide sequence of Lys-X or Arg-X located w i t h i n a loop of the p r o t e i n closed by a d i s u l f i d e bond (95,96). Reduction of d i s u l f i d e bonds are known to be quite e f f e c t i v e i n destroying the i n h i b i t o r y a c t i v i t y (77). For example, a c t i v i t y of the three i s o i n h i b i t o r s from B r a z i l i a n pink beans against both t r y p s i n and chymotrypsin was l o s t when a spe c i f i c d i s u l f i d e bond, of the 18-21 d i s u l f i d e bonds present, was reduced ( 77). SER appears to be a requirement of the binding s i t e also f o r l y s i n e - t y p e i n h i b i t o r s (Table V I I ) . There i s homology among the binding s i t e s f o r chymotrypsin i n the i n h i b i t o r s from lima bean, soybean and runner bean (Table VII). The apparent e s s e n t i a l i t y of a serine residue i n the b i n d ing s i t e f o r chymotrypsin i s also i n d i c a t e d . There appears to be much l e s s homology among the binding s i t e s of the arginine-type t r y p s i n i n h i b i t o r s (Table V I I ) . In terms of amino a c i d composition, there i s some homology among the Bowman-Birk type i n h i b i t o r s from the leguminosae as shown i n Figure 5. This appears to be so, despite the f a c t that there are some i n h i b i t o r s with molecular weights near 8000 (Bowman-Birk soybean i n h i b i t o r , lima bean i n h i b i t o r s I-IV, French bean i n h i b i t o r s 2, 3 and 4, mung bean i n h i b i t o r , kidney bean i n h i b i t o r , and Great Northern bean i n h i b i t o r s I, II and I l l b ) , and others with molecular weights near 20,000 ( B r a z i l i a n pink bean i n h i b i t o r s A, Β and C, Navy bean i n h i b i t o r and pinto bean i n h i b i tors I and II)(see 97_ f o r d e t a i l s ) . It c e r t a i n l y would be h e l p f u l to have t r y p t i c peptide maps and amino a c i d sequence ( i d e a l l y ) data on a l l of these i n h i b i t o r s . Recent immunochemical data (98) have shown that the α-amylase i n h i b i t o r s from s e v e r a l v a r i e t i e s of beans have great homology. Mechanism of Action of the

Protease and

Amylase

Inhibitors

Protease I n h i b i t o r s . There are unique and s p e c i f i c r e c o g n i t i o n s i t e s on the protease i n h i b i t o r s f o r t r y p s i n and chymotrypsin as shown i n Tables VI and VII. The best data are a v a i l a b l e f o r t r y p s i n where i t i s known that e i t h e r a s p e c i f i c a r g i n i n e or a l y s i n e residue i n the binding s i t e of the i n h i b i t o r i s r e q u i r e d . Modifi c a t i o n of the a r g i n i n e (by g l y o x y l a t i o n ) or l y s i n e (by a l k y l a t i o n , etc.) residue or t h e i r removal (99) r e s u l t s i n complete loss of



