Inactivation of Egg Trypsin Inhibitors by the Maillard Reaction

Chapter 20

Inactivation of Egg Trypsin Inhibitors by the Maillard Reaction A Biochemical Marker of Lysine and Arginine Modification 1

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Downloaded by UNIV OF MICHIGAN ANN ARBOR on April 3, 2017 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0631.ch020

Y. Kato and T. Matsuda 1

Department of Clinical Nutrition, Kawasaki University of Medical Welfare, 288 Matsushima, Kurashiki, Okayama 701-01, Japan Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan

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Ovomuocids of chicken and Japanese quail eggs inhibit trypsin by forming a stable enzyme-inhibitor complex through the arginine and lysine residues at their reactive sites, respectively. Chemically modified arginine and lysine residues of the two ovomucoids by the Maillard reaction with reducing sugars were determined by the Sakaguchi's method and the fluorometric method using fluorescamine, respectively. The decrease in trypsin inhibitory activities of chicken ovomucoid was faster than that of quail ovomucoid, and the activity loss of chicken and quail ovomuocids were corresponding to the decrease in their free guanidino and amino groups, respectively. No activity loss of chicken ovomucoid was induced by the reaction with maltose, whereas quail ovomukoid was markedly inactivated, suggesting that the arginine residue is not modified directly with the carbonyl group of reducing sugars but with some active compounds degraded from the reducing sugars at the later stages of the Maillard reaction. Food proteins are often denatured and/or chemically modified during heating, drying and storage of food. One of common protein modification events is Maillard reaction (7, 2), in which protein side chains react with carbonyl groups of reducing sugars. The initial step of this reaction is that the primary ε-amino groups of lysine side chains preferentially react as nucleophiles with carbonyl groups, resulting in the formation of stable glycosylamine intermediates such as Amadori compounds, which are detected in various processed and stored foods (3). The Amadori compounds are degraded to the deoxyosones, reactive ocdicarbonyl compounds, in neutral and acidic pH (3). Such reactive compounds interact with various food components including proteins, leading to formation of complex Maillard products. Modification of food proteins with reducing sugars through Maillard reaction has been evaluated by various analytical methods; determination of free amino groups, amino acid analysis after acid hydrolysis, detection of brownish pigments and fluorescent compounds. Some Maillard products have also been analyzed immunochemically using specific antibodies. Egg white is composed of about 10% protein and 0.5% glucose. Maillard reaction of egg white proteins with glucose proceeds during the production of dried egg white powder, resulting in the formation of brownish pigments and the alteration of powdered protein functional properties such as solubility, emulsifying activity , gelling property (4). 0097-6156/96/0631-0227$15.00/0 © 1996 American Chemical Society Lee and Kim; Chemical Markers for Processed and Stored Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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Day Figure 1. Decrease in free amino- (O) and guanioino- (Δ) groups of chicken ovomucoid by the incubation with glucose. The powdered ovomucoid was incubated with (closed symbol) or without (open symbol) glucose at 50 C for 15 days. e

COM-glu i

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Day Figure 2. Decrease in trypsin inhibitory activity of chicken- (O) and Japanese quail- (Δ) ovomucoids by the incubation with glucose. The powdered ovomucoids were incubated with (closed symbol) or without (open symbol) glucose at 50 C for 15 days. e

Lee and Kim; Chemical Markers for Processed and Stored Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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KATO & MATSUDA

