Alkali-Induced Lysinoalanine Formation in Structurally Different

12

Alkali-Induced Lysinoalanine Formation in Structurally Different Proteins

MENDEL FRIEDMAN

Downloaded by MONASH UNIV on July 17, 2013 | http://pubs.acs.org Publication Date: March 13, 1979 | doi: 10.1021/bk-1979-0092.ch012

Western Regional Research Center, Science and Education Administration, U.S. Department of Agriculture, Berkeley, CA 94710

Crosslinked amino acids have been identified in acid hydrolysates and enzyme digests of alkali-treated and heat-treated proteins. (Papers in references 1 and 2 cover this subject comprehensively). One such crosslinking derivative, lysinoalanine, has been found to cause histological (pathological) changes in rat kidneys (3, 4). These observations cause concern about the nutritional quality and safety of alkali-treated foods. Chemical changes that govern formation of unnatural amino acids during alkali treatment of proteins need to be studied and explained, and strategies to minimize or prevent these reactions need to be developed. In previous papers, we have (a) reviewed elimination reactions of disulfide bonds in amino acids, peptides, and proteins under the influence of alkali (5); (b) analyzed factors that may operate during alkali-induced amino acid crosslinking and its prevention (6); (c) demonstrated inhibitory effects of certain amino acids and inorganic anions on lysinoalanine formation during alkali treatment of casein, soy protein, wheat gluten, and wool and on lanthionine formation in wool (7, 8, 9); (d) demonstrated that protein acylation inhibits lysinoalanine formation in wheat gluten 0-8412-0478-0/79/47-092-225$05.00/0 This chapter not subject to U.S. copyright Published 1979 American Chemical Society In Functionality and Protein Structure; Pour-El, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

226

FUNCTIONALITY AND PROTEIN STRUCTURE

and soy protein (8, 9); (e) studied the transformation of lysine to lysinoalanine, and of cystine to lanthionine residues in proteins and polyamino acids (10); and (f) examined effects of lysine modification on chemical, nutritional, and functional properties of proteins (11). In this paper I report and discuss the susceptibilities of alkali-labile amino acid residues in three proteins to degradation as a function of pH.


EXPERIMENTAL Commercial casein was obtained from International Casein Corporation, San Francisco, California; commercial wheat gluten and lactalbumin from United States Biochemical Corporation, Cleveland, Ohio. Alkali Treatments. The following procedure, illustrated with casein, was also used with the other food proteins. A solution or suspension of casein (usually 0.5 gram per 50 cc of solvent or 1% w/v) in borate buffer of appropriate pH in a glass-stoppered Erlenmeyer flask was placed in a 65°C water bath. After the indicated time, the solution was dialyzed against 0.01N acetic acid with frequent changes of water plus acetic acid for about two days and then lyophilized. Amino Acid Analyses. A weighed sample (about 5 mg) of protein was hydrolyzed in 15 cc of 6N HC1 in a commercial hydrolysis tube. The tube was evacuated, placed in an acetone-dry ice bath, evacuated and refilled with nitrogen twice before being placed in an oven at 110°C for 24 hours. The cooled hydrolysate was filtered through a sintered disc funnel, evaporated to dryness at 40°C with the aid of an aspirator, and the residue was twice suspended in water and evaporated to dryness. Amino acid analysis of an aliquot of the soluble hydrolysate was carried out on a Durrum Amino Acid Analyzer, Model D-500 under the following conditions: single column Moore-Stein ion-exchange chromatography method; Resin, Durrum DC-4A; buffer pH, 3.25, 4.25, 7.90; photometer, 440 nm, 590 nm; column, 1.75 mm X 48 cm; analysis time, 105 min. Norleucine was used as an internal standard. In this system, lysinoalanine (LAL) is eluted just before histidine. The color constant of LAL was determined with an authentic sample purchased from Miles Laboratories. Some typical results are shown in Figures 1-4.

In Functionality and Protein Structure; Pour-El, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

FRIEDMAN

Lysinoalanine

Formation

227

1% CASEIN, H 0 , 2


3 HRS, 6 5 ° C

Figure 1.

Figure 2.

Amino acid analysis of a hydrolysate of casein heated in water

Amino acid analysis of a hydrolysate of casein heated in a pH 10.6 buffer. Note lysinoalanine peak.



228

FUNCTIONALITY

Figure 3.

A N D PROTEIN

STRUCTURE

Amino acid analysis of a hydrolysate of casein heated in a pH 11.2 buffer. Note lysinoalanine peak.

CASEIN, 0.1 IN N a O H , 3 HRS, 6 5 ° C

ι 10

ι 20

> 30

I 40

I 50

I 60

1

1

1

70

80

90

MIN.

Figure 4.

