Role of Electrostatic Forces in the Interaction of Soy Proteins with Lysozyme

Cereal Chem. 63(5):381-383 The nature of the interaction between lysozyme with soy protein isolate, soy proteins. The interaction between lysozyme and soy 11S was stronger soy I IS, and soy 7S fractions was studied using a turbidity method. than that of lysozyme and soy 7S, suggesting that the surface Complete dissociation of soy protein lysozyme complexes occurred at an electronegativity of soy 11 S was greater than that of soy 7S. It is proposed ionic strength of 0.12M; however, below this ionic strength the extent of that similar protein-protein interactions may occur in protein blends during dissociation was affected by pH. These observations suggested that processing. Such interactions may affect the functional behavior of electrostatic forces were involved in the interaction between lysozyme and individual protein components in protein blends. Oilseeds are an abundant source of food proteins; however, the Lysozyme-Soy Protein Interaction lack of certain functional properties limits their maximum A turbidometric method was used to study the interaction utilization in conventional foods (Kinsella et al 1985). Appropriate between lysozyme and soy proteins. The method is based on the chemical modification of oilseed proteins is a promising approach observation that the interaction of the positively charged lysozyme to improve the functional properties (Kinsella and Shetty 1979). with the negatively charged soy proteins leads to formation of an However, the economic feasibility and nutritional safety of insoluble complex at pH 8.0 in 10 mM Tris-HC1 buffer. The chemically modified proteins is yet to be determined. To overcome turbidity development under these conditions is used as a measure some of these obstacles and improve the functional properties of of the extent of interaction between lysozyme and soy proteins. In a oilseed proteins, research on the physicochemical and nutritional typical experiment, 1-ml aliquots of soy protein solutions (0.1% in properties of mixed proteins and coprecipitated protein blends is 10 mM Tris-HCl buffer) were treated with increasing amounts of being conducted (Berardi and Cherry 1979, 1981; Hayes et al 1978; lysozyme (0.1% in Tris-HC1 buffer) and the total volume was made Schmidt et al 1978). Berardi and Cherry (1979) reported that up to 2.0 ml by adding appropriate amounts of buffer. The coprecipitation of proteins from blends of cottonseed, soybean, and solutions were held for 5 min at room temperature, vortexed peanut flours produced new forms of protein preparations. These vigorously, and the turbidity was measured at 540 nm with a new proteins conceivably resulted from dissociation and Spectronic 700 spectrophotometer. subsequent reassociation of the subunit components of various In studies involving the effect of ionic strength, a constant ratio protein sources used. The hybrid proteins formed under the of lysozyme to soy protein was maintained in the final mixture, and conditions of coprecipitation influenced the functional properties the level of ionic strength was varied by adding calculated amounts of the resulting mixture when compared to the individual native of 1.OM NaCl stock solution and buffer. In these experiments, proteins (Cherry et al 1978). Incorporation of soy, whey, and yeast various solutions were added during mixing in the sequence buffer, proteins in meat as meat extenders affected the solubility, heat lysozyme, soy proteins, and NaCl stock solution. The tubes were denaturability, and emulsifying capacity (Porteous and Quinn held at room temperature for 5 min, vortexed vigorously, and the 1979). The changes in the functional behavior were attributed to turbidity at 540 nm was measured. Protein concentrations were complex formation between myosin and the soy protein quantified by the biuret method. components of the meat extenders (King 1977). To better understand and predict the functional properties of protein blends in food systems, an understanding of the RESULTS AND DISCUSSION mechanisms as well as the factors influencing the interaction The interaction of lysozyme with soy isolate, soy 1lIS, and soy 7S between proteins is desirable. Previous studies reported that fractions is shown in Figure 1. Addition of increasing amou during thermal treatment of soy protein isolate, the subunits of soy 7S fraction interact with the basic subunits of soy 11S via lysozyme to soy proteins resulted in increased turbidity. For both electrostatic forces and formed soluble complexes that reduce the soy isolate and soy 7S, the turbidity reached saturation value at a thermal coagulation of soy 11S (German et al 1982, Damodaran lysozyme-to-soy 7S weight ratio of about 1.2. In the case of soy adKinsella 1982). T better understand the electrostatic 11S, the turbidity attained saturation at a lysozyme-to-l IS weight andratio of about 0.5. The