Inactivation of Egg Trypsin Inhibitors

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About 10% of egg white proteins is a glycoprotein, ovomucoid(5). This glycoprotein with Mr of about 28,000 has been known to inhibit trypsin by forming stable enzymeinhibitor complexes (6). The amino acid sequence around the inhibitory reactive site is well conserved among ovomucoids from various avian species, and amino acid residues of reactive sites are lysine or arginine (6). Chicken ovomucoid has one reactive site of arginine for trypsin, while Japanease quail ovomucoid has two reactive sites of lysine for trypsin. Chemical modification such as acetylation of lysine residues of Japanese quail ovomucoid leads to inactivation of its inhibitory activity against trypsin (7). Therefore, it seems likely that modification with reducing sugars through Maillard reaction also inactivate Japanese quail ovomucoid with lysine-type reactive sites. In the advanced stages of Maillard reaction, protein arginine residues are also modified probably with various degradation compounds derived from sugar-lysine reaction products (8). In the present study, we investigated decrease in trypsin inhibitory activity of ovomucoids by the Maillard reaction with reducing sugars and examined relationship between the biochemical activity and the other chemical markers of the Maillard reaction. Reaction of quail and chicken ovomucoids with glucose Free amino- and guanidino-groups. As a model system of protein-sugar Maillard reaction, a powdered sample containing ovomucoid and glucose was incubated at 50 °C and 65% relative humidity for various periods from 2 to 20 days. The incubated samples were dissolved in 50 mM Tris-HCl buffer, pH 8.0 and used for chemical and biochemical analyses. Free amino- and guanidino groups of incubated ovomucoids were determined by a fluorometric method usingfluorescamine(9) and the method of Sakaguchi (10,11), respectively. A representative result on decrease in free amino- and guanidino-groups of chicken ovomucoid is shown in Figure 1. Free amino group rapidly decreased to 15% after the 2 day-incubation, and the residual free amino group was only a few percent after the 20 day-incubation. Free guanidino group also decreased but the reaction proceeded more slowly than that of amino group. About 50 and 25% of free guanidino group remained even after 5- and 15-day incubations, respectively. Similar results were obtained for Japanese quail ovomucoid. No decrease of amino and guanidino groups were observed for ovomucoids incubated in the absence of glucose. Trypsin inhibitory activity. Trypsin inhibitory activity of ovomucoids were measured with a-N-benzoyl-L-arginine p-nitroanilide as a trypsin substrate (12) for samples incubated with glucose for various periods. As shown in Figure 2, the activity of Japanese quail ovomucoid decreased rapidly for the first five days of incubation, but about 25% of the original activity remained even after the 20 day-incubation. The decreasing profile of Japanese quail ovomucoid was relatively in good agreement with that of free amino group. On the other hand, chicken ovomucoid did not loose its trypsin inhibitory activity for the 5 day-incubation, even though free guanidino group decreased to about 50% after the 5 day-incubation. Then the activity rapidly decreased to about 30% after the 10 day-incubation. It seems reasonable that TI activity loss of chicken ovomucoid did not correlate with the decrease in free amino group, because chicken ovomucoid has a reactive site of arginine. However, the TI activity loss did not necessarily correlate to the loss of the free guanidino group, especially for the first 5 days of incubation. This suggests that chemical modification of the reactive site arginine occurred with frequency lower than the other arginine residues of an ovomucoid molecule. Furthermore, Japanese quail ovomucoid which lost most of its free amino group still retained some extent of TI activity. Therefore, lysine or arginine at the reactive site of ovomucoid for trypsin inhibition might not easily be attacked by reducing sugars as compared with the these residues of the other part of a molecule, or ovomucoids modified with glucose might still retain weak affinity to trypsin.



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CHEMICAL MARKERS FOR PROCESSED AND STORED FOODS

Glu

Mal

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Glu

Mal

Figure 3. Decrease in free amino- and guanidino-groups of chicken ovomucoid by the incubation with glucose (Glu) and maltose (Mai). The powdered ovomucoid was incubated with (Glu, Mal) or without (-)sugars at 50 °C for 15 days.