Amino acid analysis of a hydrolysate of casein heated in a 0.1N NaOH solution. Note lysinoalanine peak.


12.

FRIEDMAN

Lysinoalanine

Formation

229

RESULTS AND DISCUSSION


The amino acid composition of alkali-treated casein, l a e t a l bumin, and wheat gluten are given i n Tables I-III. The results show that the following amino acids are destroyed to various extents under basic conditions: threonine, serine, cystine, lysine, and arginine, and possibly also tyrosine and histidine. The losses of these amino acids i s accompanied by the appearance of lysinoala nine and other ninhydrin-positive compounds. Inspection of the Tables reveals several interesting points. F i r s t , loss of lysine appears to level off or go through a mini mum with increasing pH. A possible explanation i s that lysinoala nine i s destroyed (besides being formed) during a l k a l i treatment, regenerating lysine. An analogous regeneration of lysine has been shown to occur when ε-cyanoethyl derivatives of lysine are subjected to alkaline conditions (12). This possibility i s supported by the following observations, (a) Time studies show that lysinoalanine formation appears to level off after about one hour when lactalbumin or soy protein i s exposed to IN NaOH at 65°C up to 8 hours (9). (b) Exposure of free- and proteinbound lysinoalanine to alkaline conditions appears not to always give quantitative recovery of lysinoalanine (13). Comparison of lysinoalanine values for wheat gluten, casein and lactalbumin treated at various pH's shows large differences in the amounts of lysinoalanine formed in the three proteins. For example, the respective values at pH 10.6 are 0.262, 0.494, and 1.04 mole per cent (ratio of about 1:2:4); at pH 11.2 the values are 0.420, 0.780, and 1.52 mole per cent; and at pH 12.5 (pH of 1% protein solution i n 0.IN NaOH), the respective values are 0.762, 0.780, and 2.62 mole per cent. (Note that the value of casein approaches that of gluten at this pH). The observed differences i n lysinoalanine content of the three proteins at different pH values are not surprising since the amino acid compo s i t i o n , sequence, protein conformation, molecular weights of protein chains, i n i t i a l formation of intra- versus intermolecular crosslinks may a l l influence the chemical reactivity of a particular protein with a l k a l i . Therefore, i t i s not surprising to find differences i n lysinoalanine content i n different proteins treated under similar conditions. These observations could have practical benefits since, for example, the lower lysinoalanine content of casein compared to lactalbumin treated under the same conditions suggests that casein i s preferable to lactalbumin in foods requiring alkali-treatment. The postulated mechanism of lysinoalanine formation (Figure 5) i s at least a two-step process. F i r s t , hydroxide ion-catalyzed elimination reactions of serine, threonine, and cystine (and to


230

FUNCTIONALITY

TABLE

A N D PROTEIN

STRUCTURE

I

E f f e c t o f pH on amino a c i d c o m p o s i t i o n o f wheat g l u t e n . Conditions: 1% wheat g l u t e n ; 65°C; 3 hours. Numbers a r e mole ( r e s i d u e ) p e r c e n t f o r e a c h amino a c i d .


pH Amino Acid

Control

9.6

10.6

11.2

12.5

13.9

ASP

3.20

3.26

3.26

3.15

3.57

2.96

THR

3.19

3.10

3.05

3.01

2.67

1.20

SER

6.81

6.75

6.64

6.55

5.30

2.24

ALA

3.91

3.95

4.07

3.86

4.40

3.78

CYS

0.976

0.691

0.00

0.00

0.00

0.00

MET

1.35

1.25

1.18

1.33

1.68

1.17

TYR

2.49

2.55

2.43

2.49

2.43

1.85

PHE

4.35

4.29

4.52

4.35

4.29

4.93

LAL

0.00

0.00

0.262

0.420

0.762

0.884

HIS

1.87

1.80

1.83

1.78

1.74

1.71

LYS

1.33

1.40

1.16

0.963

0.945

0.948

ARG

2.75

2.70

2.66

2.68

2.61

1.79


12.

FRIEDMAN

Lysinoahnine

Formation

TABLE

231

II

E f f e c t o f pH on amino a c i d c o m p o s i t i o n o f c a s e i n . Conditions: 1% c o m m e r c i a l c a s e i n ; 6 5 ° C ; 3 h o u r s . Numbers a r e m o l e p e r c e n t f o r e a c h amino a c i d .