38

XENOBIOTICS

IN F O O D S A N D

FEEDS

inhibitory activity. The b i n d i n g s i t e s f o r chymotrypsin appear to r e q u i r e l e u c i n e , t y r o s i n e or methionine. It i s probable that a s e r i n e residue attached to the l y s i n e or t y r o s i n e , l e u c i n e or methionine i s a l s o required (Table V I ) . However, t h i s does not appear to be the case f o r the a r g i n i n e - t y p e t r y p s i n i n h i b i t o r s . It appears, t h e r e f o r e , that t r y p s i n and chymotrypsin recognize and bind with the same amino a c i d residues i n the i n h i b i t o r s as with any s u b s t r a t e . However, net h y d r o l y s i s of peptide bonds does not occur at the pH optimum of the enzymes f o r reasons l a r g e l y unknown at t h i s time. S p e c i f i c h y d r o l y s i s of the peptide bond i n v o l v i n g the carboxyl group of the e s s e n t i a l amino a c i d residue does occur at low pH (around pH 4 ) . However, there i s l i t t l e current evidence to i n d i c a t e that t h i s h y d r o l y s i s i s a key step i n the i n h i b i t o r y process. Two types of data argue against i t s e s s e n t i a l i t y , (a) Complexation between i n h i b i t o r and chemically modified i n a c t i v e proteases i s o f t e n just as t i g h t as with the n a t i v e protease (12, 100, 101). (b) I n i t i a l l y , X-ray c r y s t a l l o g r a p h i c data on the complexes between t r y p s i n and p r o t e i n i n h i b i t o r s were i n t e r p r e t e d to i n d i c a t e that the complexes were probably adducts with a t e t r a h e d r a l intermediate s t a t e approaching a covalent bond (102, 103). However, b e t t e r refinements of the X-ray c r y s t a l l o g r a p h i c maps i n d i c a t e the d i s t a n c e s are too great for covalent bond formation. C-NMR studies appear to c o n c l u s i v e l y r u l e out formation of a t e t r a h e d r a l or covalent i n t e r mediate as a step i n the mechanism of i n h i b i t i o n (104-106). K i n e t i c data i n d i c a t e a conformational change may occur f o l lowing formation of the i n i t i a j . complex ( 107). Such a conforma t i o n a l change could provide s t a b i l i t y to the complex through p r e v e n t i n g i t s ready d i s s o c i a t i o n (k_2 « k_^) or i n preventing h y d r o l y s i s of the peptide bond i n the i n h i b i t o r as would occur f o r a r e g u l a r substrate (Eqn. 1). However, J i b s o n et a l .

Ε + I

v.

1

. k

- l

^

EI

2

. ^ -2

EI

f

(1)

k

(108) have r e c e n t l y shown that a conformational change probably does not occur i n t r y p s i n on binding with the Bowman-Birk soybean i n h i b i t o r or with the chick-pea t r y p s i n i n h i b i t o r . A complete explanation of why the n a t u r a l l y - o c c u r r i n g p r o t e i n protease i n h i b i t o r s are so e f f e c t i v e as i n h i b i t o r s i s s t i l l not available. α-Amylase I n h i b i t o r s . The p r o t e i n α-amylase i n h i b i t o r s form a very t i g h t complex with s a l i v a r y and p a n c r e a t i c α-amylases. For example, the complex between the red kidney bean p r o t e i n i n h i b i t o r and porcine p a n c r e a t i c α-amylase at 30°C and pH 6.9 was c a l c u l a t e d to be 3.5 χ 10~* M_ (109). The i n a c t i v e complex forms slowly, r e q u i r i n g 60 to 120 minutes to reach complete r e a c t i o n depending on pH and i n h i b i t o r and enzyme concentrations (109). The red kidney 1



2.