Chicken ovomucoid

Quail ovomucoid

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Figure 4. Decrease in trypsin inhibitory activity of chicken- and Japanese quailovomucoids by the incubation with glucose (Glu) and maltose (Mai). The powdered ovomucoids were incubated with (Glu, Mal) or without (-)sugars at 50 C for 15 days. e


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Reaction of ovomucoid with glucose and maltose Since the structure of reducing sugars was reported to affect the progress of Maillard reaction (13), the reaction of ovmucoids with glucose and maltose was investigated and the effect of reaction on TI activity of chicken and Japanese quail ovomucoids was compared. The free amino group was rapidly decreased by the reaction not only with glucose but also with maltose (Figure 3). The TI activity of Japanese quail ovomucoid was markedly decreased by the reaction with maltose, as well as glucose, during the incubation for 15 days (Figure 4). On the other hand, the TI activity of chicken ovomucoid was not affected by the reaction with maltose, though the activity was decreased considerably by the reaction with glucose. The rapid decrease in TI activity of Japanese quail ovomucoid by the reaction with maltose indicates that there is no large difference between glucose and maltose in the initial attacking efficiency against the reactive site lysine. On the contrary, no effect of maltose on TI activity of chicken ovomucoid suggests that degradation of maltose-lysine reaction products to active compounds is much slower than that of glucose-lysine reaction products. Literature Cited (1) Reynolds, T.H. Cemistry of nonenzymatic browning. I. The reaction between aldoses and amines. Adv. Food Res. 1963, 12, 1-52. (2) Namiki, M . Chemistry of Maillard reactions: Recent studies on the browning reaction mechanism and the development of antioxidants and mutagens. Adv. Food Res. 1988, 32, 115-184. (3) Ledl, F. Chemical pathways of the Maillard reaction, in "The Maillard Reaction in Food Processing, Human Nutrition and Physiology" Finot, P.Α., Aeschbacher, H.U., Hurrell, R.F., Liardon, R. Eds. Birkhauser Verlag, Basel, 1990, pp 19-42. (4) Kato, Y . , Matsuda, T., Kato, N . , Watanabe, K., Nakamura, R. Browning and insolubilization of ovalbumin by the Maillard reaction with some aldohexaoses. J. Agric. Food Chem. 1986, 34, 351-355. (5) Beeley J.G. The isolation of ovomucoid variants differing in carbohydrate composition. Biochem. J. 1971, 123, 399-405. (6) Kato I., Schrode J., Kohr, W.J., Laskowski M.Jr. Chicken ovomucoid: Determination of its amino acid sequence, determination of the trypsin reactive site, and preparation of all three of its domains. Biochemistry 1987, 26, 193-201. (7) Stevens, F.C. & Feeney, R.E. Chemical modification of avian ovomucoids. Biochemistry 1963, 2, 1346-1352. (8) Cho, R.K, Okitani, Α., and Kato, H . Polymerization of proteins and impairment of their arginine residues due to intermediate compounds in the Maillard reaction. in "Amino-carbonyl reactions in food and biological systems. Development in Food Science. 13" Fujimaki, M . , Namiki, M . , Kato, H . Eds. Elsevier, Amusterdam, 1986, pp 439-448. (9) Böhlen, O.; Stein, S.; Dairman, W.; Untenfriend, S. Fluorometric assay of proteins in nanogram range. Arch. Biochem. Biophys. 1973, 155, 213-220. (10) Sakaguchi, S. A new color reaction of protein and arginine. J. Biochem. 1925, 5, 25-31. (11) Albanese, A.A.; Irby, V.; Sur, Β. The colorimetric estimation of protein in various body fluids. J. Biol. Chem. 1946, 166, 231-237. (12) Waheed Α., Salahuddin A. Isolation and characterization of a variant of ovomucoid. Biochem. J. 1975, 47 139-144. (13) Kato, Y., Matsuda, T., Kato, N . & Nakamura, R. Maillard reaction of disaccharides with protein: suppressive effect of nonreducing end pyranoside groups on browning and protein polymerization. J. Agric. Food Chem., 1989, 37, 1077-1081.


Inactivation of Egg Trypsin Inhibitors by the Maillard Reaction

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