Polymerization of Soybean 11S Globulin Due to Reactions with Peroxidizing Linoleic Acid

Transcription

1 Agric. Biol. Chem., 42 (4), 781 `786, 1978 Polymerization of Soybean 11S Globulin Due to Reactions with Peroxidizing Linoleic Acid Kazuko SHIMADA and Setsuro MATSUSHITA Research Institute for Food Science, Kyoto University, Kyoto Received October 14, 1977 Soybean 11S globulin was polymerized by incubating with peroxidizing linoleic acid. The molar ratio of the acidic subunits to the basic subunits of 11S globulin decreased with the elapse of the incubation time. The acidic subunits were lost faster and formed polymers more easily than the basic subunits. The acidic and basic subunits in 11S globulin were fractionated by DEAE-Sephadex gel chromatography. Each of the acidic and basic subunits was allowed to react with peroxidizing linoleic acid individually. The results also showed that the acidic subunits formed polymers faster than the basic subunits. Both succinylated and acetylated 11S globulins were also submitted to the incubation with peroxidizing linoleic acid. The polymerization of the modified protein was suppressed by masking c-amino groups. Peroxidizing lipid in the processing and storage of foods damages proteins and amino acids extensively. Unsaturated fatty acids are oxidized by a free-radical mechanism, and hydroperoxides are accumulated as the primari ly stable products. Hydroperoxides decom pose easily to a variety of reactive carbonyl products. The changes in proteins reacted with peroxidizing lipid are as follows: (a) loss of enzyme activity1); (b) destruction of specific amino acids-l; and (c) polymerization, crosslinking or scission.") Damages in proteins usually result in a decrease in protein solubili ty,5) which is responsible for a most undesirable change in food systems. This loss of solubility is probably attributed to the formation of lipidprotein complex or protein polymers. Chemical modification is known to affect greatly the functionality of proteins when used in food systems. The modification has been applied to many food proteins such as fish protein concentrate,') wheat flour protein" and egg protein." This paper describes that protein polymers are formed by incubating 11S globulin, one of the main storage proteins in soybean, with pero xidizing linoleic acid and that acylated proteins prevent the polymerization of proteins. MATERIALS AND METHODS Preparation of crude ]IS globulin (cold-insoluble fraction). Soybean meal defatted under low-tempera ture was supplied from Honen Seiyu Co. The coldinsoluble fraction was prepared from aqueous extracts of the defatted soybean meal by the method of Wolf and Sly.) The lyophilized fraction was stored at -20 C until used in the experiment. The ultracentrifugal composition of the cold-insoluble fraction contained about 90% of 11S globulin. Preparation of peroxidizing linoleic acid. Linoleic acid was purchased from Tokyo Kasei Kogyo Co. Before used for experiments, linoleic acid was auto xidized in air for 3 days at 40 C. This preparation contained hydroperoxides and their secondary degraded products. Reduction and carboxyamidemethylation of HS globulin. Crude 11S globulin (150 mg) was dis solved in 5 ml of 0.05 M Tris-HCL buffer containing 8 M urea, ph 8.5, and 5µl of 2-mercaptoethanol was added in the solution for the reduction of interchain disulfide bonds, and left for several hours. The car boxyamidemethylation for preventing the protein from recombination of free sulfhydryl groups was performed as follows. The reduced protein solution was mixed with 37 mg of iodoacetoamide and a small amount of crystal of Tris(hydroxymethyl)aminomethane. Then the mixture was allowed to stand overnight in the dark. The reduced and carboxyamidemethylated protein (RCAM-protein) obtained was dialyzed against 0.12 M sodium phosphate buffer (ph 6.7) containing 6 M urea in the dark at 4 C. Ion exchange chromatography. The acidic and basic