Amino Acid

Controls No 1

(in t r i p l i c a t e ) No 2 No 3

10.6

pH 11.2

12.5

ASP

6.85

6.81

6..85

6.93

6.96

7.04

THR

4.61

4.58

4..44

4.40

4.44

3.90

SER

7.12

7.07

7.,27

7.09

6.94

4.60

GLU

18.84

18.76

19..18

19.14

19.46

20.83

PRO

11.97

12.08

12..28

12.20

12.30

12.56

GLY

3.18

3.17

3..05

3.05

3.08

3.44

ALA

4.34

4.36

4..25

4.28

4.20

4.49

VAL

6.81

6.89

6..71

6.66

6.60

6.88

MET

2.43

2.46

2..43

2.36

2.00

2.74

I LEU

4.73

4.80

4..72

4.71

4.78

4.70

LEU

9.37

9.42

9..21

9.28

9.48

9.11

TYR

3.87

3.87

3..86

3.89

3.82

3.88

PHE

4.02

3.96

3..95

4.04

4.09

4.21

LAL

0.00

0.00

0..00

0.494

0.780

2.43

HIS

2.40

2.39

2..42

2.48

2.43

2.39

LYS

6.86

6.76

6..89

6.46

6.03

4.48

ARG

2.59

2.60

2..49

2.53

2.50

2.21


232

FUNCTIONALITY

TABLE

A N D PROTEIN

STRUCTURE

III

E f f e c t o f pH on amino a c i d c o m p o s i t i o n o f l a c t a l b u m i n . C o n d i t i o n s : 1% l a c t a l b u m i n : 6 5 ° C , 3 h o u r s . Numbers a r e mole ( r e s i d u e ) p e r c e n t values o f the t o t a l accounted f o r .


Amino Acid

PH Control

9.60

10.60

11.20

12.50

13.90

ASP

12.02

11.68

11.34

12.31

12.14

15.55

THR

6.01

6.04

6.00

5.95

5.33

2.75

SER

6.18

6.11

6.17

6.08

5.44

2.26

ALA

7.40

7.78

7.74

7.50

7.97

8.22

CYS

0.952

0.558

0.190

0.00

0.00

0.00

MET

1.84

1.96

2.12

2.06

1.92

2.14

TYR

2.59

2.56

2.75

2.70

2.90

2.42

PHE

2.93

2.91

3.04

3.00

3.21

2.72

LAL

0.00

0.255

1.04

1.52

2.62

3.87

HIS

1.66

1.60

1.67

1.64

1.51

1.20

LYS

8.94

8.57

7.47

7.45

6.48

7.19

ARG

2.14

2.22

2.15

2.12

1.61

1.32


12.

FRIEDMAN

Lysinoalanine

Formation

233

Pi U

OH H CHn-C-P I · γ NH-C-P

1. RACEMIZATION

Ο

It

±

H«

2. ^-ELIMINATION


Θ

CHo-C-P

C

1

M

NH-C-P

x

o

CARBANION INTERMEDIATE

CH2=C-P +

Υ

θ

NH-C-P

DEHYDROPROTEIN

it

±

C

- H

N - P

2

3

Η

C

R

O

S

S

L

|

N

K

FORMATION

P_CH2-C-P NH-C-P II

ι

Ο

HCI

NH

4.HYDROLYSIS

2

CH -(CH )~-CH-COOH 2

2

NH-CH -CH-COOH 2

NH

P= PROTEIN SIDE CHAIN

LYSINOALANINE

2

m

Y=OH,

OR, SH, SR, S(R) , 2

Φ SSR,

Figure 5.

N(R)

3>

ΟΡΟ3Η,

etc.

Transformation of reactive protein side chains to lysinoalanine side chains via elimination and crosslinking formation.

Hydroxide ion abstracts an acidic hydrogen atom (proton) from an α-carbon atom of an amino acid residue to form an intermediate carbanion. The carbanion, which has lost the original asymmetry of the amino acid residue, can either recombine with a proton to reform a racemized residue in the original amino acid side chain or undergo the indicated elimination to form a dehydroahnine side chain. The dehydroalanine then combines with an ε-amino group of a lysine side chain to form a crosslinked protein which on hydrolysis yields free lysinoahnine (6, 9).