WHITAKER

Protease

and Amylase

Inhibitors

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bean p r o t e i n i n h i b i t o r does not i n h i b i t plant and m i c r o b i a l α-amy l a s e s ; only those from higher animals and i n s e c t s are i n h i b i t e d . It appears there i s an i n i t i a l r a p i d complex formed between the enzyme and i n h i b i t o r which i s s t i l l a c t i v e (110). Then, a much slower conformational change occurs (109,110) l e a d i n g to l o s s of a c t i v i t y . u n l i k e the p r o t e i n protease i n h i b i t o r s , complexation between the red kidney bean p r o t e i n α-amylase i n h i b i t o r and α-amylase does not appear to involve binding at the a c t i v e s i t e of the α-amylase. Evidence f o r t h i s i n c l u d e s a b i l i t y of the complex to bind maltose (a competitive i n h i b i t o r of α-amylase), s t a r c h , Sephadex and to s t i l l hydrolyze small s u b s t r a t e s . The red kidney bean α-amylase i n h i b i t o r contains 9-10% cov a l e n t l y bound carbohydrate. Removal of up to 70% of the carbo hydrate does not a f f e c t the a c t i v i t y of the i n h i b i t o r (110). The glyco groups, removed from the p r o t e i n , do not i n h i b i t α-amylase at 3.5 χ 10* times the c o n c e n t r a t i o n of the i n h i b i t o r (110). Chemical m o d i f i c a t i o n studies i n d i c a t e that h i s t i d i n e and t y r o s i n e residues i n the i n h i b i t o r may be important f o r i t s a c t i v i t y (110). In summary, our present knowledge of the mechanism of a c t i o n of the red kidney bean α-amylase i n h i b i t o r i n d i c a t e s that an i n i t i a l complex i s formed between i n h i b i t o r and enzyme which does not involve the a c t i v e s i t e of the enzyme (complex s t i l l f u l l y active). Subsequently, there i s a conformational change i n the complex which destroys the a b i l i t y of α-amylase to hydrolyze large substrates but does not prevent t h e i r binding to the enzyme. P h y s i o l o g i c a l and N u t r i t i o n a l Importance of the Protease and Amylase I n h i b i t o r s Protease I n h i b i t o r s . In animals, the p h y s i o l o g i c a l r o l e s of sev e r a l of the protease i n h i b i t o r s are w e l l known. The p a n c r e a t i c protease i n h i b i t o r s p r o t e c t the p a n c r e a t i c t i s s u e against prema ture a c t i v a t i o n of the p r o t e o l y t i c enzyme zymogens· The i n h i b i t o r s a s s o c i a t e d with the blood c l o t t i n g system prevent the pre mature a c t i v a t i o n of the p r o t e o l y t i c enzyme zymogens c i r c u l a t i n g i n the blood at a l l times and also regulate between coagulation and f i b r i n o l y s i s . They may also be a p r o t e c t i o n against pancre a t i c proteases l i b e r a t e d i n t o the blood, as i n p a n c r e a t i t i s . The protease i n h i b i t o r s i n the r e s p i r a t o r y t r a c t probably serve as a p r o t e c t i o n against proteases l i b e r a t e d by granulocytes and macro phages brought i n as a r e s u l t of i r r i t a t i o n and/or diseased con d i t i o n s of the r e s p i r a t o r y t r a c t or through i n h a l a t i o n of micro organisms . The p h y s i o l o g i c a l r o l e of the protease i n h i b i t o r s ( e s p e c i a l l y the small peptide d e r i v a t i v e s ) i n microorganisms may, i n part, be to prevent the growth of other microorganisms . They may also be important i n the r e g u l a t i o n of p r o t e o l y s i s i n the c e l l (111-113). The p h y s i o l o g i c a l r o l e of the protease i n h i b i t o r s i n higher plants i s l e s s c l e a r even though they may account f o r 5-10% of the