2 782 K. SHIMADA and S. MATSUSHITA subunits of crude 11S globulin were fractionated ac cording to the procedure of Kitamura et al.10) DEAE- Sephadex A-50 column (2.5 x 40 cm) was equilibrated with 0.12 M sodium phosphate buffer (ph 6.7) containing 6 M urea. Three hundreds mg of RCAM-protein was applied to the column, and eluted with the buffer. After eluting of the basic subunit fraction, the acidic subunit fraction was eluted stepwise with the phosphate buffer (ph 6.7) containing 0.4 M NaCL. Each of the acidic and basic subunit fraction was dialyzed against distilled water and lyophylized. A little contaminant appeared in the SDS-gel electrophoresis of both acidic and basic subunits. Acylation of crude 11S globulin. Succinylation was performed by the procedure of Groninger.11) A 5% aqueous solution of crude 11S globulin was adjusted to ph 8.0 with 1 N NaOH. Incremental amounts of succinic anhydride were added to the suspension over a 1-2 hr period with stirring, maintaining ph between 7.5 and 8.5 with 2 N NaOH. When the ratio of succinic anhydride to protein was 1: 10, 1: 20 and 1: 30 (anhy dride: protein, w/w), succinylated s-amino groups were 90 %, 73 % and 37 % of the total s-amino groups of the protein, respectively. The succinylated protein was precipitated in the isoelectric range by adjusting ph to 4.2 with I N HCL, and the supernatant was removed after centrifugation. The precipitated protein was suspended in 5-10 volumes of distilled water and neutralized with I N NaOH. The sodium salt of succinylated protein was lyophylized. Acetylation was performed by the procedure of Riordan and Vallee.12) Acetic anhydride was added slowly to a 5 % dispersion of 11S globulin in water with concurrent addition of 2 N NaOH to maintain ph 7.5. After the reaction was completed, the modified protein was recovered by a manner similar to that for succinylation. The extent of acylation of the protein was determined by the method of Kadade and Liener.13) Reaction between crude HS globulin and peroxidizing linoleic acid. In 10 ml of water containing 0.02M NaN3, 0.5 g of 11S globulin was dissolved and adjusted to ph 7.0 with 1 N NaOH. To the protein solution 0.5 ml of autoxidized linoleic acid (POV 890 meq/kg) was added. After stirring for 10 min, the reaction vessel was placed in a constant-temperature shaking bath at 40 C. The reaction solution was defatted twice with ethanol, and the defatted protein was lyophylized. A sample of zero time was withdrawn immediately after stirring for 10 min. SDS-polyacrylamide gel electrophoresis. Gels of 7.5% (w/w) polyacrylamide were prepared as described by Ornstein14) and Davis.15) Proteins were dissolved in 0.1 M sodium phosphate buffer containing 0.5 SDS and 0.02 M 2-mercaptoethanol and incubated overnight at room temperature. The samples were run at 8 ma/gel for 2.5 hr. The gels were stained with a solution of 0.2% Coomassie Brilliant Blue G-250 in water-methanol-acetic acid (5: 5: 1, v/v/v), and destained by 7 % acetic acid. The gel profiles were recorded and determined by scanning the gels with a Shimadzu dual-wavelength chromatoscanner CS-910. For measuring the amounts of the acidic and basic subunits of native 11S globulin, both subunits on a polyacrylamide gel were cut and extracted with 1 N NaOH, respectively. The amounts of the extracted subunits were determined by the method of Lowry et al.18> The molar ratio of the subunits (acidic/basic) remained unpolymerized on the 11S globulin-peroxidi zing linoleic acid system was derived from the measured value and the results of densitometric gel-scanning. Amino acid analysis. Each of the acidic and basic subunits fractionated by DEAE-Sephadex gel chro matography was hydrolyzed in 6 N HCL at 110 C for 24, 48 and 72 hr. Analysis was carried out with a Hitachi KLA-5 amino acid analyzer by the method of Spackman et al.17> Threonine and serine were esti mated by extrapolating back to zero time. For valine, the value hydrolyzed for 72 hr was adopted. Half cystine was determined as carboxyl methyl cysteine. RESULTS Polymerization of HS globulin he system containing 11S globulin and peroxidizing linoleic acid was incubated at 40 C, and protein polymers were formed. Figure l shows the SDS-gel electrophoretic profiles of the reaction mixture. The amount of each polymer increased with the incubation time and the size grew gradually. Many poly merized proteins were observed after 132 hr of incubation. Eleven S globulin consists of six acidic and six basic subunits18) and therefore the molar ratio (acidic/basic) is 1. Figure 2 shows that the remained molar ratio decreases with the incubation time. The molar ratio was 0.54 after 156 hr of incubation. Consequently, the acidic subunits were lost faster than the basic subunits on the 11S globulin-peroxidizing lino leic acid system. Polymerization of the acidic and basic subunits Each of the acidic and basic subunits iso lated by DEAE-Sephadex gel chromatography

