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Sword bean (Canavalia gladiata) seeds are a traditional food in Asian countries. In this study, we aimed to determine the optimal methods for the precipitation of sword bean proteins useful for the food development. The soaking time for sword beans was determined by comparing it with that for soybeans. Sword bean proteins were extracted from dried seeds in distilled water using novel methods. We found that most proteins could be precipitated by heating the extract at more than 90 °C. Interestingly, adding magnesium chloride to the extract at lower temperatures induced specific precipitation of a single protein with a molecular weight of approximately 48 kDa. The molecular weight and N-terminal sequence of the precipitated protein was identical to that of canavalin. These data suggested that canavalin was precipitated by the addition of magnesium chloride to the extract. Our results provide important insights into the production of processed foods from sword bean.

The formation of transparent gels by 6% bovine serum albumin (BSA), pH 7.5, was examined by one- and two-step heating methods. Heating of the BSA solutions at various NaCl concentrations produced transparent gels at 25-50mM NaCl and transparent sols at 0-20 mM NaCl (one-step heating method). The transparent sol obtained by heating without NaCl was reheated after mixing with various amounts of NaCl (two-step heating method I). The result was almost identical to that obtained by the one-step heating method. However, when the first heating was done with 10 mM NaCl, transparent gels were obtained over a wide range of NaCl concentrations with a second heating (two-step heating method II). Analyses of sols obtained at various NaCl concentrations by gel permeation chromatography and transmission electron microscopy showed the presence of linear polymers in the heated BSA sol (10 mM NaCl) and gel networks formed by the linear polymers (20 mM NaCl). The mechanism of transparent gel formation in BSA may be similar to that in ovalbumin.

We investigated the effect of the carbohydrate chain and two phosphate moieties on heat-induced aggregation of hen ovalbumin. The dephosphorylated form of ovalbumin was obtained by treating the original protein with acid phosphatase. The single carbohydrate chain was removed by digestion of heat-denatured ovalbumin with glycopeptidase F, and the resulting polypeptide without this carbohydrate chain was correctly refolded to acquire protease-resistance. Thermal unfolding can be approximated by a mechanism involving a two-state transition between the folded and unfolded states with a midpoint temperature of 76 degrees C for the original form, of 74 degrees C for the dephosphorylated form, and of 71 degrees C for the carbohydrate-free form. The conformational stability of the original form was higher than that of the carbohydrate-free form. When the three forms of ovalbumin were heated to 80 degrees C and then cooled rapidly in an ice bath, the polypeptide chains were compactly collapsed to metastable intermediates with secondary structures whose properties were indistinguishable. Upon incubation at 60 degrees C, renaturation was possible for a large portion of the intermediates of the original form, but for only a small portion of those of the carbohydrate-free form. Light scattering experiments showed that in the presence of sulfate anions, the intermediates of the carbohydrate-free form aggregated to a greater extent than did those of the original form. The intermediates of the carbohydrate-free form bound to the chaperonin GroEL with about 10-fold higher affinity than those of the original form. It follows that the carbohydrate chain and the two phosphate moieties do not affect hydrophobic collapse in the kinetic refolding of hen ovalbumin but play an important role in the slow rearrangement. They block the off-pathway reaction that competes with correct refolding by effectively decreasing surface hydrophobicity.

beta-Lactotensin (beta-LT: His-Ile-Arg-Leu) is an ileum-contracting peptide derived from residues No. 146-149 of bovine beta-lactoglobulin. The ileum-contracting activity of beta-LT was blocked by the NT1 antagonist SR48692. beta-LT was selective for the neurotensin NT2 receptor while neurotensin was selective for the NT1 receptor. beta-LT is the first natural ligand showing selectivity for the NT2 receptor. beta-LT showed hypertensive activity after intravenous administration at a dose of 30 mg/kg in conscious rats, while neurotensin showed hypotensive activity. The hypertensive activity of beta-LT was blocked by levocabastine (1 mg/kg, i.v.), an NT2 antagonist. SR48692, which blocked the hypotensive activity of neurotensin, had no effect on the hypertensive activity of beta-LT. These results suggest that the hypertensive activity of beta-LT is mediated by the NT2 receptor. It was concluded that the NT1 and NT2 receptors mediate the opposite effect on blood pressure.

Soybean 7S and 11S globulins, which are two major components of soybean protein, were mixed in various ratios. Gelation of the protein blend after the addition of glucono-δ-lactone (GDL) or calcium sulfate was studied to analyze the gelation process of tofu (soybean curd). Dynamic viscoelasticity measurements at a constant temperature as a function of time were done. Faster gelation was observed in mixtures containing 11S in a higher proportion for both the GDL and calcium systems. The value of the storage modulus of the 5:5 blend system of 7S and 11S globulins was minimum when the GDL concentration was adjusted to 17 mM, at which level the gels consisting of 7S globulin alone and 11S globulin alone had a similar storage modulus. Under the same conditions, although the gel of the soybean protein isolate showed almost the same value for the storage modulus as a mixture of 7S: 11S in the same ratio, the gelation was slower than that expected from the 7S and 11S blend. Since the use of calcium sulfate led to...

The effects of ionic strength and dithiothreitol concentration, a reducing agent, on the heat-induced gelation of a lysozyme solution were examined at pH 7. Two different heating methods were used, one involving single heating, and the other, a two-step heating process in which a sample solution was heated twice under different medium conditions. With 7.5 mM dithiothreitol, a 5% lysozyme solution gave a transparent sol, a transparent gel, a translucent gel and a turbid gel after a single heating with 0, 40, 50, and 60mM NaCI, respectively. When the transparent sol obtained without NaCI was reheated with 50mM NaCI, it produced a firm and transparent gel. This gel was at least 6 times harder than the transparent lysozyme gel obtained by the one-step heating method. High-resolution scanning electron microscopy showed that the transparent gel produced by the two-step heating method had a fine network of linear polymers composed of heat-denatured lysozyme molecules.

Aggregation occurs through hydrophobic interactions when a polypeptide chain refolds in non-native states or when genetic variants of biologically active proteins assume inappropriate conformations, as observed in the case of dysfunctional serpins. Here, using the molecular chaperone BiP from bovine liver microsomes, we characterized the hydrophobic nature of the peptide segment which is considered to be a site required for aggregation among a non-inhibitory serpin ovalbumin in a heat-denatured state. Screening of the peptide scan for binding of BiP showed that BiP-binding sites are mostly buried in the folded ovalbumin. When ovalbumin was heat-denatured, the denatured protein was recognized by the antibody that reacts with the hydrophobic surface of the amino-terminal segment of ovalbumin. This antibody significantly suppressed the binding of BiP to denatured ovalbumin. BiP also bound the immobilized peptide in an ATP-dependent manner and the peptide stimulated the ATPase activity of BiP with a Km of 165 microM and a Vmax of 0.4 nmol/min per milligram. Measurement of surface plasmon resonance showed that the peptide had a Kd of 0.52 microM by BiP, lower than that for RCMLA (Kd = 1.1 microM) and even lower than that of the peptide P10K, PLSRTLSVAAKK, (Kd = 21 microM). These results demonstrate that the aggregation-prone site on heat-denatured ovalbumin has almost the same hydrophobic nature of interacting with the molecular chaperone BiP as the conventionally known peptides that bind to the Escherichia coli chaperone DnaK.