40

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FEEDS

t o t a l p r o t e i n (114). The l e v e l of i n h i b i t o r s v a r i e s with the stage of growth, suggesting that the i n h i b i t o r s are p h y s i o l o g i c a l l y important (114). The t h i o l protease i n h i b i t o r s of the pineapple f r u i t have a c t i v i t y against the major p r o t e o l y t i c en zymes, bromelains, present i n the f r u i t (115). They may a l s o serve as a defense against i n s e c t s and microorganisms (114,116)> I n f e s t a t i o n s of potatoes with Colorado potato b e e t l e l a r v a e lead to markedly increased l e v e l s of protease I n h i b i t o r - I i n the leaves (117). The n u t r i t i o n a l importance of the protease i n h i b i t o r s i n major foods i s reasonably c l e a r . It i s known that raw soybean f l o u r i n h i b i t s growth i n r a t s , chickens and some other monogastric animals (118) and death can r e s u l t (119). It i s a l s o known that the presence of soybean i n h i b i t o r i n the small i n t e s t i n e increases the s e c r e t i o n of a hormonal pancreozymic-like substance that markedly stimulates e x t e r n a l s e c r e t i o n by the pancreas (120). The presence of a c t i v e p r o t e o l y t i c enzyme i n h i b i t o r s i n the small i n t e s t i n e increases the production and s e c r e t i o n of p r o t e o l y t i c enzymes by the pancreas, presumably to compensate f o r t h e i r l o s s by complexation (121-123). This r e s u l t s i n h y p e r p l a s i a of some of the p a n c r e a t i c c e l l s and enlargement of the pancreas. Unambiguous i n t e r p r e t a t i o n of most of the data i n the l i t e r a ture on the q u a n t i t a t i v e r o l e of the protease i n h i b i t o r s i n foods i s not p o s s i b l e . This i s because foods a l s o c o n t a i n other i n h i b i tory substances such as hemagglutinins, amylase i n h i b i t o r s , e s t r o gens and p h y t i c a c i d . Rackis (116) has suggested that the soybean t r y p s i n i n h i b i t o r appears to account f o r 30-50% of the growth r e t a r d a t i o n seen on feeding raw f l o u r and probably most of the pan c r e a t i c enlargement. Other workers have suggested that a part of the growth r e t a r d a t i o n may be due to u n a v a i l a b i l i t y of c y s t i n e , due to the poor d i g e s t i b i l i t y of the protease i n h i b i t o r s (124). Most, but perhaps not a l l , of the protease i n h i b i t o r s are destroyed by cooking of the food. The long range consequences of feeding humans low concentrations of a c t i v e protease i n h i b i t o r s are not known. α-Amylase I n h i b i t o r s . P r o t e i n s i n h i b i t o r y of α-amylase are found i n many b i o l o g i c a l f l u i d s (_9). However, only the p r o t e i n i n h i b i t o r s found i n legumes and i n wheat have been e x t e n s i v e l y i n v e s t i gated. Recently, i t has been shown that a l l i n s e c t α-amylases t e s t e d , except one, are i n h i b i t e d by the red kidney bean α-amylase i n h i b i t o r (125). Y e t t e r et a l . (126) have suggested that the wheat α-amylase i n h i b i t o r s may be a c t i v e against attack of the wheat by i n s e c t s d u r i n g storage. With one exception (see below) the plant α-amylase i n h i b i t o r s do not have any a c t i v i t y against higher plant or m i c r o b i a l amylases tested (127). The three α-amylase i n h i b i t o r s of maize have been reported to i n h i b i t maize a-amylase(s), i n d i c a t i n g a p o s s i b l e p h y s i o l o g i c a l r o l e of these i n h i b i t o r s i n maize (27).



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The n u t r i t i o n a l s i g n i f i c a n c e of the α-amylase i n h i b i t o r s i s l a r g e l y unknown. I t i s known that low l e v e l s of i n h i b i t o r y a c t i v i t y can be detected i n r e g u l a r l y cooked food products. When red kidney bean α-amylase i n h i b i t o r , f r e e of protease i n h i b i t o r s and hemagglutinins, was fed to rats i n a c a s e i n d i e t at the l e v e l s of 4.5 and 75 mg/rat/day, there was no decrease i n r a t e of growth of the r a t s i n r e l a t i o n to the c o n t r o l (128). Jaffê and Vega L e t t e (129) reported f e c a l s t a r c h from r a t s fed raw white kidney beans. Lang et a l . (130) reported a reduction of growth rate and i n creased f e c a l s t a r c h l e v e l s when r a t s were fed on a c a s e i n / s t a r c h d i e t c o n t a i n i n g p u r i f i e d wheat α-amylase i n h i b i t o r s . Bo-Linn et a l . (131) reported that α-amylase i n h i b i t o r , fed to humans as an impure preparation, had no e f f e c t on the c a l o r i c value of the starchy meal.

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