234

FUNCTIONALITY

A N D PROTEIN STRUCTURE

a lesser extent probably also cysteine) give rise to a dehydroalanine intermediate. Since such elimination reactions are secondorder reactions that depend directly on the concentration of both hydroxide ion and susceptible amino acid, the extent of lysino alanine formation should vary directly with hydroxide ion concen tration. Results i n Tables I-III show that this i s indeed the case within certain pH ranges. The dehydroalanine residue, which contains a conjugated carbon-carbon double bond, then reacts with the ε-amino group of lysine i n a second, second-order step to form a lysinoalanine crosslink. This step i s governed not only by the number of available amino groups but also by the location of the dehydroalanine and amino group potential partners in the protein chain. Only residues favorably situated to form crosslinks can do so. When convenient sites have reacted, additional lysinoalanine (or other) crosslinks form less readily or not at a l l . Each protein, therefore, may have a limited fraction of potential sites for forming crosslinked residues. The number of such sites i s presumably dictated by the protein's size, composition, conformation, chain mobility, steric factors, extent of ionization of reactive amino (or other) nucleophilic centers, etc. These considerations suggest that a cascade of reactions occurs leading to lysinoalanine residues. Thus, dehydroalanine formation i s governed not only by the absolute concentration of serine, threonine, and cystine residues but by their relative s u s c e p t i b i l i t i t i e s to base-catalyzed eliminations. Thus, results in Tables I-III show that although serine and threonine destruc tion begins to take place between pH 11 and 12, cystine residues are much more sensitive to a l k a l i , since i n the case of lactalbu min, significant amounts of cystine are destroyed even at pH 9.6 (Table I I I ) . On the other hand, reaction of the ε-amino groups with dehydroalanine to form lysinoalanine depends not only on the cited steric and conformational factors but also on the pH of the medium, which governs the concentration of reactive nonprotonated amine. Since the pK of the ε-amino groups of lysine residues i s near 10 for most proteins, complete ionization of a l l amino groups does not occur u n t i l pH 12. At pH 9 only about 10% of the amino groups are ionized, and thus available for reaction (Cf. 14) (All of the amino groups can eventually react, however, since additional amino groups are formed by dissociation of the protonated ammonium ions as the nonprotonated amino groups are used up) These results, therefore, imply that the extent of lysinoala nine formation may vary from protein to protein. Factors that favor or minimize these reactions need to be studied seprately with each proteins. ABSTRACT Lysinoalanine formation i n casein, lactalbumin, and wheat gluten was measured at 65°C at various pH's for 3 hours. Factors that control the extent of formation of the unnatural amino acid lysinoalanine during food processing and thus the degree of crosslinking i n structurally different proteins are discussed.


12. FRIEDMAN

Lysinoalanine Formation

235


LITERATURE CITED 1. Friedman, M., Ed., (1977). "Protein Crosslinking: Nutritional and Medical Consequences", Plenum Press, New York, 740 pages. 2. Friedman, M., Ed., (1977). "Protein Crosslinking: Biochemical and Molecular Aspects", Plenum Press, New York, 760 pages. 3. Woodard, C.J., Short, D.D., Alvarez, M.R. and Reyniers, J . (1975). Biological effects of N-ε-(DL-2-amino-2-carboxyethyl)L-lysine, lysinoalanine. In "Protein Nutritional Quality of Foods and Feeds", M.Friedman, Ed., Marcel Dekker, New York, Part 1, pp. 595-616. 4. Gould, D. H. and MacGregor, J . T. (1977). Biological effects of alkali-treated protein and lysinoalanine: an overview. Reference 1, pp. 29-48. 5. Friedman, M. (1973). "Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides, and Proteins", Pergamon Press, Oxford, England and Elmsford, New York, Chapter 5. 6. Friedman, M. (1977). Crosslinking amino acids--stereochemis try and nomenclature. Reference 1, pp. 1-27. 7. Finley, J . W.,Snow, J . T., Johnston, P. H. and Friedman, M. (1977). Reference 1, pp. 85-92. 8. Friedman, M. (1978). Wheat gluten-alkali reactions. In "Pro ceedings of the 10th National Conference on Wheat Utilization Research", U. S. Department of Agriculture, Science and Educa tion Administration, Western Regional Research Center, Berke ley, California 94710, ARM-W-4, pp. 81-100. 9. Friedman, M. (1978). Inhibition of lysinoalanine synthesis by protein acylation. In "Nutritional Improvement of Food and Feed Proteins", M. Friedman, Ed., Plenum Press, New York, pp. 613-648. 10. Friedman, Μ., Finley, J . W. and Yeh, Lai-Sue (1977). Reactions of proteins with dehydroalanine. Reference 1, pp. 213-224. 11. Friedman, M. (1977). Effects of lysine modification on chemi cal, physical, nutritive, and functional properties of proteins. In "Food Proteins", J . R. Whitaker and S. R. Tannenbaum, Eds., Avi, Westport, Connecticut, pp. 446-483. 12. Cavins, J . F. and Friedman, M. (1967). New amino acids derived from reactions of ε-amino groups with α,β-unsaturated compounds. Biochemistry, 6, 3766-3770. 13. Friedman, M. and Noma, A. T., manuscript in preparation. 14. Friedman, M. and Williams, L. D. (1977). A mathematical analy sis of consecutive, competitive reactions of protein amino groups. Reference 1, pp. 299-319. RECEIVED

November 3, 1978. In Functionality and Protein Structure; Pour-El, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Alkali-Induced Lysinoalanine Formation in Structurally Different

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