extent of turbidity development was used as properties of soy proteins that affect their behavior in protein a measure of the extent of interaction between lysozyme and soy blends, we studied the interaction of soy proteins with positively proteins. charged lysozyme under nondenaturing conditions. The development of turbidity upon addition of lysozyme to soy proteins may possibily be caused by electrostatic interaction MATERIALS AND METHODS between the positively charged lysozyme and the negatively charged soy proteins. This interaction, which is analogous to p H Preparation of Soy Proteins titration of proteins, decreases the net charge of the soy proteinWhole soy protein was isolated from defatted and low-heat lysozyme complex at pH 8.0. The apparent elimination of treated soy flour (Central Soya, Chicago) as described elsewhere eltrsairpuiopomedgrgtonfthecmles (Damdarn ad Kisela 181) Thesoy11Sand S gobuins via hydrophobic interaction between the nonpolar patches on the were isolated as described by Thanh and Shibasaki (1976). These sufcofteptinmlue. two protein fractions were about 90 and 70% pure, respectively, as To confirm the involvement of electrostatic interactions in judged from sodium dodecyl sulfate polyacrylamide gel complex formation between soy proteins and lysozyme, the extent electrophoresis (Utsumi et al 1984). Lysozyme was purchased from oftritydvlpetavrouNalcnnrtosws Sigma Chemical Co. (St. Louis, MO). All other chemicals used in studied. The extent of interaction of lysozyme with soy isolate and this study were of reagent grade. soy 7S decreased at higher ionic strength (Fig. 2A and B). Above 0.1M NaC1 the development of turbidity was completely 'Institute Food Science, Cornell University, Ithaca, NY 14853. suppressed even at higher lysozyme-to-soy isolate or soy 7S ratios. _____________________________________________ Because the major noncovalent force that is affected in the range of © 1986 American Association of Cereal Chemists, Inc. 0-0.2M ionic strength is the electrostatic interaction (Eagland Vol. 63, No. 5, 1986 381 1975), the results suggest that the mode of interaction between 2.5 lysozyme and soy isolate or soy 7S is electrostatic in nature. This A nonspecific interaction apparently reduces the net surface charge OM density of the soy protein-lysozyme complex, which promotes hydrophobic aggregation and development of turbidity in the 2.0system. Unlike soy isolate or soy 7S, the ionic strength dependence of C0 lysozyme-soy 11 S interaction was more complex (Fig. 2C). At zero 0 0 NaCl concentration, the development of turbidity as a function of to 1.5 lysozyme to soy 11 S exhibited a hyperbolic curve and reached maximum turbidity at a ratio of lysozyme to soy 1 S of about 0.5. At 0.075M NaCl the turbidity development was sigmoidal, and the " turbidity did not reach saturation value even at the lysozyme-to" 1.0 0 -O soy 1 lS ratio of 1.3. However, turbidity development was L markedly suppressed at 0.1 M NaCl concentration. The sigmoidal nature of lysozyme-soy 1 IS interaction at 0.05M and 0.075M NaCl 0. concentrations may be attributed to changes in the oligomeric 0.5 structure of soy I1S at various ionic strengths (Eldridge and Wolf 0.1M 1967, Koshiyama 1972). It is known that at 0.5Mionic strength, the soy I1S protein exists as an oligomeric species that contains six acidic and six basic subunits and has a sedimentation velocity of 0 0.2 0.4 0.6 0.8 1.0 1.2 11S. When the ionic strength is decreased to 0.1 M, two molecules of the l lS species associate to form a 15S species. When the ionic strength is further decreased below 0.1M, the llS species dissociates into two identical units having a sedimentation coefficient of 7S. Such changes in the oligomeric structure of soy 11S protein between 0.01 and 0.1 M ionic strength may alter its 2.5 electrostatic properties, which may affect the extent of its B interaction with lysozyme. Nonetheless, the data show that above 0.1 M ionic strength the lysozyme-soy llS interaction is 0 M suppressed, indicating involvement of electrostatic forces. 2.0The effect of pH on the dissociation of lysozyme-soy 7S and lysozyme-soy 11S complexes as a function of ionic strength is Z shown in Figure 3. In these experiments, both the lysozyme-to-soy 0 7S and lysozyme-to-soy 11S ratios were maintained at a constant Lo 1.5value of 0.8:1, and the development of turbidity at various ionic strengths in the medium was taken as the extent of complex formation. The 0.8:1 protein ratio was chosen on the basis of the data presented in Figure 1, in which both lysozyme-soy 7S and 1.0lysozyme-soy lI S systems exhibited near saturation turbidity values at the 0.8:1 protein ratio. Because precipitation of soy 11S 0-0M occurs below pH 7.0 in Tris-HC1 buffer (Thanh and Shibasaki I1976), the dissociation experiments in the case of lysozyme-soy 11S 0.52.5' 0. 1 M . • . ! ., • .. . IA 0 0.2 0.4 0.6 0.8 1.0 1.2 2. Lysozyme/Soy 7S (gig)