3 Polymerization of Soybean Protein 783 showed that the acidic subunits polymerized faster than the basic subunits. Polymerization of modified 11 S globulin On a system containing modified 11S glo bulin and peroxidizing linoleic acid, polymeri zation of proteins was examined. Figure 4 shows the SDS-gel electrophoretic profiles. Ninety per cent-succinylated protein formed little polymers even after 132 hr of incubation, and the amount of each polymerized protein gradually increased with the decrease of suc cinylated ċ-amino groups (Fig. 4-A). Figure 4-B shows the results of incubating acetylated- 11 S globulin and peroxidizing linoleic acid. The increase of polymerized proteins was observed with the decrease in the amount of acetylated s-amino groups. Polymerization of succinyl ated proteins were prevented more effectively than that of acetylated proteins. FIG. 1. SDS-polyacrylamide Gel Electrophoretic Profiles of the Reaction Products of 11S Globulin with Peroxidizing Linoleic Acid. The incubation period: A, 0 hr; B, 132 hr. For the other experimental conditions, see METHODS. a, acidic subunit; b, basic subunit. FIG. 2. Reaction of 11S Globulin with Peroxidizing Linoleic Acid. The incubation period is indicated in the figure. For the other experimental conditions, see METHODS. was incubated with peroxidizing linoleic acid. Figure 3 shows that the acidic subunits react with peroxidizing linoleic acid faster than the basic subunits. The denatured and isolated subunit fractions as well as native 11S globulin DISCUSSION The interaction between proteins and peroxidizing lipids results in the polymerization of proteins and also the chain scission of proteins. Zirlin and Karel" have noted that gelatin undergoes oxidative degradation in a freezedried model system consisting of methyl lino leate and gelatin. The polymerization of pro teins increased with rising water activity. Kanner and Karel19) have reported that the mobilization function of water is important to the radical recombination. Under the present experimental condition, that is, with an aque ous system, the loss of protein subunits seems to be due to the formation of polymerized products rather than chain scission. The polymerization of proteins is considered to occur by the interaction between a-amino groups (lysine) and aldehyde by Schiff base formation or between positively charged amino acids, mainly lysine residues, and hydropero oxides via the formation of charge complex.3,20) Therefore, the lysine residues in proteins are greatly concerned in the formation of polymers. Kitamura and Shibasaki21) reported that the acidic subunit had a slightly higher lysine con-

4 784 K. SHIMADA and S. MATSUSHITA FIG. 3. SDS-polyacrylamide Gel Electrophoretic Profiles of Incubated Acidic (A) and Basic Proteins (B) with Peroxidizing Linoleic Acid. The incubation period: 1, 0 hr; 2, 104 hr. For the other experimental conditions, see METHODS. FIG. 4. SDS-polyacrylamide Gel Electrophoretic Profiles of Incubated Succinylated (A) and Acetylated Protein (B) with Peroxidizing Linoleic Acid. Figures show the percentage of modified s-amino groups to the total a-amino groups. The incu bation period: changed 132 hr. For the other experimental conditions, see METHODS.

5 Polymerization of Soybean Protein 785 TABLE 1. AMINO ACID COMPOSITION OF ACIDIC AND BASIC SUBUNIT FRACTIONS a The protein content was measured by the micro Kjeldahl method with an conversion factor of tent than the basic subunit. Ochiai-Yanagi et a1.22, also reported that the basic subunit had a higher lysine amount than the acidic subunit. The amino acid analysis of the crude 11S globulin used in the present experiments showed that the acidic and basic subunit fractions contained 4.4 g and 5.1 g lysine re sidues per 100 g protein, respectively (Table I). Accordingly, it seems likely that the amounts of lysine residues of both subunits are nearly equal or the basic subunit has a slightly higher lysine content than the other. In view of the above described polymerization-mechanism and the lysine content of the subunits, each molecular numbers of both subunits reacting with peroxidizing linoleic acid must be equal or the basic subunits polymerize faster than the acidic ones. In practice, however, the acidic subunits polymerized faster than the basic subunits (Figs. 2 and 3). Table I shows that the basic subunits have larger amounts of the hydrophobic amino acids such as alanine, valine, isoleucine, tyrosine and phenylalanine than the acidic ones. Ac cordingly, it appears that the basic subunits aggregated by hydrophobic bonds in an aque ous system. The surface of the molecules of the aggregated basic subunits becomes small and a part of lysine residues is buried in the interior of the aggregated proteins. The basic subunits therefore reacted with peroxidizing linoleic acid slower than the acidic ones. The polymerization of the acylated proteins was extensively suppressed by masking availa ble lysine residues. The succinylated proteins were more difficult to form polymers than the acetylated proteins (Fig. 4). This may be elucidated from the following reasons. Gener ally, crude 11S globulin has a relatively large amount of acidic amino acid residues such as aspartic acid and glutamic acid (Table I). Because succinylation converts cationic amino groups to anionic residues, a whole of the protein molecule becomes further negatively charged. Contact between protein molecules and hydroperoxides or among proteins would decrease by electrostatic repulsions. Another possible reason is as follows. The added carboxyl groups of succinylated proteins form ionic attraction with neighboring positive amino acid residues such as lysine, arginine and histidine, which may inhibit competitively contact between s-amino groups (lysine) and lipids. Therefore, succinylated proteins may become more difficult to form polymers than acetylated proteins which have no additional negative charges. Acylation of proteins may afford protection of the labile lysine residues in a food system, and prevent the nonenzymatic browning and the deteriorative interaction between proteins and lipids. Especially, the functional properties, for example, solubility, emulsifying capa city, foaming property, etc., are greatly im proved by acylation. Chemical modifications of food proteins must be considered in the future. Acknowledgement. This work was supported in part by a grant from the Ministry of Education of Japan.

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